Download - Enzyme Engineering 7. Applications(1): Chemicals Synthesis 7.1 6-APA Synthesis 7.2 L-DOPA Synthesis
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 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
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
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.
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
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.
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
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?
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
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
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
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