Enzyme Engineering

7. Applications(1): Chemicals Synthesis

7.1 6-APA Synthesis

7.2 L-DOPA Synthesis

7.1 6-APA Synthesis

Production of Penicillin/Cephalosporin Antibiotics

Penicillin G

Cephalosporin

6-APA (6-aminopenicillanic acid)

* Chemical process : toxic chemicals, by-products

Penicillin Amidase

6-APA

Pen-G → 6-APA + phenylacetic acid

• Phenylacetic acid formation

→ pH become low

→ Enzyme inhibition, reversible reaction

• Therefore pH control is very important

→ Recycled reaction is better than packed-bed reaction system

6-APA

Toyo Jozo Bioreactor

-18 parallel columns

-30 L/column

-10% circulation, 6000 L/hr

-T = 30 - 36℃

-pH = 8.4 ± 0.1

-Life time = 360 cycles

(1 cycle = 3 hrs)

• Enzyme make-up/replace after enzyme deactivation

• 6-APA precipitation after reaction

• Batch reactor for 6-APA production

7-ACA (7-amino cephalosporanic acid)

Cephalosporin-C → 7-ACA

- 1979 Toyo Jozo : 2-step process (chemical + enzymatic)

- 1990 2-step enzymatic process

- 2009 1-step enzymatic process (Amicogen)

7-ACA Amidase

7-ACA Bioreactor

- Reactor = 1000 L

- Flow rate = 10,000 L/hr

- Temp. : 15 (initial) → ℃25 ℃(* To compensate the decay of Enzyme)

- Cycle = 4 hours

- T1/2 (life time) = 70 cycles

7-ADCA

Pen-G → phenylacetyl 7-ADCA → 7-ADCA(Ring expansion)

7-ADCA for semisynthetic cephalosporins

Chemical → enzymatic method

Acrylamide

- Monomer for polyacrylamide

- Made from acrylnitrile

CH2=CHCN + H2O → CH2=CHCONH2

- Before 1970

Acrylamide sulfate → ammonia → polymerization reaction

- Recently

Raney copper catalyst at 100℃

- Currently

Nitto Chemical(Japan)

- Low temperature reaction to retard enzyme deactivation

* Enzyme : nitrile hydratase

•Prof. Hideaki Yamada(Kyoto Univ.)

•Dongsuh Chem,Yongsan Chem(Korea)

Aspartame

• α-L-aspartyl-L-phenylalanine –OMe (dipeptide)

• Aspartic acid + phenylalanine → aspartame

• 200 times as sweet as sucrose

• 1965 discovered

• Low-calorie sweetener

• Reversible reaction : product insoluble

7.2 L-DOPA Synthesis

What is L-DOPA?

L-DOPA (L-3,4-dihydroxyphenylalanine) has been used drug for Parkinson

disease, neurological disorder which afflicts one out of every 1700 individuals

and is caused by deficiency of neurotransmitter dopamine. L-DOPA is a

precursor of dopamine, and since it is able to pass across the blood brain barrier

while dopamine itself cannot, it is used to increase dopamine level for the

treatment of Parkinson’s disease

About 250 tons of L-DOPA is now supplied per year and most of the current

supply is produced by chemical method. Because of the high production cost

and its high commercial value, the alternative production of L-DOPA has been

investigated

; microbial or enzymatic production.

Approaches for L-DOPA Production

Chemical Microbial Enzymatic

Chiral pool,

enantioselective homo-

geneous hydrogenation,

asymetirical hydrogenation

Complex process

Metal catalyst

Low overall yield

Low enantiomeric excess

Whole cell with Tpl activity

; Erwinia herbicola cell

Stizolobium hassjoo cell

Carbon source feeding

Separation and purification

from culture media

Long operation time

Low conversion rate

Tyrosinase

(E.C.1.14.18.1)

Two enzymatic activity

; creasolase/catecholase

Subsequent oxidation

Reducing reagent

Dehydroascorbic acid

Objective

Electroenzymatic production of L-DOPA without reducing reagent.

Instead of reducing reagent such as ascorbate,

DOPAquinone, which is a product of subsequent reaction of tyrosinase,

is re-converted to L-DOPA again at reduction potential.

Reduced production cost

Improved productivity

Oxidized L-DOPA

L-DOPA

Potential (V)

-1.0 -0.5 0.0 0.5 1.0

Cu

rren

t (A

)

-1.5e-5

-1.0e-5

-5.0e-6

0.0

5.0e-6

1.0e-5

1.5e-5

2.0e-5

2.5e-5

Buffer1mM tyrosine1 mM L-DOPA

Cyclic Voltammogram

Figure. Cyclic voltammogram of L-DOPA (WE:glassy carbon electrode, CE: Pt wire, RE: Ag/AgCl

electrode) in 50mM phosphate buffer (pH 6.5) at 20 . DOPA was oxidized to DOPAquinone at ℃

0.40V and DOPAquinone was oxidized at -0.06V and reduced to DOPA again at -0.53V

Time (Hour)

0 2 4 6 8 10

L-D

OP

A c

on

cen

trat

ion

(m

M)

0.0

0.2

0.4

0.6

0.8

1.0

Tyr

osi

ne

con

cen

trat

ion

(m

M)

0.0

0.2

0.4

0.6

0.8

1.0

Der

ivat

ives

co

nce

ntr

atio

n (

mM

)

0.0

0.2

0.4

0.6

0.8

1.0

L-DOPATyrosineDerivatives

Electroenzymatic Production of L-DOPA

Figure. Electroenzymatically synthesized L-DOPA concentration with 250 unit free tyrosinase

(WE:carbon felt, CE: ELAT, RE: Ag/AgCl electrode) in 30 ml L-tyrosine solution ( 1mM, in 50mM

phosphate buffer (pH 6.5) at 20 ) at -0.53V℃

Time (hour)

0.0 0.5 1.0 1.5 2.0

L-D

OP

A c

on

ce

ntr

ati

on

(m

M)

0.0

0.2

0.4

0.6

0.8Electroenzymatic productionEnzymatic production with ascorbic acidEnzymatic production without reducing reagent

Effect of Reducing Power

Figure. The effect of reducing power on the electroenzymatic L-DOPA synthesis (WE:carbon

felt, CE: ELAT, RE: Ag/AgCl electrode) in 30 ml L-tyrosine solution ( 1mM, in 50mM phosphate

buffer (pH 6.5) at 20 ) at -0.53V with 2000 unit free tyrosinase℃

Time (hour)

0 2 4 6

L-D

OP

A c

on

cen

trat

ion

(m

M)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 small electrode(5*3*0.6)large electrode(9*5.5*0.6)

Effect of Electrode Size

Figure. The effect of electrode size on the electroenzymatic L-DOPA synthesis (WE:carbon

felt, CE: ELAT, RE: Ag/AgCl electrode) in 30 ml L-tyrosine solution ( 1mM, in 50mM phosphate

buffer (pH 6.5) at 20 ) at -0.53V with 200 unit free tyrosinase℃

Tyrosinase Immobilized Electrode

Tyrosinase adsorbed carbon felt electrode

Adsorption of tyrosinase into the carbon felt electrode (2000 unit tyrosinase)

Dry at room temperature

Coated by Nafion® solution (5 wt% in water and alcohol)

Tyrosinase/CNPs/Polypyrrole composite

Functionalization of CNP by 1-pyrenebutyric acid

Tyrosinase immobilization on fuctionalized CNP by EDC activation, covalently

Mix the monomer pyrrole and LiClO4 for chemical polymerization

Preparation of 3-dimensional composite(1.5*1.5*0.1) by mixing the chemically

polymerizing polypyrrole and tyrosinase immobilized CNPs

Time (hour)

0 1 2 3 4 5 6

L-D

OP

A C

on

ce

ntr

ati

on

(m

M)

0.0

0.2

0.4

0.6

0.8

1.0

Time (hour)

0 1 2 3 4

L-D

OP

A c

on

cen

trat

ion

(m

M)

0.0

0.2

0.4

0.6

0.8

1.0

Tyrosinase adsorbed carbon felt electrodeCNP-Ty-Ppy composite electrode

Electroenzymatic L-DOPA Synthesis with Tyrosinase-Immobilized Electrodes

Figure. Electroenzymatically produced L-DOPA

concentration with immobilized tyrosinase

(WE:tyrosinase immobilized electrode, CE: ELAT,

RE: Ag/AgCl electrode) in 30 ml L-tyrosine solution

( 1mM, in 50mM phosphate buffer (pH 6.5) at 20 ) ℃

at -0.53V, 2000 unit tyrosinase was immobilized.

Figure. Electroenzymatically produced L-DOPA

concentration (WE:CNP/Ty/Ppy composite electrode,

CE: ELAT, RE: Ag/AgCl electrode) in 300 ml L-tyrosine

solution ( 1mM, in 50mM phosphate buffer (pH 6.5) at

20 ) at -0.53V, ℃ 2000 unit tyrosinase was immobilized.

Reuse number

0 2 4 6 8 10

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

Tyrosinase adsorbed carbon felt electrodeTyrosinase-Carbon nanoparticles-Polypyrroe composite electrode

Operational Stability;Tyrosinase Immobilized Electrode

Figure. Operational stability of tyrosinase immobilized electrodes as function of reuse number.

Relative activity was determined from the synthesized L-DOPA concentration in 30ml L-tyrosine

solution (1mM, in 50mM phosphate buffer (pH 6.5) ) at -0.53V for 4 hours .

Reaction Type Productivity(mg/Lh)

Conversion rate

(%)Remark Ref.

Chemical synthesis 44 Several reaction steps 1

Immobilized tyrosinase

Batch reactor1.7

Long time operation (170hrs)

Low operational stability2

Immobilized tyrosinase

Batch reactor4.5 1.8 Low conversion rate 3

Erwinia herbicola culture 1800 7.34

Low conversion rate

Substrate mixture feeding

(pyruvate,ammonia, catechol)

4

Stizolobium hassjoo culture 3.13 Long time operation (over 10days) 5

Free tyrosinase in batch

Electroenzymatic134.66 68.3

High conversion rate

Short operation time (1hr)

This study

Immobilized tyrosinase

Batch, Electroenzymatic39.08 99.1

High conversion rate

Short operation time (4hr)

Good operational stability

This study

Ref.) [1] Catalysis Comm. 5; 631, [2] Biotechnol. Bioeng. 51; 141, [3] EMT 40; 683, [4] J. Biotech. 115; 303, [5] EMT 30; 779

Table. L-DOPA production on different reaction type

The reason why the electroenzymatic system can show the high conversion rate and productivity?

Hypothesis

; The reason for the enhanced conversion rate and productivity was efficient electron transfer

from electrode to DOPAquinone. In terms of reaction rate, the electrical reduction of

DOPAquinone to L-DOPA predominated over the oxidation of L-DOPA by catecholase activity

in tyrosinase/CNPs/Ppy composite.

(1) The tyrosinase was covalently attached on the carbon nanoparticles which play roles as not

only an immobilization support but also electron carriers in the composite electrode.

(2) The tyrosinase, which was immobilized on the electron carrier, converted L-DOPA to

DOPAquinone by its catecholase activity, and the DOPAquinone was directly reduced to L-

DOPA by electrons from the electrode.

(3) Therefore, by-product DOPAquinone did not accumulate in the reactor and the conversion rate

increased up to 99.1%.

Summary

L-DOPA can be synthesized in electroenzymatic system. In electroenzymatic

system for L-DOPA production, by-product DOPAquinone was reduced to L-DOPA

by electrons from cathode. The electrical reducing power was more efficient to

enzymatic L-DOPA synthesis than reducing reagent, ascorbic acid.

In electroenzymatic L-DOPA synthesis, the conversion rate and productivity by

tyrosinase/CNPs/Ppy composite electrode was 95.9 % and 134.7 mg/Lh,

respectively. When the reactor was scaled up to 10 times, the conversion rate was

maintained.

Based on the kinetic constants k1, k2, ke electrical reduction of DOPAquinone to L-

DOPA was faster than oxidation of L-DOPA by catecholase activity.

May 30, 2012

홍 은 영

Novel bioreduction system for the production of chiral alcohols

IntroductionIntroduction

The enzymes catalyze chemical reactions specifically on three levels: chemo-, regio-, and stereo-selectivity.

COBE

rac-4-chloro-3-hydroxybutanoate etheyl ester

O

O OH

Cl CHBE

(R)-4-chloro-3-hydroxybutanoate etheyl ester

O

O OH

Cl

CHBE

(S)-4-chloro-3-hydroxybutanoate etheyl ester

O

O OH

Cl

Asymmetric synthesis

Kinetic resolution

CHBE(S)-4-chloro-3-hydroxybutanoate etheyl ester

Asymmetric Synthesis vs. Kinetic ResolutionAsymmetric Synthesis vs. Kinetic Resolution

O

O OH

Cl

CHBE(S)-4-chloro-3-hydroxybutanoate etheyl ester

O

O OH

Cl

COBErac-4-chloro-3-hydroxybutanoate etheyl ester

O

O OH

Cl

50% maximum yield

100% maximum yield

O

O O

Cl

COBE4-chloro-3-oxobutanoate etheyl ester

Designing a bioreduction systemDesigning a bioreduction system

Recombinant DNA(1)carbonyl reductases(2)Cofactor regeneration enzymes (3) host–vector systems for co-expression of two enzyme genes.

Problems when using wild type microbial cells• Multiple reductases with opposite stereoselectivities toward carbonyl compounds in one microorganism

sometimes lower the optical purity of chiral alcohol.• the cellular content of the carbonyl reductase giving a desired enantiomer is relatively low, because the

enzyme gene might be expressed constitutively, although the physiological roleof carbonyl reductases remains unknown.

Screening of carbonyl reductaseScreening of carbonyl reductase

ketopantoyl lactone (KPL; a), ketopantoic acid (KPA; b)White triangles Yeasts, white circles molds, white squares bacteria, black squaresactinomycetes, black circles basidiomycetes.

pH 4-6 pH 7-8

Characterization and cloning of carbonyl reductase

Structures of plasmids Solid bars ARI, S1, or GDH genes, solid lines vector plasmid DNA. ARI The ARI gene, S1 the S1 gene, GDH the GDH gene, Apr ampicillin-resistance gene, Kmr kanamycin-resistance gene, Ptac tac promoter, Plac lac promoter

Characterization and cloning of carbonyl reductaseCharacterization and cloning of carbonyl reductase

AR1 -GDH

S1 -GDH

Development of bioreduction processDevelopment of bioreduction process

Why use two-phase aqueous/organic solvent system?n-butyl acetate/water diphasic system

Both COBE and CHBE are poorly soluble and unstable in water Both COBE and CHBE are inhibitory to the enzyme.

Development of bioreduction processDevelopment of bioreduction process

AR1 –GDH converted (R)-COBE in 16 h (molar yield: 94%, optical purity: 92% e.e.). S1 –GDH converted (R)-COBE in (molar yield: 96%, optical purity: 100% e.e.) The industrial production of (S)- CHBE with a bioreduction system was started in 2000 byKaneka Corp

Promising reactions for the production of other Promising reactions for the production of other chiral alcoholschiral alcohols

d-ephedrin, one of the four stereoisomers of ephedrin. Its main pharmacological use is as a decongestant or anti-asthmatic compound, although recent reports indicate its potential in obesity control.

Conclusion and future prospectsConclusion and future prospects

Assessment of the work and Further researchAssessment of the work and Further research

•Alcohol dehydrogenase and formate dehydrogenase, can be used for the same purpose.• • The direct-evolution technique, such as site-directed utagenesis, random mutagenesis, and DNA shuffling, is an effective method for the improvement or expansion of carbonyl reductase functions. Alteration of cofactor requirement, substrate specificity, stereospecificity, enzyme stability, and so on might be expected.

A New Detergentless Micro-Emulsion System

Using Urushiolas an Enzyme Reaction System

Yoo, Chang-Hyuk

2012. 05. 30.

Introduction

Urushiol -Alkenylphenol-Natural monomeric oil-A major portion of the oily constituent of urushi sap from Rhus vernicifera-Catecholic head group with a C-15 tail with varying degrees of saturation.

Urushi sap-Japanese lacquer-Containing 60-70% urushiol, 20-30% water, 10-15% gum and polysaccharides, and less than 1% of enzymes and proteins

Laccase-A copper containing oxidoreductase that uses oxygen-Polymerization of Urushiol

Urushiol

Enzymein water

Organic solvent

2-propanol

Fig. 2. Possible mechanism of 2-propanol in formation ofdetergentless micro-emulsion.

Reverse micelle

DMS (detergentless micro-emulsion system)\

-Proposed by Martineck et al.-Enzyme reaction system for water insoluble substrate-2-propanol carries out the surfactant-like function to create a stable phase separation between water and hydrophobic organic solvent media-Reaction surface area : up to 100 m2/ml-Easy remove of 2-propanol from the product-Hexane and toluene as the organic solvent media

Introduction

In this study,

-Use of urushiol, water and 2-propanol to create DMS

-Study of the formation of micro-emulsion by conductance measurements

-Use of the dynamic light scattering method to measure the mean water droplet diameter

-A novel concept of using a natural oil urushiol (hydrophobic solvent/substrate)

Introduction

Fig. 4. Conductance measurements of solutions.(a) 2-Propanol, (b) n-propanol, and (c) ethanol were added in increments to the original solution of 10% (v/v) water and 90% (v/v) urushiol.

Conductance measurement

Normal water in oil

Fig. 5. DLS measurement showing the observed peak of mean water droplet diameter at the transitionary phase where unstable seperation of smaller micro-droplets begins to appear.

Fig. 6. DLS measurement showing the mean diameter of water droplets at the mole fraction of 0.41.Near singular peaks of radius of 46nm can be seen.

Dynamic Light Scattering

Fig. 7. The mean hydrodynamic radius of water droplets at differing molar ratios of 2-propanol, which shows rapid decrease in the diameter.

Dynamic Light Scattering

Fig. 8. Relative enzyme activity of laccase analyzed by measuring absorbance at 530nm using syringaldezine as substrate in a DMS system [ Laccase activity ◆(abs/min)]

Relative Enzyme Activity

Fig. 9. Increase in viscosity of urushiol polymerization in the bubble column reactor with 32% (v/v) addition of various organic solvents.X water, ● THF, ▲ acetonitrile, ■ ethanol, 2-◆propanol.

Urushiol Polymerization

Fig. 3. Schematic diagram of the DMS bubble column reactor for urushiol polymerization.A (Ice bath), B (Condenser), C (100ml reactor tube, D (Urushiol/Water/2-propanol mixture),E (Air hose), F (Flux).

Summary

In this study,

-Use of urushiol, water and 2-propanol to create DMS

-Mole fraction of 2-propanol for the formation of micro-emulsion : 0.4 ~ 0.44 (L2 phase)

-Relative enzyme activity of laccase : 0.4 ~0.46

-A novel concept of using a natural oil urushiol (hydrophobic solvent/substrate)

-Suggest that other fats and oil could be used to created a similar DMS

발표자 : 이선기

Introduction

Enzyme catalysis has been known for well over a century, it has been largely restricted to aqueous systems

Non aqueous enzymology

Enzymes catalysis in Organic solvent(nonaqueous)1.Synthesis of different polymers2.Modification of different polymers

1) Synthesis of polyaromatic compounds

2) Synthesis of polysaccharides & polyesters

3) Transesterification of polyhydroxy compounds

4) Future Prospects

Contents

This article reviews some of the recent work on tailoring structural and functional properties of polymers

1-1. Synthesis of polyaromatic compounds

The architecture of polyaromtic compounds synthesiszed by enzyme-catalysed reactions is strongly dependent on the type of the reaction system used to synthesize the polymer

a. mono-and biphasic solvent

b. Langmuir trough (Air-water

interface)

c. reverse-micellar system (water/oil)

containing a detergent

- water : surfactant ratio size

Horseradish peroxidase(HSP),

hydrogen peroxide

1-1. Synthesis of polyaromatic compounds

Polymer’s molecular weight and polydispersity

Composition of the reaction medium

polar solvent(ethanol, acetone …) + buffered aqueous phase

Organic solvent growth of oligomer and polymer chains

enzyme activity

1-2. Properties of polyaromatic compounds

Potentially electrically conductive under doped condition, although their properties have not yet been studied in any detail

Polyaromatic compounds are formed by aromatic ring-ring linking at ortho positions and this linkage results in a conjugated backbone for the polymer

The conductive and fluorescent properties of these polymers, coupled with the ability to incorporate derivatizable functional groups, provide excellent opportunities for polyphenols in biosensor applications

1-2. Properties of polyaromatic compounds

An easier way to prepare such conjugates is to generate copolymers with a controlled density of the desired functional groups

2. Synthesis of polysaccharides & polyesters

Polysaccharide

Using cellulase enzymes in organic-solvent-aqueous-buffer

systems and -cellobiosyl fluoride as the monomer, a crystalline

form of cellulose has been synthesized.

Polyester

The synthesis of polyesters is accomplished using mammalian

and microbial enzymes in organic solvents, supercritical fluids or

in bulk without solvents

3. Transesterification of polyhydroxy compounds

Transesterification: alcohol + ester → different alcohol + different ester

Many polyhydroxylated compounds are insoluble in organic media and hence standard nonaueous enzymology is unable to sustain catalytic transformations.

For example, amylose(an organic solvent-insoluble polysaccharide) can, when deposited as a thin film, be regioselectively acylated by catalysis in organic solvents using an enzyme preparation of proteases

3. Transesterification of polyhydroxy compounds

methylene-stretching mode of the alkyl chain (2920 and 2850 cm-)carbonyl group (1693–1760 cm-)

amylose treated with the vinyl esters(900 and 1000 cm-)

fatty-acid ester to an isooctane solution containing solubilized subtilisin was pipetted onto a thin layer of amylose deposited onto zinc-selenide slides.

4. Future prospects

The biocatalysts are still too expensive for applications to be economical recombinant enzymes immobilized enzymes Site-directed mutagenesis There are a number of conditions in which conventional chemical methods oraqueous enzymology are unable to support catalytic synthesis or transformations.

Under these circumstances, the development of suitable techniques based on enyme-catalysed reactions in organic solvents for the

specialized synthesis and selective modification of macromolecules are both desirable and cost effective.

Summary

Synthesis and modification by enzyme in organic solvent

(J. Akkara, unpublished)(Ref. 24; J. Akkara, unpublished)(Refs 15,20; J. Akkara, unpublished)

Polyaromatic compounds >> polysaccharides , polyester

Nowadays, other compounds?

Jung Ho Ahn5.30.2012

Introduction

Cis dihydrodiols

Potential chiral building blocks for the synthesis of: Polymers Bioactive aza sugars Cis amino-alcohols

Intermediates in the microbial metabolism of aromatic hydrocarbons Toluene, indene, naphthalene

Use of mutants lacking cis dihydrodiol dehydrogenase activity Accumulation of desired cis diol in the

cultivation medium

Commonly use Pseudomonas species for the production of large array of cis dihydrodiols

`

TDO=toluene dioxygenase

Rhodococcus sp. MB 5655

Gram positive bacteria

Efficiently utilize toluene for growth

Offer good tolerance to hydrocarbons present in the cultivation medium

Hypothesis Cis dihydrodiol dehydrogenase blocked mutant of MB 5655 may prove to be an attractive cis diol producer with the potential to produce an array of other interesting cis dihydrodiols

Methods

Mutation

Wild type cells Form large colonies

Mutant cells Form small colonies

Cell culture

Nitrosoguanidine(NTG)

solution

10 min

20 min

30 min

Mutagenized cells

Incubate

Cell plating

Toluene vapor

NTG = mutation inducing chemical = mutagen

Mutant evaluation

Inoculate

Culture tube containing

toluene

Toluene cis glycol

concentration in each sample

foundSpectrophotometric assay

Result

Cell viability after treatment of NTG

Viable cell count reduction of 2 orders of magnitude after 30 min exposure to NTG

Commonly considered a necessary target when employing mutation inducing chemicals

Cell viability after treatment of NTG

Ratio of small to large colonies

2000 small clones were evaluated

Clone MA 7249 Accumulated large

amounts of toluene cis glycol in the cultivation medium

Only one to exhibit toluene cis glycol accumulation

Confirm using HPLC analysis

Toluene cis glycol production in bioreactor

H-NMR

C-NMR

MA 7249 toluene dioxygenase bioconversion range

Evaluated 18 substrates

Screening studies performed in the presence of toluene to ensure induction of dioxygenase

TDO=toluene dioxygenase

Toluene bioconversion potential

Achieved linear toluene cis glycol production rate 0.253 g/L/hr

Max. toluene cis glycol conc.=18g/L

Cultivation time (hr)

Toluene bioconversion potential

Cell viability maintained throughout the duration of the bioconversion phase

Continual increase in biomass concentration

Active glucose consumption

Conclusion

Successfully generated toluene cis glycol dehydrogenase mutant of Rhodococcus NTG mutagensis First to report this mutant in Rhodococcus

MA 7249 excellent candidate for large production of toluene cis glycol in bioreactor 18 g/L (unoptimized) Lack of cis glycol toxicity Good tolerance to toluene and potentially

to other solvents

Further suggestions

Strain lack of dehydrogenase activity lead to deficient coafactor regneration

Lack of glucose catabolite repression on toluene dioxygenase(TDO) induction Allow co-factor recycling via glucose

metabolism

Toluene +

NADH + H+

Toluene cis glycol

+ NAD+

Toluene catechol +

NADH + H+

Volker Höllrigl, Frank Hollmann, Andreas C. Kleeb, Katja Buehler and Andreas Schmid (2008) Applied Microbiology and Biotechnology 81:263-273

효소공학 2012. 06. 04 이하은

Alcohol dehydrogenase (ADH): Catalyze reversible interconversion of primary and secondary alcohols into corresponding aldehydes and ketones, or oxidation of aldehyde into carboxylic acid

NAD+

NAD+

NADH

NADHO

H

RR OH

1o alcohol Aldehyde

Oxidation

Reduction

NAD+

NAD+

NADH

NADH

R1 R2

2o alcohol Ketone

Oxidation

Reduction

OH

R1 R2

O

O

H

R

Aldehyde

NAD+

NAD+

NADH

NADH

Oxidation

Reduction

R

OH

O

Carboxylic acid

Chiral active site of ADH: useful for organic synthesis of enantiomerically pure molecules

1) Asymmetric reduction

2) Kinetic resolution

Ketone (pro-chiral)

R1 R2

O NAD+

NAD+

NADH

NADHR1 R2

(S)-alcohol (chiral)

OH

NAD+

NAD+

NADH

NADHR1 R2

(R)-alcohol (chiral)

OH

R1 R2

OH (R)-specif ic ADH

R1 R2

(S)-alcohol (chiral)

OH

Ketone (pro-chiral)

R1 R2

O

(S)-specif ic ADH

R1 R2

(R)-alcohol (chiral)

OH

Ketone (pro-chiral)

R1 R2

O

Racemic alcohol(S 50%: R 50%)

Method for efficient cofactor regeneration is required Expensive Electrochemical / enzymatic regeneration

Stability of enzyme must be maintained Process condition ≠ Native condition Instability Organic solvent, pH, elevated temperature Lower activity and stability

ADH from extremophiles Increased stability due to rigid structure with many ionic interactions and disulfide bonds

Enzyme isolated from alkane-degrading thermophile: TADH is thermostable

NAD-dependent: useful for replacing the NADP-dependent Thermoanaerobium brockii (TbADH) which has been most studied so far (NADPH 5x more expensive than NADH)

Recombinant expression in E. coli : Convenient preparation of the enzyme

Biochemical characterization of TADH with its applicability for the production of chiral alcohols and carboxylic acids was carried out in this research.

Preparation of TADH E. coli expression Purification by heat treatment and anion exchange

Enzyme activity assay Measure consumption (substrate reduction) or

formation (substrate oxidation) of NADH by UV-spectrophotometer

Enantiomeric excess measured by gas chromatography

Dismutase activity measured by gas chromatography

Purity of TADH Heat treatment (lane 1):

65% Anion exchange (lane 2):

95% Dehydrogenase activity

occurs only by TADH Coomassie staining (lane 3) Dehydrogenase specific

activity stain (lane 4)

Optimum temp and pH 60℃ : trade-off between

activity and stability (Hollmann et al. 2005, Tetrahedron Asymmetry)

pH 6 for reduction, pH 9 for oxidation

Red Oxi

Reaction

Substrate Km (mM) Vmax (U mg-1)

Kcat / Km (mM-1 s-1)

Oxidation

Cyclohexanol

2.09±0.2620.78±0.5

524.7

NAD+ 0.13±0.01520.78±0.5

5397.06

Reduction

Cyclohexanone

3.68±0.26 15.14±0.2 10.2

NADH 0.01±0.0015 15.14±0.2 3760.78

Steady state kinetics of TADH

Preference for primary alcohol for aliphatic substrate

Sterically large aromatic alcohols at generally low rate

Cyclic alcohols are good substrates, but ring substitution with the exception of the 3-position decreases activity

Increasing C

1o alcohol

2o alcohol

Cyclic

Aromatic

Higher activity toward aldehydes than ketones

Activity decrease towards bulkier substituents, especially if the bulky group was close to the ketone function

For aliphatic ketone, activity is generally low without respect to chain length

Increasing C

Alipathic aldehyde

Cyclic aldehyde

O

Aliphatic ketone

Ketones with bulky ring

Acetophenone

Asymmetric reduction Prochiral ketone (S)-alcohol (99%

enantiomeric excess)

Kinetic resolution Racemic 3-methylcyclohexanone (1S,3S)-

3-methylcyclohexanol 98.7% diastereomeric excess

O

(S)

OH

2-Pentanone (S)-2-Pentanol

O O

(R)

(S)

OH

(S)

(rac)-3-Methylcyclohexanone

(1S,3S)-3-Methylcyclohexanol

(R)-3-Methylcyclohexanol

Additives Metal ions and iron-chelator have no

influence Divalent ion chelator, sulfide-

reducing agent and detergents reduce activity by 90%: zinc binding is important in structural stability

Triton X100 doubles activity Upto 1M Urea activity increases

Solvents Water soluble (log Po/w: -1.35~0.05):

Approx. 20% activity except acetone (50%)

Water immiscible (log Po/w: 0.73~8.25): Activity increases upto 80% as log Po/w increases, except hexadecane with 35% activity

Butyraldehyde n-butanol + butyric acid There should be no net change of the NAD redox state:

NADH formed by oxidation to acid is used up in the simultaneous reduction to alcohol

In reality, NADH accumulates because the alcohol is oxidized back to aldehyde (reversible reaction) while oxidation to acid is irreversible Dismutase activity measured by NADH accumulation increases at higher pH

With cofactor regen, excess of butyric acid to alcohol increases from 4.4% (1.1:1) to 38% (2.23:1)

TADH shows a broad substrate spectrum Highest conversion rates with primary alcohols and

aldehydes, as well as some cyclohexane derivatives Substrate spectrum is similar to the TbADH that it can

convert bigger substrates than yeast ADH Opposite enantioselectivity ((S)-preferred) to TbADH

TADH is stable in organic solvents over a broad range of log Po/w values Water miscible solvent: can be used as a co-substrate

for cofactor regeneration Water immiscible solvent: can be used to solubilize

substrates and products when substrate/product are water-insoluble, or to separate products from enzyme when it is inhibitory to the enzyme

When coupled to cofactor regeneration and given enough time, dismutase activity allows synthesis of carboxylic acid with 99.9% purity

Significance ADH can catalyze many types of reactions applicable in

industry, with high activity and enantioselectivity TADH is thermostable and uses a cheaper cofactor than

conventional TbADH The research explores many environmental factors such as

pH, various substrates, additives and solvents, and measures kinetic and enantioselectivity parameters

Shortage Trends between environmental factors (substrate size,

additive/solvent properties) and the activity were not found

Since there is no enzyme structure, it is difficult to give a molecular explanation of the results: This could be a future work

Cofactor regeneration is still a limiting factor to efficient reaction