DISCOVERY AND CHARACTERIZATION OF MICROBIAL ESTERASES FOR ... · DISCOVERY AND CHARACTERIZATION OF...

DISCOVERY AND CHARACTERIZATION OF MICROBIAL ESTERASES FOR FIBER MODIFICATION

by

Lijun Wang

A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science

Department of Chemical Engineering and Applied Chemistry University of Toronto

© Copyright by Lijun Wang 2009

ii

Discovery and Characterization of Microbial Esterases for Fiber

Modification

Lijun Wang

Masters of Applied Science

Department of Chemical Engineering and Applied Chemistry University of Toronto

2009

Abstract

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and

then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble

form. These were screened for activity using a range of both commercial and natural substrates.

Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any

activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase

activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of

pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge

there is only one other published report of arylesterases from archaea. The synthetic capability

of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this

project provides an arsenal of industrially significant activities that can extend the antioxidant

properties of lignin-derived molecules in a broader range of renewable products.

iii

Acknowledgments

I would like to thank my supervisor Prof. Emma Master for her guidance and encouragement. I

would like to thank the following people from SPiT lab (University of Toronto) that helped me

with gene selection, gene cloning, and protein purification and for providing guidance on

experimental techniques: Valentina Mavisakalyan, Alexander Yakunin, Greg Brown, and

Michael Proudfoot. I would also like to thank the following people that helped me with

experimental techniques, solving problems, allowed me access to their equipment, and for

providing guidance and encouragement throughout my project: Alex Tsai, Jacqueline McDonald,

Sonam Mahajan, Elizabeth Srokowski, Angelika Duffy, and everyone else in BioZone. Further,

I would like to thank Ping Hay Lam for his hard work as summer research student and his

contribution to this project. Finally, I would like to thank my family and friends for their

support and encouragement over the course of the project.

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Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgments.......................................................................................................................... iii

Table of Contents........................................................................................................................... iv

Abbreviations................................................................................................................................ vii

List of Tables ............................................................................................................................... viii

List of Figures ................................................................................................................................ ix

List of Appendices .......................................................................................................................... x

1 Introduction ................................................................................................................................ 1

1.1 Plant Biomass: Opportunities and Challenges for Renewable Materials ........................... 1

1.2 Thesis Objectives ................................................................................................................ 2

2 Literature Survey........................................................................................................................ 3

2.1 Lignocellulose Structure and Composition......................................................................... 3

2.2 Lignocellulose-active Enzymes .......................................................................................... 4

2.3 Carboxyl Esterases and Lipases.......................................................................................... 6

2.3.1 Distinguishing Lipases from Carboxyl Esterases ................................................... 6

2.3.2 Carboxyl Esterase and Lipase Structure ................................................................. 6

2.3.3 Proposed Mechanism of Esterases and Lipases...................................................... 8

2.3.4 Classification of Esterases and Lipases .................................................................. 8

2.3.5 Applications of Carboxyl Esterases and Lipases .................................................... 9

2.3.5.1 Applications in the Detergent Industry..................................................... 9

2.3.5.2 Applications in the Pharmaceutical Industry.......................................... 10

2.3.5.3 Applications in the Food Industry .......................................................... 10

2.3.5.4 Other Industrial Applications ................................................................. 10

2.3.6 Esterase and Lipase Catalyzed Transesterification............................................... 11

v

2.3.7 Potential of Carboxyl Esterases and Lipases for Lignocellulose Modification.... 12

3 Bacterial Genome Mining and Recombinant Expression of Putative Arylesterases ............... 15

3.1 Introduction....................................................................................................................... 15

3.2 Materials and Methods...................................................................................................... 16

3.2.1 Materials ............................................................................................................... 16

3.2.2 Sequence Selection of Putative Arylesterases ...................................................... 17

3.2.3 Gene Cloning ........................................................................................................ 17

3.2.4 Protein Purification ............................................................................................... 17

3.2.5 Primary Enzyme Assays Using p-Nitrophenyl (pNP) Substrates......................... 18

3.2.6 Primary Screen Using Natural Substrates............................................................. 19

3.2.7 General Properties of Arylesterases...................................................................... 19

3.2.7.1 Optimal pH and pH Stability .................................................................. 19

3.2.7.2 Thermal Inactivation .............................................................................. 19

3.2.8 Enzyme Kinetics ................................................................................................... 20

3.2.9 Construction of A Phylogenetic Tree ................................................................... 20

3.3 Results and Discussion ..................................................................................................... 20

3.3.1 Identification and High Throughput Production of Putative Arylesterases.......... 20

3.3.2 pH Optima of Purified Esterases........................................................................... 21

3.3.3 Thermal Inactivation of Purified Esterases........................................................... 25

3.3.4 Kinetic Analysis of Purified Esterases.................................................................. 25

3.3.5 Sequence Analyses for Improved Prediction of Arylesterases ............................. 25

3.4 Conclusions....................................................................................................................... 30

4 Solvent Stability of Bacterial Carboxyl Esterases ................................................................... 36

4.1 Introduction....................................................................................................................... 36


4.2.1 Materials ............................................................................................................... 38

vi

4.2.2 Effect of Detergents and Reducing-agents on Enzyme Stability.......................... 38

4.2.3 Effect of Organic Solvents on Enzyme Stability .................................................. 38

4.2.4 Effect of Ionic Liquids (ILs) on Enzyme Stability ............................................... 39


4.3.1 Effects of Detergents, Chelators and Reducing-agents on Hydrolysis Activities 40

4.3.2 Effects of Organic Solvents on Hydrolysis Activities .......................................... 41

4.3.3 Effects of Ionic Liquids (ILs) on Hydrolysis Activities ....................................... 45

5 Isolation and Characterization of Two, Solvent-Tolerant, Thermophilic Archaeal Esterases. 49

5.1 Introduction: Extreme Environments: A Source of Unique Biocatalysts ......................... 49



5.3.1 Esterase Activity with p-Nitrophenyl Substrates .................................................. 51

5.3.2 Effect of Additives and Organic Solvents on Esterase Stability........................... 57

5.3.3 Kinetic Properties.................................................................................................. 61

6 Conclusions and Recommendations ........................................................................................ 62

6.1 Summary ........................................................................................................................... 62

6.2 Recommendations............................................................................................................. 63

Bibliography ................................................................................................................................ 65

Appendices................................................................................................................................... 75

vii

Abbreviations

BLASTp

BTB

CAZymes

CBM

CE

DMSO

EDTA

GGL

GH

GlcA

GT

HEPES

HSL

IL

ITC

Lif

LiP

MeGlcA

MnP

NPS

PAF-AH

PCR

PL

pNP

SDS

SDS-PAGE

Basic Local Alignment Search Tool: protein-protein BLAST

Bromothymol blue

Carbohydrate-active enzymes

Carbohydrate Binding Module

Carbohydrate Esterase

Dimethyl Sulfoxide

Ethylenediaminetetraacetic Acid

Glycerol Glucose α-Lactose

Glycoside Hydrolase

α-D-glucosyluronic acid

Glycosyl Transferase

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Hormone-Sensitive Lipases

Ionic Liquids

Isothermal Titration Calorimetry

Lipase-specific foldase

Lignin Peroxidase

4-O-methyl-α-D-glucosyluronic acid

Manganese Peroxidase

Ammonium Phosphate Sulfate

Platelet-Activating Factor Acetylhydrolase

Polymerase Chain Reaction

Polysaccharide Lyase

p-Nitrophenol

Sodium Dodecyl Sulfate

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

viii

List of Tables

Table 2.1.Differences between lipases and esterases...................................................................... 6

Table 2.2. Summary of Enzymes Involved in Polymer Synthesis and/or Modification............... 14

Table 3.1. Activities (µmol•min-1•mg protein-1) of enzymes at 0.5 mM concentration of each substrate. ....................................................................................................................................... 23

Table 3.2. General properties of 8 characterized proteins. ........................................................... 27

Table 3.3. Kinetic parameters of 8 characterized proteins............................................................ 28

Table 3.4. Kinetic parameters of the arylesterase EstA from Lactobacillus helveticus CNRZ32 and EstB from Lactobacillus casei LILA ..................................................................................... 28

Table 4.1. Effect of detergents, inhibitor, and disulfide reducing agent on enzyme stability. ..... 41

Table 4.2. Effect of various organic solvents on enzyme stability. .............................................. 43

Table 4.3. Effect of ionic liquids on enzyme stability. ................................................................. 48

Table 5.1. Specific activities of AF_est1 and AF_est2 on different substrates. ........................... 51

Table 5.2. Effects of various surfactants on AF_est1 and AF_est2 stability. ............................... 57

Table 5.3. Effects of various organic solvents on AF_est1 and AF_est2 stability. ...................... 58

Table 5.4. Effects of ionic liquids on AF_est1 and AF_est2 stability. ......................................... 60

ix

List of Figures

Fig. 2.1. Schematic representation of the α/β fold. ........................................................................ 7

Fig. 2.1. Crystal structure of a lipase from Candida rugosa (PDB: 1TRH) .................................. 7

Fig. 2.3. Schematic representation of esterification (a) and transesterification (b) reactions...... 11

Fig. 3.1. The distribution of enzyme activities that were detected using a variety of substrates……………………………………………………………………………..…………. 23

Fig. 3.2. Thermal inactivation of RP_est1 … .............................................................................. 29

Fig. 3.3. Kinetics of RP_est1.. ..................................................................................................... 29

Fig. 3.4. Amino acid sequence alignment of 8 characterized bacterial proteins. ........................ 31

Fig. 3.5. Amino acid sequence alignment of bacterial proteins that were active on phenyl acetate........................................................................................................................................................ 32

Fig. 3.6. Amino acid sequence alignment of proteins that were not active on any tested substrates....................................................................................................................................... 33

Fig. 3.7. A phylogenetic tree of 39 enzymes that showed activity on tested substrates. ............. 35

Fig. 4.1. Effect of DMSO on enzyme activity. ............................................................................ 46

Fig. 5.1. Substrate preference of AF_est1 and AF_est2 on pNP-esters....................................... 52

Fig. 5.2. Amino acid sequence alignment of AF_est1 and AF_est2 with other esterases from Archaeoglobus fulgidus. ............................................................................................................... 53

Fig. 5.3. Amino acid sequence alignment of AF_est1 and AF_est2 with an arylesterases from Sulfolobus solfataricus.................................................................................................................. 54

Fig. 5.4. pH profiles of AF_est1 (black) and AF_est2 (grey)...................................................... 55

Fig. 5.5. Effects of pH on AF_est1 (black) and AF_est2 (grey) stability.................................... 55

Fig. 5.6. Effect of thermal inactivation of AF_est1 and AF_est2................................................ 56

x

List of Appendices

Appendix A.1 Amino acid sequence alignment of proteins that were active on phenyl acetate. . 75

Appendix A.2 Amino acid sequence alignment of proteins that were not active on any substrates............................................................................................................... 78

Appendix A.3 Amino acid sequence alignment of AF_est1 and AF_est2 with 8 bacterial esterases that were characterized in Chapter 3. .................................................... 85

Appendix B.1 Summary of the 208 genes targeted for cloning, protein expression, purification, and biochemical characterization. .................................................... 87

Appendix B.2 The schematic representations of pNP-esters, phenyl acetate, and tributyrin. ...... 93

Appendix B.3 SDS-PAGE (15%) of the purified bacterial esterases that were characterized in this study. .............................................................................................................. 94

Appendix B.4 A standard curve of p-nitrophenol at pH 7. ........................................................... 95

Appendix B.5 Absorbance reading at 400 nm of the residual activity for PP_est3 after 5 h preincubation in organic solvents. Absorbance reading of blanks for organic solvents mixed in 50 mM posstassium phosphate buffer at pH 7......................... 96

Appendix B.6 SDS-PAGE (15%) of the purified archaeal esterases that were characterized in this study. .............................................................................................................. 98

1

1 Introduction

1.1 Plant Biomass: Opportunities and Challenges for Renewable Materials

It is now generally accepted that current consumption rates of easily extracted petroleum

resources are not sustainable given the finite nature of these reserves and detrimental

environmental impacts associated with its usage. This acknowledgement has led to a renewed

interest in developing petroleum-displacing sources of energy, chemicals and polymeric

materials.

The inherent polymeric structure and carbon-rich composition of lignocellulosic biomass means

it is an important resource for the production of renewable bioproducts [1-4]. In addition to

dedicated crops for energy and chemicals, lignocellulose can be obtained from a variety of

industrial wastes, including straw, hulls, stems, and stalks from agricultural crops, slash and

sawdust from forest industries, and municipal solid wastes (paper, cardboard, yard trash, wood

products) [5]. Although a large fraction of this biomass is currently disposed of by burning or

landfill, much of it could be converted into higher-value renewable products [6].

Thermochemical conversion and bioconversion are two strategies aimed at diversifying the range

of products generated from lignocellulose. In both cases, most research efforts have been aimed

at depolymerizing biomass to building blocks for biofuel production. However, it is anticipated

that the synthesis of high-value products from plant fiber is an important means to reducing

biofuel costs and stabilizing biofuel markets. The importance of high-value products from plant

biomass is perhaps particularly relevant to Northern climates where the biomass feedstocks are

slower to grow.

Cellulose, hemicellulose and lignin possess many active functional groups that are susceptible to

chemical reactions. Modifications on celluloses have been increasingly investigated over last

decades in order to confer new properties to fiber surfaces, such as water retention and surface

reactivity [2,3,7]. However, extensive chemical modification of cellulose can compromise the

integrity of cellulose microfibrils, such as in the case of super-adsorbing hydrogels [8].

Therefore, coating or lamination is used when retention of fiber strength is required [4]. These

coatings are used to improve the compatibility of cellulose fibrils with typical hydrophobic

2

matrix polymers [4]. While conventional coatings include synthetic resins [9], the common β-

(1,4)-linked backbone structure of cellulose and hemicellulose can be harnessed to create

biodegradable alternatives to synthetic resins. For example, xyloglucan endotransglycosylases

have been used to covalently link various functional groups to xyloglucan, which is then used to

coat cellulose fibrils [4, 10-12]. In this way, entirely “green” biocomposites can be synthesized

using both renewable compounds and catalysts. Similarly, lipases have been used to acetylate

cellulose, as well as catalyze regio-specific addition of aliphatic side-groups to xyloglucan

oligosaccharides [12]. Indeed, given their specificity and mild reaction requirements, enzymes

are ideal catalysts for plant fiber engineering since corresponding substrates are typically highly

functionalized, and resulting products are more likely to retain desired degree of polymerization

and crystallinity characteristics. Still, significant challenges to realizing the full potential of

lignocellulose fiber and its polymeric components include 1) extraction of intact biopolymers

from plant fibers; 2) specific, reproducible modification of biopolymer chemistry and properties,

and 3) the limited repertoire of industrially relevant enzymes to perform corresponding catalyses.

1.2 Thesis Objectives

The overall objective of my Master’s thesis is to increase the arsenal of industrially significant

enzymes for plant polymer engineering, taking advantage of microbial genome sequences and a

high-throughput protein production platform. More specifically, my objectives are to:

1. Discover novel carboxyl esterases (particularly arylesterases) from microorganisms with

publicly available genome sequences.

2. Recombinantly produce and characterize new esterases on the basis of substrate specificity,

optimal pH, pH stability, thermal inactivation, kinetics as well as solvent tolerance in a variety of

detergents, organic solvents, and ionic liquids.

3

2 Literature Survey

2.1 Lignocellulose Structure and Composition

Lignocellulose is the major structural component of plants and is comprised of the two most

abundant polymers on earth, cellulose and lignin [1]. In addition to cellulose and lignin,

lignocellulose contains different hemicelluloses, as well as varying amounts of pectin, protein

and waxes [2]. Cellulose is a linear polymer of β-(1,4)-D-glucose, where the repeating unit is

cellobiose. These glucan polymers adhere to each other through hydrogen bonding and

hydrophobic interactions to form crystalline microfibrils; microfibrils also associate to form

highly organized cellulose fibrils [3]. Hemicellulose represents approximately 20 to 35 % of the

lignocellulosic biomass, and has a branched structure consisting of a variety of sugars including

pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids [4]. This

class of molecules is also called crosslinking polysaccharides given their interaction with both

lignin and cellulose. Like cellulose, backbone sugars of hemicellulose are linked through β-

(1,4)-linked glycosidic bonds. Xylans are the most abundant hemicelluloses, which predominate

in secondary walls of hardwoods and the fibrous fraction of agricultural feedstocks. Xylan

consists of a homopolymeric backbone chain of β-(1,4)-D-xylopyranose units, and can be

branched by short α-D-glucosyluronic acid (GlcA), 4-O-methyl-α-D-glucosyluronic acid

(MeGlcA), α-L-arabinosyl, O-acetyl, feruloyl, or coumaroyl groups [3]. The frequency and

composition of these side groups depend on the source of xylan [5]. Mannan is the primary

hemicellulose in softwood fiber; the backbone structure of mannan consists of β-(1,4)-linked

mannose or a combination of glucose and mannose residues. The mannan backbone can be

substituted with side chains of α-1,6-linked galactose residues, resulting in four subfamilies:

linear mannan, glucomannan, galactomannan, and galactoglucomannan [6]. While xylan and

mannan are the major hemicelluloses in secondary cell walls of wood fiber, xyloglucan is the

primary hemicellulose in seeds and primary cell walls of plants [7]. Xyloglucan is composed of

a linear β-(1,4)-glucan backbone branched at C-6 with α-linked xylopyranosyl residues; most

xyloglucans contain XXXG or XXGG oligosaccharide repeats, where X and G refer to an

unbranched β-D-Glcp residue and an α-D-Xylp(1→6)-β-D-Glcp segment, respectively [7].

4

Lignin is a complex phenolic polymer that is composed of a combination of coniferyl alcohol,

sinapyl alcohol and/or para-coumaryl alcohol [2]. These monolignols are incorporated into

lignin in the form of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively [9].

Unlike cellulose and hemicellulose, the lignin structure is highly irregular. The relative

abundance of each monolignol depends on the plant species, cell type, as well as individual cell

wall layers; they are also influenced by developmental and environmental affects [9]. Generally,

hardwood lignins contain a mixture of G and S units and traces of H units, whereas softwood

lignins consist of mainly G units with low amounts of H units. In contrast, grass lignins show

comparable levels of G and S units and comparatively high amounts of H units [9].

Cellulose, hemicellulose and lignin are associated by a variety of covalent and non-covalent

linkages. The conventional model of lignocellulose describes hemicellulose as a crosslinking

polysaccharide that is hydrogen bound to cellulose and covalently linked to lignin [2,3]. While

hemicellulose is believed to impart flexibility to cellulose fibrils, lignin is believed to participate

in resistance to plant pathogens and facilitate water transport.

2.2 Lignocellulose-active Enzymes

Thermochemical and bioconversion are two approaches to processing biomass for the production

of sugars or the extraction of polymeric components from lignocellulose. Bioconversion

processes typically include an initial physical/chemical pretreatment of biomass followed by

biological conversion of resulting sugars and biopolymers to fuels, chemicals, and new materials.

In part because of lower temperatures and the specificity of enzyme-catalyzed reactions, key

advantages of bioconversion strategies are i) the potential to selectively extract lignocellulose

polymers and sugars, ii) low corrosive impact on process equipment; iii) minimal formation of

inhibitors to downstream fermentation processes, and iv) corresponding bioprocesses are often

renewable and have low energy requirements.

Carbohydrate-active enzymes (CAZymes) have been classified into over 100 glycoside

hydrolase (GH) families, 91 glycosyl transferase (GT) families, 16 carbohydrate esterase (CE)

families, 21 polysaccharide lyase (PL) families and 55 carbohydrate binding modules (CBM)

(http://www.cazy.org/). Renewed interest in applying these enzymes for the production of

fermentable sugars from biomass reflects widespread concern over greenhouse gases emitted

5

from transportation fuels, and efforts to increase societal and economic security through the

production of domestic biofuels [1]. Specific areas of research towards bioconversion of

lignocellulose to fermentable sugars includes reducing the cost of enzyme production,

discovering and engineering hydrolytic enzymes with increased specific activity on cellulosic

substrates, and development of enzyme suites that are tailored for the hydrolysis of specific

feedstocks [1]. Examples of CAZymes that are already used in industrial applications include

amylases, pectinases, and pectate lyases used for food processing, cellulases in laundry

detergents and by textile industries, and xylanases used for pulp bleaching [1,10]. Amylases and

cellulases are also used for biofuel production. Notably, these applications have harnessed the

depolymerization activity of CAZymes. Despite their industrial relevance, comparatively few

applications have been developed for CAZymes that have the potential to modify carbohydrates

through synthetic catalyses. An exception includes the development of a xyloglucan

endotransglycosylase to graft new chemistry to the surface of cellulose microfibrils [8,11].

Similarly, lipase enzymes have been used to acetylate cellulose, as well as catalyze the regio-

specific addition of aliphatic side-groups to xyloglucan oligosaccharides [11,12]. Further,

glycosyl transferases can synthesize novel oligosaccharides, some having significant clinical

relevance [13, 14]. However, these examples have yet to transfer to an industrial scale.

Lignin degradation is mainly catalyzed by extracellular, oxidative enzymes [15]. Lignin

peroxidases (LiPs), manganese peroxidases (MnPs), and laccases are the most studied enzymes

involved in ligninolysis. These enzymes require low molecular weight mediators to carry out

lignin degradation [16]. Other reductive enzymes such as cellobiose dehydrogenase, aryl alcohol

oxidases, and aryl alcohol dehydrogenases might also participate in ligninolysis [15]. Lignin that

is recovered during the conversion of biomass to fermentable sugars is typically burned to help

fuel the process. However, the development of higher-value products from lignin could improve

the economics of biofuel production from lignocellulose feedstocks. For instance, the biological

antioxidant properties of lignin could be further harnessed in functional foods, cosmetics, as well

as a broader range of lubricant oils [17]. To extend the utilization of lignin, it will be important

to adjust its water solubility characteristics. For instance, enzymatic esterification of lignin-

derived phenolic acids with aliphatic molecules can increase the miscibility of the product in

food with higher fat content. The synthetic potential of carboxyl esterases and lipases has been

demonstrated for this application [18].

6

2.3 Carboxyl Esterases and Lipases

2.3.1 Distinguishing Lipases from Carboxyl Esterases

Lipases and esterases are hydrolytic enzymes that cleave ester linkages through the addition of a

water molecule, producing a carboxylic acid and an alcohol. Esterases and lipases are lipolytic

enzymes but exhibit different substrate preferences (Table 2.1). While esterases catalyze the

hydrolysis of short to medium length aliphatic esters that are partially soluble in water, lipases

typically display maximal activity towards water insoluble long-chain esters. Interfacial

activation and deviant Michaelis-Menten kinetic curves are additional phenomena that are used

to distinguish lipases from esterases [19]. These phenomena have been correlated to the

presence of a flexible hydrophobic α-helix domain (lid or flap) in many lipases, which are

thought to expose the active site and direct substrate binding at the interface between water and

solvent phases [19] . Lipases also typically contain comparatively high numbers of non-polar

residues, Val, Leu and Ile [20]. It has been suggested that increased hydrophobic content may

improve lipase binding to hydrophobic substrate aggregates [20]. Finally, lipases have been

distinguished from esterases by having optimal activity at comparatively alkaline pH, and by

distinct surface electrostatic potential distributions at corresponding pH optima [21].

Table 2.1.Differences between lipases and esterases [19].

2.3.2 Carboxyl Esterase and Lipase Structure

Esterases and lipases acquire the α/β hydrolase fold, a structural conformation that is highly

conserved among other hydrolytic enzymes. The α/β hydrolase fold typically consists of 8 β-

strands connected by 6 α-helices (Fig. 2.1). An example of crystal structure for Candida rugosa

lipase is shown in Fig. 2.2. A conserved feature of α/β hydrolases is the nucleophilic elbow,

7

which displays a sharp γ turn with a nucleophilic serine residue positioned between a β-strand

and an α-helix [23]. Most of these enzymes also contain a catalytic triad consisting of a serine,

histidine and aspartate residue. Glutamate replaces aspartate in some lipases. In primary

sequences, the catalytic serine is usually part of the conserved pentapeptide Gly-X-Ser-X-Gly,

where X represents any amino acid. Exceptions include GDSL family esterases, where the

catalytic serine is presented in the conserved Gly-Asp-Ser-(Leu) motif found near the N-terminus

of the enzyme [24]. As mentioned above, the 3D structure of many lipases also reveals a lid

(flap) structure that covers the active site of the enzyme in the absence of substrate.

Fig. 2.1.Schematic representation of the α/β fold. Six α helices and 8 β strands are shown by cylinders and arrows, respectively. The catalytic triad is indicated by black dot. (A modified representation of Fig. 1 from [19])

Fig. 2.2. A crystal structure of a lipase from Candida rugosa (PDB: 1TRH). The structure was displayed by NewCartoon generated in Visual Molecular Dynamics program (Version 1.8.5).

8

2.3.3 Proposed Mechanism of Esterases and Lipases

Esterases and lipases share the same catalytic mechanism for ester hydrolysis [19]. The

mechanism involves four steps: 1) formation of a tetrahedral intermediate that is stabilized by the

catalytic His and Asp residues once the substrate is bound to the active serine (in lipases, this

step also involves movement of the lid structure); 2) the formation of an acyl-enzyme complex

after the release of the alcohol; 3) nucleophilic attack of the acyl-enzyme complex by water,

forming a second tetrahedral intermediate, and 4) the release of the acid product and free

enzyme. The reverse reaction (transesterification) is achieved by substituting water for an

alcohol, thereby forming new ester compounds.

2.3.4 Classification of Esterases and Lipases

Bacterial esterases and lipases were classified by Aprigny and Jaeger (1999) based mainly on a

comparison of primary amino acid sequences and some biological properties including how the

enzyme is secreted, requirements for lipase-specific foldases, and potential relationship to other

enzyme families [24]. A total of 53 sequences were compared and classified into 8 different

families. Family I (block L in ESTHER database, http://bioweb.ensam.inra.fr/ESTHER)

contains true lipases and is further divided into 6 subfamilies. Subfamilies I.1 and I.2 appear to

require a lipase-specific foldase (Lif) for proper folding and contain two conserved aspartic acid

residues involved in the Ca2+ binding and two cysteine residues that form a disulphide bridge

[24]. However, Subfamily I.1 and I.2 differ in size; subfamily I.1 enzymes range from 30 to 32

kDa, while subfamily I.2 are roughly 33 kDa and contain an insertion sequence that forms an

anti-parallel double β-strand at the protein surface. By contrast, subfamily I.3 are typically 50 to

60 kDa, and do not contain cysteine residues; subfamily I.4 are approximately 20 kDa, and

subfamily I.5 are approximately 45 kDa and usually display maximal activity between pH 7 to 9

and 30 to 50 °C [24,25]. The conserved pentapeptide sequence in Bacillus lipases from

subfamily I.4 and I.5 is characterized by Ala-X-Ser-X-Gly, whereby the first glycine residue is

replaced by an alanine residue. Finally, subfamily I.6 is usually secreted as a precursor protein

and cleaved extracellularly by a specific protease with processing beginning at the N-terminus.

Lipases and esterases with the conserved sequence Gly-Asp-Ser-(Leu) motif are classified in

family II. Family III (block L in ESTHER database) includes lipases that possess the conserved

9

catalytic triad and show 20 % identity with the human PAF-AH (platelet-activating factor

acetylhydrolase). By contrast, family IV (block H in ESTHER database) includes hormone-

sensitive lipases (HSL) that show significant sequence similarity to the mammalian HSL. The

remaining families also contain the conserved catalytic triad but are distinguished by sequence

similarity to other functional enzymes, including epoxide hydrolases, dehalogenases and

haloperoxidase for family V (block X in ESTHER database), eukaryotic lysophospholipases for

family VI (block X in ESTHER database), eukaryotic acetylcholine esterases and intestine/liver

carboxylesterases for family VII (block C in ESTHER database), and class C β-lactamases for

family VIII.

2.3.5 Applications of Carboxyl Esterases and Lipases

Esterases and lipases comprise a large group of hydrolytic enzymes, which function in both

hydrolysis and synthesis of ester bonds. They can be utilized in pharmaceutical synthesis, food

processing, detergent treatment, as well as kinetic resolution [26]. The interest in these enzymes

resides in the fact that they do not require any cofactors, are easy to acquire, show high regio-

and stereospecificity, and are usually stable and active in organic solvents [19].

2.3.5.1 Applications in the Detergent Industry

Owing to their ability to hydrolyze fats, esterases and lipases are the major enzyme additives in

industrial laundry and household detergents. For instance, the first commercial lipase,

LipolaseTM was introduced by Novo Nordisk in 1994, which was isolated from the fungus

Thermomyces lanuginosus and recombinantly expressed in Aspergillus oryzae [27]. In addition,

Genencor International introduced two bacterial lipases used as detergent additives in 1995:

LumafastTM from Pseudomonas mendocina and LipomaxTM from Pseudomonas alcaligenes [27].

Favorable enzymes properties for this application include low substrate specificity and high

tolerance to pH shifts, temperature and solvents. All these requirements can be resolved either

by screening naturally stable enzymes or enhancing enzyme properties through protein

engineering [28].

10

2.3.5.2 Applications in the Pharmaceutical Industry

Esterases and lipases catalyze a wide variety of chemo-, regio-, and stereo-specific

transformations, thereby providing great opportunities in organic syntheses. One example

includes synthesis of optically pure (S)- and/or (R)-ketoprofen [2-(3-benzoylphenyl) propionic

acid] by Trichosporon brassicae esterase. This product is used in the reduction of inflammation

and relief of pain caused by arthritis, sunburn, menstruation, and fever [29]. Another anti-

inflammatory drug, naproxen, is synthesized by carboxyl esterase NP from Bacillus subtilis [30].

(S)-naproxen generated in this process is 99 % enantiomerically pure, and the reaction yield is in

excess of 95 %. Other examples include stereospecific conversions in the production of

pharmaceutical intermediates such as taxol synthesis, throumboxane-A2-antagonist,

acetylcholine esterase inhibitors, anti-cholesterol drugs, anti-infective drugs, Ca2+ channel

blocker drugs, K channel blocking drugs, anti-arrhythmic agents and antiviral agents [31].

2.3.5.3 Applications in the Food Industry

The physical properties and nutritional value of triglycerides is greatly influenced by the

position, chain length and degree of unsaturation of the fatty acids [27]. Since fats and oils are

important constituents of foods, higher value fats can be synthesized using esterases or lipases in

a relatively inexpensive way. For instance, these enzymes can alter the position of fatty acid

chains in the glyceride substrate and replace one or more of fatty acids with more desirable ones.

A typical example in the food industry includes upgrading palm oil to achieve properties similar

to cocoa butter used in the production of chocolate [32]. Here, lipases from Rhizomucor miehei

have been used to substitute undesired palmitic acids with desired stearic acids. Other examples

include lipase-catalyzed removal of fat from meats, and extraction of polyunsaturated fatty acids

from plants and animals to be used as neutraceuticals.

2.3.5.4 Other Industrial Applications

In addition to the above-mentioned applications, esterases and lipases are employed in many

other industries, including perfume industries, pulp and paper processing, and degradation of

synthetic materials [31, 32]. A sub-group of esterases, in particular the arylesterases and feruloyl

esterases, are interesting candidates for the production of flavors and anti-oxidant additives from

lignin-derived phenolic acids [31]. For example, feruloyl esterases along with xylanases can

11

release ferulic acid from xylan, which can be converted to vanillin [19]. Feruloyl esterases

belong to carbohydrate esterase family 1, and have been isolated from many fiber-active

microorganisms, including Aspergillus niger, and Fusarium oxysporum [31]. In addition,

cholesterol esterases and polyurethenase have been used to degrade synthetic pollutants

including plastics, polyurethane, and polyesters. These enzymes were successfully isolated from

Pseudomonas chlororophis and Pseudomonas aeruginosa [33, 34].

2.3.6 Esterase and Lipase Catalyzed Transesterification

Esterases and lipases have demonstrated ability in organic chemical synthesis, catalyzing a wide

range of chemo-, regio-, and stereo-selective transformations. Corresponding (trans-)

esterification reactions are favored in the absence of water, whereby an ester bond is synthesized

between an alcohol and a carboxylic acid (or carboxylic ester) by synthesizing the acid or

exchanging the organic group (R3) of the ester with the organic group (R2) of the alcohol (Fig.

2.3). For instance, the commercial lipase, PS “Amano” SD, originated from Burkholderia

cepacia, catalyzes a variety of transesterification reactions and successfully resolves many

racemic mixtures [35].

Fig..2.3. Schematic representation of esterification (a) and transesterification (b) reactions. In an esterification, an ester is formed between a carboxylic acid and an alcohol by releasing a water molecule; in a transesterification, a new ester is form by exchanging the organic group (R3) of an ester with the organic group (R2) of the alcohol.

As mentioned, water content determines the reaction equilibrium (ie., synthesis vs. hydrolysis),

the distribution of products in the reaction vessel, as well as the thermo-inactivation of

biocatalysts. Researchers have demonstrated that enzymes show higher stability at elevated

temperatures in low water environments compared to aqueous solutions [36]. Organic solvents

12

are thus used as reaction media because they not only favor (trans-)esterification, but also

increase the solubility of organic substrates, thereby resulting in higher reaction rates.

Accordingly, in transesterification reactions, the only water present in the system is the water

bound to the enzyme itself; this water is essential to maintain the structural integrity of the

enzyme. A general agreement previously stated by Klibanov et al. is that enzymes can function

in predominantly nonaqueous environment or low water environment as long as the essential

water layer around them is not stripped off [36].

Enzyme activities can be correlated to the hydrophobicity of an organic solvent [36]. Log Poct is

defined as the logarithm of the partition coefficient of a solvent with respect to the octanol/water

two-phase system, and shows good correlation with enzyme activity [36]. Generally, organic

solvents with log P ≤ 2 are not good choices for reaction media because polar solvents may strip

off the essential water layer around the biocatalyst. Rather, organic solvents with log P ≥ 4 are

most suitable [36]. The effect of organic solvents with log P between 2 and 4 depends on the

nature of the biocatalyst. An exception includes the pancreatic lipase, which functions in all

tested organic solvents with log P ranging from -1.3 to 13.7. Nevertheless, this enzyme still

performs better in hydrophobic organic solvents compared to hydrophilic organic solvents.

Lanne et al. (1987) explain that the outer part of the pancreatic lipase is more hydrophobic than

other lipases, thereby binding its essential water more tightly [37].

2.3.7 Potential of Carboxyl Esterases and Lipases for Lignocellulose Modification

Canada has an abundance of underutilized biomass that can be harnessed for the production of

sustainable energy and materials. Our biomass resource provides an opportunity to reduce our

demand for petroleum feedstocks while satisfying the need to generate renewable and

environmental friendly products. Moreover, valorization of natural biopolymers using enzymes

represents a “green” process technology, ensuring that the product as well as its synthesis, are

environmentally sustainable.

To date, lipases, esterases, proteases, nitrilases and glycosidases have been developed for the

specific non-destructive functionalization of polymer surfaces (Table 2.2) [38]. Owing to their

broad specificity, high solvent-tolerance, and ability to catalyze transesterification reactions,

esterases and lipases display particular benefits for the production of value-added lignocellulosic

13

polymers. While enzymatic transesterification of sugars and short oligosaccharides has already

been demonstrated [39], similar examples of directly modifying long fibers rather than short

oligosaccharides are lacking. Similarly, there are few examples where enzymes have been used

to adjust the solubility characteristics of lignin-derived phenolics to harness their valuable

antioxidant properties in a broader range of products. An exception is the use of feruloyl

esterases to catalyze the esterification of phenolic acids with n-butanol and glycerol, for the

production of antioxidant nutritional supplements [20,40].

Given the current interest in conversion of lignocellulose-derived sugars to biofuel, arylesterases

that modify lignin and lignin-derived phenolic acids are particularly interesting targets for the

production of high-value co-products from biomass [41]. To advance enzyme-catalyzed

transesterification of lignin for the production of lignin derivatives with altered miscibility, it is

necessary to find arylesterases with new substrate specificities and high performance in different

industrial conditions. The following chapters will describe efforts to isolate and characterize

new arylesterases from different microorganisms. These data will be used to assess the potential

of each enzyme for synthetic modification of lignin.

14

Table 2.2. Summary of Enzymes Involved in Polymer Synthesis and/or Modification [38].

15

3 Bacterial Genome Mining and Recombinant Expression of Putative Arylesterases

3.1 Introduction

Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) similarly hydrolyze carboxyl ester bonds and

generally exhibit broad substrate specificity. Under non-aqueous conditions, the reverse reaction

can be catalyzed, whereby esterification of hydroxyl groups on sugars, phenolics and alcohols

has been demonstrated [1-3]. Historically, lipases and esterases were distinguished by

preference for medium to long-chain triacylglycerols and short-chain fatty acyl esters,

respectively. Structural analyses of lipases also revealed the presence of a peptide loop that was

correlated to transformation of water-insoluble substrates and activation at lipid-water interfaces.

However, exceptions to these generalizations have been observed, leading some to suggest that

lipases should constitute any carboxyl ester hydrolase that has the capacity to hydrolyze long-

chain triacylglycerols.

The regioselectivity and stereospecificity of lipase and esterase catalyzed transesterification

reactions offer advantages over chemical syntheses, which often demonstrate poor selectivity and

formation of undesirable side products [4]. While several examples of enzyme-catalyzed

transesterification of sugars and fatty acids have been reported, there are few examples of

enzymatic esterification of phenolic compounds. Exceptions include the production of

antioxidant nutritional supplements through feruloyl esterase esterification of phenolic acids with

n-butanol and glycerol, respectively [5, 6]. Accordingly, there is considerable opportunity to

identify new arylesterases with ability to synthesize novel chemicals from lignin-derived

aromatic substrates. The development of value-added compounds from lignin is an important

means to reducing the cost biofuels from lignocellulose feedstocks, and creating new products

for forest and agricultural sectors [7]. At the same time, the multitude of genome sequences that

are publicly available enables the identification of new enzymes with targeted activities.

To date, there are 3056 isolated genomes, of which 923 are completed, 1143 are on draft

assembly, and 990 are in progress (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html).

Most of these genomes were annotated based on the sequence similarity to proteins that are

biochemically characterized [8]. While this approach is quick and inexpensive, 40 to 60 % of

16

sequences fail to be assigned a function, and many open reading frames are incorrectly annotated

[9,10]. In addition to sequence similarity-based approaches, comparative genomics [11-15],

two-hybrid assays [16-18], microarrays [19-22], and three-dimensional protein structures [23-

25], are being applied to improve functional predictions of gene products. Arguably, the most

definitive approach to assign a molecular function to a predicted open reading frame is to isolate

and biochemically characterize the corresponding protein [26]. While heterologous production

of proteins and the development of appropriate activity screens is still challenging, high

throughput platforms for protein synthesis and characterization have successfully identified

erroneous annotations and enzyme activities initially missed by conventional sequence

comparisons [26].

Given the industrial significance of arylesterases and the difficulty of distinguishing esterases

from lipases based on sequence comparisons alone [27], high throughput production and

characterization of predicted esterases and lipases was conducted. Microbial genome sequences

were selected based on the environmental niche of the corresponding microorganism, and the

availability of genomic DNA from the American Type Culture Collection (ATCC). Bacterial

genomes were initially analyzed to maximize successful protein expression in E.coli [28, 29].

3.2 Materials and Methods

3.2.1 Materials

All bacterial genomes were purchased from the ATCC. The reagents for the autoinduction

medium were purchased from Bioshop (Canada). The commercial pNP-esters (pNP-acetate,

pNP-propanioate, pNP-butyrate, pNP-capraote, pNP-caprate, pNP-laurate, pNP-palmitate, pNP-

benzoate, and paraoxon) and natural substrates (phenyl acetate, olive oil, and tributyrin) were

purchased from Sigma (Canada). The chemicals used in preparing universal buffers (phosphoric

acid, acetic acid and boric acid) were also purchased from Sigma (Canada). All chemicals are

pure at molecular level and sterilized before use. The pNP-ester stock solutions (10 mM) used

for primary screens were prepared in DMSO.

17

3.2.2 Sequence Selection of Putative Arylesterases

A biochemically characterized and structurally resolved bacterial arylesterase (Swiss-Prot

accession: P22862) was identified by searching the BRENDA and Swiss-Prot databases. The

corresponding protein sequence served as a parent sequence for BLASTp search of the following

15 translated bacterial genomes: Agrobacterium tumefaciens C58, Clostridium acetobutylicum

824D-S, Clostridium thermocellum 27405D, Escherichia coli K12, Nitrosomonas europaea,

Pseudomonas aeuginosa, Pseudomonas putida KT2440, Pseudomonas syringae, Rhodococcus

sp. RHA1, Rhodopseudomonas palustris CGA009, Ralstonia solanacearum 11696D-S,

Streptomyces avermitilis, Streptomyces coelicolor, Sinorhizobium meliloti, and Thermotoga

maritima. In order to capture novel esterase activities, an E-value of 0.01 was used to first filter

enzyme targets. Corresponding sequences were then aligned to P22862 to confirm the presence

of conserved catalytic residues and sequence motifs [27, 30].

3.2.3 Gene Cloning

Forward and reverse primers were designed and synthesized for each gene target; all primers

were flanked by BseRI restriction sites (www.idtdna.com). The genes were amplified from

bacterial genomes purchased from the ATCC; PCR was performed using a proof reading DNA

polymerase (Pfx polymerase) and the following PCR cycles: denaturation at 95 °C for 15 sec,

annealing at 53 °C for 30 sec, and elongation at 68 °C for 1 min. PCR products were purified

using the QIAquick PCR purification kit (Qiagen), digested using BseRI, and then ligated to

p15Tv-L (GenBank accession EF456736) using T4 DNA ligase (Fermentas). Ligation products

were transformed into Escherichia coli DH5α by electroporation; plasmids were isolated from

transformants using the Qiaprep spin miniprep kits (Qiagen) and sequenced at TCAG DNA

sequencing facility (Toronto).

3.2.4 Protein Purification

Briefly, E. coli transformants were cultured at 37 °C in autoinduction media [31] containing 100

µg/mL of ampicillin. Autoinduction medium contained 1 mM MgSO4, 1x metal mix, 1x GGL,

and 1x NPS. The 1000x trace metal mixture contained 0.1 M FeCl3･6H2O, 1 M CaCl2, 1 M

ZnSO4･7H2O, 1 M MnCl2･4H2O, 0.2 M CoCl2･6H2O, 0.1 M CuCl2･2H2O, 0.2 M NiCl2･6H2O,

18

0.1 M Na2MoO4･5H2O, and 0.1 M Na2SeO3･5H2O. The stock of 20x NPS contained 0.5 M

(NH4)2SO4, 1 M KH2PO4, and 1 M Na2HPO4, while 50x GGL contained 0.5 % glycerol, 0.05 %

glucose, and 0.2 % α-lactose.

Protein expression was induced by reducing the temperature to 20 °C. The cells were harvested

after overnight incubation, suspended in binding buffer (300 mM NaCl, 50 mM HEPES, pH 7.5,

5 % Glycerol, 5 mM imidazole), and lysed by sonication. Cell extracts were collected by

centrifugation and incubated with Ni resin (Qiagen) for 2 h. The resin was then washed with 200

mL of washing buffer (0.5 M NaCl, 50 mM HEPES, pH 7.5, 5 % Glycerol, 30 mM imidazole)

and eluted with approximately 10 mL elution buffer (0.5 M NaCl, 50 mM HEPES, pH 7.5, 5 %

Glycerol, 250 mM imidazole). Protein concentrations were measured using the Bradford assay

and their purities were evaluated by 15 % SDS-PAGE.

3.2.5 Primary Enzyme Assays Using p-Nitrophenyl (pNP) Substrates

The standard assay was performed in 96-well plate format at 37 °C; final reaction volumes were

200 µL and contained 1 µg of purified protein. Reactions were prepared using an automated

liquid handler (TECANTM Freedom EVO 100 MCA) and product formation was measured using

a microplate spectrophotometer (TECANTM Infinite M200). Product absorbency was initially

measured at a specified wavelength after 30 min at 37 °C. Reactions showing positive activity

were then assayed every minute for the first 6 min, followed by every 5 min up to 30 min. The

amount of enzyme added was also adjusted to ensure that initial reaction rates were measured,

(i.e., 0.05 µg for RP_est1, 0.25 µg for RP_est2, 0.5 µg for RP_est3, 0.5 µg for PP_est1, 0.5 µg

for PP_est2, 0.5 µg for PP_est3, 0.5 µg for PA_est1 and 1 µg for SAV_est1). Enzyme dilutions

were prepared in 50 mM potassium phosphate buffer (pH 7). Reactions were prepared by mixing

1 volume of the 10 mM pNP-ester stock solution with 9 volumes of 100 mM sodium phosphate

buffer (pH 7), containing 2.5 % Triton X-100, followed by immediate vortexing [32]. The final

pNP-ester concentration in the reaction was 0.5 mM. Liberation of pNP was measured at 400

nm and one enzymatic unit is defined as the amount of enzyme required to produce 1 µmol of

pNP per min. pNP was thus used to generate a standard curve (0.002 µmol to 0.1 µmol). All

purified proteins were assayed at pH 6, 7 and 8 on pNP-acetate, pNP-caprate, pNP-palmitate, and

pNP-benzoate.

19

3.2.6 Primary Screen Using Natural Substrates

Phenyl acetate, olive oil and tributyrin were used as natural substrates. The production of acids

was detected by Bromothymol blue (BTB) as described by Mertinez-Martines et al. [33] with

minor modifications. In brief, 30 mM of each substrate was prepared in 5 mM sodium phosphate

buffer (pH 7.3) containing 0.01 % of BTB. Initial reactions contained 1 µg of purified protein.

Reactions were prepared in 96-well plates, and were incubated for 30 min at 37 °C, followed by

absorbance measurement at 616 nm. The specific activity of enzymes on phenyl acetate was

calculated by generating a standard curve using acetic acid (1.75 µmol to 17.5 µmol).

3.2.7 General Properties of Arylesterases

3.2.7.1 Optimal pH and pH Stability

The effect of pH was investigated using the Britton and Robinson's universal buffer system (50

mM phosphoric acid, acetic acid and boric acid, pH adjusted to 4 to 11 with NaOH) [34]. pNP-

acetate was used to evaluate optimal pH. To examine enzyme stability at different pH values

(pH 6, pH 7, pH 8, pH 9, pH 9.5), residual enzyme activity was measured after 24 h of

incubation at 37 °C. Residual enzyme activity was measured using standard reaction conditions

and pNP-acetate as the substrate. In brief, 10x concentrated enzymes (i.e. 0.5 µg/100 µL for

RP_est1, 2.5 µg/100 µL for RP_est2, 5 µg/100 µL for RP_est3, 5 µg/100 µL for PP_est1, 5

µg/100 µL for PP_est2, 5 µg/100 µL for PP_est3, 5 µg/100 µL for PA_est1 and 10 µg/100 µL for

SAV_est1) were preincubated in each pH buffer for 24 h at 37 °C. The residual activity was

measured by transferring 10 µL aliquots back to standard conditions, as described in section

3.2.5.

3.2.7.2 Thermal Inactivation

Residual enzyme activity was measured after incubation at 22 °C, 30 °C, 37 °C, 50 °C, 55 °C,

and 70 °C for up to 5 h. Enzyme samples were incubated in 50 mM potassium phosphate buffer

(pH 7). Residual enzyme activity was measured using standard reaction conditions and pNP-

acetate as the substrate. In brief, 10X concentrated enzymes (i.e. 0.5 µg/100 µL for RP_est1, 2.5

µg/100 µL for RP_est2, 5 µg/100 µL for RP_est3, 5 µg/100 µL for PP_est1, 5 µg/100 µL for

PP_est2, 5 µg/100 µL for PP_est3, 5 µg/100 µL for PA_est1 and 10 µg/100 µL for SAV_est1)

20

were preincubated at each temperature for up to 5 h. The residual activity was measured by

transferring 10 µL aliquots back to standard conditions, as described in section 3.2.5.

3.2.8 Enzyme Kinetics

Kinetic parameters were obtained at 37 °C and using the optimal pH and preferred substrate for

each enzyme. Substrate concentrations ranged from 0.05 mM to 1 mM with 50 µM increments

(i.e., 20 different substrate concentrations). The amount of each enzyme used per each reaction

was 0.05 µg for RP_est1, 0.1 µg for RP_est2, 0.5 µg for RP_est3, 0.5 µg for PP_est1, 0.5 µg for

PP_est2, 0.25 µg for PP_est3, 0.5 µg for PA_est1 and 0.5 µg for SAV_est1. Initial rates of each

reaction at different substrate concentration were measured every minute for total of 10 min and

rates of product formation (V) were determined using the pNP standard curve. The kinetic curve

was generated by plotting velocity against substrate concentration using the software GraphPad

Prism5 (GraphPad Software, Inc.). In this way, non-liner regression was used to determine the

kinetic parameters Vmax and KM using the Michaelis-Menten model.

3.2.9 Construction of a Phylogenetic Tree

Protein sequences were aligned using MUSCLE, and 500 bootstrapped data sets were generated

using SEQBOOT. Protein distance matrices were obtained using the PHYLIP 3.69 Neighbor-

Joining algorithm PRODIST (100 iterations), and trees were drawn using NEIGHBOR and a

consensus tree was generated using CONSENSE. The tree was viewed by PhyloWidget

(http://www.phylowidget.org/).

3.3 Results and Discussion

3.3.1 Identification and High Throughput Production of Putative Arylesterases

The objective of this study is to identify and isolate new arylesterases, given their potential to

modify phenolic acids and thereby synthesize high-value compounds from lignin. The ability of

certain esterases to modify hydroxyl groups present on sugars also means these enzymes could

be used to create new compounds from polysaccharides, including biomass-derived

hemicellulose and cellulose.

21

A total of 202 gene targets from 15 bacteria were selected based on overall sequence similarity to

a biochemically characterized arylesterase, as well as presence of conserved catalytic residues

and sequence motifs [35]. While lipases and esterases are difficult to distinguish at the sequence

level, differences in catalytic properties can be used to differentiate these enzymes. For instance,

lipases can be distinguished from esterases by their ability to hydrolyze insoluble substrates and

by interfacial activation [35]. Previous analyses also suggest that lipases can be distinguished

from esterases by optimal activity of the former at alkaline pH, and by correlating the

electrostatic potential of enzyme surfaces to pH optima [30]. In addition, preference for aliphatic

and aromatic substrates can be used to distinguish lipases, carboxyl esterases and arylesterases.

Therefore, to identify arylesterases encoded by the gene targets, each gene was cloned for

heterologous expression in E.coli. A total of 180 genes were successfully cloned and 84 of them

were expressed as soluble proteins in E.coli. All 84 soluble proteins were purified and then

screened using colorimetric assays; 46 proteins showed activity on pNP-acetate at pH 8 while 38

proteins did not exhibit any activity towards the test substrates (Fig. 3.1). Phenyl acetate is a

general substrate to identify arylesterase and 18 proteins showed activity towards this substrate.

The specific activity of these 18 proteins on aliphatic pNP-esters and phenyl acetate was

compared to evaluate whether they demonstrate arylesterase-like activity or lipase-like activity

(Table 3.1). All 18 bacterial enzymes showed significantly higher specific activity towards

phenyl acetate than the aliphatic pNP-esters, and none of these enzymes hydrolyzed olive oil to

detectable levels, which is a common screen for lipase activity. Accordingly, these 18 bacterial

enzymes can be classified as arylesterases [36-38]. Six arylesterases that were also active on

medium-chain aliphatic substrates were selected for further biochemical characterization. Two

enzymes with activity towards pNP-acetate only were also characterized for comparison. All of

the eight enzymes preferentially hydrolyzed short- and/or medium- chain substrates, which is

typical of esterases [35]. Notably, none of the eight esterases exhibited paraoxonase-like

activity. The general properties of these 8 enzymes are summarized in Table 3.2.

3.3.2 pH Optima of Purified Esterases

Given the structural similarity of phenyl acetate and pNP-acetate, and challenges associated with

changing the pH of BTB assays, pNP-acetate was used to measure the optimal pH and pH

stability of selected enzymes. Previous analyses that correlate electrostatic potential distribution

22

of enzyme surfaces to pH optima suggest that lipases can be distinguished from esterases by

optimal activity of the former at alkaline pH [39]. Notably, the pH optima of esterases isolated

in this study were either neutral or alkaline (Table 3.2). According to biochemical data for

arylesterases listed in the BRENDA enzyme database (http://www.brenda-enzymes.info), neutral

or basic pH optima were reported for both eukaryotic arylesterases [40, 41] and bacterial

arylesterases [36, 37]. This suggests that arylesterases may represent a unique subfamily of

esterases that share properties of both esterases and lipases. All esterases except RP_est3 and

SAV_est1 were stable in both acidic to alkaline conditions. Differences in pH stabilities likely

result from proton activation and deactivation of titratable amino acid residues [36]. By

comparing pH optima to predicted pI values of each enzyme, it appeared that the enzymes

isolated in this study are most active when a net negative charge is attained. This result is

consistent with the electrostatic study published by Petersen et al. [39], which explains that while

products are deprotonated once released from the enzyme active site, lipases and esterases can

still carry a negative potential and retain an active conformation. At a high pH, the active site

may also carry a negative potential, which repulses the product. Supporting analyses such as

predicting the electrostatic potential distribution of enzymes will be performed when

corresponding x-ray structures are solved.

23

Fig. 3.1. The distribution of enzyme activities that were detected using a variety of substrates. All assays were performed using standard conditions, and were incubated for 30 min at 37 °C. The number of enzymes that were active on each substrate is shown in parentheses.

Table 3.1. Activities (µmol•min-1•mg protein-1) of enzymes using 0.5 mM of substrate. Microorganisms ID Substrates

pNP acetate

pNP caprate

pNP palmitate

Phenyl acetate

b2593 0.46±0.01 - - - Escherichia coli K12 b2799 0.16±0.02 - - - NE0279 0.14±0.02 - - - Nitrosomonas

europaea NE2298 0.37±0.02 - - 3.17±0.27

PA1469 0.1±0.01 - - - PA2934 0.38±0.01 - - 3.25±0.86 PA2949 0.66±0.02 1.04±0.4 0.2±0.01 2.33±0.43

Pseudomonas aerigonosa

PA3132 0.25±0.01 - - - PP0364 0.48±0.02 - - - PP3645 0.8±0.01 1.02±0.16 4.96±0.44

Pseudomonas putida KT2440

PP5253 0.91±0.02 - - 5.83±0.49

24

PSPTO3914 0.23±0.00 - - - PSPPH3482 0.24±0.02 - - 3.27±0.18

Pseudomonas syringae PSPPH4522 0.49±0.02 - - 5.22±0.19

RHA1_ro00320 0.2±0.02 - - 3.13±0.29 RHA1_ro01401 0.19±0.00 - - 3.47±0.47 RHA1_ro03350 0.24±0.02 - - - RHA1_ro02607 0.41±0.02 - - 4.22±0.06 RHA1_ro04513 0.19±0.02 - - - RHA1_ro03520 0.06±0.02 - - - RHA1_ro01338 0.38±0.01 - - - RHA1_ro03603 0.23±0.01 - - - RHA1_ro08044 1.29±0.01 - - - RHA1_ro08081 1.61±0.01 - - 6.27±0.12

Rhodococcus sp. RHA1

RHA1_ro10146 1.46±0.02 - - - RPA0348 2.24±0.10 1.74±0.05 - 7.36±0.49 RPA1163 0.31±0.01 - - - RPA2105 0.1±0.02 0.6±0.01 - - RPA3430 0.63±0.01 0.68±0.01 - 3.53±0.37

Rhodopseudomonas palustris CGA009

RPA4646 1.2±0.02 0.24±0.00 - 9.06±0.24 RSc1135 0.23±0.02 - - - Ralstonia

solanacearum 11696D-S

RSc2250 0.05±0.01 - - -

SAV512 0.45±0.00 - - - SAV875 0.1±0.02 - - - SAV2722 0.13±0.02 - - - SAV3810 0.15±0.02 - - -

Streptomyces avermitilis

SAV5173 0.13±0.02 - - - SCO1989 0.1±0.02 - - - SCO3439 0.35±0.01 - - 3.55±0.24 SCO3566 0.33±0.01 - - 3.09±1.07 SCO3690 0.83±0.02 - - 4.8±0.20 SCO6712 0.03±0.01 - - -

Streptomyces coelicolor

SCO7440 0.3±0.02 - - 3.02±0.41 Sinorhizobium meliloti RA0563 0.14±0.01 - - -

TM0111 0.18±0.01 - - - Thermotoga maritima TM1350 0.68±0.03 - - -

25

All assays were performed using standard conditions, and were incubated for 30 min at 37°C. n=3; errors indicate standard derivation.

3.3.3 Thermal Inactivation of Purified Esterases

RP_est1, PP_est2 and PP_est3 exhibited the highest half-lives at 50 °C (Table 3.2). While

RP_est2 was stable at 50 °C after 5 h incubation, the half-life of the enzyme at 55 °C was 4 h.

By contrast, 10 min at 50 °C reduced RP_est3 and PA_est1 activity by 80 %, while PP_est1 was

entirely inactivated. Thermal inactivation is likely due to protein denaturation at high

temperature.

3.3.4 Kinetic Analysis of Purified Esterases

Activity data for lipases with soluble substrates typically display either hyperbolic or deviant

Michaelis-Menten kinetics [42, 43]. Since kinetic data collected in this study obeyed the

Michaelis-Menten equation, kinetic data further support classifying the corresponding enzymes

as esterases (Table 3.3). In comparison to EstA from Lactobacillus helveticus CNRZ32 and

EstB from Lactobacillus casei LILA [36] (Table 3.4), most esterases characterized in this study

showed comparatively low catalytic efficiency with pNP-esters. Two explanations might

account for this: 1) enzyme kinetics were not performed at the optimal temperature of each

enzyme owing to the instability of pNP-esters at high temperatures and alkaline pH, and 2) the

structure of the aromatic moiety of substrates is important for binding by the arylesterases

characterized in this study. The latter is supported by comparing the specific activity of each

enzyme using pNP-acetate and phenyl acetate, which differ only by a nitro functional group at

position 4 of the phenyl moiety (Table 3.1). Thus, future experiments will include enzyme

kinetics at optimal temperature with phenyl acetate as well as other phenolic esters such as

phenyl butyrate and phenyl palmitate.

3.3.5 Sequence Analyses for Improved Prediction of Arylesterases

The amino acid sequences for the eight characterized esterases were aligned by using the

MUSCLE algorithm (http://www.ebi.ac.uk/Tools/muscle/). All proteins except for RP_est1

exhibit the catalytic triad Ser-Asp-His with Ser in the conserved Gly-X-Ser-X-Gly pentapeptide

sequence (Fig. 3.2). By contrast, the primary sequence of RP_est1 contained two analogous

pentapeptide sequences, one upstream and one downstream of the conserved pentapeptide

26

sequence. At this stage, it is unclear whether one or both of the Ser residues participate in the

catalytic mechanism of RP_est1; the significance of this unusual sequence feature will be

explored in future studies using site-directed mutagenesis and 3D structure determination. Most

of these eight characterized enzyme targets also contain the HG sequence, which constitutes a

conserved oxyanion hole that contributes to charge stabilization of reaction intermediates [27].

Instead, RP_est1, PP_est1 and SAV_est1 contain TA, PG, and HY sequences, respectively. The

structure of the parent protein used in this study (Swiss-Prot number: P22862) predicts two Phe

residues that contribute to the substrate-binding site of this enzyme [44]. Neither Phe residue

was identified in predicted sequences of the eight enzymes characterized in this study. Rather,

random amino acid residues are presented at these two positions. However, a conserved Phe

residue was identified near the C-terminal end of the proteins. Additional experiments, including

site-directed mutagenesis, are required to elucidate the significance of a conserved Phe residue at

this position.

In an attempt to improve future predictions of arylesterases based on primary protein sequence

information, enzymes that were biohemically characterized, that were active on phenyl acetate,

and that were not active on any of the test substrates were aligned (Fig. 3.4, 3.5 and 3.6).

Unfortunately, no distinguishing features were revealed. A phylogenetic tree of 39 active

enzymes was also constructed, to determine whether enzymes active on phenyl acetate clustered

together (Fig. 3.7). However, clustering according to enzyme activity was not observed. Future

bioinformatics analyses will attempt to compare these proteins based on predicted secondary and

tertiary structures. In this way, correlations between substrate preference and the hydrophobicity

of residue in the active site cleft might be supported, which could strengthen Hidden Markov

Models for arylesterases. The difficulty to identify new motifs that distinguish arylesterases,

esterases and lipases underline the importance of heterologous protein production and

characterization efforts for accurate annotation of open reading frames in publicly available

sequence databases.

27

Table 3.2. General properties of 8 characterized proteins. Properties RP_est1 RP_est2 RP_est3 PP_est1 PP_est2 PP_est3 PA_est1 SAV_est1

ID RPA0348 RPA4646 RPA3430 PP0364 PP5253 PP3645 PA2949 SAV512

Molecular weight 32.4 kDa 26.2 kDa 27.2 kDa 26.3 kDa 29.9 kDa 31.1 kDa 34.8 kDa 28.5 kDa

Predicted pI 5.28 5.31 5.55 5.03 4.92 5.09 6.09 5.41

Substrate preference on pNP substrates

pNP acetate to pNP caproate

pNP acetate pNP caproate

pNP acetate

pNP acetate

pNP caproate

pNP butyrate

pNP acetate

Optimal pH 7 9 9 8 to 9 9 8 to 9 7 to 9 8

pH stability1 4 to 9 5 to 10 8 to 10 5 to 11 6 to 8 4 to 10 6 to 8 7 to 10

Half life at 50 °C 5 h Stable after 5 h2

<5 min 5-10 min 4 h 5 h <5 min 2 h

1. Residual activity was greater than 80 % with respect to the highest activity. 2. The half-life at 55 °C was 4 h. n=6, standard derivation are within 20 % of the averaged value. The half life was interpreted from the corresponding thermal inactivation curve. An example of thermal inactivation curve is shown in Fig. 3.2.

28

Table 3.3. Kinetic parameters of 8 characterized proteins.

The kinetics were performed at 37 °C. Kinetic parameters were calculated using GraphPad Prism5 (GraphPad Software, Inc.). All kinetic curves obeyed the Michaelis-Menten model and showed R2 of fitness above 0.96. n=3, errors indicate standard deviation. An example of a kinetic curve is shown in Fig. 3.3.

Table 3.4. Kinetic parameters of the arylesterase EstA from Lactobacillus helveticus CNRZ32 and EstB from Lactobacillus casei LILA EstA1 EstB1 Substrate Km

(mM) Specific activity at Vmax (µmol•min-1•mg protein-1)

Vmax/Km Km (mM)

Specific activity at Vmax (µmol•min-1•mg protein-1)

Vmax/Km

pNP-acetate 0.16 120 750 1.9 20 11 pNP-butyrate 0.17 150 880 0.38 17 45 pNP-caproate 0.065 3.4 51 0.029 16 550

Data from reference [36]. Kinetic assays were conducted at 30 °C for EstA and 35 °C for EstB. Reactions were performed at protein concentrations of 0.064 to 3.0 µg protein/ml. Kinetic constants were calculated from Hyperbola (Hyperbol.fit) program of Sigma Plot 3.0.

Proteins ID Assayed conditions Parameters pH pNP substrates Km (mM) Specific activity at Vmax

(µmol•min-1•mg protein-1) Vmax/Km kcat (s-1) kcat / Km(s-1•mM-1)

RP_est1 RPA0348 7 pNP butyrate 0.5±0.0 139.8±2.7 279.6 75.5 151.0 RP_est2 RPA4646 9 pNP acetate 1.4±0.2 120.6±9.9 83.2 52.7 37.6 RP_est3 RPA3430 9 pNP caproate 2.1±0.3 11.1±1.0 5.2 5.0 2.4 PP_est1 PP0364 8 pNP acetate 1.4±0.1 8.8±0.5 6.4 3.8 2.7 PP_est2 PP5253 9 pNP acetate 1.1±0.1 25.0±1.0 22.5 12.4 11.3 PP_est3 PP3645 8 pNP caproate 0.2±0.0 23.3±0.5 136.8 12.1 60.5 PA_est1 PA2949 8 pNP butyrate 5.4±1.7 38.6±9.6 7.2 22.4 4.1 SAV_est1 SAV512 8 pNP acetate 0.3±0.0 6.0 ±0.2 22.2 2.8 9.3

29

Fig. 3.2. Thermal inactivation of RP_est1. The residual activities of enzymes were measured after preincubation at corresponding temperatures for up to 5 h. Error bars indicate standard derivation.

Fig. 3.3. Kinetics of RP_est1. Rate of hydrolysis of pNP-butyrate in the range of 0 to 0.95 mM performed at pH 7 and 37 °C. Error bars indicate standard derivation.

30

3.4 Conclusions

In this study, a total of 180 genes were successfully cloned and 84 of them were expressed as

soluble proteins in E.coli. All 84 soluble proteins were purified and then screened using

colorimetric assays; 46 proteins showed activity on pNP-acetate at pH 8 while 38 proteins did

not exhibit any activity towards the test substrates. Amino acid sequence alignment revealed that

most of the proteins, whether they were active or not, contained the conserved catalytic triad,

suggesting that additional, unidentified biochemical features are required for esterases and lipase

activity. Meanwhile, 8 bacterial esterases have been characterized. All of the enzymes showed

optimal activity at neutral or alkaline pH and did not show paraoxonase activity. To our best

knowledge, all 6 arylesterases (RP_est1, RP_est2, RP_est3, PP_est2, PP_est3, and PA_est1)

represent the first arylesterases to be isolated and characterized from Rhodopseudomonas

palustris, Pseudomonas putida, and Pseudomonas aeuginosa, respectively. PP_est3

demonstrated the highest thermal stability and solvent stability, suggesting that it is the best

bacterial candidate for subsequent transesterification trials.

31

Fig. 3.4. Amino acid sequence alignment of 8 characterized bacterial proteins. The 8 bacterial protein sequences were aligned with the parent sequence (P22862). The putative catalytic triad residues and the putative residues contributed to oxyanion hole are highlighted. The identical and similar residues are marked by (*) and (:), respectively.

32

Fig. 3.5. Amino acid sequence alignment of bacterial proteins that were active on phenyl acetate. The putative catalytic triad residues and the putative residues contributed to oxyanion hole are highlighted. The identical and similar residues are marked by (*) and (:), respectively. The full-length sequence alignment is provided in Appendix A.1.

33

TM0820 27 KNA---GIRKVLFLYGGGSIKKNGVYDQVV...SVVDSAKAVAAGALYEGDIWDAF 122 TM0423 26 SRF---GERAFVVIDDFVD--KNVLGENFF...VEEETDVVV--GIGGGKTLDTAK 99 ATu2215 19 GEN---RVRHGFFTRQGGV--SEGLYQGLN...VHSPDVVVV--DDNYDGNRPQAD 99 PP0624 14 PAP--ASVRACVTTRQGGV--SLPPYETFN...VADADPTVV--AE------ADAS 91 PA3301 22 PSA--TAVGAVMLSHGMAE--HAGRYERLA...HPELPIFLL--GHSMGSYISMAY 121 PP4551 22 LPA-TPVKAVVLLAHGMAE--HAGRYQRLG...FPCTPLFLF--GHSMGSYIAQAY 122 PSPPH0357 30 GSP---EGLPVVFIHGGPG--SGCDAHSRC...LGIDKWVLF--GGSWGSTLALAY 119 PP3523 8 GKP-------------------MSKFDFQL...LGLDTVEHI--ETTLLGMITLNG 65 ATu2497 7 GRT-DAEAPTILLSSGLGG--SSAYWLPQI...LNLEKFHFM--GHALGGLIGLDI 97 RPA3731 24 GDV-DPDKPVALLLHGTGF--VGEIWDEIA...LGLSGIYGI--GHSAGATDLLLT 112 PSPPH1999 23 GPK---DGQPVIALHGWLD--NANSFARLA...LGWQRFALL--GHSLGAIISVLL 111 SAV0974 18 GSG-----PPVVFSHGWPL--NADAWDVQL...LDLRDAVVV--GHSTGGGEVARY 104 ATu5389 18 GPK---DAQPIVFHHGWPL--SSDDWDAQM...LDLKNAVHI--GHSTGGGEVARY 106 PA2717 18 GPR---DAQVIYFHHGWPL--SSDDWDAQM...LGVRGAIHV--GHSTGGGEVVHY 106 PA3053 60 APK-KANGRTILLMHGKNF--CAGTWERTI...LGVARASVI--GHSMGGMLATRY 151 PP2201 59 AQG-QANGHNVVLMHGKNF--CAATWETTI...LGVKQTIVL--GHSTGGMLATRY 150 SAV0923 13 GRP---DGPVVLLAHGFGC--DQNMWRLVV...LDLRDVAFV--GHSVSAMVGVLA 104 ATu2061 31 GPG-----PVLLMLHGFSD--TSRSFSIIE...MGVSRFFVL--GHSMGAMTAIEL 114 SAV2105 28 GTG-----PLVLLVHGFPE--SWYSWRHQL...LGERTAVIV--GHDWGSNIAATS 116 RPA2773 21 GEG-----PLVVLCHGWPE--LSYSWRHQI...LGETRAAII--GHDWGAPVAWHA 109 SAV141 23 GQG-----PLVLLLHGWPE--SWYSWRHQF...LGEEQAVVV--GHDWGAPVAWTT 111 RHA05146 24 GDG-----EPLVLIHGTAL--SHAIWRGFG...LDLPSAHVL--GYSLGGRVALAL 113 RHA00069 20 GEG-----ETLLLVHGMAG--SSATWRAVL...LDIERVTVI--GQSLGGGVAMQF 105 SAV1746 41 GEG-----PVLVLIHGIGD--SSATWAELI...LDIESATLV--GHSLGGGVAMQF 126 PP4540 17 GQG-----EPLVLLHGLGS--SCQDWELQV...LHTGPVHFV--GLSMGGMVGFQF 102 PSPPH2566 19 GQG-----HPVVLIHGVGL--NKEMWGGQI...LQLPQATVV--GFSMGGLVARAF 104 PA0231 17 GPA---GAPVLLLSNSLGT--DLGMWDTQI...LELPRVHFC--GLSMGGLIGQWL 104 PP1380 17 GPD---DAPVLVLSNSLGT--DLGMWDTQI...LDIQKAHFV--GLSMGGLIGQWL 104 PA3586 44 GRD---SDPALLLVMGLGG--QLIHWPDEV...LDIPQAHVL--GASMGGMIAQHI 154 PP2804 21 GHE---AAPAVLLIVGLGL--QLTYWPQAL...LHLEAAHVV--GMSMGGMIAQTL 125 SAV0519 14 GGC----GRPVVLLHGLAG--HAGEWDTLA...LALHRPVLV--GQSLGGHTAMLT 100 SC0267 17 GTG-----PTLVFLHYWGG--SARTWDLVV...AGITDYVLV--GHSMGGKVAQLV 101 SC5943 22 GDG-----VPLLLVHGGES--DRTQFATLR...LGLARAHLL--GTSFGGAVAQHA 107 PSPPH0033 17 GTG-----PVVLLGHSYLW--DKAMWSAQI...LNIERCSIV--GLSVGGMWGAIA 103 RPA1568 16 GKPFDASLPAAVFIHGAGF--DHSVWALQT...TGAQPAKLI--GHSMGSLIALEA 106 SAV7198 43 GKG-----PAVLLLHGFPH--TWELWTDVM...LGAGPAAVV--GIDAGTPPALLL 128 SAV1548 17 GPHDGDGGVPLVFIHGWTA--DRHRWDHQM...LEIDRFIPV--GHSMGGMIAQTL 105 SAV4410 11 GTQ-RDTSLPLVLVHGHPF--DRTMWAPQL...LKADRFVLA--GLSMGGQIAMEC 100 TM0820 280 IAHGAGLAIV...QFERFAKKIFGFE...SLKDAG---IPEEDIDKIVDNVMLLVE 365 TM0423 267 YLHGEKVAIG...EEVGLPTTLAEIG...AEKACDKNETIHNEPQPVTSKDVFFAL 350 ATu2215 218 TKAGVKAENL...----GLCTYPDEH...RFYSYR-RTTHRAEPDYGRQISAIAIL 362 PP0624 202 IRLAARGVTA...--------VYGGG...TVSDAR-FFSYRRTPQGGRFASLVWLD 244 PA3301 242 IIGGERDPVS...LGD-LADALRGAG...TYPEARHELFNESNRDAVTQDLIDWLE 301 PP4551 243 VIGGECDPVS...LTH-LADALRATG...VYPEARHEVLNETNRGEVIADILGWLE 302 PSPPH0357 259 IVHGRYDVIC...AWE-LHQNWPGSE...VIREAGHSASESGIADALVRAAAEIAR 313 PP3523 97 KFYGCL----...-------------...MVDGLG---------PAMRFDILPK-- 118 ATu2497 203 VVATKDDLLV...SLR-LAEGLPQSE...LLDFGA-HAVNITEPDLFNTRLLQFLL 256 RPA3731 246 IATAELSWPI...ATR-ASALIPGAS...AFDGVG-HCVAQEAPTQLLTALEEFAA 299 PSPPH1999 231 LMVASDGMLA...QRQELLSALPFDV...LAGGHHLHLNDEQGARSVAHCINRFFA 282 SAV0974 218 IAHGDADQIV...SAPKSAELVKDAT...VYSGAP-HGLTGAYEQEFNADLLNFIR 272 ATu5389 223 VLHGEDDQIV...AARKSIKLLKNGQ...TYPGFS-HGMLTVNADVLNQDLLAFIK 276 PA2717 21 VMHGDDDQIV...SGVLSAKLLRNGT...TYPGFP-HGMPTTQAEVINADLLAFIR 275 PA3053 263 LLIGEKDNTA...GKD-AARRIPQAT...EFPDLG-HTPQIQAPERFHQALLEGLQ 332 PP2201 262 LLIGDKDTTA...GPQ-VARLIPKGE...TFEGMG-HAPQIEEPVRFNRTLVEWLG 331 SAV0923 211 VLECAQDVIA...GAY-VHAAIPGSR...TLDATG-HCPQLSAPDATAQAITAFLG 264 ATu2061 215 CLAGSEDPLF...-RQQLFEAFPLAQ...TMPGHG-HNPHWENPQGVSTLVTEFFA 268 SAV2105 268 FIGGGLDAST...AIEAYPVTLPGLV...ILDGCG-HWLQQERPQDTNRLLTDWLA 324 RPA2773 255 FIAGSNDPVI...HLAAINRVLPNLK...IIDGAG-HWIQQEKPAEVNAALIEFLK 313 SAV141 265 YVVGDRDMVT...EIF-RGQGGPGNP...VLPGCG-HWTQQERPVEVNAALLDFLT 336 RHA05146 213 LFVGSNDA--...DTQRVAALIPGAS...ILPGFD-HSTAVAASPEVLAAVEPFLA 265 RHA00069 223 LIWGDSDGII...GYA-AHEAIPGSR...VLDGVG-HYPHLEDPAAVVEIIDDFVS 276 SAV1746 245 LLWGDRDSVV...AYG-AHEAMPGSR...IFEGAG-HFPFHTDPARFLALVEEFTG 298

34

PP4540 206 VIAADHDYT-...LKERYVALIPNAR...VVDDSR-HATPLDQPEVFNQTLLQFLA 258 PSPPH2566 210 IATGELDPGS...ARE-LAMRISGAE...ILPDQR-HMMPVESPRLVNQVLLDFFE 263 PA0231 209 IVAGSHDAVT...ARF-MQARIADAQ...LVEFAAAHLSNVEAGDAFSRRLVDFLL 261 PP1380 209 IVAGTQDVVT...GRF-MQAGIQGAE...YVDFPAAHLSNVEIGEAFSRRVLDFLL 261 PA3586 265 VIHGTADPLL...GVH-VAAHIRGSE...LIPGLA-HRFQEAFKEPLLAAVVPYLK 318 PP2804 238 VIHGDKDLMV...GFA-TASAIRGAN...LLPGMG-HDLPTCLSGTLLSLLTGHMR 291 SAV0519 203 TVLGQSGIIS...--ETMLRRLPVAT...SVPGAG-HDVHLERPAVLRHLIQEFLE 255 SC0267 202 VVAGEHDQVE...LRDNLVPYLARAD...VVPRTG-HLIPLEAPADLADAVTAFAT 256 SC5943 203 IVHGTQDRLA...GALLMERRIPNAE...PIEGGR-HGIAVEFADTVAHRVREFLG 256 PSPPH0033 215 VMCGDADIPR...TRE--MASLIGCP...LVPEAG-HIANLENPDFVSGALMTFLA 267 RPA1568 210 FILGERDMMT...GKT-LAAAISGSR...ILKGAG-HTMMVERPDEVLKALQQ--- 260 SAV7198 248 -AVGSHPVGA...QLRPFADDLTGH-...LVEDCG-HIIPLHRPDALLALLHPFLK 298 SAV1548 204 LVHGYYDIQL...MLR-MAKDYPDAV...IVDAG--HELPVEKPAELTSALDRFVT 256 SAV4410 203 VVVGADDTFT...AAA-MHAALPGAT...VIDGAA-HLPNLERPEEFNAALGEFLF 256 Fig. 3.6. Amino acid sequence alignment of proteins that were not active on any substrates. The putative catalytic triad residues and the putative residues contributed to oxyanion hole are highlighted. The identical and similar residues are marked by (*) and (:), respectively. The full-length sequence alignment is provided in Appendix A.2.

35

Fig. 3.7. A phylogenetic tree of 39 enzymes that showed activity on tested substrates. The 8 characteried proteins were highlighted. RP_est1: RPA0348; RP_est2: RPA4646; RP_est3: RPA3430; PP_est1: PP0364; PP_est2: PP5253; PP_est3: PP3645; PA_est1: PA2949; and SAV_est1: SAV512.

36

4 Solvent Stability of Bacterial Carboxyl Esterases

4.1 Introduction

Esterases are valuable catalysts for biotechnological applications since many are able to both

hydrolyze and synthesize ester linkages, depending on the reaction condition. Further, they

usually exhibit high regio- and stereo-specificity under mild reaction conditions, making them

ideal tools for green chemistry, particularly when applied for the production of chiral pure

compounds. While reactivity under mild conditions is attractive for energy and environmental

considerations, commercial applications typically demand enzymes that are stable at high

temperature, acidic or basic pH, or in the presence of organic solutions. For instance, non-

aqueous conditions are required to force lipases and esterases to function as synthetic catalysts,

rather than catalyze hydrolysis reactions. Further, many lipase and esterase substrates are

lipophilic, suggesting that reaction efficiencies can be improved in the presence of an organic

solvent. As a result, esterases that are stable in detergents, organic solvents, or ionic liquids are

particularly useful for most industrial applications [1]. Given the impact of temperature on

substrate solubility and reaction rates, thermal stable enzymes are also desirable [2,3]. Notably,

a high temperature process can improve reaction performance by reducing solvent viscosity and

minimizing microbial contamination [4].

While enzyme immobilization can stabilize enzyme activity [5, 6], enzymes with innate stability

are preferred for industrial applications. However, predicting temperature and solvent stability

from primary sequence and protein structural analyses is often inconclusive, particularly since

less than 1 % differences in protein sequences can lead to significant changes in the temperature

stability [7]. Accordingly, two strategies that are frequently carried out to optimize enzyme

performance include: 1) screening for natural solvent- and/or thermostable enzymes [8-10], and

2) random mutagenesis and selection of temperature stable or solvent tolerant enzymes

[4,11,12]. For example, solvent-stable lipases and proteases were previously isolated from

organic solvent-tolerant microorganisms grown on solvent-rich media [1,13]. It was

hypothesized that the enzymes secreted during growth on these carbon sources must be solvent-

stable [14]. Lipase LST-03 from Pseudomonas aeruginosa was isolated in this way [13], which

has a longer half-life in the presence of organic solvents like DMSO and cyclohexane than in the

37

absence of an organic solvent. Solvent tolerant enzymes have also been discovered by

measuring residual enzyme activity after pre-incubation in different solvents [15]. This approach

typically uses heterologously expressed and purified proteins, and incorporates high throughput

enzyme screens in the presence of different solvents. More recently, ionic liquids (ILs) have

become attractive solvents for enzyme-catalyzed reactions. ILs represent a specific class of

organic solvents, and are considered green solvents due to their low volatility, thermostability,

high polarity, and recyclability [16]. Many studies have demonstrated the potential of ILs to

promote enzyme-catalyzed synthetic reactions [16-19]. In particular, lipases and esterases have

been used with ILs, and exhibit comparatively high (enantio)selectivity and operational stability

compared to reactions performed in organic solvents [20-23].

While several publications demonstrate the synthetic potential of lipases and optimization of

these catalyses [24-26], less effort has focused on synthetic reactions catalyzed by esterases,

including arylesterases. Exceptions include transesterification of a phenolic acid by a feruloyl

esterase from Fusarium oxysporum, and esterification of glycerol with sinapic acid by a feruloyl

esterase from Aspergillus niger [27, 28]. Phenolic acids, including sinapic acids, are derived

from lignin, which is a by-product generated during the production of biofuels, as well as pulp

for paper. The production of value-added materials from lignin is anticipated to off-set the cost

of biofuels, and increase the long term viability of future biorefineries whose main product is

bioenergy [29]. Accordingly, to increase the arsenal of enzymes capable of synthesizing new

and industrially relevant chemicals from lignin, 15 publicly available bacterial genomes were

mined for putative arylesterase-encoding genes. Corresponding genes were recombinantly

expressed and characterized as described in Chapter 3. This chapter reports the stability of eight

esterases in 7 detergents, 14 organic solvents and 4 ILs. The stability of these esterases was

compared to that of two commercial lipases (PS “Amano” SD and AY “Amano” 30). This

analysis is the first step to identifying new enzymes and reaction conditions that synthesize new

products from lignin.

38


4.2.1 Materials

Ionic liquids, including [BMIM]BF4, [BMIM]PF6, [BMIM]CF3SO3 were purchased from

Solvent Innovation GmbH (Germany) and Cyphos 109 was kindly provided by Cytec Industries

Inc. (Canada). Tween 20, Tween 60, Tween 80, Triton X-100, SDS, EDTA, and 2-

mercaptoethanol were purchased from Sigma (Canada). Solvents, including methanol, ethanol,

isopropanol, 1-butanol, tert-amyl alcohol, acetone, acetonitrile, tetrahydrofuran, 1,6-dixaone,

DMSO, toluene, p-xylene, hexane, cyclohexane were also purchased from Sigma (Canada). PS

“Amano” SD and AY “Amano” 30 were kindly supplied by AMANO Enzyme Inc. The

recombinant enzymes used in this study were recombinantly expressed and purified as described

in Chapter 3.

4.2.2 Effect of Detergents and Reducing Agent on Enzyme Stability

The effect of several detergents on enzyme stability was analyzed by preincubating 0.5 µg to 1

µg of each enzyme in 50 mM potassium phosphate buffer (pH 7), containing 1 % (v/v) of Tween

20, Tween 60, Tween 80 or Triton X-100, or 1 mM of SDS, EDTA, or 2-mercaptoethanol.

Residual activity was measured after 5 h incubation at 37 °C. The enzyme amount added to each

reaction was adjusted to ensure that initial reaction rates were measured. The standard activity

assay was performed to measure residual activity, as described in Chapter 3 (Section 3.2.5). In

brief, 10X concentrated enzymes (i.e. 0.5 µg/100 µL for RP_est1, 2.5 µg/100 µL for RP_est2, 5

µg/100 µL for RP_est3, 5 µg/100 µL for PP_est1, 5 µg/100 µL for PP_est2, 5 µg/100 µL for

PP_est3, 5 µg/100 µL for PA_est1 and 10 µg/100 µL for SAV_est1) were preincubated in each

detergent for 5 h preincubaton at 37 °C. The residual activity was measured by transferring 10

µL aliquots back to the standard conditions. The effect of the small amount of detergent in the

10 µL aliquot on background absorbency and enzyme activity was evaluated and found to be

negligible.

4.2.3 Effect of Organic Solvents on Enzyme Stability

Residual enzyme activity was measured using the standard assay following 5 h of perincubation

at 37 °C in the presence of the following solvents: methanol, ethanol, isopropanol, 1-butanol,

39

tert-amyl alcohol, acetone, acetonitrile, tetrahydrofuran, 1,6-dixaone, DMSO, toluene, p-xylene,

hexane, cyclohexane. Organic solvents were prepared in 15 %, 30 % or 50 % (v/v) in 50 mM

potassium phosphate buffer (pH 7). The effect of DMSO on enzyme activity was studied in

more detail. Here, 0.5 µg to 1 µg of each enzyme was incubated in buffer containing 0 to 40 %

of DMSO with 5 % increments. Residual activity was measured after 24 h incubation at 37 °C.

Specific activities were measured in the presence of 5 % to 95 % of DMSO; pNP-acetate was

used as the substrate. In brief, 10X concentrated enzymes (i.e. 0.5 µg/100 µL for RP_est1, 2.5

µg/100 µL for RP_est2, 5 µg/100 µL for RP_est3, 5 µg/100 µL for PP_est1, 5 µg/100 µL for

PP_est2, 5 µg/100 µL for PP_est3, 5 µg/100 µL for PA_est1 and 10 µg/100 µL for SAV_est1)

were preincubated in each organic solvent mixture for 5 h preincubaton at 37 °C. The residual

activity was measured by transferring 10 µL aliquots back to the standard conditions, as

described in section 3.2.5. Slight changes in absorbance readings that could result from

partitioning of substrate between phases was subtracted (Appendix B.5).

4.2.4 Effect of Ionic Liquids (ILs) on Enzyme Stability

Three imidazolium-based ILs ([BMIM]BF4, [BMIM]PF6, [BMIM]CF3SO3), and one

phosphonium-based IL (Cyphos 109) were used to study the effect of ILs on enzyme stability.

The imidazolium ILs were selected since they are most commonly used in lipase-catalyzed

synthetic reactions [17-26]. Enzymes were incubated for 3 h at 37 °C in the presence of 40 %,

50 %, 60 %, 70 %, 85 %, or 100 % of each solvent mixed with DMSO. Residual enzyme

activity was measured using the standard activity assay. In brief, 10X concentrated enzymes (i.e.

0.5 µg/100 µL for RP_est1, 2.5 µg/100 µL for RP_est2, 5 µg/100 µL for RP_est3, 5 µg/100 µL

for PP_est1, 5 µg/100 µL for PP_est2, 5 µg/100 µL for PP_est3, 5 µg/100 µL for PA_est1 and 10

µg/100 µL for SAV_est1) were preincubated in each IL cosolvent for 3 h preincubaton at 37 °C.

The residual activity was measured by transferring 10 µL aliquots back to the standard

conditions, as described in section 3.2.5.

40


This chapter extends the work described in Chapter 3, focusing on enzyme stability in a variety

of detergents and organic solvents. This analysis was conducted to identify the best enzyme

candidates for future applications in synthetic reactions.

4.3.1 Effects of Detergents, Chelators and Reducing agents on Hydrolysis Activities

The effect of detergents (Tween 20, Tween 60, Tween 80, Triton X-100, and SDS), chelator

(EDTA), and disulfide reducing agent (2-mercaptoethanol) on enzyme activity is summarized in

Table 4.1. Esterases from Rhodopseudomonas palustris showed higher stability compared to

esterases from Pseudomonas putida, Pseudomonas aeruginosa and Streptomyces avermitilis.

Notably, preincubation of RP_est1 with Triton X-100 and SDS appeared to have an activation

effect. Similarly, while SDS inhibited most of esterases, RP_est1 was activated by over 75 %.

Increases in enzyme activity after incubation may be explained by partial unfolding of enzymes

or possible complexes formed between enzymes and micelles [30, 31]. Additional experiments

such as Isothermal Titration Calorimetry (ITC) are required to confirm this hypothesis. PA_est1

activity was reduced following incubation with detergents, but was not inhibited by EDTA or 2-

mercaptoethanol. Instead, the activity of PA_est1 was 20 to 30 % higher after incubation with

EDTA and 2-mercaptoethanol compared to the control. These observations suggest that the

enzyme is not a metalloprotein and probably does not contain cysteine disulfide bridges that are

required for enzyme activity [32, 33]. Indeed, the primary sequence of PA_est1 contains only

one cysteine residue that is unable to form a disulfide bond. To confirm if the enzymes are

metalloproteins, additional experiments should be performed as described by Hogbom et al. [34]

based on detection of chemiluminescence produced by luminol in the presence of certain metal

catalysts when mixed with a base and an oxidant, and color production of some metal complexes

of the chelator 4-(2-pyridylazo)resorcinol. PP_est2 and RP_est3 also contain only one cysteine

residue and SAV_est1 does not contain any cysteine residues. However, their stability was

slightly affected in the presence of 1 mM 2-mercaptoethanol. The presence of disulfide bridges

in esterases that contained more than one cysteine residue and were affected by EDTA (i.e.,

RP_est2, and PP_est1) will be evaluated using corresponding structural information, which is

currently being acquired. The activity of all esterases isolated from P. putida decreased after

41

preincubation with the four nonionic detergents, except for PP_est3, which was stable in the

presence of Triton X-100. The stability profiles of RP_est1 and RP_est2 were similar to the

commercial lipases PS-SD and AY-30, which were stable in the presence of detergents and

slightly inhibited by EDTA and 2-mercaptoethanol.

Table 4.1. Effect of detergents, inhibitor, and disulfide reducing agent on enzyme stability.

Residual activities were measured after 5 hr incubation at 37 °C. n= 6; errors indicate standard derivation. The values of Critical Micelle Concentration (CMC) for nonionic and ionic detergents were obtained from [39] and [40], respectively.

4.3.2 Effects of Organic Solvents on Hydrolysis Activities

All esterases were more stable in non-polar solvents (log P> 2.5) than in polar solvents (log P<

0.89) (Table 4.2). This is consistent with the general agreement that water miscible polar

solvents can strip off the water layer that forms around many proteins, which is essential for

activity [15]. In contrast, non-polar solvents may protect the microenvironment of enzymes,

thereby preventing loss of the water layer. Among the esterases that were screened in this study,

RP_est3 was the most stable in polar solvents. Even after preincubation with 50 % of all tested

Residual activity (%) Detergents CMC at 22°C

RP_ est1

RP_ est2

RP_ est3

PP_ est1

PP_ est2

PP_ est3

PA_ est1

SAV_ est1

None n/a 100 100 100 100 100 100 100 100 1 % v/v Tween 20

0.042 mM

131±5 96±14 64±8 31±8 17±1 82±24 24±4 78±9

1 % v/v Tween 60

0.022 mM

136±39 93±9 88±7 40±10 89±2 78±26 101±15 90±5

1 % v/v Tween 80

0.028 mM

135±9 100±4 91±8 64±9 80±3 50±3 32±8 70±1

1 % v/v Triton X-100

0.20 mM

174±8 93±16 90±9 27±8 58±3 110±10 25±7 67±16

1 mM SDS 5.29 mM

177±3 95±1 73±9 30±8 58±6 53±6 40±4 28±7

1 mM EDTA

n/a 93±1 85±9 80±21 40±8 66±15 98±9 130±16 88±4

1 mM 2-Mercaptoethanol

n/a 89±35 86±4 88±13 44±11 85±8 95±10 123±9 82±13

42

solvents, RP_est3 retained more than 50 % of its original activity. While solvent stability is

important during the equilibration step of enzymatic reactions performed in organic solvents,

enzyme activity in the presence of organic solvents must be evaluated independently. This is

because solvent stability is not well correlated to enzyme activity in the presence of the

corresponding solvent.

None of the esterases exhibited higher stability in organic solvents compared to the commercial

lipases PS-SD and AY-30. This could be attributed to the lid-like structure that many lipases

display close to the active site [35-37]. This lid structure, and surrounding non-polar residues,

could reduce the accessibility of polar solvents to active site residues.

Since lipase-catalyzed transesterification reactions are typically performed in up to 40 % DMSO,

the standard activity assay was performed in the presence of 0 to 95 % DMSO (Fig. 4.1). While

RP_est3 and PP_est3 were activated by preincubation with 15 to 30 % or 30 to 50 % of DMSO,

respectively, the specific activity of these enzymes decreased rapidly when measured in the

presence of more than 10 % DMSO. By contrast, in the presence of 40 % DMSO, PP_est2,

RP_est2, RP_est1 and SAV_est1 exhibited 70 %, 46 %, 66 %, 39 % of residual activity. Their

performance could be further improved by lyophilization and/or immobilization. DMSO is used

as a co-solvent to prevent hydrolysis reactions when using substrates that are soluble in aqueous

solutions. Accordingly, the relative stabilities and activities of arylesterases in non-polar

solvents including hexane, butanol and isopropanol might be more relevant to predicting their

ability to catalyze the esterification of lignin subunits with aliphatic molecules.

43

Table 4.2. Effect of various organic solvents on enzyme stability.

Residual activity (%) Organic solvents

LogP Conc. (v/v%) PS-SD AY-30 RP_

est1 RP_ est2

RP_ est3

PP_ est1

PP_ est2

PP_ est3

PA_ est1

SAV_ est1

None - - 100 100 100 100 100 100 100 100 100 100 15 105±10 85±5 112±25 83±6 138±12 42±10 78±3 100±14 77±11 103±17 30 105±8 69±4 118±18 94±6 171±7 32±7 54±2 111±11 28±7 103±4

DMSO -1.22

50 116±12 18±7 16±2 110±3 70±44 0±3 0±0 121±11 25±6 4±2 15 86±5 96±4 112±14 83±6 72±7 92±53 75±6 87±18 35±7 37±13 30 100±12 104±9 132±17 84±8 66±4 0±1 3±0 70±31 22±4 1±1

Methanol -0.76

50 105±12 75±14 139±26 3±0 68±10 0±2 0±1 103±9 20±3 1±1 15 102±12 57±9 86±2 85±7 98±8 0±3 0±0 97±8 35±8 6±2 30 111±26 35±5 12±2 7±2 91±11 1±2 0±0 78±6 33±4 1±1

1,6-Dixaone -0.42

50 125±26 4±5 0±2 5±1 114±5 5±4 0±0 1±1 38±7 5±1 15 87±4 29±46 109±8 85±2 72±4 0±2 47±2 86±29 21±2 123±8 30 70±12 98±22 111±7 3±0 84±13 0±2 0±1 110±38 20±2 2±2

Acetonitrile -0.34

50 95±13 12±9 0±1 3±1 84±12 0±21 0±5 0±3 23±5 0±12 15 94±5 66±11 94±11 98±5 70±5 0±3 12±7 115±14 18±6 53±24 30 110±16 38±8 79±17 6±2 72±10 0±3 0±1 95±14 19±3 0±1

Isopropanol -0.28

50 116±22 3±5 2±1 3±1 88±12 0±3 0±1 21±22 15±4 0±1 15 91±3 63±2 104±5 87±5 76±5 0±1 56±5 103±15 20±2 26±10 30 104±16 49±2 124±11 67±2 80±4 0±2 1±0 89±12 16±4 0±1

Ethanol -0.24

50 108±15 14±7 3±1 3±1 73±11 0±2 0±0 34±8 25±7 1±2 15 89±5 111±12 99±6 84±9 71±4 6±3 53±10 105±11 36±11 118±15 30 91±14 109±32 75±24 94±4 66±5 0±2 7±16 117±19 22±3 128±18

Acetone -0.23

50 96±30 72±10 172±12 3±0 72±9 0±2 0±0 86±8 19±4 4±3

44

15 89±3 105±12 118±4 81±3 79±9 0±1 0±1 108±21 24±3 119±16 30 87±3 115±8 119±8 3±0 84±12 0±2 0±1 89±7 24±4 6±1

Tetrahydro-furan

0.49

50 88±15 50±51 15±4 4±1 104±10 0±1 0±0 0±0 32±6 2±2 15 61±13 17±3 2±0 86±4 70±4 2±1 4±2 111±16 22±3 1±1 30 79±11 13±7 1±1 90±4 62±7 0±1 1±1 125±27 23±3 1±1

1-Butanol 0.8

50 114±27 20±6 2±1 88±28 68±12 2±2 1±0 94±20 24±4 1±1 15 96±8 34±11 86±14 94±4 80±4 0±3 0±0 125±11 26±3 0±2 30 86±19 4±5 4±1 99±4 69±7 0±3 0±0 133±23 25±4 0±1

Tert-amyl alcohol

0.89

50 106±8 0±6 3±0 87±33 72±16 0±6 0±0 164±26 24±4 0±3 15 95±51 119±4 101±7 83±6 101±9 38±5 15±12 44±57 105±3 111±16 30 100±55 131±13 122±7 83±8 119±6 94±18 99±5 81±62 130±7 127±18

Toluene 2.5

50 97±36 182±20 181±14 82±17 132±39 148±15 115±5 174±12 160±54 167±31 15 81±9 137±11 120±7 73±2 87±35 176±45 51±17 88±31 107±5 104±18 30 64±14 250±90 118±6 69±9 127±10 120±11 94±6 135±9 121±49 106±33

p-Xylene 3.1

50 65±28 323±70 169±14 79±14 116±40 320±183 117±7 194±34 178±54 135±31 15 73±8 154±10 112±5 72±8 125±8 11±15 42±47 63±56 126±17 116±16 30 85±7 190±30 140±6 77±8 131±18 176±74 78±35 56±41 142±21 147±15

Cyclohexane 3.2

50 77±25 20013 187±17 74±7 162±70 356±127 126±9 166±38 166±75 208±16 15 89±9 93±3 105±10 78±6 115±8 32±36 62±41 43±58 95±20 132±8 30 85±12 142±20 116±10 76±9 130±6 300±101 66±32 85±64 138±12 143±8

Hexane 3.9

50 97±17 195±14 165±12 79±14 147±55 285±70 120±14 202±22 143±74 187±19

Residual activities were measured after 5 h incubation at 37 °C. The column “conc. v/v %” indicates the percentage of corresponding organic solvents in 50 mM potassium phosphate buffer (pH 7). n= 6; errors indicate standard derivation.

45

4.3.3 Effects of Ionic Liquids (ILs) on Hydrolysis Activities

Ionic liquids offer new options for retaining enzyme activity during synthesis reactions that must

be performed in the absence of water. In this study, esterase stability was evaluated using three

typical imidazolium-based ILs and one phosphonium-based IL. A co-solvent system containing

DMSO and the selected IL was generated. None of the recombinant or commercial enzymes was

active in 40 % to 100 % of the phosphonium-based IL (Cyphos 109). Therefore, only the results

for imidazolium-based ILs are shown (Table 4.3). [BMIM]PF6 significantly destabilized all

esterases for P. putida and S. avermitilis. By contrast, RP_est2 and RP_est3 from R. palustris

retained 40 % and 60 % of activity after preincubation with 85 % of this IL. In general,

increasing the fraction of IL in DMSO/IL co-solvents to at least 85 % increased measured

residual activities (Table 4.3). An exception was SAV_est1, which retained 85 % activity in 100

% of DMSO. Overall, RP_est3 displayed higher stability than the commercial lipases PS-SD

and AY-30, suggesting that this enzyme is a particularly interesting target for application

development. Notably, RP_est3 appeared to be activated by preincubation with [BMIM]BF4 and

[BMIM]CF3SO3. A similar result was observed for AY-30 and PP_est3 with [BMIM]CF3SO3,

albeit to a lesser extent. The mechanism behind IL stabilization of enzymes remains unclear

[38]. However, two recent spectroscopic studies of protein thermostability in ILs suggest that a

stabilization effect may result from alteration in the protein hydration level and structural

compaction as well as other additional factors such as free volume contributions, ionic

interactions and confinement effects [38].

Owing to their limited commercial availability and the extensive use of mechanistically related

lipases as synthetic catalysts, the arylesterases characterized in this study constitute an important

set of new, industrially relevant catalysts. Their ability to catalyze hydrolysis reactions in the

presence of organic solvents, as well as esterification of lignin-derived compounds is currently

being evaluated.

47

Fig. 4.1. Effect of DMSO on enzyme activity. Enzyme activity was measured in the presence of 5 to 95 % DMSO at 37 °C using pNP-acetate as the substrate. n= 6; error bars indicate standard deviation.

48

Table 4.3. Effect of ionic liquids on enzyme stability. Residual activity (%) Ionic

liquids Conc. (v/v% in DMSO)

PS-SD

AY-30 RP_est1 RP_est2 RP_est3 PP_est1 PP_est2 PP_est3 PA_est1 SAV_est1

None - 100 100 100 100 100 100 100 100 100 100 100% DMSO

- 0±0 16±3 5±2 4±2 59±8 0±1 8±1 0±4 22±2 85±3

40 3±0 70±11 9±2 6±4 67±3 13±8 9±1 11±5 36±1 14±1 50 3±0 72±14 9±3 6±4 74±7 13±8 8±1 13±8 31±6 12±2 60 3±0 78±14 10±3 5±3 84±9 14±7 11±0 18±12 41±5 15±2 70 24±16 73±19 11±3 20±19 102±11 17±12 13±1 14±14 47±8 15±2 85 60±19 91±15 13±4 78±20 184±16 18±10 16±1 25±17 47±8 24±4

[BMIM]BF4

100 12±3 105±19 20±6 84±14 205±53 30±19 23±2 39±27 60±3 37±8 40 0±1 19±8 1±2 3±4 43±18 0±6 0±2 0±13 20±18 0±2 50 0±1 14±5 0±5 4±3 20±12 0±8 0±7 0±12 22±21 0±4 60 23±2 7±14 0±3 22±18 41±21 0±27 0±8 0±39 23±11 1±5 70 35±4 16±18 5±4 61±8 81±12 2±13 0±8 0±2 23±16 0±1 85 20±7 33±28 41±19 61±11 77±12 0±28 0±4 42±28 20±2 6±4

[BMIM]PF6

100 12±5 32±40 2±3 10±5 11±18 0±8 0±4 0±4 10±15 0±4 40 3±0 82±14 11±2 6±4 84±10 16±9 13±1 13±±53 34±9 13±1 50 4±0 93±8 11±2 6±6 94±17 20±11 15±1 18±10 50±7 14±1 60 6±1 100±8 13±2 9±2 108±22 25±14 18±2 56±22 54±7 16±1 70 44±9 116±7 21±3 35±12 119±13 27±16 20±2 84±44 61±11 19±3 85 40±3 135±14 51±9 44±19 176±19 42±21 37±6 120±44 81±13 28±4

[BMIM]CF3SO3

100 34±6 145±11 41±10 63±10 177±14 67±16 96±10 121±25 64±19 27±1 Residual activities were measured after 3 h incubation at 37 °C. The column “conc. v/v % in DMSO” indicates the percentage of corresponding ILs in DMSO during the incubation. n= 6; errors indicate standard derivation.

49

5 Isolation and Characterization of Two, Solvent-Tolerant, Thermophilic Archaeal Esterases

5.1 Introduction: Extreme Environments: A Source of Unique Biocatalysts

Archaea represent one of the three domains of living organisms. They are single celled

microogranisms, and display unique properties that the other domains do not share. For instance,

archaea are often found in extreme habitats such as high temperature or highly acidic or basic

environments. Organisms that withstand these environments, and indeed thrive in them, are

called extremophiles. For instance, Archaeoglobus fulgidus are hyperthermophiles and can be

found in hydrothermal vents and hot springs. Enzymes that are isolated from extremophiles

often exhibit superior performance in industrial applications [1]. Amongst extremophiles,

thermophilic archaea have attracted significant attention, especially for their production of

thermophilic enzymes [1]. A key example is Taq polymerase from Thermus aquaticus, an

enzyme ubiquitously used in PCR reactions, a core molecular biology technique [2]. There are

several advantages to performing biocatalyses at an elevated temperature, including enhanced

reaction rates, increased substrate solubility and decreased risks of contamination [1]. In

addition, thermophilic enzymes usually exhibit high resistance against many organic solvents

and detergents and are more resistant to proteolytic attack [1].

Despite the industrial relevance of temperature stable enzymes that can be retrieved from

hyperthermophilic archaea, most industrial enzymes that are currently used were isolated from

either bacteria or fungi. Archaeal enzymes that have received most attention to date include

amylases, cellulases, xylanases, and proteases; a handful of lipases and esterases have also been

reported [1,3]. The industrial interest in lipases and esterases resides in the fact that they

typically do not require cofactors, show high regio- and stereospecificity, are often

comparatively stable and active in organic solvents, and can perform both hydrolytic and

synthetic reactions [4].

More lipases than carboxyl esterases have been described to date, and at the time of writing, only

one example of an arylesterase from an archaeal species had been reported [5]. Additional

examples of arylesterases isolated from thermophilic archaea will enable the identification of

50

sequence motifs and structural features that determine substrate specificity and thermostability

within this class of enzymes. Understanding these biochemical determinants will greatly

facilitate subsequent optimization of substrate preference and stability of arylesterases, and

possibly other carboxyl esterases and lipases.

Herein, we report the cloning, recombinant expression, and characterization of two thermostable

esterases from A. fulgidus. The purified enzymes were most active on phenyl acetate and pNP-

acetate in comparison to other pNP-esters and retained over 80 % of activity after 5 h incubation

at up to 85 °C and in the presence of several detergents and organic solvents. These two

esterases exhibited significantly lower thermal inactivation than the bacterial esterases described

in Chapter 3. In contrast to the bacterial enzymes, they were also comparatively stable in both

polar and nonpolar solvents.


Genes for this study were isolated from the Archaeoglobus fulgidus genomic DNA purchased

from the ATCC (Cat. # 49558). Please refer to Chapter 3 for details regarding gene cloning,

recombinant expression in E. coli, protein purification, and enzyme characterization using pNP

substrates, as well as phenyl acetate. To evaluate enzyme activity at optimal pH, pNP-palmitate

was used as the substrate, since it is difficult to monitor pH-acetate at pH 10 and pH 11. Please

refer to Chapter 4 for details regarding enzyme stability measurements in the presence of

detergents, organic solvents and ionic liquids.


Six putative esterase genes from A. fulgidus were cloned and expressed in E. coli. Of these, two

were soluble, three were insoluble and one protein was not expressed. The two soluble proteins

(AF_est1 and AF_est2) were biochemically characterized to determine their substrate preference,

optimal pH, kinetic parameters, pH stability, thermal inactivation, as well as their stability in the

presence of various detergents, organic solvents, and ionic liquids.

51

5.3.1 Esterase Activity with p-Nitrophenyl Substrates

Lipases and esterases share similar conserved sequence motifs, and can be differentiated based

on substrate preference. Therefore, to investigate whether AF_est1 and AF_est2 were esterases

or lipases, the activities of these enzymes were screened using a series of short to long-chain

pNP-esters. Consistent with esterase activity, both enzymes showed preference for short-chain

pNP-esters, with the highest activity towards pNP-acetate (Fig. 5.1). To a lesser extent, AF_est1

and AF_est2 also hydrolyzed long-chain pNP-esters including pNP-palmitate, an activity that

was not observed by another esterase (Est-AF) isolated from A. fulgidus [6]. A comparison of

specific activities on all of the substrates tested indicated that both esterases preferred phenyl

acetate, a substrate that is commonly used to identify arylesterase (Table 5.1). Many

arylesterases are also paraoxonases, and are not inhibited by organophosphates, like paraoxon [7-

9]. However, AF_est1 and AF_est2 did not show any activity towards paraoxon, and so are not

paraoxonases.

Table 5.1. Activities (µmol•min-1•mg protein-1) of enzymes using 0.5 mM of substrate. Esterases Specific activities of the test substrates

pNP

acetate pNP

caproate pNP

palmitate pNP

benzoate Phenyl acetate

Tributyrin

Olive oil

AF_est1 0.74±0.01 0.36±0.01 0.08±0.02 n.a1 7.04±0.29 n.a n.a AF_est2 0.81±0.01 0.59±0.00 0.15±0.00 n.a 7.93±0.33 n.a n.a

1. n.a means not active.

All assays were performed using standard conditions, and were incubated for 30 min at 37°C. n=3; errors indicate standard derivation.

AF_est1 shared 20 % sequence identity with AF_est2 in a global alignment. The alignment of

AF_est1 and AF_est2 with three known archaeal esterases confirmed the presence of a

conserved catalytic triad sequence, consisting of Ser, Asp, and His, where the serine residue is

located in the conserved Gly-X-Ser-X-Gly pentapeptide sequence (Fig. 5.2). An X-ray structure

has been solved for the esterase from A. fulgidus (AFEST; O28558) [10]. AF_est1 and AF_est2

shared 8.6 % and 19.8 % sequence identity with AFEST, respectively. Based on the alignment

and positions of catalytic triad predicted from X-ray structure of AFEST, it showed that Ser95,

His210, Asp238 constitute the catalytic triad in AF_est1, and that Ser98, His214, and Asp243

constitute the catalytic triad in AF_est2. AF_est1 and AF_est2 sequences were also aligned to an

52

arylesterase isolated from Sulfolobus solfataricus (Fig. 5.3). Notably, the pentapeptide sequence

of S. solfataricus is slightly downstream compared to AF_est1 and AF_est2; AF_est1 and

AF_est2 shared 2.3 % and 13.2 % identity to this arylesterase, respectively. An alignment of

AF_est1 and AF_est2 to the eight bacterial esterases described in Chapter 3 showed that the

putative catalytic triad was perfectly aligned for all of them, except for RP_est1, which contained

two analogous pentapeptide sequences, one upstream and one downstream of the conserved

pentapeptide sequence. Features that distinguished the archaeal enzymes from the bacterial

enzymes were not identified by primary sequence comparisons (Appendix A.3). Future

bioinformatic analyses will attempt to identify residues that contribute to the thermal inactivation

of the archaeal enzymes.

Fig. 5.1. Substrate preference of AF_est1 and AF_est2 on pNP-esters. Activities of AF_est1 (black) and AF_est2 (grey) with various pNP-esters were determined photometrically at 400 nm at 37 °C. Enzymes were diluted in 50 mM potassium phosphate buffer (pH 7) and adjusted to ensure that initial reaction rates were measured in the kinetic cycle. n= 6; error bars indicate standard deviation.

53

Fig. 5.2. Amino acid sequence alignment of AF_est1 and AF_est2 with other esterases from Archaeoglobus fulgidus. Three esterases were listed by their Swiss-Prot accession numbers. The putative catalytic triad residues were highlighted. The identical and similar residues were marked by (*) and (:), respectively.

54

Fig. 5.3. Amino acid sequence alignment of AF_est1 and AF_est2 with an arylesterases from Sulfolobus solfataricus. The arylesterase was listed by their Swiss-Prot accession numbers) The putative catalytic triad residues were highlighted. The identical and similar residues were marked by (*) and (:), respectively.

AF_est1 had an optimal activity at pH 9.5 whereas AF_est2 showed highest activity between pH

9 and 10 (Fig. 5.4). Both enzymes also showed high stability in alkaline buffers, even at pH 11

(Fig. 5.5). These results indicate that AF_est1 and AF_est2 are more stable at alkaline pH than

the previously reported esterase from A. fulgidus, which lost activity above pH 8 [6]. AF_est1

and AF_est2 were also stable at high temperatures. Even after 5 h at 90 °C, AF_est1 and

AF_est2 retained 42 % and 52 % of activity, respectively (Fig. 5.6). Moreover, the activities of

AF_est1 and AF_est2 activities increased slightly after 2 h incubation at 75 to 84.5 °C. This

activation effect decreased for AF_est2 after 5 h incubation. In comparison, the thermal

stabilities of AF_est1 and AF_est2 were 4 to 5 times greater than Est-AF, an esterase previously

isolated from A. fulgidus [6]. However, both of them were not as stable as the arylesterase

isolated from S. solfataricus [5], which retained 52 % of its activity after 50 h of incubation at 90

°C.

55

Fig. 5.4. pH profiles of AF_est1 (black) and AF_est2 (grey). The activity was measured with 0.5 mM pNP-palmitate at 37 °C in the Britton and Robinson’s universal buffers with different pH. n= 6; error bars indicate standard deviation.

Fig. 5.5. Effects of pH on AF_est1 (black) and AF_est2 (grey) stability. The residual enzyme activity was measured with pNP-acetate at 37 °C after 24 h incubation in the Britton and

56

Robinson’s universal buffers with different pH at 37 °C. n= 6; error bars indicate standard deviation.

Fig. 5.6. Effect of thermal inactivation on AF_est1 and AF_est2. Enzymes were diluted in 50 mM potassium phosphate buffer (pH 7). The residual enzyme activity was measured with pNP-acetate at 37 °C after 2 h (black) and 5 h (grey) incubation in different temperatures. n=6; error bars indicate standard deviation.

57

5.3.2 Effect of Additives and Organic Solvents on Esterase Stability

Esterases, including arylesterases have significant industrial potential, since they have the

capacity to catalyze hydrolytic as well as synthetic reactions. To promote synthetic catalyses,

reactions are performed in the absence of water, and instead in organic solvents or more recently,

ionic liquids. Accordingly, enzymes that are stable in the presence of detergents, organic

solvents, and ionic liquids are particularly suitable for biotechnological applications. The

stability of AF_est1 and AF_est2 was investigated in the presence of detergents, a chelating

agent, a disulfide reducing agent, conventional organic solvents, and ionic liquids. Both

esterases retained over 85 % of activity after 5 h incubation in the presence of detergents, EDTA

and 2-mercaptoenthanol (Table 5.2). AF_est1 was slightly more stable than AF_est2, and

appeared to be activated in the presence of 1 mM SDS. Increases in enzyme activity after

incubation may be explained by partial unfolding of enzymes or possible complexes formed

between enzymes and micelles [5, 11].

Table 5.2. Effects of various surfactants on AF_est1 and AF_est2 stability. Residual activity of (%) Organic solvents CMC at 22°C

AF_est1 AF_est2 None n/a 100 100 1 % v/v Tween 20 0.042 mM 100±2 91±8 1 % v/v Tween 60 0.022 mM 113±20 102±16 1 % v/v Tween 80 0.028 mM 96±14 87±10 1 % v/v Triton X-100 0.20 mM 114±11 99±7 1 mM SDS 5.29 mM 131±1 101±8 1 mM EDTA n/a 114±1 104±20 1 mM 2-Mercaptoethanol n/a 105±16 93±11

Residual activities were measured after 5 h incubation at 37 °C. n= 6; errors indicate standard derivation. The values of Critical Micelle Concentration (CMC) for nonionic and ionic detergents were obtained from [20] and [21], respectively.

The destabilizing effects of the organic solvents were investigated using three solvent

concentrations (Table 5.3). With the exception of isopropanol, tetrahydrofuran, and acetonitrile,

AF_est1 and AF_est2 retained 70 % of activity after 5 h of incubation in 50 % of each solvent

(Table 5.3). This is consistent with the observation that thermophilic enzymes are typically

solvent tolerant, giving a great potential for these enzymes to catalyze reactions in organic media

58

[1,12]. Furthermore, both esterases were more stable in non-polar solvents with log P> 2.5 than

in polar solvent with log P< 0.89. A possible explanation for this observation was described in

Section 4.3.2, whereby polar solvents may strip off the essential water layer around the protein,

leading a destabilizing effect. By contrast, hydrophobic organic solvents appear to stabilize the

water layer at the protein surface, leading to reduced enzyme flexibility [13]. The activation

effect of several solvents is likely due to an improved packing of the polar side chains at the

protein surface [13].

While both the archaeal and bacterial esterases were reasonably stabile in non-polar solvents,

AF_est1 and AF_est2 exhibited higher stability in polar organic solvents compared to the

bacterial esterases. Similar to AFEST (O28558), the thermophilic carboxyl esterase from A.

fulgidus, AF_est1 and AF_est2 might contain additional intramolecular ion pairs, fewer loop

extensions, and a lower ratio of hydrophobic to charged amino acids at the protein surface,

compared to the bacterial enzymes [9]. Crystallization trials for AF_est1 and AF_est2 have

begun to test these predictions. Unfortunately, the solvent stability of AFEST and other archaeal

esterases has not been well characterized. Nevertheless, structural characteristics that lead to

solvent stability are often correlated to thermal stability.

Table 5.3. Effects of various organic solvents on AF_est1 and AF_est2 stability. Residual activity of (%) Organic solvents log P Conc. (v/v%)

AF_est1 AF_est2 None - - 100 100

15 91±7 92±9 30 93±3 85±3

DMSO -1.22

50 83±3 86±13 15 100±8 88±27 30 84±4 97±5

Methanol -0.76

50 81±26 90±7 15 90±11 106±6 30 96±9 94±8

1,6-Dixaone -0.42

50 79±18 64±5 15 77±11 75±10 30 78±6 85±2

Acetonitrile -0.34

50 54±8 46±8 15 62±11 69±7 Isopropanol -0.28 30 51±8 56±3

59

50 42±11 35±4 15 103±18 92±4 30 74±5 85±17

Ethanol -0.24

50 69±16 59±10 15 91±9 104±12 30 80±7 93±6

Acetone -0.23

50 68±12 63±7 15 95±13 113±13 30 75±5 72±4

Tetrahydrofuran 0.49

50 16±4 11±2 15 68±7 66±9 30 69±8 66±19

1-Butanol 0.8

50 72±4 54±36 15 74±3 74±3 30 71±11 79±16

Tert-amyl alcohol 0.89

50 91±1 62±41 15 109±5 121±27 30 124±6 115±26

Toluene 2.5

50 138±22 129±60 15 108±11 124±30 30 134±11 120±35

p-Xylene 3.1

50 144±17 130±58 15 128±10 134±28 30 160±29 129±30

Cyclohexane 3.2

50 113±70 135±71 15 103±22 116±19 30 169±44 134±31

Hexane 3.9

50 116±40 130±52 Residual activities were measured after 5 h incubation at 37 °C. The column “conc. v/v %” indicates the percentage of corresponding organic solvents in 50 mM potassium phosphate buffer, pH 7 during the incubation. n= 6; errors indicate standard derivation.

Ionic liquids (ILs) are intriguing alternatives to organic solvents given their low vapor pressure,

and potentially reduced effect on enzyme stability. Indeed, several studies have already

demonstrated the ability of lipases and esterases to catalyze synthetic reactions in the presence of

ionic liquids [14-17]. In this study, the impact of three imidazolium ILs ([BMIM]CF3SO3,

[BMIM]PF6, and [BMIM]BF4) and one phosphonium IL (Cyphos 109) on AF_est1 and AF_est2

stability was evaluated. The ILs were selected based on previous reports demonstrating their

60

ability to promote lipase-catalyzed synthetic reactions [14-17]. Little or no enzyme activity was

retained after incubation in the phosphonium-based ionic liquids, and so only results using the

imidazolium-based ILs are shown (Table 5.4). Both AF_est1 and AF_est2 exhibited higher

stability in the presence of [BMIM]CF3SO3 than the other two ILs. In most cases, the stability of

AF_est1 and AF_est2 increased with increasing concentrations of ionic liquids. Since previous

studies of lipase-catalyzed transesterification reactions in ILs used 100 % of the IL, or co-

solvents using up to 50 % of an organic solvent, the current results suggest that both AF_est1 and

AF_est2 are ideal candidates for application development. Accordingly, subsequent studies will

evaluate the ability of AF_est1 and AF_est2 to catalyze the transesterification of phenolic acids

or sugars with short-chain aliphatic compounds.

Table 5.4. Effects of ionic liquids on AF_est1 and AF_est2 stability. Residual activity (%) Ionic liquids Conc.

(v/v% in DMSO) AF_est1 AF_est2 None - 100 100 100% DMSO - 12±0 12±2

40 22±3 12±2 50 24±0 13±1 60 36±3 23±10 70 56±11 58±7 85 81±13 70±11

[BMIM]BF4

100 56±22 58±10 40 10±12 9±4 50 32±12 18±5 60 60±26 38±12 70 57±14 47±3 85 35±3 49±20

[BMIM]PF6

100 8±9 9±14 40 29±2 17±0 50 38±9 30±4 60 96±16 56±14 70 109±21 76±7 85 116±8 96±4

[BMIM]CF3SO3

100 138±28 109±44 Residual activities are measured after 3 h incubation at 37 °C. The column “conc. v/v % in DMSO” indicates the percentage of corresponding ILs in DMSO during the incubation. n= 6; errors indicate standard derivation .

61

5.3.3 Kinetic Properties

Enzyme kinetic parameters were measured to obtain more detailed characterization of AF_est1

and AF_est2. The enzymatic reactions were performed at 37 °C to facilitate the detection of

enzymatic hydrolysis of pNP-acetate at low substrate concentrations; autohydrolysis of pNP-

acetate is detected at higher temperatures [18]. The Km and kcat of AF_est1 and AF_est2 were

3.8 mM and 5.1 s-1, and 3.7 mM and 10.3 s-1, respectively. These kinetic parameters are

approximately 10 to 20 times lower than the thermophilic esterase from Bacillus acidocaldarius

and Sulfolobus shibatae, which were characterized using pNP-acetate at 37 °C (0.183 mM and

145 s-1, 0.013 mM, and 56.1 s-1, respectively) [18, 19]. The comparatively low activity of

AF_est1 and AF_est2 of pNP-acetate is not surprising given their preference for phenyl acetate.

The reaction conditions such as substrate preparation and reaction buffer may also be a factor.

The thermal and pH lability of phenyl acetate has complicated the collection of kinetic data using

this substrate. Nevertheless, efforts to obtain these data are still underway.

In conclusion, AF_est1 and AF_est2 were highly thermostable and were active at a wide range of

temperatures. This is especially important for industrial enzymes, which typically experience

high temperature conditions. Both AF_est1 and AF_est2 preferred short chain pNP-esters, and

were most active on phenyl acetate. In addition, they were stable in a variety of detergents and

organic solvents, a property required for organic syntheses. Amino acid sequence analysis

revealed that AF_est1 and AF_est2 contained the putative catalytic triad Ser, Asp and His, where

Ser was located in the conserved peptapeptide Gly-X-Ser-X-Gly. Future experiments will

investigate their potential to catalyze the transesterification of simple sugars or phenolic acids

with aliphatic compounds; crystallization trials are also underway.

62

6 Conclusions and Recommendations

6.1 Summary

Carboxyl esterases and lipases display broad but distinct substrate specificities, can perform

chemo-, regio-, and stereospecific hydrolytic and synthetic reactions, and are active under mild

reaction conditions. These properties make this class of enzymes important catalysts for

synthetic transformation of highly functionalized substrates, including biomass-derived

polymers. For example, arylesterases could be used to alter the solubility of lignin and lignin-

derived phenolic acids so their antioxidant properties can be harnessed in a broader range of

products.

In the present work, a total of 202 gene targets from 15 bacterial genomes were selected (Chapter

3). Exactly 180 genes were successfully cloned and 84 of them were expressed as soluble

proteins. Forty-six proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not

exhibit any activity towards all substrates. Accordingly, the high throughput platform used for

protein synthesis successfully produced roughly 50 % of the novel enzyme targets, and more

than 50 % of proteins produced in soluble form were also active on predicted substrates. Among

the 46 active proteins, 18 proteins showed arylesterase activity. Six arylesterases that were also

active on medium-chain aliphatic substrates were selected for further biochemical

characterization. Two enzymes with activity towards pNP-acetate only were also characterized

for comparison. To our best knowledge, all 6 arylesterases (RP_est1, RP_est2, RP_est3, PP_est2,

PP_est3, and PA_est1) represent the first arylesterases to be isolated and characterized from

Rhodopseudomonas palustris, Pseudomonas putida, and Pseudomonas aeuginosa, respectively.

Further, a comparative sequence analysis of active and inactive proteins did not reveal sequence

features that would allow us to improve our prediction of arylesterases. Almost all of proteins,

either active or not active, contained the residues that comprise the catalytic triad and the HG

oxyanion sequence.

In addition to general properties (substrate specificity, optimal pH, pH stability, thermal

inactivation, kinetics), enzyme stability was assessed in a variety of detergents, organic solvents,

63

and ionic liquids (Chapter 4). In particular, RP_est1 and PP_est3 which showed the best kinetics

data were stable at 50 °C and were also stable in a range of solvents.

Two archreal esterases from Archaeoglobus fulgidus were also studied along with the bacterial

esterases (Chapter 5). These enzymes displayed high temperature stability and were stable in a

variety of detergents and organic solvents, satisfying a required property for organic syntheses.

In contrast to the bacterial enzymes described in Chapter 3, these two esterases exhibited

significantly lower thermal inactivation and were also comparatively stable in both polar and

nonpolar solvents, and ionic liquids. Notably, only one other arylesterase from an archaeal

species has been reported in the published literature.

The X-ray structural determination of all 10 characterized proteins (8 bacterial esterases and 2

archaeal esterases) is in progress. In the meanwhile, the potential for the most promising

enzymes, particularly PP_est3, AF_est1 and AF_est2, to catalyze the transesterification of simple

sugars and lignin-derived phenolic acids with hydrophobic compounds will be evaluated.

6.2 Recommendations

To expand on the discoveries presented in this study, the following research objectives are

recommended:

1. Obtain kinetic parameters for all enzymes using phenyl acetate at the optimal enzyme

temperature, as well as pH.

2. Evaluate structural changes of proteins at different temperatures by Circular Dichroism

and then correlate the results of thermal inactivation to thermostability.

3. Perform additional bioinformatic analyses to compare the active and non-active enzymes

isolated in this study and further attempt to identify sequence features that are correlated

to arylesterase activity. Moreover, compare the archaeal esterases with the bacterial

esterases to predict amino acid residues that contribute to the temperature and solvent

tolerance demonstrated by AF_est1 and AF_est2.

4. Analyze solved x-ray structures of the targeted enzymes to identify sequence and

structural features that contribute to substrate specificity.

64

5. Evaluate the unusual sequence feature of RP_est1 using site-directed mutagenesis and 3D

structure determination.

6. Perform additional experiments to explain the increase in RP_est1 activity after

incubation in 1 % SDS solution.

7. Follow the published procedures by Hogbom, et al. to confirm if 10 characterized

enzymes are metalloproteins.

8. Measure the hydrolytic activity of enzymes in the presence of organic solvents.

9. Follow published procedures to evaluate the potential of PP_est3, AF_est1 and AF_est2

to catalyze the transesterification of phenolic acids, as well as simple sugars. This might

require immobilizing the enzymes onto a solid support, such as celite. Here, methods

developed by a fourth year thesis student (Ping Hay Lam) can be followed.

10. Evaluate the enantiomeric selectivity of the new arylesterases.

65

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Appendices

Appendix A.1 Amino acid sequence alignment of proteins that were active on phenyl acetate.

RPA0348 ------------------------------------------------------------ RPA4646 ------------------------------------------------------------ PA2949 -----------------------------------------------MKRFLLGLVLLLA SCO3439 -----------------------------------------------------MGGLGAG SCO3566 ------------------------------------------------------MTGSST PA2934 -----------------------------------------------------MILDRLC SCO7440 -----------------------------------------------------------M RHA02607 ------------------------------------------------------------ RHA00320 ------------------------------------------------------------ SCO3690 ------------------------------------------------------------ NE2298 ------------------------------------------------------------ RHA01401 ------------------------------------------------------------ PP5253 ------------------------------------------------------------ PSPPH4522 MDKYNHAQAKARFDPKELIPLCPASSVPAGKYDEPMFQKITPEQYAHSSRLNYAKRQLPE PSPPH3482 ------------------------------------------------------------ RPA3430 ------------------------------------------------------------ PP3645 ------------------------------------------------------------ RHA08081 ------------------------------------------------------------ RPA0348 --------------MLAQGQRFEGLLCGDDSSRCEVVEMAKLTQFLISGACALLITATSA RPA4646 ----------------------------MSEAVMAGAEAFAFDGG---KTGVLLVHGFTG PA2949 VAAGVLYFVPATLLASVRTVERGLAGLSEHSVQVDNLEIAYLEGGSEKNPTLLLIHGFGA SCO3439 GALAGLGWAYADGGPLVADGKPPRAPAHEGAPLRDLPEVRSREGR--PEHTLTVATTRPV SCO3566 SPGQSPSPQHPNPTSVVRLGIPGGPEVTHRDVAANGARFHIAELG--DGPLVLLLHGFPQ PA2934 RGLLAGIALTFSLGGFAAEEFPVPNGFESAYREVDGVKLHYVKGG--QGPLVMLVHGFGQ SCO7440 TDDATAPSSSCDQNPVPGLPLHDLAGFTHRWVDAEGIRLHAVEGGRPAGPTVVLLAGFPQ RHA02607 --------------------------MREGSIRVGDRTITYLEAGDPGGPLVLHNHGGPS RHA00320 --------------MTTTKFDKAVGRYVHLTIDDVEYRVYYEESG--SGIPLLLQHTAGC SCO3690 ----------------------------MDVRDRNHVTV----TGRADGPVVMLAHGFGC NE2298 -----------------------------------MASIHIETTG--NGPDLVMLHGWAM RHA01401 ---------------------------MGTITTQDGTEIFYKDWG--TGQPIVFSHGWPL PP5253 ---------------------------MSTFVTRDGTSIYFKDWG--SGKPVLFSHGWPL PSPPH4522 LSAAAEGMHEPVHQSACPINPTSRGICMSTFNAKDGTEIYYKDWG--EGKPVLFSHGWPL PSPPH3482 ----------MSSSNLQTHSAQSFVQAPNKIATVAGIAFAYRESGLRGGVPLLLLNYWGA RPA3430 ----------------------------MPSFDHAGVNIAYLDEG--EGEPILLIHGFAS PP3645 -----------MACLPAAPDLQGAPSMIQPVAERTPAGTSYLDVG--QGQPVVLIHGVGL RHA08081 -----------------MTTTTDQSPEIAKTIDVNGIATNYHDVG--EGAPVVLIHGSGP RPA0348 IADPIAPAGKAIGQDGGGHT-IYQVDANGISIGYKLI---GQGAPMVMIM---GLGG--- RPA4646 SPQSMRYLGERLGE-----R-GYSI------LGPRLP---GHGVSPAA-----MAKT--- PA2949 DKD------NWLRFARPLTE-RYHV------VALDLP---GFGDSSKPQ----QASY--- SCO3439 VGGRRLHLDTYNGQLPGALL-RIRPGDRLRILLRNRMLPSGVPLNALP-----PLCA--- SCO3566 ------FWWTWRHQLVALADAGFRA------VAMDLR---GVGGSDRT-----PRGY--- PA2934 ------TWYEWHQLMPELAK-RFTV------IAPDLP---GLGQSEPP-----KTGY--- SCO7440 ------TWWAWRKVMPGLAA-RFRV------IAIDLP---GQGHSERP-----RGGY--- RHA02607 SRLEAELFDSH-----AKAN-GLRF------VCADRP---GIGGSDP------QPGR--- RHA0032 DGR---QYRHLL-EDEEITS-HFRV------ITWDLP---YHGRSLPPLSVRWWEQEYNL SCO3690 DQN---LWRL---VTPILER-DFTV------VLFDHV---GSGNSNLSA----WDPERYG NE2298 ------HSGVWDGVVESLSQ-RFRL------HQVDLP---GHGASRDC------------ RHA01401 SAD---DWDTQMLFFLQ--H-GYRV------IAHDRR---GHGRSTQT-----GDGH--- PP5253 DAD---MWDSQMEFLAS--R-GYRA------IAFDRR---GFGRSSQP-----WNGY--- PSPPH4522 DAD---MWEYQMEYLSS--R-GYRT------IAFDRR---GFGRSSQP-----WTGY--- PSPPH3482 VLD---DFDPR--IVDGLAT-RHHV------IAIDYR---GAGLSTG------TAPL--- RPA3430 NKNVNWVYPSWLSELKR--T-GRRV------IAIDNR---GHGESSKLYD---PNDY--- PP3645 NKE---MWGG---QFVGLAN-DYRV------IAYDML---GHGQSRVP-----AADT---

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RHA0808 GVT---AWANWRTTIPHLAE-KFRV------IAPDIL---GFGYTERPD----GVEY--- RPA0348 TADNWPPQV---IEALSKNHQLILMDNRGMGHTTANDTPFSYPLFAADVIGL---LDA-- RPA4646 TANDWVNCAEDALLELSAKCDKTFVAGLSMGGTLSLYLAASHPDKVAGVIPINAAVQI-- PA2949 DVGTQAERVANFAAAIGVRR-LHLAGNS-MGGHIAALYAARHPEQVLSLALI--DNAG-- SCO3439 DKAADGTEHAHSAAGGPLHC-APEAGHQVVDGHTIIQQALATNLHTHGLQVSPSGSAD-- SCO3566 DPAGLALDITGVIRSLGEPD-AALVGHD-LGGYLAWTAAAMRPKLVRRLAVSSMPHPRRW PA2934 SGEQVAVYLHKLARQFSPDRPFDLVAHD-IGIWNTYPMVVKNQADIARLVYMEAPIPD-- SCO7440 DTHTVASRVQTALTALDVPK-YWLVGHD-VGAWVAFSLALKYEERLHGVALLDAGIPG-- RHA02607 TFEGWTDDLLLLADSFGAQR-FAVTGWSEGGPWALAAAAYLDPARLVNVVCIAGGNYGTF RHA00320 TQDFFMKFVVALAEALDCED-AVYMGSS-MGGNLAPDLALHYPGVFRAVIALEAALHS-- SCO3690 SLDGYVDDVLELCRELALGP-VTFVGHS-VSAMMGVLAAVREPEAFAGLVLL---APS-- NE2298 ALDSL-DQMTEVIADRLPGR-YSVCGWS-LGGQVAIRLALQAPERVQQLVLV---AST-- RHA01401 DMDHYADDLAELTAHLDLQD-AIHVGHSTGGGEVVHYLARHGESRVSKAALI--SAVP-- PP5253 DYDTFADDIAQLIEHLDLRD-VTLVGFSMGGGDVSRYIARHGSERVAGLVLL--GAVT-- PSPPH4522 DYDTFADDIAELIEHLDLHD-VTLVGFSMGGGDVTRYIAKYGSERVAKLALL--GSVT-- PSPPH3482 TIGEMARDTLELVRAMGYRQ-VDLIGFS-MGGFVAQDLALKAPDLVRKVILAGTGPAG-- RPA3430 TLEAMASDAVALMDHLGIAR-ADVMGYS-MGARIGANLARRQEQRVRSVVL---GGLG-- PP3645 PLEGYADQLAELLDHLQIAQ-ATVIGFS-MGGLVARAFALNYPQRLAALVVL-------- RHA08081 NSTTWTQHLVGLLDALGLDT-VSIVGNS-FGGSLALNIATKHPERVDRLVLM--GSVG-- . RPA0348 -----LGIKRSDVLGYSMGSTITQQLLLQYPD------------RFNKALIH-----ATS RPA4646 ----------------------------DSPD-----LAGLAYARGLPEFVP-------- PA2949 -----VMPARKSELFEDLERGENPLVVRQPED----------FQKLLDFVFVQQPPLPAP SCO3439 -----NVFVRLDP------LEDHQYAYDIPFD----------HPAGLHWYHP-------- SCO3566 -----RSAMLGDVRQSRAGSYVWGFQRPWVPE----RQLTADDGALVGRLLH-----DWS PA2934 -----ARIYRFPAFTAQGESLVWHFSFFAADDRLAETLIAGKERFFLEHFIK----SHSS SCO7440 -----ITLPDSIPTDPDRAWKTWHFAFHLVPE-LPETLLTGRERDYVDWFLK----VKTL RHA02607 GSNWAAKYLSSVDALGGRLELHFHPGFTLMYD-----VLGISATHFADRYAKAITQSACT RHA00320 -----PGFYLDYWFHPEISNDSKPAAMYALTS---------------------------- SCO3690 -----PCFVDDPDTGYRGG-----FSAADIEE-----LLDSLDANYLGWSGAMAPVIMGN NE2298 -----PCFVRRAD--WPWGMEDSTLTL-FMEN------LARDYTQTLNRFLT---LQVSG RHA01401 -----PLMVKTEA--NPGGLPKDVFD--DLQA-----QLAANRSKFYRDLPS-GPFYGFN PP5253 -----PVFGKRDD--NPDGVDLSVFE--GIRA-----GLRADRAQFIADFAT---PFYGL PSPPH4522 -----PFFLKTDD--NPEGVDKSVFD--GIKE-----GLLKDRAQFISDFAT---PFYGL PSPPH3482 -----------GHGIERIGALSWPLILKGLLR--------LRDPKVDMFFTS---TLNGR RPA3430 -----MGLLSNEG--RPGENVARALEADALDD---------------------------- PP3645 -----NSVFNRTPEQSAGVIARAAQAAELGPD-------ANVDAALDRWFSR-----EYK RHA08081 -----VPFEITDGLDAVWGFEPSLPAMRKLLD-----VFAYDRSLVNDELAE-------- RPA0348 TDGSNVAKALH-GRVPTDPIVARQVEATTHWKTPLDKL---------PSIDN----QVML RPA4646 GIGSDMVDTST-KELAYDQVPVTCVKQVMGLAATARALL--------PRIKC----PTLV PA2949 LKRYLGERAVA-ASAFNAQIFEQLRQRYIPLEPEL------------PKIEA----PTLL SCO3439 ---HHHGSTTH-QAWSGLAGPIVVEGDIDHVPEIAGMRE--------RTIVL----SMLR SCO3566 GPRLLDDDAVT-AYRRAMCIPSTAHCSVEPYRWLVRSLARPDGIQFYRRMKRPVRVPTLH PA2934 NTEVFSERLLD-LYARSYAKPHSLNASFEYYRALNESVRQNAE-LAKTRLQM----PTMT SCO7440 SPDTFDGAEID-HYAAAVAAEGGLSASLAYYRDAAESARRNHDALERGHLTV----PVLG RHA02607 ADREVLSDEKV-LDAFLRAGRECFRHGADGLVVDATMLYKAWP-FDMTKVTR----PVHF RHA00320 PTAPEVGRRET-TWVYSQGSPPVFKGDLHYYSVEHDLRETA------QNIDTSKT-AVYI SCO3690 PERPELGEELTNSFCRTDPDIARVFARVTFLSDNRADL---------APVRV----PTLV NE2298 SEDQARVLAWL-RKSILRGQPPTPATLQAGLKILQTSDLRA----ELNQVSQ----PVLL RHA01401 RPGVESSEAII-ANWWRQGMMGGAKAHYDGIVAFSQTDFTE----DLKKITV----PVLV PP5253 NHGQQVSQGVQ-TQTLNIALMASIKGTLDCVTAFSETDFRP----DMAKVDV----PTLV PSPPH4522 NKGQKVSEGVQ-AQTLNIALLASLKGTLDCVTAFSATDFRA----DMAKIDV----PTLV PSPPH3482 RAARNYLSRVK-ERTVDRDKPPTPRLLLRQLKAIKAWGKQPAQ--DLARLRV----PVLI RPA3430 ---VTDPVGRT-FRAFADQTRSDRKALAACMRGSRGLMSREDA----AQIAV----PVLI PP3645 AANPAQVAAI--RQVLASNDPQGYHTTYSLFA--TQDMYRAD---DLGSIQV----PTLI RHA08081 LRLAAATRPGV-QEAFSAMFPAPRQQGVDEMAVDETLI---------AGLTN----DTLI : RPA0348 VVGTADNVVGTESSKTIASAIPG-AWLVQ--FKGATHHLMYETPEGF-SAAALTFFETNE

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RPA4646 INSRVDHVLSPANATVIANHVGS-SRIETLWLDNSYHVATIDNDKDLIAAEIDSFVTRNL PA2949 LWGDRDRVLDVSSIEVMRPLLKR-PSVVI--MENCGHVPMVERPEET-AQHYQAFLDGVR SCO3439 LDGNGEN-----------------PTAVV--LPTGGDDPFTTVPAVP-TRMVATLNGQLR SCO3566 LHGSLDPVMRTRSAAGSGQYVEAPYRWRL--FDGLGHFPHEEDPVAF-STELINWLKDPE PA2934 LAGGGHGGMGTFQLEQMKAYADD-VEGHV--LPGCGHWLPEECAAPM-NRLVIDFLSRGR SCO7440 VSGSHGSI--PDMAASIGPWAAN-ATGAV--IPQAGHFIPDEQPEAT-VKVLTAFIDYER RHA02607 WQGSADTLVPEIINKTVADKTPG-AVWHP--ISGGGHFIAVSHANDI-LALVANDLAPVQ RHA00320 LNGEYDFATGPEEAAELGAQIEG-AKVVP--MAGLGHFPMSEDAAQF-FRCIKPVLWEIR SCO3690 AQCSSDAIAPREVGAFVHAQIPA-SRLVT--LNATGHCPQLSAPQET-AAAIAAFAGGAD NE2298 IHGRNDVITPAGAADWMQQHLPR-ARLVL--FPHCGHAPFLSFPEQF-VSCFDAL----- RHA01401 MHSEDDQIVPYADAGPLSAKLLANGSLKT--YQDFPHGMPTTQAETI-NADLLAFLKDSY PP5253 IHGDDDQIVPFETTGKQAAELIRGAELKV--YAGAPHGFAVTHAQQL-NEDLLAFLQR-- PSPPH4522 IHGDGDQVVPFEASGKRAAELIKGAELKV--YPGAPHGFAVTHAEAL-NKDLLTFVQG-- PSPPH3482 VVGDSDVMVASELSRDMSQRLPQ-AQLVV--YQDAGHGCVFQYHADF-VSTALEFLA--- RPA3430 AVGTDDDVAG--SAHELGDIIPG-SQVLD--IPRRDHMRAVGD-RVY-KEGVVDFLARRP PP3645 ATGELDSGSTPAMTRQLAACIPG-ARSVV--LAEQRHMMPVEAPREV-NKMLLDFLTQAR RHA08081 VHGRDDQVIPLSNSLRLLELIDR-SQLHV--FGRCGHWVQIEHSARF-NSLIADFLSE-- RPA0348 TVTPKIEPNASVAPPPTQP RPA4646 ------------------- PA2949 NAQVAGR------------ SCO3439 PK----------------- SCO3566 PDR---------------- PA2934 ------------------- SCO7440 AE----------------- RHA02607 N------------------ RHA00320 DSAVPSKVLSATQ------ SCO3690 ------------------- NE2298 ------------------- RHA01401 EN----------------- PP5253 ------------------- PSPPH4522 ------------------- PSPPH3482 ------------------- RPA3430 ------------------- PP3645 TLTESAKGIVA-------- RHA08081 -------------------

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Appendix A.2 Amino acid sequence alignment of proteins that were not active on any substrates.

TM0820 ----------------------------------MENFVFHNPTKIVFGRGTIPKIGEEI TM0423 ---------------------------------MITTTIFPGRYVQGAGAI--NILEEEL ATu2215 ---------------------------------------MQNATLPLPLQS--PLLAA-S PP0624 ----------------------------------------MSDLTQA------LLFPD-W PA3301 -------------------------------------MPAAAYWLPASDET--PLYTRHW PP4551 -------------------------------------MPHDAFWLPASEHC--SLYVHQW PSPPH0357 -----------------------------MQTLYPQIKPYARHDLAVEQPH--VLYVDES PP3523 -----------------------------------------------M-----EAFNQ-E ATu2497 -----------------------------------------------------MHFEV-H RPA3731 ----------------------------------MHLIHRHRLWIDNAGVR--IALVR-W PSPPH1999 -----------------------------------MTVAVEEVRLSLGHIE--LAAHL-Y SAV0974 ----------------------------------------MGTFTTDDGTE--IFYKD-W ATu5389 ----------------------------------------MGFVTTKDGTE--IFYKD-W PA2717 ----------------------------------------MGYVTTKDGVE--LFYKD-W PA3053 MSLPRLSLSLCLALACGPALASQAPVYGERLEGFDYAYPVHYLDFTSQGQPLSMAYLD-V PP2201 --MLRATALALACLASSSAIAAESPTYGPQLEGFSYPHPLKHFDFKSQGQALQMGYMDVP SAV0923 -----------------------------------------------MDIRRRNNVVV-T ATu2061 -----------------------------MTAPSLDNWEKRKRFVELPRKRQ-MAFIDTG SAV2105 ------------------------------MPQTPRTTALTHRLVSSPAGR--IHLVE-Q RPA2773 -------------------------------------MASTRSTITANGIE--LFLRE-Q SAV141 -----------------------------------MEGSLTHRFVDVNGVR--LHIAE-Q RHA05146 ----------------------------------MQTGRVQHATNPVDGVR--IAYKT-V RHA00069 --------------------------------------MSQLQHLALHGDA--VAYRL-S SAV1746 -----------------MVDVPPPRPRRALRLRPVGDGELRLHHRVVHGYR--RAYRM-A PP4540 -----------------------------------------MAYFEHEGCT--LHYEE-Y PSPPH2566 -----------------------------------------MIQLTAEHTPAGTSYLA-T PA0231 -----------------------------------------MPTVRLADGD--LNYSL-E PP1380 -----------------------------------------MAHLQLADGV--LNYQI-D PA3586 --------------MRAFIFLAALLCSLSSFAAARCDARVPTETVELGDVR--LAYQS-I PP2804 --------------------------------------MSTDRFCELAGERR-LCYRS-H SAV0519 --------------------------------------------MTRGGVR--LSCRD-W SC0267 --------------------------------------MSTTQHTLV------PVYDHRT SC5943 ------------------------------------MTKHRQETVRAGELD--VGFLD-S PSPPH0033 -----------------------------------------MPDLLIDGKT--LHYAD-Q RPA1568 ------------------------------------------MQIKVNGTD--TFVAT-G SAV7198 ---------------MCLSPLVTTPVSPPVSPTWRDRSMTVLRRVSVNGVE--LNVAL-A SAV1548 -----------------------------------------MPQLAVDGAG--MTYDD-E SAV4410 -----------------------------------------------MAPF--LAYED-K TM0820 KNA---GIRKVLFLYGGGSIKKNGVYDQVV--DSLKKHGIEWVEVSGVKPNPVL-----S TM0423 SRF---GERAFVVIDDFVD--KNVLGENFF-------SSFTKVRVNKQIFG--------- ATu2215 GEN---RVRHGFFTRQGGV--SEGLYQGLN--VGLGSHDEPEKVQENRRRV--------- PP0624 PAP--ASVRACVTTRQGGV--SLPPYETFNLGDHVG-DDPAAVAENRRRLS--------- PA3301 PSA--TAVGAVMLSHGMAE--HAGRYERLA--AALNAAGYHFYAIDQRGHG-RT-----A PP4551 LPA-TPVKAVVLLAHGMAE--HAGRYQRLG--QALSEAGFALFAADQRGHG-RT-----A PSPPH0357 GSP---EGLPVVFIHGGPG--SGCDAHSRC---YFDPNLYRIVTFDQRGCG-RS-----T PP3523 GKP-------------------MSKFDFQL-------------AYTIKPHN--------- ATu2497 GRT-DAEAPTILLSSGLGG--SSAYWLPQI--EALS-DHFRIVTYDHRGTG-RT-----G RPA3731 GDV-DPDKPVALLLHGTGF--VGEIWDEIA--NGLA-DRYTVYALDRRGHG-AS-----D PSPPH1999 GPK---DGQPVIALHGWLD--NANSFARLA--PRL--EGLQVVALDLAGHG-HS-----D SAV0974 GSG-----PPVVFSHGWPL--NADAWDVQL--RLVAENGYRAVAHDRRGHG-RS-----G ATu5389 GPK---DAQPIVFHHGWPL--SSDDWDAQM--LFFLSKGYRVVAHDRRGHG-RS-----A PA2717 GPR---DAQVIYFHHGWPL--SSDDWDAQM--LFFLAEGFRVVAHDRRGHG-RS-----S PA3053 APK-KANGRTILLMHGKNF--CAGTWERTI--DVLADAGYRVIAVDQVGFC-KS-----S

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PP2201 AQG-QANGHNVVLMHGKNF--CAATWETTI--DALSKAGYRVIAPDQVGFC-TS-----S SAV0923 GRP---DGPVVLLAHGFGC--DQNMWRLVV--PALA-DDFRVVLFDYVGSG-RS-----D ATu2061 GPG-----PVLLMLHGFSD--TSRSFSIIE--PYF--QEYRLIAPDLPGHG-AS------ SAV2105 GTG-----PLVLLVHGFPE--SWYSWRHQL--PALAAAGYRAVAVDVRGYG-RS-----S RPA2773 GEG-----PLVVLCHGWPE--LSYSWRHQI--GALADAGYHVVAPDMRGFG-RS-----S SAV141 GQG-----PLVLLLHGWPE--SWYSWRHQF--GALAAAGYRVVAPDQRGYA-RS-----E RHA05146 GDG-----EPLVLIHGTAL--SHAIWRGFGYVAALR-DRYRLILVDLRGHG-CS-----D RHA00069 GEG-----ETLLLVHGMAG--SSATWRAVL--PQLA-RRYRVLAPDLPGHG-DS-----A SAV1746 GEG-----PVLVLIHGIGD--SSATWAELI--PDLA-RTHTVIAPDLLGHG-AS-----D PP4540 GQG-----EPLVLLHGLGS--SCQDWELQV--PLFS-RHYRVILMDIRGHG-RS-----D PSPPH2566 GQG-----HPVVLIHGVGL--NKEMWGGQI--VGLA-TNYQVIAYDMLGHG-AS-----P PA0231 GPA---GAPVLLLSNSLGT--DLGMWDTQI--PALT-AHFRVLRYDTRGHG-AS-----L PP1380 GPD---DAPVLVLSNSLGT--DLGMWDTQI--PLWS-QHFRVLRYDTRGHG-AS-----L PA3586 GRD---SDPALLLVMGLGG--QLIHWPDEV-VSALCEQGFRVIRYDNRDVGLSAWNVPVP PP2804 GHE---AAPAVLLIVGLGL--QLTYWPQAL-IDGLVERGFRVITLDNRDAG-RS-----F SAV0519 GGC----GRPVVLLHGLAG--HAGEWDTLA--GALS-PRYRVIAVDQRGHG-AS-----E SC0267 GTG-----PTLVFLHYWGG--SARTWDLVV--DRL--PGRAVVAVDFRGWG-RS-----R SC5943 GDG-----VPLLLVHGGES--DRTQFATLR--AGLG-AGIRAISYDQRDSG-IT-----V PSPPH0033 GTG-----PVVLLGHSYLW--DKAMWSAQI--DTLA-SRYRVIVPDLWGHGDSS-----G RPA1568 GKPFDASLPAAVFIHGAGF--DHSVWALQT--RWFAHHGFAVLAPDLPGHG-RS------ SAV7198 GKG-----PAVLLLHGFPH--TWELWTDVM--AGLS-ERYQVIAPDLRGFG-AS-----T SAV1548 GPHDGDGGVPLVFIHGWTA--DRHRWDHQM--AHFA-DKRRVVRLDLRGHG--------- SAV4410 GTQ-RDTSLPLVLVHGHPF--DRTMWAPQL--AAFA-PSRRVIAPDLRGYG-AS-----P TM0820 KV-------------HEAVEVAKKEKVEAVLGVGGG-SVVDSAKAVAAGALYEGDIWDAF TM0423 -----------------------GECSDEEIERLSGLVEEETDVVV--GIGGGKTLDTAK ATu2215 --------------------TQWFGLSADRLATVHQ-VHSPDVVVV--DDNYDGNRPQAD PP0624 --------------------EQFTIQPAWLKQVHGRVVADADPTVV--AE------ADAS PA3301 EADELGHFAD-----QGGWGKVVGDLASLNHHIRQQ-HPELPIFLL--GHSMGSYISMAY PP4551 ELGNLGLFAR-----HHGWNAVVNDLGCVAQHIGQQ-FPCTPLFLF--GHSMGSYIAQAY PSPPH0357 PH-------------ASLENNTTWKLVEDLERIREH-LGIDKWVLF--GGSWGSTLALAY PP3523 ---------------PA---YDETDAAQARLHLREK-LGLDTVEHI--ETTLLGMITLNG ATu2497 G--------------EVPTEGGISAMADDVLEIVSA-LNLEKFHFM--GHALGGLIGLDI RPA3731 K--------------PGR--YHFADFASDLAAAIET-LGLSGIYGI--GHSAGATDLLLT PSPPH1999 HR-------------PAGSSYALADYAFDVLQVAEQ-LGWQRFALL--GHSLGAIISVLL SAV0974 QP-------------WNG--NHMDTYADDLSQLIGA-LDLRDAVVV--GHSTGGGEVARY ATu5389 QV-------------ADG--HDMDHYAADAFAVVQA-LDLKNAVHI--GHSTGGGEVARY PA2717 QV-------------WDG--HDMDHYADDVAAVVER-LGVRGAIHV--GHSTGGGEVVHY PA3053 K--------------PAHYQYSFQQLAANTHALLER-LGVARASVI--GHSMGGMLATRY PP2201 K--------------PAHYQYSFQQLADNTHALLEQ-LGVKQTIVL--GHSTGGMLATRY SAV0923 LSAW-----------SEQRYSSLEGYALDVLEVCEE-LDLRDVAFV--GHSVSAMVGVLA ATu2061 ---------------SVGHGFHVADFAETIDRFLTL-MGVSRFFVL--GHSMGAMTAIEL SAV2105 RP-------------NAVHAYRMLDLVEDNVAVVHA-LGERTAVIV--GHDWGSNIAATS RPA2773 AP-------------QAVEAYSIFDLVGDMVALVAE-LGETRAAII--GHDWGAPVAWHA SAV141 QP-------------PDVASYTLLHLVGDVIGLIEE-LGEEQAVVV--GHDWGAPVAWTT RHA05146 KP-------------HDESAYAMDLVSGDVLAVLDH-LDLPSAHVL--GYSLGGRVALAL RHA00069 KP-------------RGD--YSLGAFAAWLRDLLNE-LDIERVTVI--GQSLGGGVAMQF SAV1746 KP-------------RAD--YSVAAYANGVRDLLAS-LDIESATLV--GHSLGGGVAMQF PP4540 KP-------------ADG--YQIATFSADLLALLEH-LHTGPVHFV--GLSMGGMVGFQF PSPPH2566 RP-------------DPD--TGLPGYAEQLRELLEH-LQLPQATVV--GFSMGGLVARAF PA0231 VT-------------PGP--YAIGQLGADVLALLDA-LELPRVHFC--GLSMGGLIGQWL PP1380 VT-------------EGP--YSIEQLGRDVLALLDG-LDIQKAHFV--GLSMGGLIGQWL PA3586 SSRLTYEVVRYRLGLPVSAPYTLTDMAGDALHLLDA-LDIPQAHVL--GASMGGMIAQHI PP2804 FTDVAPPTPLQQFLRKRTPGYDLGDMASDVIGLMDA-LHLEAAHVV--GMSMGGMIAQTL SAV0519 RF-------------PRE--VSRAAYVADVVAVLDQ-LALHRPVLV--GQSLGGHTAMLT SC0267 AL-------------PGP--YTLGRFADDTLAVLAD-AGITDYVLV--GHSMGGKVAQLV SC5943 NP-------------PVP--YTPEVLADDLVDLLDA-LGLARAHLL--GTSFGGAVAQHA

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PSPPH0033 FP-------------EGT--RNLDDLARHALALLDH-LNIERCSIV--GLSVGGMWGAIA RPA1568 ---------------GGEALKTIAEMADWIAALLDA-TGAQPAKLI--GHSMGSLIALEA SAV7198 RA-------------KTG--YDAGSLADDAAALLET-LGAGPAAVV--GIDAGTPPALLL SAV1548 --E------------SGGSARTIDELAGDVIALLDH-LEIDRFIPV--GHSMGGMIAQTL SAV4410 VV-------------PGI--TPLSVFAEDIAALLDE-LKADRFVLA--GLSMGGQIAMEC TM0820 IGKY-----QIEKALPIFDVLTISATGTEMNGNAVITNEKTKEKYGVSSKA----LYPKV TM0423 AVAY-----KLKKPVVIVPTIASTDAPCSALSVIYTPNGEFKRYLFLPRNPDVVLVDTEI ATu2215 AMVT-ATP-GFVLGVLSADCGPILFADAQAGVVGAA----HAGWKGALFGV----LENTI PP0624 WTRQ---P-GIACTVMTADCLPALFCDRAGTRVAAA----HAGWRGLAGGV----LEATL PA3301 LLHH---S-CSLQGAILSGSNYQPQALYRIARL-------IARFERWRQGP----LGKSA PP4551 LLHH---S-GSLHGAILSGSNYQPAVLYRFARL-------IARLESWRQGP----LGKSA PSPPH0357 AQTH---P-DRVHALILRGIFLARQQEIDWFYQAGASRLFPDYWQDYVAPI----PLDER PP3523 TTLA---------------------------------------------DR----KREAE ATu2497 ALRQ---P-RLIDRLVLINAWSKADPHS------------GRCFDVRIELL----EKSGV RPA3731 AKLL---P-DRIARLFVMEPTVMDPSVVPGR-------------DARLSDQ----GEAAV PSPPH1999 ASSL---P-ERVTRLALIDGLLPLTGKAESA---------AERMGAAMQAQ----LELAN SAV0974 IGRH---GSGRVAKAVLVGAVPPIMLKTDANPEGLP----IEAFDAIRAGV----LSNRA ATu5389 VAKHGQKA-GRVAKAVLVSAVPPLMVKTTANPEGLP----IEVFDGFRSAL----AANRA PA2717 IARY---PDDPVPKAAIISAVPPLMVKTEGNPGGLP----KSVFDDLQAQL----AANRA PA3053 ALLY---P-RQVERLVLVNPIGLEDWKALGVP--------WRSVDDWYRRD----LQTSA PP2201 ALMY---P-QQVERLAMVNPIGLEDWKALGVP--------YRTVDQWYARE----LKLDA SAV0923 AQKA---P-ERFSRLVMVAPSPRYID--------------EDGYRGGFSAE----DIDEL ATu2061 AARR---S-SSVRGLALISGTLEPDFGTESK---------LARDILALRDP----IKPAG SAV2105 ALVR---P-DVFRAVGLLSVPYTPRGGPRPSEIFAGMGGDEEFYVSYFQEP----GRAEA RPA2773 AQFR---P-DLFAAVAGLSVPPPWRGKGPPLDQLRAAGI-TNFYWQYFQKL----GVAET SAV141 AMLR---P-DKVRAVAGLSIPPILPGGMVPPSITRTQYG-EGFYQVYFQQP----GVADA RHA05146 AVGA---P-ERLESLIVGGGSSRPQAGAFD----------RLFFPGCIDVL----ERDGM RHA00069 SYQH---P-ELCDRLVLIGSGGLGP---------------DVNWTLRLLAA----PGSEF SAV1746 AYQF---P-ERTERLILVSAGGV-----------------GREVNPVLRAV----SLPGA PP4540 AVDH---P-QWLRSLCIVNSAPEVKRRT------------RSDWAWWLKRW----SLARI PSPPH2566 ALEF---P-QLLAGLVILNSVFNRS---------------PEQRAGVIART----SQAAE PA0231 GIHA---G-ERLGRLVLCNTAAKIAS--------------DEVWNTRIDTV----LKGGE PP1380 GIHA---G-ERLHSLTLCNTAAKIAN--------------DEVWNTRIDTV----LKGGQ PA3586 ADMA---P-QRLLSLTLVMTSSGAEGLPAPS---------ESLLRLLARRE----AASRE PP2804 AARY---P-ERVSTLTSIFSTTGSLRVGQPA---------LKALFKLLRRP----PRNQH SAV0519 AAAH---P-HLAHALVLVEAGPGGPNPRVQAE--------IGGWLDAWPTP----FPSRE SC0267 AATR---P-AGLRAIVLVGSGPAKPAARV-----------TPEYREALSH-----AYDSA SC5943 ARRH---P-ERVASLVLVATTPSYAMG-------------SAAIDELLEMS----HEDRQ PSPPH0033 ALLA---P-ERITGLVLMDTYLGKESEA------------KKAYYFSLLDK----LEQAG RPA1568 AARH---P-AKVASLALIGTTSTMAVGP------------DLLKAAEANDP----AAIAM SAV7198 ALRR---P-GLVRRLVVMESLLGRLPGAESFLAG------GAPWWFGFHAV----PGLAE SAV1548 ALAH---P-ERIERLVLVNSIS------------------RMTYSRGRGLL----MAAST SAV4410 YRLF---P-ERIAGLVLADTFPTAETEAG-----------KRARGAMADRL----LREGM TM0820 SIIDPSVQFTLPKEQTVYGAVDAISH----------ILEYYFDGSSPEISNEIAEGTIRT TM0423 VAKAPARFLVAGMGDALATWFEAESC--------KQKYAPNMTGRLGSMTAYALARLCYE ATu2215 EAMVKLGASRERIAASLGPSISQDNY---------------------------------- PP0624 DRLALPP-------EEVLVW-LGPAI---------------------------------- PA3301 LIDFLSF-------GSFNKA-FKPNR-------------TAFDWLSRDP----------- PP4551 LIEWLSF-------GSFNNA-FKPNR-------------TAFDWLSRDP----------- PSPPH0357 DNILAAFHKRLTGPDQIAQMHAAKAW---------------STWEGRCATLRPN------ PP3523 KLVRDYIH---------------------------------------------------- ATu2497 DAFVKAQPLFLYPAAWMSEHQERLAR---------------------------------- RPA3731 SGARRRQAEFASAAAAFERYRSAPAF---------------APWSEYALRTYISHGFAHQ PSPPH1999 KKKPVYQDQDRAIQARMKGV-VAVSR---------------EAAELLAQR----------

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SAV0974 QYYWDLS-------DQFYGF-NRPGA-------------EGTDGVR-------------- ATu5389 QFFRDVPA------GPFYGF-NRDGA-------------KVQEGVI-------------- PA2717 QFYQDIPA------GPFYGY-NRPGA-------------QPSEGIV-------------- PA3053 EGIRQYQQA-----TYYAGE-WRPEF---------------------------------- PP2201 EGVRAYERK-----TYYAGR-WKPEY---------------------------------- SAV0923 LTSLD---------SNYLGWSATMAP---------------------------------- ATu2061 GFLHDWYSC------------SRPVN---------------------------------- SAV2105 EIEPDVR-------GWLAGF-YAALSADTMPAPDAPDPHFVRRGGTLRERFPAGRLPAWL RPA2773 EFERDVASTMR---GMLCGGFADPGR-----------SLFVPEGRGFIGRSAASLPLPPW SAV141 EFAKDIP-------NSFRRF-LVGASGDN-PLGREPSPLVIPDGLGLLDIMPESPALPAW RHA05146 DAFLAQW-------NARRSWPIDAGT---------------------------------- RHA00069 VLPLVAPSAVRDAGNKVRGWLAAVGI---------------HSVRG-------------- SAV1746 HLMLSTL--------RLPGMRFQVGM-------FARLMRLLDTDLGQDA----------- PP4540 LSVETVGK------GLAERLFPKPQQ---------------------------------- PSPPH2566 HGPDANAG------EALSRWFSREYQ---------------------------------A PA0231 QAMRDLRD------ASVARW-FTAGF--------------------------------AE PP1380 QAMVDLRD------ASIARW-FTPGF--------------------------------AQ PA3586 QAVEQQADLL----AALGSPEVRDDR---------------------------------- PP2804 ESVRDYVDIMGLI-GSRLEW-NEPAL-------------------------------RDY SAV0519 SAVEFLG-------GGLVGEGWAAGL---------------------------------E SC0267 ESVAGAR-------DHVLTATELPAA---------------------------------- SC5943 QAAADYF--------------FTPEG---------------------------------- PSPPH0033 AFPEPLL-------DIVVPIFFRPGI-----------------------------DPQSP RPA1568 MTIWGLGPD-----AEIGGN-LAPGL---------------------------------- SAV7198 TVLEGNE-------ARYLDWFLDTGT--------------------------LGDGVRPA SAV1548 LVPFKLFVA-----ANIQRA-FAPGH---------------------------------- SAV4410 RGYAD---------EVLHRM-VAPYA---------------DADV--------------- TM0820 IMKMTERLIEKPDDYEARANLA----WSATIALNGTMAVGRRGGEWACHRIEHSLSALYD TM0423 TLLEYGVLAKRSVEEKSVTPALEKIVEANTLLSGLGFESGGLAAAHAIHNGLTVLENTHK ATu2215 --EVGPEYVARFTDIDPAYADY---------FAATERQGHALFDLKQFT----I-DRL-- PP0624 ------GPQAFEVGLEVRDAFTAVHPQAARAFVDGERPGKLLADIYELA----R------ PA3301 --QEVDRYVADPLCGFRCSNQL----WVDLLGGLADITPP--THLRQIDAD--L-PLL-- PP4551 --GEVDQYVNDPLCGFRCTNQL----WLDLLQGLAQISQP--GNLAQIDPN--L-PML-- PSPPH0357 --PQVVDRFAEPHRALSIARIE----CHYFMNKAFLEENQLIRDMPKIA----HLPAI-- PP3523 ------------------------------------------DALKELR----VLSTV-- ATu2497 ------DDAHGVAHFQGKTNVL----RRIAALRAFDID----ARLGEIG----N-PVL-- RPA3731 PDGRVRLLCSPDVETEILRPIF----EAMEQVYRGDERGNPFGWLRELG----C-PVR-- PSPPH1999 --GLMPVPGGYTWRSDSRLTLP-------SAIRFTDQQAM--AFVHGIR----C-PTQ-- SAV0974 --RAFWLWS----MQVGLKAAY----DCIEQFSEQDFT----EDLRRID----V-PTF-- ATu5389 --QNWWRQG----MMGGAKAHY----DGIKAFSETDQT----EDLTAIT----V-PTL-- PA2717 --RNWWRQG----MMGGAKAHY----DGIVAFSQTDFS----DDLKRID----I-PVL-- PA3053 --DRWVQMQAGMYRGKGRESVA----WNSALTYDMIFTQPVVYELDRLQ----M-PTL-- PP2201 --ERWVQMLVGLNKGPGHEAVA----WNSALIYDMIFTQPVYHEFKHLQ----M-PTL-- SAV0923 --VIMDNPDRPELGEELTASFCATDPDIARAFARTTFLSDSRQDLKSVA----V-PTL-- ATu2061 --EEFLFRMKRDAANMAATTWH----GVLKAFAETDLH----YSASKIN----V-PVL-- SAV2105 SEADLDVYAGEFERTGLSGALN----RYRAMDRDWEDLAP--FDGAPVR----Q-PSL-- RPA2773 LTEAELAFFIEQYKQSGFRGGL---NWYRNIDRNWELTSP--WQGAPIH----Q-PAA-- SAV141 LTEEDIQAYAEDFALHGERAFTGAFNWYRNIERNNELLAP--FRGRGID----V-PAL-- RHA05146 ------RAAFMANDAQALAAYM----RRSGVEPGVDD-----DVLRGIA----V-PTL-- RHA00069 --DEMWNAYSSLSDSDTRQAFL----RTLRAVVDHRGQAV--SALSRLYLNEGL-PTQ-- SAV1746 --PELLTLVDALPDVTSRSAFI----RTLRAVVDWRGQAV--TMLDRCYLTEGM-PTL-- PP4540 --ADLRQKMAQRWARNDKRAYL----KSFDAIVDWGVQ----ERIGQIH----C-PTL-- PSPPH2566 ANPAQIAAIRQNLASNDPQGYL----TTYKLFATQDMYRA--EDLGDIR----A-PTL-- PA0231 REPAQVERIVAMLAATSPQGYA----ANCAAVRDADFR----EQLGLVQ----A-PTL-- PP1380 AQAEQAQRICQMLAQTSPQGYA----GNCAAVRDADYR----EQLGRIQ----V-PAL-- PA3586 -QQLLLQAARSYDRAFNPEGVQ----RQLLAILAEPSRV---PLLNRLQ----V-PTL--

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PP2804 AMQAWPRGGGEASNLGTARQIG-------AIINSGDRT----QELQRIR----C-PTL-- SAV0519 ERDGWWPCFDREIMVASLAEIAQRSFW---------------DEWARIT----C-PVL-- SC0267 ---LKARIVTDSRNVTDAARIE----WPLRGIAQDIT-----EHTRRID----V-PAL-- SC5943 -QAGQRARPAGTLTRRTPEQSV----RRHDAARRHEIR----DGLGGIT----A-PTL-- PSPPH0033 VYQAFRSALAGMNAEQLRQTVV---PLGRMIFGRDDWL----GLLEQLN----ADTTL-- RPA1568 ----WMHGGSVRVLEANRPGVIFNDLSACNDYKDAL------AAAAKVT----V-PTL-- SAV7198 VRDAFVRAYTGREALRCAFSYY----RALPVSATQIERA---FDTARLT----V-PTM-- SAV1548 -PREEVREYIRASSATPRDVVM----TYYAAMRSFDVL----DRVGEIR----M-PTL-- SAV4410 --QAHVHRMMTGTDPEGAAAAL----RGRAQRPDYR------DLLTRVS----V-PAL-- TM0820 IAHGAGLAIV--------FPAWMKYVYRKNPAQFERFAKKIFGFEGEGEELILKGIEAFK TM0423 YLHGEKVAIGVLASLFLTDKPRKMIEEVYSFCEEVGLPTTLAEIG-------------LD ATu2215 TKAGVKAENL--------------------------GLCTYPDEH--------------- PP0624 IRLAARGVTA------------------------------VYGGG-------------LC PA3301 IIGGERDPVS--------QGKR----------LGD-LADALRGAG-------------LR PP4551 VIGGECDPVS--------AGKR----------LTH-LADALRATGNRHVQ--------LR PSPPH0357 IVHGRYDVIC--------PLDN----------AWE-LHQNWPGSE-------------LQ PP3523 KFYGCL------------------------------------------------------ ATu2497 VVATKDDLLV--------PYTR----------SLR-LAEGLPQSE-------------LC RPA3731 IATAELSWPI--------YKEM----------ATR-ASALIPGAS-------------RI PSPPH1999 LMVASDGMLA----------------------QRQELLSALPFDV-------------ER SAV0974 IAHGDADQIV--------PLVA----------SAPKSAELVKDAT-------------LK ATu5389 VLHGEDDQIV--------PIAD----------AARKSIKLLKNGQ-------------IK PA2717 VMHGDDDQIV--------PYEN----------SGVLSAKLLRNGT-------------LK PA3053 LLIGEKDNTA--IGKDAAPAELKARLGNYAQLGKD-AARRIPQAT-------------LV PP2201 LLIGDKDTTA--IGSDIAPPEVKARLGNYAELGPQ-VARLIPKGE-------------LI SAV0923 VLECAQDVIA--------PREV----------GAY-VHAAIPGSR-------------LV ATu2061 CLAGSEDPLF--------DDSH-----------RQQLFEAFPLAQ-------------TV SAV2105 FIGGGLDAST-------QWLAD----------AIEAYPVTLPGLV-------------SS RPA2773 FIAGSNDPVI--------SDKMSGK-------HLAAINRVLPNLK-------------QK SAV141 YVVGDRDMVTSLRGPDGGPSLS----------EIF-RGQGGPGNP-------------LS RHA05146 LFVGSNDA----------ERIG----------DTQRVAALIPGAS-------------LT RHA00069 LIWGDSDGII--------PVAH----------GYA-AHEAIPGSR-------------LA SAV1746 LLWGDRDSVV--------PVRH----------AYG-AHEAMPGSR-------------LE PP4540 VIAADHDYT---------PIQ-----------LKERYVALIPNAR-------------LV PSPPH2566 IATGELDPGS--------TPEM----------ARE-LAMRISGAE-------------VA PA0231 IVAGSHDAVT--------TPDN----------ARF-MQARIADAQ--------------- PP1380 IVAGTQDVVT--------TPEH----------GRF-MQAGIQGAE--------------- PA3586 VIHGTADPLL--------PVMH----------GVH-VAAHIRGSE-------------LK PP2804 VIHGDKDLMV--------ATNG----------GFA-TASAIRGAN-------------LV SAV0519 TVLGQSGIIS--------PQES------------ETMLRRLPVAT-------------AA SC0267 VVAGEHDQVE--------PAGV----------LRDNLVPYLARAD-------------FV SC5943 IVHGTQDRLA--------PYE-----------GALLMERRIPNAE-------------LC PSPPH0033 VMCGDADIPR--------PPEE----------TRE--MASLIGCP-------------YV RPA1568 FILGERDMMT--------PTKN----------GKT-LAAAISGSR-------------TV SAV7198 -AVGSHPVGA--------ALER----------QLRPFADDLTGH---------------- SAV1548 LVHGYYDIQL--------PVSQ----------MLR-MAKDYPDAV-------------VR SAV4410 VVVGADDTFT--------PVAD----------AAA-MHAALPGAT-------------LR TM0820 NWLKKVGAPVSLKDAG---IPEEDIDKIVDNVMLLVEKNLKPKGASLGRIMVLEREDVRE TM0423 GVSDEDLMKVAEKACDKNETIHNEPQPVTSKDVFFALKAADRYGRMRKNLT--------- ATu2215 ----------RFYSYR-RTTHRAEPDYGRQISAIAILED--------------------- PP0624 ----------TVSDAR-FFSYRRTPQGGRFASLVWLDPR--------------------- PA3301 QVTLK-----TYPEARHELFNESNRDAVTQDLIDWLEQALRHRRDHSTKERT-------- PP4551 ----------VYPEARHEVLNETNRGEVIADILGWLEQALALGRPARSE----------- PSPPH0357 ----------VIREAGHSASESGIADALVRAAAEIARNLLDLPPEEA-------------

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PP3523 ----------MVDGLG---------PAMRFDILPK------------------------- ATu2497 ----------LLDFGA-HAVNITEPDLFNTRLLQFLLPADQT------------------ RPA3731 ----------AFDGVG-HCVAQEAPTQLLTALEEFAAA---------------------- PSPPH1999 ----------LAGGHHLHLNDEQGARSVAHCINRFFAAS--------------------- SAV0974 ----------VYSGAP-HGLTGAYEQEFNADLLNFIRG---------------------- ATu5389 ----------TYPGFS-HGMLTVNADVLNQDLLAFIKG---------------------- PA2717 ----------TYPGFP-HGMPTTQAEVINADLLAFIRG---------------------- PA3053 ----------EFPDLG-HTPQIQAPERFHQALLEGLQTQP-------------------- PP2201 ----------TFEGMG-HAPQIEEPVRFNRTLVEWLGK---------------------- SAV0923 ----------TLDATG-HCPQLSAPDATAQAITAFLGATR-------------------- ATu2061 ----------TMPGHG-HNPHWENPQGVSTLVTEFFAEVMTDASPVRVETAADKR----- SAV2105 H---------ILDGCG-HWLQQERPQDTNRLLTDWLASLPG------------------- RPA2773 L---------IIDGAG-HWIQQEKPAEVNAALIEFLKENY-------------------- SAV141 AVAPQLQGPVVLPGCG-HWTQQERPVEVNAALLDFLTRIDGSAPR--------------- RHA05146 ----------ILPGFD-HSTAVAASPEVLAAVEPFLASV--------------------- RHA00069 ----------VLDGVG-HYPHLEDPAAVVEIIDDFVSTTPART----------------- SAV1746 ----------IFEGAG-HFPFHTDPARFLALVEEFTGTTRPAHWSREHWRELLRAGRPGS PP4540 ----------VVDDSR-HATPLDQPEVFNQTLLQFLAAASTSQGSLSPC----------- PSPPH2566 ----------ILPDQR-HMMPVESPRLVNQVLLDFFEKTGLDKLATAHNSIKGIVA---- PA0231 ----------LVEFAAAHLSNVEAGDAFSRRLVDFLLSA--------------------- PP1380 ----------YVDFPAAHLSNVEIGEAFSRRVLDFLLAH--------------------- PA3586 ----------LIPGLA-HRFQEAFKEPLLAAVVPYLKAHRQASVVQL------------- PP2804 ----------LLPGMG-HDLPTCLSGTLLSLLTGHMR----------------------- SAV0519 ----------SVPGAG-HDVHLERPAVLRHLIQEFLEENAAPRGPARRPGRGRDRGPGRL SC0267 ----------VVPRTG-HLIPLEAPADLADAVTAFATAV--------------------- SC5943 ----------PIEGGR-HGIAVEFADTVAHRVREFLGVEGHG------------------ PSPPH0033 ----------LVPEAG-HIANLENPDFVSGALMTFLARVNQQQG---------------- RPA1568 ----------ILKGAG-HTMMVERPDEVLKALQQ-------------------------- SAV7198 ----------LVEDCG-HIIPLHRPDALLALLHPFLKD---------------------- SAV1548 ----------IVDAG--HELPVEKPAELTSALDRFVTGQVGGVQLVG------------- SAV4410 ----------VIDGAA-HLPNLERPEEFNAALGEFLFSLSGV------------------ TM0820 ILKLAAK------------------- TM0423 -------------------------- ATu2215 -------------------------- PP0624 -------------------------- PA3301 -------------------------- PP4551 -------------------------- PSPPH0357 -------------------------- PP3523 -------------------------- ATu2497 -------------------------- RPA3731 -------------------------- PSPPH1999 -------------------------- SAV0974 -------------------------- ATu5389 -------------------------- PA2717 -------------------------- PA3053 -------------------------- PP2201 -------------------------- SAV0923 -------------------------- ATu2061 -------------------------- SAV2105 -------------------------- RPA2773 -------------------------- SAV141 -------------------------- RHA05146 -------------------------- RHA00069 -------------------------- SAV1746 AAGRPDTVRNRAVERDLRQASERSAT PP4540 --------------------------

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PSPPH2566 -------------------------- PA0231 -------------------------- PP1380 -------------------------- PA3586 -------------------------- PP2804 -------------------------- SAV0519 RGGGRHLQPPPPA------------- SC0267 -------------------------- SC5943 -------------------------- PSPPH0033 -------------------------- RPA1568 -------------------------- SAV7198 -------------------------- SAV1548 -------------------------- SAV4410 --------------------------

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Appendix A.3 Amino acid sequence alignment of AF_est1 and AF_est2 with 8 bacterial esterases that were characterized in Chapter 3.

AF_est2 -----------------------------------------MTLRTKDGLTLYTRRWDVE PP_est1 -----------------------------------------------------------M RP_est1 ------------------------MLAQGQRFEGLLCGDDSSRCEVVEMAKLTQFLISG- RP_est2 -----------------------------------------MSEAVMAGAEAFAF--DGG PA_est1 MKRFLLGLVLLLAVAAGVLYFVPATLLASVRTVERGLAGLSEHSVQVDNLEIA-YLEGGS AF_est1 -----------------------------------------MPYAD-NGVKIYYEVEDGG SAV_est1 ----------------------------------MTSQHVVGRFFDVQGGQVYAHVREGD PP_est2 ----------------------------------------MSTFVTRDGTSIY-FKDWGS RP_est3 -----------------------------------------MPSFDHAGVNIA-YLDEGE PP_est3 ------------------------MACLPAAPDLQGAPSMIQPVAERTPAGTS-YLDVGQ AF_est2 SPRAVICLVHGLGEHSG------RYEHVARFFNENG-----ISF------AAFDLRGHGR PP_est1 R--NRLVLLPGWGLGTA------ALEPLAASLRAQD-----ARLQVEL--MPLPELAHS- RP_est1 ---ACALLITATSAIAD------PIAPAGKAIGQDGGGHTIYQVDANGISIGYKLIGQGA RP_est2 K--TGVLLVHGFTGSPQ------SMRYLGERLGERG-----YSI------LGPRLPGHGV PA_est1 EKNPTLLLIHGFGADKD------NWLRFARPLT-ER-----YHV------VALDLPGFGD AF_est1 E--PAIVFVHGWTANMN------FWREQREYFK-GK-----HRM------LFIDNRGHGK SAV_est1 G--PALIFLHYWGGSRR------TWIPVLQRLD-PG-----QGF------VAYDQRGWGG PP_est2 G--KPVLFSHGWPLDAD------MWDSQMEFLASRG-----YRA------IAFDRRGFGR RP_est3 G--EPILLIHGFASNKNVNWVYPSWLSELKRTG--------RRV------IAIDNRGHGE PP_est3 G--QPVVLIHGVGLNKE------MWGGQFVGLA-ND-----YRV------IAYDMLGHGQ : . . AF_est2 SE--GKRGHAEYQQLMDDITLFLQSLDYDCPKI-LYGHSM-GGNLALNYILRYDPDIAGG PP_est1 ----------DVQAWIDHLDRKL------PNDVWLGGWSL-GGMLASALAHKRGDHCCGL RP_est1 PMVMIMGLGGTADNWPPQV---IEALSKNHQLILMDNRGM-GHTTANDTPFSYPLFAADV RP_est2 SP--AAMAKTTANDWVNCAEDALLELSAKCDKTFVAGLSM-GGTLSLYLAASHPDKVAGV PA_est1 SSK-PQQASYDVGTQAERVANFAAAIG--VRRLHLAGNSM-GGHIAALYAARHPEQVLSL AF_est1 SDKPFNRSFYEFDNFVSDLHAAVKDAS--FDRFVLVGHSF-GTMISMRYCVEHPGRVEAL SAV_est1 ST--SVPGPYDLEQLADDAQRVVDALG--YSRYVLVGHSM-GGKVAQILAARKPAGLRGV PP_est2 SS--QPWNGYDYDTFADDIAQLIEHLD--LRDVTLVGFSMGGGDVSRYIARHGSERVAGL RP_est3 SSKLYDPNDYTLEAMASDAVALMDHLG--IARADVMGYSM-GARIGANLARRQEQRVRSV PP_est3 SR--VPAADTPLEGYADQLAELLDHLQ--IAQATVIGFSM-GGLVARAFALNYPQRLAAL : . .: * . AF_est2 IISAPFLALPKELPKHLFFILKLLNVVAPSIQLSNGIDPNLISRDR--EVVEAY------ PP_est1 LTLASNPSFLARPD----WPHG------MAEDTFGTFLDGCRNHTQ--VTLKRFRTL--- RP_est1 IGLLDALGIKRSDVLG--YSMGSTITQQLLLQYPDRFNKALIHATS--TDGSNVAKA--- RP_est2 IPINAAVQIDSPDLAGLAYARG-------LPEFVPGIGSDMVDTST--KELA-------- PA_est1 ALIDNAGVMPARKSE-------------LFEDLERGENPLVVRQPE--DFQKLLDFVF-- AF_est1 VLIGGGARIQSLHRYG--YPIG-----RLFATLAYGISARIIANMAFGRKAGELRDW-GL SAV_est1 VLVAPAPP----------APIG------VTEQVQETVSHAYDNEEAVLQSIDLM------ PP_est2 VLLGAVTPVFGKRDD---NPDG------VDLSVFEGIRAGLRADRA--QFIADFATPFYG RP_est3 VLGGLGMGLLSNEGRP-----G--------ENVARALEADALDDVT--DPVGRTFRAFAD PP_est3 VVLNSVFNRTPEQSAG---------------VIARAAQAAELGPDA--NVDAALDRWFSR AF_est2 ---VSDPLVHDKISPRFILQSLE------------AGKWA--LENADRLRKPILLIHGTA PP_est1 ---CSDGALQPRTLLRQLGVGVPETDPLYLATGLEVLAKLDTREALQAYDGPQLHLFAGS RP_est1 ---LHGRVPTDPIVARQVEA---------------TTHWKTPLDKLPSIDNQVMLVVGTA RP_est2 ----YDQVPVTCVKQV-------------------MGLAATARALLPRIKCPTLVINSRV PA_est1 ---VQQPPLPAPLKRYLGERAVAASAFNAQIFEQLRQRYIPLEPELPKIEAPTLLLWGDR AF_est1 KEALENTPKHAALNTLW------------------TLTTVDLRDIAREIEKPTLIVVGKE SAV_est1 ---LTRGGLTPELRRQVVEDSLRGGDEARLEWPR-RGLVQDVSAGVSAIEVPVLVLAGSH

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PP_est2 L--NHGQQVSQGVQTQTLNIALMASIKGTLDCVT-AFSETDFRPDMAKVDVPTLVIHGDD RP_est3 QTRSDRKALAACMRGS-------------------RGLMS--REDAAQIAVPVLIAVGTD PP_est3 EYKAANPAQVAAIRQVLASNDPQGYHTTYSLF---ATQDMYRADDLGSIQVPTLIATGEL : . AF_est2 DQITSYRASQEFAKRAGELCKFVS--YEGFYHEPHNEPEKERVLADMLKWIEEVI----- PP_est1 DALVP-AEAAKALSELLPDVEVGM--VEDSSHAFLLEYPQE-LAAGIKSFLHESGDD--- RP_est1 DNVVG-TESSKTIASAIPGAWLVQ--FKGATHHLMYETPEG-FSAAALTFFETNETVTPK RP_est2 DHVLS-PANATVIANHVGSSRIETLWLDNSYHVATIDNDKDLIAAEIDSFVTRNL----- PA_est1 DRVLD-VSSIEVMRPLLKRPSVVI--MENCGHVPMVERPEE-TAQHYQAFLDGVRNAQVA AF_est1 DALLP-VSKSEELSRLIKNSKMVI--VPDAGHCVMLEQPEI-VNRVLEEFIHTFSAMLIR SAV_est1 DKVDPPTVLADHLLPLIPTATLTV--LKDTGHLSPLEVPDQ-VAAHIGAFVAQL------ PP_est2 DQIVPFETTGKQAAELIRGAELKV--YAGAPHGFAVTHAQQ-LNEDLLAFLQR------- RP_est3 DDVAG-SA--HELGDIIPGSQVLD--IPRRDHMRAV-GDRV-YKEGVVDFLARRP----- PP_est3 DSGST-PAMTRQLAACIPGARSVV--LAEQRHMMPVEAPRE-VNKMLLDFLTQARTLTES * * :. AF_est2 -------------- PP_est1 -------------- RP_est1 IEPNASVAPPPTQP RP_est2 -------------- PA_est1 GR------------ AF_est1 A------------- SAV_est1 -------------- PP_est2 -------------- RP_est3 -------------- PP_est3 AKGIVA--------

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Appendix B.1 The progress of 208 gene targets in gene cloning, protein expression and purification, and protein activity assay.

Full Name Protein ID Cloned Expressed Purified Active

activity AF_0865 1 1 1 1 AF_1706 1 insoluble AF_1753 1 1 1 1 AF_2336 1 insoluble AF_1537 1 0

Archaeoglobus fulgidus AF_0675 1 insoluble

Atu1572 1 0 Atu1814 1 0 Atu5389 1 1 1 0 Atu3664 1 0 Atu2061 1 1 1 0 Atu2497 1 1 1 0 Atu2679 1 0

Agrobacterium tumefaciens C58 Atu2215 1 1 1 0

CA_C2688 0 CA_P0097 0 CA_P0133 0 CA_C2936 0

Clostridium acetobutylicum 824D-S CA_C0816 0

Cthe_3133 0 Clostridium thermocellum 27405D Cthe_1390 0

b2593 1 1 1 1 Escherichia coli K12 b2799 1 1 1 1

NE2298 1 1 1 1 NE0456 0 NE2295 0 NE2346 0 NE1455 0

Nitrosomonas europaea NE0279 1 1 1 1

PA2717 1 1 1 0 PA4152 1 0 PA3226 1 0 PA0480 1 0 PA2949 1 1 1 1 PA0231 1 1 1 0

Pseudomonas aerugonosa

PA3053 1 1 1 0

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PA3586 1 1 1 0 PA3301 1 1 1 0 PA3509 1 insoluble PA3132 1 1 1 1 PA0829 1 0 PA5057 1 insoluble PA3429 1 0 PA3994 1 insoluble PA3695 1 0 PA5080 1 insoluble PA2934 1 1 1 1 PA2856 1 0

PA1469 1 1 1 1

PP5253 1 1 1 1 PP4021 1 insoluble PP3645 1 1 1 1 PP4540 1 1 1 0 PP0553 1 insoluble PP1380 1 1 1 0 PP0364 1 1 1 1 PP2201 1 1 1 0 PP4624 1 0 PP4164 1 insoluble PP5028 1 insoluble PP2804 1 1 1 0 PP1979 1 0 PP4551 1 1 1 0 PP5117 1 0 PP2318 1 insoluble PP3523 1 1 1 0

Pseudomonas putida KT2440 PP0624 1 1 1 0

PSPPH_4522 1 1 1 1 PSPPH_3123 1 insoluble PSPPH_2566 1 1 1 0 PSPPH_1434 1 0 PSPPH_0033 1 1 1 0 PSPPH_3013 1 0 PSPPH_2235 1 insoluble PSPPH_1999 1 1 1 0

Pseudomonas syringae

PSPPH_3482 1 1 1 1

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PSPPH_0357 1 1 1 0 PSPPH_3478 1 0 PSPPH_0378 1 insoluble PSPPH_2039 1 insoluble

PSPTO_3914

1 1 1 1 RHA1_ro01401 1 1 1 1 RHA1_ro02101 0 RHA1_ro01338 1 1 1 1 RHA1_ro02074 1 insoluble RHA1_ro08044 1 1 1 1 RHA1_ro03350 1 1 1 1 RHA1_ro04327 1 0 RHA1_ro03976 1 0 RHA1_ro10146 1 1 1 1 RHA1_ro08081 1 1 1 1 RHA1_ro05146 1 1 1 0 RHA1_ro10061 1 0 RHA1_ro10040 1 insoluble RHA1_ro03603 1 1 1 1 RHA1_ro02771 1 0 RHA1_ro01727 0 RHA1_ro06181 1 0 RHA1_ro03968 1 0 RHA1_ro00069 1 1 1 0 RHA1_ro08672 1 insoluble RHA1_ro06216 1 insoluble RHA1_ro06804 1 0 RHA1_ro01528 1 0 RHA1_ro06793 1 0 RHA1_ro08833 1 0 RHA1_ro04298 0 RHA1_ro04713 0 RHA1_ro01411 1 insoluble RHA1_ro03520 1 1 1 1 RHA1_ro09014 1 insoluble RHA1_ro01171 1 insoluble RHA1_ro00320 1 1 1 1 RHA1_ro04639 1 insoluble RHA1_ro00371 1 0 RHA1_ro04540 1 insoluble

Rhodococcus sp. RHA1 RHA1_ro02607 1 1 1 1

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RHA1_ro04883 1 insoluble RHA1_ro05212 1 0 RHA1_ro06688 1 0 RHA1_ro05164 1 insoluble RHA1_ro02879 1 insoluble RHA1_ro04513 1 1 1 1 RHA1_ro00365 1 insoluble RHA1_ro02153 1 0

RHA1_ro01863 1 0 RPA2105 1 1 1 1 RPA3430 1 1 1 1 RPA2773 1 1 1 0 RPA2836 1 0 RPA1568 1 1 1 0 RPA2734 1 insoluble RPA0348 1 1 1 1 RPA3731 1 1 1 0 RPA1647 1 0 RPA1163 1 1 1 1

Rhodopseudomonas palustris CGA009 RPA4646 1 1 1 1

RSp1418 0 RSp1148 0 RSc0298 0 RSc1135 1 1 1 1 RSc3346 1 insoluble RSc2250 1 1 1 1 RSc1396 1 0 RSc1841 1 insoluble RSp0196 0 RSc1561 0

Ralstonia solanacearum 11696D-S RSc1717 0

SAV974 1 1 1 0 SAV1548 1 1 1 0 SAV923 1 1 1 0 SAV141 1 1 1 0 SAV4410 1 1 1 0 SAV4596 1 0 SAV3810 1 1 1 1 SAV4968 0 SAV512 1 1 1 1 SAV7198 1 1 1 0

Streptomyces avermitilis SAV2105 1 1 1 0

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SAV519 1 1 1 0 SAV1256 1 0 SAV4779 1 0 SAV1746 1 1 1 0 SAV620 1 insoluble SAV3624 1 insoluble SAV5173 1 1 1 1 SAV1968 1 insoluble SAV5668 1 0 SAV298 1 insoluble SAV297 1 insoluble SAV875 1 1 1 1 SAV5711 1 insoluble SAV2164 1 0

SAV2722 1 1 1 1 SCO0465 1 insoluble SCO5943 1 1 1 0 SCO1097 1 insoluble SCO0939 1 insoluble SCO3566 1 1 1 1 SCO2886 1 insoluble SCO7245 1 insoluble SCO3690 1 1 1 1 SCO0267 1 1 1 0 SCO4746 1 0 SCO0427 1 insoluble SCO7205 1 insoluble SCO3233 1 0 SCO6277 1 insoluble SCO4479 1 insoluble SCO6697 1 insoluble SCO7440 1 1 1 1 SCO0878 1 insoluble SCO6612 1 insoluble SCO4392 1 0 SCO6966 1 0 SCO0526 1 insoluble SCO6339 1 0 SCO1989 1 1 1 1 SCO5986 1 0 SCO3171 1 insoluble

Streptomyces coelicolor SCO4395 1 insoluble

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SCO3439 1 1 1 1 SCO6712 1 1 1 1 Sinorhizobium meliloti RA0563 1 1 1 1

TM_1350 1 1 1 1 TM_0423 1 1 1 0 TM_0820 1 1 1 0

Thermotoga maritima TM_0111 1 1 1 1 Total 208 186 86 86 48

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Appendix B.2 The schematic representations of pNP-esters, phenyl acetate, and tributyrin.

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Appendix B.3 SDS-PAGE (15%) of the purified bacterial esterases that have been characterized in this study.

Enzymes were run at 10 µg scale. Lane 1: Molecular mass standards; lane 2: RP_est1; lane 3: RP_est2; lane 4: RP_est3; lane 5: PP_est1; lane 6: PP_est2; lane 7: PP_est3; lane 8: PA_est1; lane 9: SAV_est1. Proteins were stained using the comassive blue staining procedure.

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Appendix B.4 A standard curve of p-nitrophenol at pH 7.

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Appendix B.5 Absorbance reading at 400 nm of the residual activity for PP_est3 after 5 h preincubation in organic solvents.

1 2 3 4 5 6 7 8 9 10 11 12 A Buffer blank Buffer blank Control 0.1737 0.1769 0.6739 0.6693 0.6284 B Substrate blank Substrate blank Tert-amyl alcohol 0.2565 0.2523 0.745 0.7165 0.7242 0.7998 0.6134 0.6595 0.7316 0.8554 0.8678 C Std 10uM Std 10uM 1,6-Dixaone 0.2043 0.2032 0.6803 0.6681 0.6276 0.5529 0.55 0.5301 0.2705 0.2611 0.2565 D Std 25uM Std 25uM DMSO 0.2491 0.253 0.6828 0.5966 0.6485 0.6963 0.6987 0.6289 0.6984 0.7526 0.7084 E Std 50uM Std 50uM Toluene 0.3519 0.3472 0.7015 0.7666 0.7508 0.2298 0.2259 0.22 0.8923 0.9447 0.9714 F Std 75uM Std 75uM P-xylene 0.4585 0.4334 0.4335 0.4609 0.4562 0.7696 0.7875 0.8065 0.9885 1.0852 1.1407 G Std 100uM Std 100uM Hexane 0.5694 0.5393 0.2495 0.2339 0.2309 0.7768 0.797 0.7844 0.9711 1.0212 1.0343 H Std 500uM Std 500uM Cyclohexane 2.5616 2.3794 0.4979 0.4854 0.4786 0.5008 0.4886 0.4626 0.7621 1.0678 1.0508

15 % v/v organic solvent



Absorbance reading of blanks for organic solvents mixed in 50 mM potassium phosphate buffer at pH 7.

1 2 3 4 5 6 7 8 9 10 11 12 A 50 mM Potassium phosphate, pH 7 0.1533 0.1489 0.1501 B Tert-amyl alcohol

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0.1471 0.307 0.2292 C 1,6-Dixaone 0.1387 0.1409 0.1464 D DMSO 0.1493 0.1414 0.1408 E Toluene 0.1283 0.1335 0.1369 F P-xylene 0.1287 0.1314 0.1379 G Hexane 0.1241 0.1209 0.12 H Cyclohexane 0.1337 0.1224 0.1222 15 % v/v organic solvent 30 % v/v organic solvent 50 % v/v organic solvent

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Appendix B.6 SDS-PAGE (15%) of the purified archaeal esterases that have been characterized in this study.

Enzymes were run at 10 µg scale. Lane 1: Molecular mass standards; lane 2: AF_est1; lane 3: AF_est2. Proteins were stained using the comassive blue staining procedure.

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