Review

In vitro engineering of microbial enzymes wapplications: Prospects and perspectives

Swati Joshi, Tulasi Satyanarayana Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New De

This review covers protein engineering of mic In vitro evolution tools for making alterations

cal proped into

denite information on redesigning industrial enzymes. This review focuses on

enzymes. Scien-mes which canevailing ineratures, e

and others are a few of the requisite properties. The ultimate goalis to redesign the proteins in such a way that the industrial pro-cesses can be carried out in a more economic and greener way.In protein engineering, various methods are employed in modify-ing the target proteins. These are mainly rational design, directedevolution and semi-rational approaches for designing andconstructing novel proteins. The rational methods require priorknowledge of the aa sequence, 3D-structure and the knowledge

0 0

substitute; HRPL, high-redox potential laccases; IvAM, in vivo assembly of mutantlibraries; LTM, look-through mutagenesis; MD, molecular dynamics; Mod-PCR,mutagenic oligonucleotide-directed PCR; PDB, protein data bank; PGs, polygalac-turonases; PLs, polygalacturonate lyases; RACHITT, random chimeragenesis ontransient templates; REase, restriction endonuclease; SCP, single cell protein; SDM,site directed mutagenesis; SeSaM, sequence saturation mutagenesis; SLAC, smalllaccases; spCSR, short-patch compartmentalized self-replication; StEP, staggeredextension process. Corresponding author. Tel.: +91 11 24112008; fax: +91 11 24115270.

E-mail address: tsnarayana@gmail.com (T. Satyanarayana).

Bioresource Technology 176 (2015) 273283

Contents lists availab

Bioresource T

journal homepage: www.elsing activity in the alkaline or acidic environments, highperformance in non-aqueous media, increased protease resistance

PCR, error prone polymerase chain reaction; GSSM, gene site saturation mutagen-esis; GLNBP, galacto-N-biose/lacto-N-biose I phosphorylase; HMFS, human milk fatural biocatalysts and to develop process-specictists have been attempting to generate enzywithstand harsh and unfavourable conditions prtrial processes. The tolerance to high or low temp

Abbreviations: 2 -O-meNTP, 2 -O-methylribonucleoside triphosphates; aa,amino acid; CBM, cellulose binding module; CBM, combinatorial benecialmutagenesis; CGTase, cyclodextrin glycosyltransferase; CODH, carbon monoxidedehydrogenase; CSAT, combinatorial active site saturation test; CSSM, coevolving-site saturation mutagenesis; DB, disulphide bridge; EG III, endoglucanase III; ep-http://dx.doi.org/10.1016/j.biortech.2014.10.1510960-8524/ 2014 Elsevier Ltd. All rights reserved.indus-xhibit-Microbial enzymesIndustrial biotechnology

the recent developments in improving the characteristics of various biotechnologically importantenzymes.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

The tailoring of the microbial enzymes has become a trend inthe eld of protein engineering to overcome the limitations of nat-Protein engineeringIn vitro evolution enzymes, there is a lack of Industrially relevant biophysico-chemi Protein engineered enzymes that enter

a r t i c l e i n f o

Article history:Received 16 September 2014Received in revised form 28 October 2014Accepted 29 October 2014Available online 13 November 2014

Keywords:Site directed mutagenesisrobial enzymes with multiple uses.in the enzymes are discussed.erties have been kept in focus.the market are also cited.

a b s t r a c t

The discovery of a novel enzyme from a microbial source takes anywhere between months to years, andtherefore, there has been an immense interest in modifying the existing microbial enzymes to suit thepresent day needs of the industry. The redesigning of industrially useful enzymes for improving theirperformance has become a challenge because bioinformatics databases have been revealing new factson a day-to-day basis. Modication of the existing enzymes has become a trend for ne tuning ofbiocatalysts in the biotech industry. Hydrolases are employed in pharmaceutical, biofuel, detergent, foodand feed industries that signicantly contribute to the global annual revenue, and therefore, the emphasishas been on engineering them. Although a large data is accumulating on making alterations in microbialh i g h l i g h t sith multifarious

lhi 110 021, India

le at ScienceDirect

echnology

evier .com/locate /bior tech

This method mimics natural selection in vitro and reduces the timerequired for evolution from millions of years to few weeks or

urcemonths. The directed evolution can be employed by using anon-recombinant or recombinant approach. Non-recombinantapproach includes techniques such as SeSaM (Gupta and Farinas,2010) and ep-PCR (Song et al., 2012). Recombination baseddirected evolution involves techniques such as fragment shufing(Bei et al., 2009) and random chimeragenesis on transient tem-plates (RACHITT) (Coco et al., 2001). There are both advantagesand disadvantages of using either rational or directed evolutionmethodology as a sole approach for protein engineering experi-ments. Thus, a combination of both the techniques would be morea promising approach to improve the properties and functions ofthe proteins. Depending on the number of sites chosen to mutate,mutagenesis can be divided into single site and multisite satura-tion mutagenesis. The popular single site saturation mutagenesistechniques include Mod-PCR, where degenerate primers are usedto introduce mutations in DNA sequence elements (Chiang et al.,1993), codon cassette mutagenesis where a universal mutageniccassette is used to introduce a single codon at the site of mutation(Kegler-Ebo et al., 1994). Multisite saturation mutagenesis is morecomplex process than the single site saturation mutagenesis. Dur-ing the past few years, several attempts have been made to evolveand simplify multisite saturation mutagenesis. A Quik ChangeMulti Site-Directed Mutagenesis Kit sold by Stratagene Company(Stratagene, La Jolla, CA, USA) is employed for multisite saturationmutagenesis. Latest development in multisite saturation techniquehas been reported by a German group of researchers, which hascome up with a cost effective Omni Change methodology(Dennig et al., 2011), wherein ve independent sites can besaturated simultaneously in four simple steps. These techniquesare becoming more and more useful to introduce changes in theproteins of interest. In this review, an attempt has been made toupdate the recent advances in ameliorating enzyme properties byprotein engineering in order to achieve improved performance.

2. Redesigning microbial enzymes

Since the very beginning of evolution, nature has beenconstantly evolving proteins for survival and better adaptation ofliving organisms to their environments. The nature evolvedenzymes are, however, not always best for industrial purposes.Some additional features are required to suit their applicabilityin industries. Sometimes two or three specic properties areneeded in one protein at the same time. Moreover, natural evolu-tion takes a very long time for the protein to acquire the desirableproperties. This is where in vitro evolution takes the charge as itaids in tailoring and evolving microbial enzymes at a much fasterrate (Fig. 1).of the structurefunction aspects of the target proteins. So far,95,052 protein structures have been solved and are available inthe PDB. When mutations carried out by computational andrandom mutagenesis are compared, often the best mutants havebeen developed by either of the methods. Computationally devel-oped muteins are obtained seldom by random mutagenesis, whichexplains the importance of rational design in terms of eitheranalysing the effect of a single residue on protein stability or onfolding and function of the mutein (Wunderlich et al., 2005). Thedirected evolution is a powerful tool to achieve more ambitiouscharacteristics in proteins of interest. Unlike the rational designmethodology, directed evolution does not require prior knowledgeof the primary sequence of proteins and their function or structure.

274 S. Joshi, T. Satyanarayana / BioresoDifferent industrial processes require enzymes with uniqueproperties to operate in the physicochemical environment ofthe process. For example, a protease must exhibit tolerance todetergents and bleaching agents in order to be used as a detergentadditive (Joshi and Satyanarayana, 2013). On the other hand,an ideal xylanase suitable for the paper and pulp industryshould be cellulase-free and thermo-alkali-stable (Verma andSatyanarayana, 2012). In order to generate biocatalysts that suitdiverse industrial applications, various molecular approaches havebeen employed (Table 1, see Supplementary le, Table S1). In thisreview, we have discussed recent achievements in in vitro tailoringof various industrial enzymes.

2.1. In vitro changes in properties of laccases

The industrial importance of laccases arises from the fact thatthese versatile enzymes can oxidize both toxic and non-toxic sub-stances. Laccases are utilized in a plethora of processes includingpharmaceutical, textile, wood processing, food processing andchemical industries (Koschorreck et al., 2009; Sherif et al., 2013).It is known that subtle molecular changes in protein structure bymutations can signicantly alter their biochemical properties(Ema et al., 2005; Mullaney et al., 2012; Verma andSatyanarayana, 2013). Mutagenesis has been the tool of choice tounleash secrets of biochemical properties of microbial laccases.Protein termini are known to have a signicant role in determiningenzyme properties (Wang et al., 2009).

Autore et al. (2009) found out the role of C-terminal extensionof 16 aa residues in the stability and catalytic activity of Pleurotusostreatus laccase (POXA1b). C-terminal truncated laccase wasgenerated and properties of the full length and truncated enzymeswere compared. The removal of C-terminal led to reduced stabilityat alkaline pH (pH 10), while increased stability at acidic pH (pH 5).As C-terminal is cleaved by proteolysis and is absent in maturelaccases, removal of C-terminal eliminates the obstruction ofT2/T3 channel, which is the route for oxygen entry and exit ofwater molecule, thereby aiding the catalysis by laccases (Autoreet al., 2009). Bacillus licheniformis laccase CotA harbouring K316Nmutation exhibited higher activity than the wild type enzyme,while the mutation D500G led to 11.4-fold higher expression(Koschorreck et al., 2009). The presence of glycine at position500 creates a large space between Gly500 and Met502 residuesand brings about structural changes in the protein that result inefcient protein folding and production of higher quantities ofthe enzyme. A combinatorial approach of random mutagenesis(IvAM) [Table S1] and SDM have resulted in 34,000-fold improve-ment in total activity of high-redox potential laccase (HRPL)(Mate et al., 2010) (Fig. 2). The mutations have been introducedin a fusion product of the prepro-leader sequence of the yeasta-factor mating pheromone and basidiomycete PM1 HRPL i.e.a-PM1. Initially mutations were introduced by mutagenic PCRfollowed by combined use of mutagenic PCR and in vivo DNAshufing. The mutations that signicantly improved a-PM1 prop-erties but not selected for the nal rounds of mutagenesis, weretraced and introduced by SDM in the nal stage of the experiment.Mutations in both the prepro-leader sequence and PM1 regioncontributed to the improvement in properties of a-PM1. Themutations in the prepro-leader sequence have been known toaffect targeting of proteins to endoplasmic reticulum and secretionof the protein. Small laccases (SLAC, multicopper oxidases) fromStreptomyces coelicolor have also been subjected to mutagenesisfor understanding the effect of sequence variation (Sherif et al.,2013). Seventeen aa residues have been found to be importantfor the activity of SLACs. About ten histidine residues have beenshown to coordinate copper during catalysis. Among the mutations

Technology 176 (2015) 273283studied, Y229A and Y230A increased SLAC activity by 10-fold.Higher activity of Y229A and Y230A was attributed to accumula-tion of higher copper amount than the wild type enzyme.

y adabio-tion

urce2.2. Alterations in phytases

Phytases are the enzymes which hydrolyze phytic acid in orderto ameliorate phosphorus availability and assimilation, and miti-gating anti-nutritional properties in monogastrics and the associ-ated environmental problems. Most of the mutagenesis studiescarried out to tailor phytase of choice focus on two aspects: ther-mostability and activity at low pH. Based on crystal structure of

Fig. 1. The power of in vitro over the natural evolution for developing the industriallniche of the microbe harbouring the gene. In contrast, in in vitro evolution, variouscertain residues of the enzyme, which is several times faster than the natural evolu

S. Joshi, T. Satyanarayana / Bioresothe Aspergillus niger phyA, muteins K301E, K300E, D262H, E228K,K94, K91E and Q50P were constructed and expressed heterolo-gously in Pichia pastoris (Kim et al., 2006). These mutations weredirected to lower the pH optima of phyA from 5.5 to match thepH of the stomach (Kim et al., 2006). Among the muteins expressedin P. pastoris, the mutation E228K shifted the pH optimum towardsacidic range. Optimal pH of this mutein was 3.8. The shift in the pHpossibly resulted from lowering of pKa value of acid/base catalysisalong with creation of a more favourable microenvironment forbinding negatively charged substrate. Additionally, this muteinexhibited 266% higher hydrolysis of soy phytate than the wild type.Similarly in another mutagenesis study, the pH stability of aphytase was targeted. In an attempt to obtain a versatile and morepreferred phytase, a semi-rational strategy to alter the propertiesof fungal phytase was attempted (Bei et al., 2009). An array ofchimeric phytase sequences have been constructed by swappingthe regions of Aspergillus fumigatus ATCC 13073 phytase (Afp) andA. niger NRRL 3135 phytase (Anp) genes (Fig. 3). The resultantchimeric phytases were cloned into pGAPZaA vector andtransformed into P. pastoris X33. The analysis of different chimericproteins thus generated revealed regions II and VI to be responsiblefor the difference in specic enzyme activity at pH 5.0, whileregions IV and V of Anp contribute to its second pH optima at2.5. The effects of substitutions were found to be additive in nature,except the effect of regions V and VI.

Mullaney et al. (2012) performed mutation studies to alterdisulphide bridges of A. niger phytase. The phytase containing5 disulphide bridges (DB) was altered at a disulphide bridgeforming cysteine residues and the mutated genes were expressedin P. pastoris. The effect of removal of every disulphide bond wasstudied in terms of altered optimum temperature, pH optima andKm. Alteration in DB 2 led to complete loss of activity, whileabolishing disulphide bond 1, 3/4 and 5 lowered the optimumtemperature to 53, 37 and 42 C, respectively. The pH prole ofthe mutated phytases was changed in such a way that a peak atpH 2.5 was abolished simultaneously with the removal of DB at1, 3 and 5. The removal of DB 5 led to the elimination of peak atpH 5.0. The K of recombinant phytase was not signicantly

pted enzymes. Natural evolution depends on rare mutagenic event occurring in thetechniques are used in the laboratory to force random and directed mutagenesis of(Please refer Abbreviations and Table S1 for the acronyms used in the gure.).

Technology 176 (2015) 273283 275m

changed upon alteration of disulphide bridges, but the turnoverof the recombinant phytase was signicantly lowered (Mullaneyet al., 2012).

Farhat-Khemakhem et al. (2013) deciphered the mechanism ofthermostabilization of Bacillus subtilis US417 phytase (PHY US417)by employing both mutagenesis and computer aided structuralinvestigation. PHY US417 differs from PhyC in R257P substitutionand is more thermostable than PhyC. It was found that Pro 257was present in a surface loop joining 2 of the 6 characteristicb-sheets. The P257 residue has, therefore, been presumed to alterthe local thermal exibility of the surface loop, thereby contribut-ing to higher thermostability of PHY US417 (Farhat-Khemakhemet al., 2013). By in depth understanding of the amino acid residuesresponsible for a particular character of the protein, improvementsin the enzyme properties can be attained through minimumin vitro manipulations.

2.3. Improvement of a-amylases by tailoring their protein sequences

Amylases are main workhorses of starch processing industry.Most of the processes carried out in starch processing industryrequire high operational temperatures which necessitate the useof adequately thermostable amylases. Amylases have also beensubjected to in vitro alterations which have improved this classof enzymes for industrial applications. Recently, Kachan andEvtushenkov (2013) introduced two point mutations (Gly211Valand Asn192Phe) in the Bacillus sp. 406 a-amylase, which increasedthe Tm of the enzyme from 52 C to 55.5 C and 58.8 C. Thesemutations had an additive effect on Tm as it was increased by 26 Cin comparison with the wild type a-amylase. The mutationGly211Val improved the packing of hydrophobic core of the

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urceTable 1Microbial enzymes and alterations in their properties attained through various in vitr

Target enzyme Technique used (see Table S1) Microbial source

Laccase Site directed mutagenesis(SDM)

Streptomyces coelicolor

Laccase Rational design and SDM Bacillus licheniformis

Xylanase (Bcx) Rational and SDM B. circulans

GH-11 xylanase Introduction of disulphidebridge

ND

b-Glucosidase (BglC) Family shufing, sitesaturation, and SDM

Thermobida fusca

Tyrosine phenol-lyase Random mutagenesis,reassembly and activityscreening

Symbiobacterium toebi

Lipase B Molecular dynamics (MD)simulation and SDM

Candida antarctica

Xylanase Look-through mutagenesis(LTMTM) combinatorial

Hypocrea jecorina

276 S. Joshi, T. Satyanarayana / Bioresoenzyme, thereby increasing thermostability. On the other hand,mutation Asn192Phe established a triple interaction betweenPhe192, Tyr195 and His237 residues along with the removal ofan amide group of Asp193 that led to higher thermostability ofthe mutein. By adopting a unique and novel mutagenesis strategy,Wang et al. (2012) employed coevolving-site saturation mutagen-esis (CSSM) [Table S1] and mutated functionally correlated varia-tion sites of B. subtilis CN7 amylase (Amy7C). The mutation ofthese hot spot target sites improved thermostability of Amy7C by8 C. The technique used in this study can also be used for generat-ing hot spot focussed libraries for improving other industriallyimportant enzymes. Rahimzadeh et al. (2012) targeted asparaginedeamidation to improve thermostability of a Ca2+-independentamylase (BKA) of Bacillus sp. KR-8104. This research team replacedall the potential deamidation sites by site specic mutagenesis.Among fourteen variants tested, four showed higher thermostabil-ity than the wild type enzyme. The replacement at position 112was the most promising as it resulted in a 5-fold increase in t1/2of BKA at 70 C. Ozturk et al. (2013) attempted to improve the oxi-dative sensitivity of a-amylase (AmyC) of Thermotoga maritima. Forthe applicability of the a-amylase as detergent additive, oxidativestability is a major concern. The oxidation sensitive methionineresidue was targeted in the vicinity of active site. With the helpof solved crystal structure of AmyC, Met43, Met44, Met55 andMet62 residues have been modied. The substitution of the

benecial mutagenesis(CBMTM)

2

Bgl-licMB Splicing-by-overlap extension B. amyloliquefaciens(Bgl) and Clostridiumthermocellum (licMB)

2pin

Endo-b-glucanase PCR-based gene truncation Bacillus subtilis JA18 Inec

Phytase Semi-rational SDM technique Aspergillus niger 113 Ins

Phytase Fragment shufing A. niger NRRL 3135and A. fumigatus ATCC13073

Idc2

Polymerase I Short-patchcompartmentalized self-replication (spCSR)

Thermus aquaticus Gthhniques.

hanges attained through mutagenesis Applicability ofthe variantproduct

References

0-fold increase in the activity than the nativenzyme

Degradation ofphenoliccompounds

Sherif et al.(2013)

creased expression level and improvedecolorizing efciency

Dimerization ofphenolic acids

Koschorrecket al. (2009)

esidues involved in thermostabilization wereentied and N52Y showed thermostabilizingffect

Biomassdegradation

Joo et al.(2011)

x-xyl-SS3 displayed increasedermostability and thermoactivity

Biorening ofAgricultural by-products

Paes andODonohue(2006)

crease in half-life from 12 to 1244 min Bioconversion ofcellulosicbiomass

Pei et al.(2011)

roduction of L-tyrosine and its derivatives Production of L-tyrosine and itsderivatives

Rha et al.(2009)

nhancement of thermostability Detergentindustries

Le et al.(2012)

creased thermostability (enzyme showedctivity even after heating at 100 C for

Hemicellulosedegradation

Hokansonet al. (2011)

Technology 176 (2015) 273283methionine residues led to a little loss in residual activity, butthe muteins were less oxidation sensitive than the wild type AmyC.Mehta and Satyanarayana (2013) showed the importance of N-ter-minal region spanning 128 aa residues in thermostabilization andsubstrate preference of a maltogenic amylase (Gt-Mamy) of theextremely thermophilic bacterium Geobacillus thermoleovorans.More specically, the residue D109 has been shown to be respon-sible for the molecular features of this amylase. In this study, dele-tion of 128 aa residues from N-terminus led to monomerization ofGt-Mamy, which in turn led to alteration in various biochemicalproperties apart from the substrate preference (Fig. 4). Mabrouket al. (2013) increased the afnity and catalytic efciency of a mal-togenic amylase from Bacillus sp. US149 (MAUS149) towards b-cyclodextrin as compared to that of the wild type enzyme. It wasfound that the residue D46 forms a salt bridge with the residueK282 to maintain a particular orientation of the catalytic site. Inmutein D46V, where a charged aa had been replaced by anuncharged aa, access to the catalytic cleft got altered, and hence,the substrate selectivity.

2.4. Alteration in properties of xylanases

In the paper and pulp industry, xylanase treatment step is usedafter the alkali extraction of the pulp at elevated temperatures.This requires thermostable and alkalistable xylanases (Verma and

0 min)

.7 and 20-fold higher Kcat/Km than that of thearental Bgl and licMB, respectively along withcreased thermostability at 80 C

Brewing andanimal-feedindustries

Sun et al.(2011)

creased thermostability and catalyticfciency of truncated enzyme forarboxymethylcellulose

Beer and fruitjuice processing

Wang et al.(2009)

creased specic activity and afnity toodium phytate

Food and feedindustry

Tian et al.(2011)

entication of different fragmentsontributing to specic activity at pH 5 and pH.5

Dephytinizationof phytate

Bei et al.(2009)

eneration of single polypeptide behaving asree different enzymes

RecombinantDNA technology

Ong et al.(2006)

urceS. Joshi, T. Satyanarayana / BioresoSatyanarayana, 2012, 2013). This has led to intensive research forthe improvement of xylanases stable at higher temperatures andalkaline pH. Song et al. (2012) utilized ep-PCR in combination withadapted staggered extension process (StEP) [Table S1] in order togenerate an efcient xylanase that can degrade wheat straw andsolubilise arabinoxylans better than the native xylanase. GH11xylanase from Thermobacillus xylanilyticus (Tx-Xyn) was mutatedby this approach for developing two mutants (S27T and Y111T)with 2.3- and 2.1-fold higher efciency in hydrolyzing wheat strawand solubilizing arabinoxylans than the native enzyme. Higherefciency of the muteins was linked to the efcient binding ofxylanase to the substrate due to improvement in the secondaryligand binding site, reduced surface hydrophobicity and alterationin the exibility of thumb region that resulted from mutation at aaposition 111 (Paes and ODonohue, 2006). Utilizing the fact thatthermostable xylanases possess more arginine residues on thesurface than their mesophilic counterparts, a metagenomic GH11xylanase was mutated in order to improve its thermostability(Verma and Satyanarayana, 2013). Metagenomic xylanase withfour arginine substitutions (MxylM4) exhibited t1/2 of 150 min at80 C which is 20 min higher than the parent xylanase. Chenet al. (2010) made ve arginine substitutions in xylanase B fromA. niger F19 along with the introduction of a disulphide bridge.These modications increased the optimum temperature of thisxylanase by 5 C. The t1/2 of xylanase B improved enormously bythese modications in the primary sequence of protein. Themutated xylanase B exhibited t1/2 of 170 min which is higher thanthe wild type xylanase; these changes have, however, led to adecline in Vmax and Km.

Fig. 2. The combination of rational and directed approaches of mutagenesis. Schematic phigh-redox potential laccases (HRPL). Adopted from Mate et al. (2010).Technology 176 (2015) 273283 277Zhang et al. (2010) used a combinatorial approach to alter ther-mostability of the xylanase XT6 of Geobacillus stearothermophilus.The sequence family shufing along with the consensus basedsemi-rational approach was employed in mutagenesis. The opti-mum temperature of the mutated xylanase was 10 C higher thanthe native xylanase. The mutated enzyme differed in 13 aa moie-ties from the wild type that exhibited 52-fold improvement inhalf-life and 80% enhancement in catalytic efciency. These muta-tions possibly generated additional hydrophobic interaction andsalt bridges resulting in increased thermostability of the xylanase.Chen et al. (2012) used SDM to modify the xylanase (XynR8)obtained from unpuried rumen fungal cultures. Mutations N41Dand N58D improved thermostability and kinetic parameters (Vmax,Kcat and Km) of XynR8. Mutations N41D and N58D possibly led tohydrogen bond formation and increased rigidity and stability ofthe catalytic cleft which in turn improved overall performance ofthe enzyme (Chen et al., 2012). The self priming polymerase chainreaction (Table S1) was used for shufing different modulesbetween Cex xylanase of Cellulomonas mi and XylA xylanase ofThermomonospora alba (Wang and Xia, 2007). The Cex has opti-mum activity at 40 C, while XylA at 80 C. After module shufing,the resultant chimeric xylanase CXC-X4,5 exhibited a markedincrease in its thermal prole and attained optimum activity at65 C, a 25 C rise in optimum temperature. CXC-X4,5 receivedmodule 4 and module 5 from XylA that conrmed the thermo-stabilizing role of these modules, and therefore, these modulescan further be utilized to alter thermal prole of other lessthermostable benecial xylanases (Wang and Xia, 2007).

resentation of articial evolution pathway used to introduce benecial mutations in

regg ov

urceFig. 3. The domain shufing strategy for constructing the chimeric phytase. Sevenamplications including formation of heteroduplex and homoduplex molecules durinAfp gene, while open bars show Anp gene. Source Bei et al. (2009).

278 S. Joshi, T. Satyanarayana / Bioreso2.5. Mutations in lipases

Lipases are used in multiple industrial applications rangingfrom fat and oil processing, synthesis of pharmaceuticals and nechemicals, production of cosmetics to paper manufacture and foodprocessing. Kumari and Gupta (2013) improved catalytic efciencyof Yarrowia lipolytica lipase 12 by a single mutation Phe148Leu.Phe 148 is a component of the oxyanion hole which plays a crucialrole in the stabilization of substrate-enzyme complex. The substi-tution of Phe 148 with Leu enhanced binding and catalytic ef-ciency of Lip12. Ema et al. (2005) used SDM based onmechanistic details of the enzyme, which altered the biochemicalproperty of Burkholderia cepacia lipase. This study suggested thatthe enantioselectivity of the lipases can be altered by a singlemutation in the proximity of the enzyme active site. An increasein enantioselectivity of the B. cepacia lipase for secondary alcoholswas achieved by a point mutation. Engstrom et al. (2010) reversedthe enantioselectivity of Candida antarctica lipase A (CalA) bymutagenesis. The (R)-selectivity muteins were generated from(S)-selectivity of the wild type lipase by using combinatorial activesite saturation test (CSAT) approach (Table S1). The muteins F233Gand Phe149Tyr/Ile150Asn/Phe233Gly (YNG) proved very promis-ing and exhibited large improvement in enantiomeric ratio(E value) for seven different esters as compared to the wild type(Engstrom et al., 2010). Not every study results in a successfuloutcome but may provide some insight and future directions toachieve the desired goals. The mutations W115Q and M72S causeda steep decline in the specic activity of lipase B of C. antarctica(CalB), but the mutation M83I slightly increased thermostabilityas compared to the wild type CalB; this gave some clue to theresearchers for further work on thermostabilization and peroxidestability of CalB (Irani et al., 2013).

Modelling and docking techniques have revolutionized directedmutagenesis as these provide researchers a bypass from theions along with conserved regions are present in both the genes. Sequential PCRerlapping PCR were performed in order to obtain chimeric ORFs. Hatched bars show

Technology 176 (2015) 273283screening of thousands of colonies for the desired change by muta-genesis. With the help of prior in silico analysis of binding energiesof wild type lipase and mutant lipase, directed mutations werecarried out to alter Rhizomucor miehei lipase for improving the per-formance in producing human milk fat substitute (Zhang et al.,2013). Among tailored muteins, Asp256Ile/His257Leu displayed1.82-fold improvement in oleic acid incorporation for catalysis oftripalmitin and oleic acid. Dror et al. (2014) enhanced the stabilityof G. stearothermophilus lipase T6 by using two approaches in par-allel: ep-PCR and structure guided consensus methodology(Table S1). The muteins exhibited an increased half-life in 70%methanol. It was suggested that Q185L assists the close lid confor-mation of lipase, which could in turn limit the exposure of theactive site to increased methanol concentration. The increasedstability due to H86Y and A269T was attributed to the formationof new hydrogen bonds.

2.6. Alterations in chitinases

Chitin hydrolyzing enzymes have been employed in a widerange of applications including synthesis of chitooligosaccharidesand N-acetyl D-glucosamine, single-cell protein (SCP), fungal andyeast protoplast isolation, biocontrol of pathogenic fungi, treatmentof chitinous waste and prevention of malaria transmission (Vahedet al., 2013). In search of an ideal chitinase, biotechnologists havetailored various microbial chitinases. An aa residue was identiedin the chitin binding domain of chitinase A1 from Bacillus circulansWL-12 and substituted it with different aa residues (Hara et al.,2013). Besides Trp687, the residue Gln679 was also identied tobe involved in chitin binding, while Q679A showed very poor chitinbinding. To decipher the role of catalytically important residues ofBacillus thuringiensis WB7 chitinase, Chai et al. (2009) generatedtwelve muteins of WB7 chitinase. Among them, muteins F201L,G203D, D205N, D207E, D207N, W208C and E209D were inactive

urceS. Joshi, T. Satyanarayana / Bioresoshowing signicance of these aa residues for enzymatic activity ofchitinase. The mutation G203A lowered the optimum temperatureby 10 C, indicating the importance of G203 for higher Topt(Chai et al., 2009). Vahed et al. (2013) used a different approachfor improving chitinolytic activity of the chitinase from Bacilluspumilus SG2. Mutagenesis by UV irradiation and nitrous acid treat-ments increased chitinolytic activity of B. pumilus SG2. To decipherthe changes occurred at molecular level, whole operon that con-tained two chitinase genes ChiL and ChiS ORFs was sequenced.The sequencing of mutated operon revealed that the increasedchitinolytic activity was due to single nucleotide change (G? A)in ChiLI gene which changed glutamate for glycine in the catalyticregion. Boer et al. (2007) used mutagenesis to conrm the role oftwo carboxylic acid residues E172 and D170 which are putativeproton donor and stabilizer in the reaction mechanism of GH-18family chitinase (Chit42) of Trichoderma harzianum. In an attemptto alter transglycosylation properties of Serratia marcescenschitinase, Aronson et al. (2006) modied tryptophan residue(Trp 167) present at 3 subsite. The mutein thus generated wasable to form higher oligosaccharides from tetra- and penta-saccharides, which could be due to the increased retention timeof glycosyl fragment at the active site.

Fig. 4. Monomerization of Gt-Mamy by mutagenesis. Schematic presentation of chain Asubstrate entry site. Important aa residues essential for binding of smaller substrate (b-cposition. Solid arrows represent entry and exit of high afnity substrates, while dotted arTechnology 176 (2015) 273283 2792.7. Mutagenesis studies on proteases

Proteases share approximately 60% of the total enzyme marketin various industries (Joshi and Satyanarayana, 2013). Proteaseshave been used since long for multifarious applications rangingfrom detergent additive to pharmaceuticals. Protein engineeringhas also been exploited as a powerful tool to tailor this remarkableclass of enzymes. Rajput et al. (2012) replaced prosequence of theB. licheniformis keratinase (keratin specic protease) with that ofprosequence of B. subtilis keratinase and reported that the resultingchimeric enzyme revealed better attributes than the native prote-ase. It was found that the novel chimeric protein Ker ProBPBL hast1/2 of 45 min at 80 C whereas native B. licheniformis protease wasunstable beyond 60 C. The chimeric keratinase also showedimprovement in K:C ratio (activity on keratin and casein sub-strate). Strausberg et al. (1995) reported that the removal of cal-cium binding loop from subtilisin BPN resulted in thedestabilization of protease (Strausberg et al., 1995). Later Straus-berg group used directed mutagenesis and screened a subtilisinwhich was stable and active even in strong chelating environment.A mutein that exhibited 1000-times higher stability in such envi-ronment was generated healing the Achilles heel (Strausberg

and B of dimeric Gt-Mamy of G. thermoleovorans. Circles show aa moieties at theyclodextrin) both in chain B and A have been depicted by their symbols followed byrows are for low afnity substrates. Adopted from Mehta and Satyanarayana (2013).

urceet al., 1995). A pharmaceutically important NS3 protease of theDengue virus type2 had also been mutated (Valle and Falgout,1998). In this study, 46 single aa substitutions were made andthe effect of each substitution on self cleavage of NS2B-NS3 wasstudied. It was revealed that the substitution of ultra conservedresidues resulted in the loss of cleavage activity. The replacementof the conserved residue with residue that is conserved in otheravivirus proteases did not restore the cleavage activity (Valleand Falgout, 1998).

2.8. SDM of pectinases

This group of enzymes encompasses a diverse and complexpopulation of hydrolases involved in degradation of pecticsubstances. Primarily saprophytes and phytopathogens producepectinases for penetration through the plant cell wall. Bioscouringpotential of all three classes of pectinases [pectin esterases (PEs),polygalacturonases (PGs) and polygalacturonate lyases (PLs)] isused for various purposes (Sharma et al., 2013). Despite the devel-opment of efcient bioscouring processes, there is a persistingdemand for an ideal thermo-alkali-stable pectinase in the market.In an attempt to improve biotechnologically important propertiesof pectate lyase, Solbak et al. (2005) used directed mutagenesisapproach and developed a novel pectate lyase that contained eightpoint mutations at Ala118, Ttyr190, Ala 197, Ser208, Ser263,Asn275, Tyr309 and Ser312 positions. The mutated pectate lyaseexhibited 16 C higher melting temperature as compared to thenative pectate lyase. It was also observed that a lower enzymedosage was required to perform bioscouring at elevated tempera-tures. While Xiao et al. (2008) used melting temperature guidedalignment technique (Table S1) to carry out SDM to improve theproperties of pectate lyase from Xanthomonas campestris. In thisstudy, a 6 C increase in melting temperature of the enzyme wasattained by a point mutation (R236F) without any change incatalytic efciency of the enzyme. Concomitantly a 23-foldincrease in t1/2 of the enzyme was achieved at 45 C.

2.9. In vitro tailoring of cellulases

Cellulose hydrolysing enzymesmake amulti-component systemthat functions synergistically. The components of cellulose hydro-lytic machinery include at least three types of cellulases e.g. endo-glucanases or endocellulases, cellobiohydrolases or exocellulasesand b-glucosidases (Voutilainen et al., 2009). Most of the fungalcellulases are active at acidic pH that limits their use in neutraland alkaline conditions. Therefore, most of the protein engineeringresearches in cellulases have been directed towards improving thecatalytic efciency of these enzymes at higher pH. Voutilainenet al. (2009) employed a dual approach for altering the thermosta-bility and activity of GH-7 family cellulase ofMelanocarpus albomy-ces Cel7B (Ma Cel7B). An additional disulphide bond (G4C/M70C)was introduced close to the active site groove of Ma Cel7Balong with a known thermostabilizing mutation (S290T). Thecombination of the mutations resulted in 4 C increase in unfoldingtemperature (temperature at which initiation in the disassembly ofnon-covalent linkages takes place). The incorporation of a cellulosebinding module (CBM) along with a linker from Trichoderma reeseiCel7A at the C-terminus improved both thermostability and activityofMaCel7B. Ni et al. (2010) studied the effect of a few aamoieties onthe activity of endoglucanase from a termite Reticulitermes speratus(RsEG). Muteins G91A, Y97W and K429A were generated usingSDM, and a synergistic effect was observed on the activity of themuteins as double and triplemuteinsweremore active than the sin-

280 S. Joshi, T. Satyanarayana / Bioresogle muteins. The increased activity possibly resulted from reducedaggregation, proper folding and increased solubility of the muteins.Recently Ortiz et al. (2013) achieved improvement in insolublecellulose hydrolysing capacity of two thermophilic endoglucanasesby joining a carbohydrate binding domain from the family 2a tocatalytic domain of the endoglucanases. The catalytic domainsof Cel9A from Alicyclobacillus acidocaldarius and Cel5A fromT. maritimawere joined to the family 2a CBD. The resultant chimericendoglucanase exhibited 3-fold higher cellulose hydrolysing abilitythan its wild type counterpart (Ortiz et al., 2013). Hahn and Kim(2012) used error-prone rolling circle amplication (Table S1)to get Bacillus amyloliquefaciens endoglucanase muteins, whichdisplayed enhanced activity. The mutation responsible for thisenhancement in the enzyme activity was traced to E289V. Changet al. (2012) reported a key mutation Y61G in a hyperthermostableb-1,4-endoglucanase from T. maritima that led to the generation of amutein with increased catalytic activity without a loss in thermo-stability. This was attributed to a change in facile dissociation ofcleaved sugar residue at the reducing end.

2.10. Mutagenesis in DNA polymerases

The recombinant DNA technology (RDT) would not havereached where it is today, in case DNA polymerases would havefailed to amplify DNA with high processivity and delity. Withthe discovery of DNA polymerases active at high temperature,there has been a revolutionary change in the biotech industry. Inview of the importance of DNA polymerases, tremendous researchefforts are being made to nd out the role of DNA polymerases inpathology of certain diseases along with the development of DNApolymerases with novel and improved properties for their applica-tion as RDT workhorses. There are many reports on diseases causeddue to mutations in DNA polymerases but the number of deliberatemutations is not as many. Vega et al. (2010) coupled a DNA bindingdomain to the C-terminus of the bacteriophage u29 DNA polymer-ase, a DNA polymerase capable of isothermal DNA amplicationwith high processivity and delity. It was observed that the addi-tion of helix-hairpin-helix domain increases the DNA binding ef-ciency of the hybrid u29 DNA polymerase as compared to its wildtype counterpart, without compromising the replication rate. Thisstudy also resulted in improved amplication prociency of u29DNA polymerase that can be utilized as a powerful tool in genom-ics. DNA polymerases are also known for their faithful recognitionof their substrates, and thus, used with exceptionally high specic-ity. On the contrary, Ghadessy et al. (2004) developed a DNA poly-merase which has a very broad substrate spectrum and usesunnatural nucleotides. This new polymerase extends mismatchespromiscuously while maintaining high turnover and bypassesblocking lesions such as thymidine dimers, abasic sites and thebase analog 5-nitroindole. Fa et al. (2004) utilized directed evolu-tion to generate DNA polymerase capable of incorporating unnatu-ral substrates during DNA replication. The phage based selectionsystem was used for screening a library of 6 107 members. Themuteins SfM18 (I614Y:E615G), SfM19 (I614E:E615G) and SfM30(I614E:E615A) have been screened for their ability to incorporate20-O-methylribo-nucleoside triphosphates (20-O-meNTP) oppositea DNA template. Among these muteins, SfM19 was 1000 timesmore efcient in incorporating 20-O-meNTP than the wild typepolymerase. Ong et al. (2006) developed a variant of DNA polymer-ase I from Thermus aquaticus that catalyzed reactions as DNA poly-merase, RNA polymerase and reverse transcriptase. A polypeptidebehaving as three different enzymes was generated by spCSR,where only a short region under investigation was diversiedand replicated. Two denite motifs of the DNA polymerase I havebeen involved in substrate recognition in the active site for direc-ted modication (Ong et al., 2006). This novel molecule has paved

Technology 176 (2015) 273283the way for the use of spCSR technique for generating other mole-cules for biotechnological applications and enzymatic synthesis oftailored nucleic acids.

an indirect involvement of cystein residue (Cys 295) and directinvolvement of a histidine residue (His261) in coordinating nickel

ment) with a leucine (L). An amylase used in the detergent industrymust possess oxidation resistance. Attempts have been made to

urcein the C-cluster of CODH II of Carboxydothermus hydrogenoformanshas been conrmed through mutagenesis studies by a Japanesegroup of researchers (Inoue et al., 2013).

In one study, starch debranching enzyme, pullulanase fromBacillus deramicans was subjected to SDM based on the structureguided consensus approach. The half-lives of muteins D503F,D437H and D503Y were 2-fold higher at 60 C than the nativepullulanase. A double mutant D437H/D503Y exhibited a largerchange in thermostability than single mutants (Duan et al.,2013). Pei et al. (2011) generated a thermostable variant ofb-glucosidase (VM2) employing a combinatorial approach ofmutagenesis. The mutein VM2 exhibited mutations at three keysites (L444Y, G447S and A433V). Apart from higher thermostability(144-fold improved half-life than the parent counterpart BglC),VM2 exhibited improvement in catalytic efciency bothagainst p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose(Pei et al., 2011).

Han et al. (2014) showed as to how glycosylation can alter theenzymatic properties by incorporating three additional N-glycosyl-ation sites in the elastase from Pseudomonas aeruginosa. Amongthree point mutations (I38T, A69T and N266T) in glycosylationsites at N36, N67 and N264 positions, I38T mutation yielded amutein with higher rate of peptide synthesis in 50% dimethyl-sulphoxide. In aqueous medium, I38T mutation decreased2.11. Redesigning of restriction endonucleases

To cut target DNA sequence at the desired site, all molecularbiotechnologists bank upon the wonderful molecular scissorsknown as restriction endonucleases (REases). Restriction enzymescatalyze hydrolysis of the phosphodiester bond in a site specicmanner yielding 30-hydroxyl and 50-phosphoryl termini. New factsabout these enzymes have come into light by mutagenesis studieson REases. It is very well known that most of the REases requireMg2+ for catalysis and inhibited by the presence of Ca2+. An excep-tion to this, R.KpnI exhibits high delity catalysis in the presence ofCa2+. It also shows complete inhibition of promiscuous cleavageinduced by Mg2+. It has been shown that the insertional mutationleads to the disruption of spatial arrangement of ExDxD and HNHmotifs (which are involved in Ca2+ co-ordination and nucleophilicactivation of R.KpnI) and impairs both Ca2+intranetbinding andDNA cleavage activity (Nagamalleswari et al., 2012). Recently, bya single mutation (D148E) in KpnI, Vasu et al. (2013) eliminatedthe property of Ca2+ mediated cleavage by KpnI. With this change,a high delity with Mg2+ was imparted to this REase. It was provedby biophysical methods that these mutations lead to changes ingeometry of Ca2+ binding active site. Promiscuous activity of KpnIwas found to be related to the plasticity of active site in coordinat-ing different metal ions.

2.12. Other enzymes

There are various other important enzymes which have not yetbeen explored by in vitro methods. Among these, carbonic anhyd-rases (CAs), carbon monoxide dehydrogenases (CODHs), b-glucosi-dases and b-fructofuranosidases are a few to name. CODHs belongto biotechnologically signicant group of enzymes as they ndapplication in cost effective production of hydrogen which itselfis a potential clean future fuel and an efcient electron donorfor reductive processes such as biodesulfurization (Sipma et al.,2006). Reports on mutations in CODH are very scanty. Recently,

S. Joshi, T. Satyanarayana / Bioresothermostability of the elastase, while N226T increased it. Theintroduction of glycosylation site at N36 was, therefore, favourablein terms of industrial application of the elastase.substitute oxidation prone methionine and cysteine residues tomeet this requirement. Purastar OxAm and Duramyl areengineered amylases from Genencor Intl. which show oxidationstability (Araujo et al., 2010).

A phytase (Phy9X) from Escherichia coli, which is sold under thebrand name Quantum Phytase by Syngenta Animal NutritionInc., is another success story of the protein engineering (Garrettet al., 2004). The gene site saturation technique was employedfor introducing benecial changes in Phy9X. The mutein Phy9Xaccumulated mutations at 8 sites (Q84W, Y277D, W68E, K97C,R181Y, N226C, A95P and S168E). The mutation Q84W contributedKoyama et al. (2013) improved the thermostability of thegalacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) by randommutagenesis. GLNBP is used for industrial production of lacto-N-biose I. Among ve muteins generated by ep-PCR, C236Y andD576V exhibited slight improvement in the thermostability. Adouble mutant C236Y/D576V exhibited 20 C higher thermostabil-ity than the native enzyme. In addition to the formation of a newhydrogen bond between phenolic oxygen of Y236 and E319, themutation C236Y provides a bulky hydrophobic side chain at posi-tion 236, which in turn lls solvent accessible cavity leading toenhancement in thermostability of the enzyme.

2.13. Engineered microbial enzymes in the market

Global industrial enzyme sales will cross US $7.1 billion by 2018(http://www.bccresearch.com/market-research/biotechnology/enzymes-industrial-applications-bio030h.html, last accessed onAugust 28th, 2014). The engineered restriction endonucleasesexhibiting high substrate specicity, low star activity and highdelity have already made their way to the market. In 1995,Gist-Brocades introduced an engineered lipase with the brandname Lipomax. This is a variant of Pseudomonas alcaligenes lipasedeveloped by protein engineering (Misset, 1997). Lipomax

(M21L mutein) is insensitive to anionic surfactants and exhibitsbetter wash performance than its native counterpart. Lipolaseultra is oxidation resistant lipase derived from that of Humicolalanuginosa. Lipolase ultra is an advanced mutein of Lipolase(Novo Nordisk) in which negatively charged aa residues in the lipidcontact zone of the enzyme have been replaced with the positivelycharged and hydrophobic aa residues in addition to D96L muta-tion. These changes improved both surface activity of the lipaseand better performance in the presence of surfactants (Misset,1997). Among detergent proteases, primarily subtilisins (serineproteases) from Bacillus have been commercialized. These enzymessuffer from sensitivity to oxidizing agents such as H2O2. Theprimary cause of this sensitivity is the presence of oxidationsensitivity of the surface exposed methionine residues whichresults in decreased efciency of the proteases in the presence ofoxidizing agents found in various commercial bleaches. Theengineered oxidation resistant variants of subtilisin with the tradename Maxapam (Gist Brocades) and Everlase (Novozyme) arenow in the market (Walsh, 2006). Similarly subtilisin BPN, anengineered industrial protease (with Y217L mutation) derivedfrom B. amyloliquefaciens has made entry to the market and is soldunder the brand name Purafect Prime (http://www.google.nl/patents/WO2010088161A1?cl=en, last accessed on August 28th,2014). Purafect Prime exhibits alkali stability because of thereplacement of Y217 (positioned close to the substrate bindingregion of the enzyme and prone to ionization in alkaline environ-

Technology 176 (2015) 273283 281signicantly to enhance thermostability of the Phy9X due to 3 basechanges. Many more engineered proteins are expected to reach themarket in the near future.

applications. In: Nierstrasz, V., Cavaco-Paulo, A. (Eds.), Advances in TextileBiotechnology. Woodhead Publishing Ltd., Cambridge, pp. 331.

Bei, J., Chen, Z., Fua, J., Jiang, Z., Wang, J., Wang, X., 2009. Structure-based fragmentshufing of two fungal phytases for combination of desirable properties. J.

Chang, Y.S., Ko, T.P., Huang, J.W., Wu, T.H., Lin, C.Y., Luo, W., Li, Q., Ma, Y., Huang,

urceC.H., Wang, A.H., Liu, J.R., Guo, R.T., 2012. Enhanced activity of Thermotogamaritima cellulase 12A by mutating a unique surface loop. Appl. Microbiol.Biotechnol. 95, 661669.

Chen, X., Xu, S., Zhu, M., Cui, L., Zhu, H., Liang, Y., Zhang, Z., 2010. Site-directedmutagenesis of an Aspergillus niger xylanase B and its expression, puricationand enzymatic characterization in Pichia pastoris. Process Biochem. 45, 7580.

Chen, Y.C., Chiang, Y.C., Hsu, F.Y., Tsai, L.C., Cheng, H.L., 2012. Structural modelingand further improvement in pH stability and activity of a highly-active xylanasefrom an uncultured rumen fungus. Bioresour. Technol. 123, 125134.

Chiang, L.W., Kovari, I., Howe, M.M., 1993. Mutagenic oligonucleotide-directed PCRamplication (Mod-PCR): an efcient method for generating random basesubstitution mutations in a DNA sequence element. PCR Methods Appl. 2, 210217.

Coco, W.M., Levinson, W.E., Crist, M.J., Hektor, H.J., Darzins, A., Pienkos, P.T., Squires,C.H., Monticello, D.J., 2001. DNA shufing method for generating highlyrecombined genes and evolved enzymes. Nat. Biotechnol. 19, 354359.

Dennig, A., Shivange, A.V., Marienhagen, J., Schwaneberg, U., 2011. OmniChange:the sequence independent method for simultaneous site-saturation of vecodons. PLoS One 6, e26222.

Dror, A., Shemesh, E., Dayan, N., Fishman, A., 2014. Protein engineering by randomBiotechnol. 139, 86193.Boer, H., Simolin, H., Cottaz, S., Soderlund, H., Koivula, A., 2007. Heterologous

expression and site-directed mutagenesis studies of two Trichoderma harzianumchitinases, Chit33 and Chit42, in Escherichia coli. Protein Expr. Purif. 51, 216226.

Chai, W., Sha, L., Zhou, J., Huang, Z., Guan, X., 2009. Functional analysis of active siteresidues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis.World J. Microbiol. Biotechnol. 25, 21472155.Aronson, N.N., Halloran, B.A., Alexeyev, M.F., Zhou, X.E., Wang, Y., Meehan, E.J., 2006.Mutation of a conserved tryptophan in the chitin binding cleft of Serratiamarcescens chitinase A enhances transglycosylation. Biosci. Biotechnol.Biochem. 70, 243251.

Autore, F., Vecchio, C.D., Fraternali, F., Giardina, P., Sannia, G., Faraco, V., 2009.Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase:analysis by site-directed mutagenesis. Enzyme Microb. Technol. 45, 507513.3. Conclusions

Protein engineering is an exponentially growing eld of molec-ular biology, where several developments have taken place inin vitro manipulations of microbial enzymes by rational, directedand combinatorial approaches. By these approaches, the desiredchanges can be brought into the biocatalysts in a shorter time. Asthere is no universal strategy to alter an enzyme, multipleapproaches have to be considered to develop more sophisticatedand selective strategies to mimic natures evolutionary processesfor introducing process-specic properties in industrial biocata-lysts. These techniques can signicantly contribute to the develop-ment of biocatalysts with novel desired characteristics in order tosuit the diverse biotechnological applications.

Acknowledgements

We gratefully acknowledge nancial assistance from theUniversity Grants Commission (UGC) and Council of Scientic &Industrial Research (CSIR), Govt. of India, New Delhi, while writingthis review.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.10.151.

References

Araujo, R., Casal, M., Cavaco-Paulo, A., 2010. Part I Technologies involved in textilebiotechnology. Design and engineering of novel enzymes for textile

282 S. Joshi, T. Satyanarayana / Bioresomutagenesis and structure guided consensus of Geobacillus stearothermophiluslipase T6 for enhanced stability in methanol. Appl. Environ. Microbiol. 80,15151527.Duan, X., Chen, J., Wu, J., 2013. Improving the thermostability and catalyticefciency of Bacillus deramicans pullulanase by site-directed mutagenesis.Appl. Environ. Microbiol. 79, 40724077.

Ema, T., Fujii, T., Ozaki, M., Korenaga, T., Sakai, T., 2005. Rational control ofenantioselectivity of lipase by site-directed mutagenesis based on themechanism. Chem. Commun. 37, 46504651.

Engstrom, K., Nyhlen, J., Sandstrom, A.G., Backvall, J.E., 2010. Directed evolution ofan enantioselective lipase with broad substrate scope for hydrolysis of r-substituted esters. J. Am. Chem. Soc. 132, 70387042.

Fa, M., Radeghieri, A., Henry, A., Romesberg, F., 2004. Expanding the substraterepertoire of a DNA polymerase by directed evolution. J. Am. Chem. Soc. 126,17481754.

Farhat-Khemakhem, A., Ali, M.B., Boukhris, I., Khemakhem, B., Maguin, E., Bejar, S.,Chouayekh, H., 2013. Crucial role of Pro 257 in the thermostability of Bacillusphytases: biochemical and structural investigation. Int. J. Biol. Macromol. 54, 915.

Garrett, J.B., Kretz, K.A., ODonoghue, E., Kerovuo, J., Kim, W., Barton, N.R.,Hazlewood, G.P., Short, J.M., Robertson, D.E., Gray, K.A., 2004. Enhancing thethermal tolerance and gastric performance of a microbial phytase for use as aphosphate-mobilizing monogastric-feed supplement. Appl. Environ. Microbiol.70, 30413046.

Ghadessy, F.J., Ramsay, N., Boudsocq, F., Loakes, D., Brown, A., Iwai, S., Vaisman, A.,Woodgate, R., Holliger, P., 2004. Generic expansion of the substrate spectrum ofa DNA polymerase by directed evolution. Nat. Biotechnol. 22, 755759.

Gupta, N., Farinas, E.T., 2010. Directed evolution of CotA laccase for increased substratespecicity using Bacillus subtilis spores. Protein Eng. Des. Sel. 23, 679682.

Hahn, V.V., Kim, K., 2012. Improvement of cellulase activity using error-pronerolling circle amplication and site-directed mutagenesis. J. Microbiol.Biotechnol. 22, 607613.

Han, M., Wanga, X., Yana, G., Wang, W., Taoa, Y., Liua, X., Caoa, H., Yu, X., 2014.Modication of recombinant elastase expressed in Pichia pastoris byintroduction of N-glycosylation sites. J. Biotechnol. 171, 37.

Hara, M., Sugimoto, H., Uemura, M., Akagi, K.I., Suzuki, K., Ikegami, T., Watanabe, T.,2013. Involvement of Gln679, in addition to Trp687, in chitin-binding activity ofthe chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J.Biochem. 154, 185193.

Hokanson, C.A., Cappuccilli, G., Odineca, T., Bozic, M., Behnke, C.A., Mendez, M.,Coleman, W.J., Crea, R., 2011. Engineering highly thermostable xylanasevariants using an enhanced combinatorial library method. Protein Eng. Des.Sel. 24, 597605.

Inoue, T., Takao, K., Yoshida, T., Wada, K., Daifuku, T., Yoneda, Y., Fukuyama, K., Sako,Y., 2013. Cysteine 295 indirectly affects Ni coordination of carbon monoxidedehydrogenase-II C-cluster. Biochem. Biophys. Res. Commun. 441, 1317.

Irani, M., Tornavall, U., Genheden, S., Larsen, M.W., Kaul, R.H., Ryde, U., 2013. Aminoacid oxidation of Candida antarctica lipase B studied by molecular dynamics andsite directed mutagenesis. Biochemistry 52, 12801289.

Joo, J.C., Pack, S.P., Kimc, Y.H., Yooa, Y.J., 2011. Thermostabilization of Bacilluscirculans xylanase: computational optimization of unstable residues based onthermal uctuation analysis. J. Biotechnol. 151, 5665.

Joshi, S., Satyanarayana, T., 2013. Biotechnology of cold-active proteases. Biology 2,755783.

Kachan, A.V., Evtushenkov, A.N., 2013. Thermostable mutant variants of Bacillussp.406 a-amylase generated by site-directed mutagenesis. Cent. Eur. J. Biol. 8,346356.

Kegler-Ebo, D.M., Docktor, C.M., DiMaio, D., 1994. Codon cassette mutagenesis: ageneral method to insert or replace individual codons by using universalmutagenic cassettes. Nucleic Acids Res. 22, 15931599.

Kim, T., Mullaney, E.J., Porres, J.M., Roneker, K.R., Crowe, S., Rice, S., et al., 2006.Shifting the pH prole of Aspergillus niger PhyA phytase to match the stomachpH enhances its effectiveness as an animal feed additive. Appl. Environ.Microbiol. 72, 43974403.

Koschorreck, K., Schmid, R.D., Urlacher, V.B., 2009. Improving the functionalexpression of a Bacillus licheniformis laccase by random and site-directedmutagenesis. BMC Biotechnol. 9, 12.

Koyama, Y., Hidaka, M., Nishimoto, M., Kitaoka, M., 2013. Directed evolution toenhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase.Protein Eng. Des. Sel. 26, 755761.

Kumari, A., Gupta, R., 2013. Phenylalanine to leucine point mutation in oxyanion 1hole improved catalytic efciency of Lip12 from Yarrowia lipolytica. EnzymeMicrob. Technol. 53, 386390.

Le, Q.A., Joo, J.C., Yoo, Y.J., Kim, Y.H., 2012. Development of thermostable CandidaAntarctica lipase B through novel in silico design of disulde bridge. Biotechnol.Bioeng. 109, 867876.

Mabrouk, S.B., Ayadi-Zouari, D., Hlima, H.B., Bejar, S., 2013. Changes in the catalyticproperties and substrate specicity of Bacillus sp. US149 maltogenic amylase bymutagenesis of residue 46. J. Ind. Microbiol. Biotechnol. 40, 947953.

Mate, D., Garcia-Burgos, C., Garcia-Ruiz, E., Ballesteros, A., Camarero, S., Alcalde, M.,2010. Laboratory evolution of high-redox potential laccases. Chem. Biol. 17,10301041.

Mehta, D., Satyanarayana, T., 2013. Dimerization mediates thermo-adaptation,substrate afnity and transglycosylation in a highly thermostable maltogenicamylase of Geobacillus thermoleovorans. PLoS One 8, e73612.

Misset, O., 1997. Developments of new lipases. In: Ee, J.H., Misset, O., Bass, E.J. (Eds.),Enzymes in Detergency. Marcel Dekker Inc., New York, pp. 107133.

Mullaney, M., Sethumadhavan, K., Boone, S., Lei, X.G., Ullah, A.H.J., 2012. Elimination

Technology 176 (2015) 273283of a disulde bridge in Aspergillus niger NRRL 3135 phytase (PhyA) enhancesheat tolerance and optimizes its temperature versus activity prole. Adv. Biol.Chem. 2, 372378.

Nagamalleswari, E., Vasu, K., Nagaraja, V., 2012. Ca2+ binding to the ExDxD motifregulate the DNA cleavage specicity of a promiscuous endonuclease.Biochemistry 51, 89398949.

Ni, J., Takehara, M., Watanabe, H., 2010. Identication of activity related amino acidmutations of a GH9 termite cellulase. Bioresour. Technol. 101, 64386443.

Ong, J.L., Loakes, D., Too, S.J.K., Holliger, P., 2006. Directed evolution of DNApolymerase, RNA polymerase and reverse transcriptase activity in a singlepolypeptide. J. Mol. Biol. 361, 537550.

Ortiz, V.R., Heins, R.A., Cheng, G., Kim, E.Y., Vernon, B.C., Elandt, R.B., Adams, P.D.,Sale, K.L., Hadi, M.Z., Simmons, B.A., Kent, M.S., Tullman-Ercek, D., 2013.Addition of a carbohydrate-binding module enhances cellulase penetration intocellulose substrates. Biotechnol. Biofuels 6, 93.

Ozturk, H., Ece, S., Gundeger, S., Evran, S., 2013. Site-directed mutagenesis ofmethionine residues for improving the oxidative stability of a-amylase fromThermotoga maritima. J. Biosci. Bioeng. 116, 449451.

Paes, G., ODonohue, M.J., 2006. Engineering increased thermostability in thethermostable GH-11 xylanase from Thermobacillus xylanilyticus. J. Biotechnol.125, 338350.

Pei, X.Q., Yi, Z.L., Tang, C.G., Wu, Z.L., 2011. Three amino acid changes contributemarkedly to the thermostability b-glucosidase BglC from Thermobida fusca.Bioresour. Technol. 102, 33373342.

Rahimzadeh, M., Khajeha, K., Mirshahia, M., Khayatianb, M., Schwarzenbacherc, R.,2012. Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 a-amylase. Int. J. Biol. Macromol. 50, 11751182.

Rajput, R., Tiwary, E., Sharma, R., Gupta, R., 2012. Swapping of pro-sequencesbetween keratinases of Bacillus licheniformis and Bacillus pumilus: alteredsubstrate specicity and thermostability. Enzyme Microb. Technol. 51,131138.

Rha, E., Kim, S., Choi, S.L., Hong, S.P., Sung, M.H., Song, J.J., Lee, S.G., 2009.Simultaneous improvement of catalytic activity and thermal stability oftyrosine phenol-lyase by directed evolution. FEBS J. 276, 61876194.

Sun, J., Wang, H., Lv, W., Ma, C., Lou, Z., Dai, Y., 2011. Construction andcharacterization of a fusion b-1,31,4-glucanase to improve hydrolyticactivity and thermostability. Biotechnol. Lett. 33, 21932199.

Tian, Y.S., Peng, R.H., Xu, J., Zhao, W., Gao, F., Fu, X.Y., Xiong, A.S., Yao, Q.H., 2011.Mutations in two amino acids in phyI1s from Aspergillus niger 113 improve itsphytase activity. Mol. Biol. Rep. 38, 977982.

Vahed, M., Motalebi, E., Rigi, G., Noghabi, K.A., Soudi, M.R., Sadeghi, M., et al., 2013.Improving chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.J. Microbiol. Biotechnol. 23, 15191528.

Valle, R.P.C., Falgout, B., 1998. Mutagenesis of the NS3 protease of dengue virus type2. J. Virol. 72, 624632.

Vasu, K., Nagamalleswari, E., Zahran, M., Imhof, P., Xu, S.Y., Zhu, Z., Chan, S.H.,Nagaraja, V., 2013. Increasing cleavage specicity and activity of restrictionendonuclease KpnI. Nucleic Acids Res. 41, 98129824.

Vega, M.D., Lazaro, J.M., Mencia, M., Blanco, L., Salas, M., 2010. Improvement of u29DNA polymerase amplication performance by fusion of DNA binding motifs.Proc. Natl. Acad. Sci. U.S.A. 107, 1650616511.

Verma, D., Satyanarayana, T., 2012. Molecular approaches for amelioratingmicrobial xylanases. Bioresour. Technol. 117, 360367.

Verma, D., Satyanarayana, T., 2013. Improvement in thermostability ofmetagenomic GH11 endoxylanase (Mxyl) by site-directed mutagenesis and itsapplicability in paper pulp bleaching process. J. Ind. Microbiol. Biotechnol. 40,13731381.

Voutilainen, S.P., Boer, H., Alapuranen, M., Janis, J., Vehmaanpera, J., Koivula, A.,2009. Improving the thermostability and activity of Melanocarpus albomycescellobiohydrolase Cel7B. Appl. Microbiol. Biotechnol. 83, 261272.

Walsh, G., 2006. Protein engineering: case studies of commercialized engineeredproducts. Enzyme Microb. Technol. 39, 235251.

Wang, C., Huang, R., He, B., Du, Q., 2012. Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis. BMC

S. Joshi, T. Satyanarayana / Bioresource Technology 176 (2015) 273283 283Sharma, N., Rathore, M., Sharma, M., 2013. Microbial pectinase: sources,characterization and applications. Rev. Environ. Sci. Biotechnol. 12, 4560.

Sherif, M., Waung, D., Korbeci, B., Mavisakalyan, V., Flick, R., Brown, G., et al., 2013.Biochemical studies of the multicopper oxidase (small. laccase) fromStreptomyces coelicolor using bioactive phytochemicals and site-directedmutagenesis. Microb. Biotechnol. 6, 588597.

Sipma, J., Henstra, A.M., Parshina, S.N., Lens, P.N.L., Lettinga, G., Stams, A.J.M., 2006.Microbial CO conversions with applications in synthesis gas purication andbio-desulfurization. Crit. Rev. Biotechnol. 26, 4165.

Solbak, A.I., Richardson, T.H., McCann, R.T., Kline, K.A., Bartnek, F., Tomlinson, G.,Tan, X., Parra-Gessert, L., Frey, G.J., Podar, M., Luginbhl, P., Gray, K.A., Mathur,E.J., Robertson, D.E., Burk, M.J., Hazlewood, G.P., Short, J.M., Kerovuo, J., 2005.Discovery of pectin-degrading enzymes and directed evolution of a novelpectate lyase for processing cotton fabric. J. Biol. Chem. 280, 94319438.

Song, L., Siguier, B., Dumon, C., Bozonnet, S., ODonohue, M.J., 2012. Engineeringbetter biomass-degrading ability into a GH11 xylanase using a directedevolution strategy. Biotechnol. Biofuels 5, 3.

Strausberg, S.L., Alexander, P.A., Gallagher, D.T., Gilliland, G.L., Barnett, B.L., Bryan,P.N., 1995. Directed evolution of a subtilisin with calcium-independentstability. Nat. Biotechnol. 13, 669673.Bioinform. 13, 263.Wang, Q., Xia, T., 2007. Enhancement of the thermostability and hydrolytic activity

of GH10 xylanase by module shufing between Cellulomonas mi Cex andThermomonospora alba XylA. World J. Microbiol. Biotechnol. 23, 10471055.

Wang, Y., Yuan, H., Wang, J., Yu, Z., 2009. Truncation of the cellulose binding domainimproved thermal stability of endo-b-1,4-glucanase from Bacillus subtilis JA18.Bioresour. Technol. 100, 345349.

Wunderlich, M., Martin, A., Staab, C.A., Schmid, F.X., 2005. Evolutionary proteinstabilization in comparison with computational design. J. Mol. Biol. 351, 11601168.

Xiao, Z., Bergeron, H., Grosse, S., Beauchemin, M., Garron, M.L., Shaya, D., Sulea, T.,Cygler, M., Lau, P.C., 2008. Improvement of the thermostability and activity of apectate lyase by single amino acid substitutions, using a strategy based onmelting-temperature-guided sequence alignment. Appl. Environ. Microbiol. 74,11831189.

Zhang, J.H., Jiang, Y.Y., Lin, Y., Sun, Y.F., Zheng, S.P., Han, S.Y., 2013. Structure-guidedmodication of Rhizomucor miehei lipase for production of structured lipids.PLoS One 8, e67892.

Zhang, Z.G., Yi, Z.L., Pei, X.Q., Wua, Z.L., 2010. Improving the thermostability ofGeobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis. Bioresour. Technol. 101, 92729278.

In vitro engineering of microbial enzymes with multifarious applications: Prospects and perspectives1 Introduction2 Redesigning microbial enzymes2.1 In vitro changes in properties of laccases2.2 Alterations in phytases2.3 Improvement of -amylases by tailoring their2.4 Alteration in properties of xylanases2.5 Mutations in lipases2.6 Alterations in chitinases2.7 Mutagenesis studies on proteases2.8 SDM of pectinases2.9 In vitro tailoring of cellulases2.10 Mutagenesis in DNA polymerases2.11 Redesigning of restriction endonucleases2.12 Other enzymes2.13 Engineered microbial enzymes in the market

3 ConclusionsAcknowledgementsAppendix A Supplementary dataReferences