1 Enzyme Technology and Biocatalysis OH1

Enzyme Technology and Biocatalysis
Enzymtechnologie und Biokatalyse

MOL.931 + CHE.891, 2 VO
WS 2021/22, Wolfgang Kroutil
wolfgang.kroutil@uni-graz.at
Slides
Slides can be downloaded via Dropbox Link (sent by email)
Major parts of the lecture are based

on the book: Biotransformations
in Organic Chemistry
Kurt Faber, 7th edition
2018.
VO dates See
Wed. 8:15-9:45, for December online
Maybe in January: Seminarraum SR 03.K1,
Universitätsplatz 1, Kellergeschoß
VO Enzyme Technology and Biocatalysis, Kroutil

Other recent books
Applied Biocatalysis: From Fundamental Science to Industrial
Applications, 2016
Editor(s): Lutz Hilterhaus, Andreas Liese, Ulrich Kettling, Garabed
Antranikian
Biocatalysis: An Industrial Perspective, 2018

Editors: Gonzalo de Gonzalo, Pablo Domínguez de María
Biocatalysis: Biochemical Fundamentals and Application

2018
Peter Grundwald

Contents
Introduction and Background Information

 Introduction:
Motivation
Advantages and Disadvantages of Biocatalysts
 Enzyme Properties and Nomenclature:

Mechanistic Aspects
Classification and Nomenclature
Coenzymes
Enzyme Sources

Contents
Biocatalytic Applications
 Hydrolytic Reactions:
Mechanistic and Kinetic Aspects
Hydrolysis of the Amide Bond
Ester Hydrolysis: Esterases and Proteases, Lipases
Hydrolysis of Nitriles
 Reduction Reactions:
Recycling of Cofactors
Reduction of Aldehydes and Ketones
Reduction of C=C-Bonds Using Whole Cells
 Oxidation Reactions:
Oxygenation Reactions
Mono-Oxygenation: Hydroxylation of Alkanes
Di-Oxygenation: Dihydroxylation of Aromatics

Contents
 Formation of Carbon-Carbon Bonds:

Aldol Reactions
 Addition and Elimination Reactions:

Cyanohydrin Formation
Addition of Water and Ammonia
 Transfer Reactions:
Transamination
Special Techniques
 Enzymes in Organic Solvents
Ester Synthesis

Biocatalysis = application of
enzymes/biocatalysts for the
transformation of also non-natural
substrates in organic synthesis

Some write:
Want to create catalysts as good as enzymes
Use enzymes!!
before 1990: enzyme preparation only for specialists,
isolation from Nature, wild type microorganisms
>1992: enzyme engineering techniques,
mainly hydrolytic enzymes
>2000: DNA synthesis improved, 2003: one gene: 15000 Euro
>2015: DNA synthesis: one gene: 100 Euro;

target: 1 $/gene?
Enzymes in Organic Synthesis
Only selected examples shown
Time
1980 1990 2000 2010
Lipases, Esterases,
ADHs, Aldolases, BVMO, P450
HNL, nitrile hydratase, nitrilase
Transaminases
Biocatalytic retrosynthesis
N. Turner, E. O‘Reilly, Nat. Biotechnol. 2013, 9, 285 IREDs,…
Lipase as biocatalyst for industrial purposes
Seifenfabrikant, 40, 4, (1920)
Enantioselectivity of lipase: Z.physiol. Chemie, 140, 203, (1904)

Biocatalysis in Organic Synthesis (Redox)
Early example: Reichenstein process
O OH OH OH
OH HO HO O chemical
[H2] Acetobacter steps
HO = HO HO HO vitamin C
OH OH OH OH
OH HO HO HO
OH O OH OH
D-glucose D-sorbitol L-sorbose
T. Reichstein, A. Grüssner,
Eine ergiebige Synthese der L-Ascorbinsäure (C-Vitamin),
Helv. Chim. Acta 1934, 17, 311–328.
Tadeus Reichstein,
Nobel prize 1950, medicine
Wild type strain
Non natural reaction: e.g. for aspartame
aspartame
since 1988, 2500 t/a
example: Acrylamide
O
CN Rhodococcus sp. O
NH 2
H2O >30000 t/a OH
> 400 g/L H2O
H2O H2O
Cu-catalysis
Kupfer-Katalyse

Acrylamide acrylamide market data
• Very important monomer for non-ionic and ionic polyacrylamides

– Flocculants for water treatment and mining
– Thickeners for enhanced oil recovery
– Retention aids and dry strength improvement in paper
making
– Nappies
• Global annual production

– total 500.000 t (calc. 100%)
Common Prejudices Against Enzymes
 „Enzymes are sensitive”.

 „Enzymes are expensive”.
 „Enzymes are only active on their natural substrates”.
 „Enzymes work only in their natural environment”.

Advantages of Biocatalysts
 Enzymes are very efficient catalysts.
 Enzymes are environmentally acceptable.

 Enzymes are biodegradable
 Enzymes are made (in general) from renewables
 Enzymes act under mild conditions.
 Enzymes are compatible with each other.
 Enzymes are not bound to their natural role.
 Enzymes can catalyze a broad spectrum of reactions.
 Enzymes are tunable
The trouble with metallorganic- or organo-catalysts:

Examples of applications of enzymes
washing powder
synthesis of
pharmaceuticals
synthesis of flavour
and fragrances
Stonewashed
jeans VO Enzyme Technology and Biocatalysis, Kroutil
Disadvantages of Biocatalysts
 Enzymes are provided by nature in only one enantiomeric form.
 Enzymes require narrow operation parameters.
 Enzymes display their highest catalytic activity in water.
 Enzymes are prone to inhibition phenomena.
Not valid anymore:

 Enzymes are bound to their natural cofactors.

Types of selectivities displayed by enzymes
 Chemoselectivity (e.g. reduction of carbonyl versus C=C)
 Regioselectivity (e.g. differntiaton between two ketone groups))
 Enantioselectivity
 Diastereoselectivity

Biological effects of enantiomers
(Scheme 1.1)

Enantiomeric excess (ee)
H HO
HO H
N N
(S)-Pyridyl-2-ethanol (R)-Pyridyl-2-ethanol
ee = 0% Racemic
ee = 100% Enantiopure
H 2N SH HS NH 2
HO2 C Me Me CO 2H
H Me Me H
S-penicilamine R-penicilamine
ANTIART HRITIC TOXIC

Structure of a Candida antarctica lipase B mutant
Figure 1.1
Structure of a Candida antarctica lipase B mutant bearing an inhibitor
(yellow) bound to the active site (left). Structural water molecules are
depicted as red balls (right).
Schematic representation of the 'lock-and-key' mechanism
(Figure 1.2)
E. Fischer, Einfluβ der Konfiguration auf die Wirkung der Enzyme.

Ber. Ges. Dtsch. Chem., 27 (1894), p. 2985

Schematic representation of the 'induced-fit' mechanism
(Figure 1.3)
Enzyme Enzyme
A X A B
X X X
Substrate Substrate
No Induced Fit Inactive Enzyme Induced Fit Active Enzyme
Koshland DE. Application of a theory of enzyme specificity to protein synthesis.

Proc. Natl. Acad. Sci. USA. 1958, 44, 98-104.

Schematic representation of binding forces within a protein structure
(Scheme 1.2., adapted)
H H HOH
H H O
O H H
H H O- NH3+ O
O Salt Bridge
O H H
O
H H
O
O Van der Waals/
H H H H
O London dispersion
O
H H H H
O O
H HN
H
O
H
H H O
O
H H H Stacking
O
O
H H
Hydrogen Bonding

Why is an enzyme an excellent cat?
Active site: gets the reagents close together

Working modes of a (bio)catalyst
1) Gets the reagents close together
2) Activates substrate
3) Activates reagent
4) Stabilises transition state
5) May provide alternative mechanism
6) Employing metal ions for activation
7) Chiral recognition

What makes an enzyme a special catalyst?
• Substrate binding
• Exact positioning of reagents
shape of active site
• Activation
synergistic catalysis
• Stabilisation of TS
• Product (ideally) not tightly
bound
• Flexible backbone
• Chiral environment
Classification of enzymes (Table 1.5, extended)
7. Translocases* movement of ions or molecules across

membranes or their separation within membranes
a Theestimated utility of an enzyme class for the transformation of nonnatural substrates ranges
from +++ (very useful) to (little use)
* Since August 2018 VO Enzyme Technology and Biocatalysis, Kroutil
Common coenzymes required for biotransformations
(Table 1.6)

Characterization of a catalyst

Characterization of a catalyst (overview)
measuring biocatalyst performance.
All methods are dependent upon the conditions used, and therefore, should not
be quoted in isolation.
* TTN = Total Turnover number: Measured over the lifetime of the biocatalyst.
** Achievable product concentration and productivity
M. Dias Gomes, J. M. Woodley, Molecules 2019, 24, 3573

Characterization of a catalyst (overview)
Complementary Metrics
• Product concentration
• Productivity
defined as the amount of product produced
per volume of reactor per time
• Enzyme stability
M. Dias Gomes, J. M. Woodley, Molecules 2019, 24, 3573

Definitions of Protein Stability
Thermodynamic Kinetic
Ku
N U N D
kd,obs
• Reversible Unfolding • Irreversible Deactivation

• Ku, DGu, Tm • kd,obs, t1/2 = ln 2 / kd,obs
0
1
-0.5
Fraction unfolded
y = -0.0351x
0.8 -1 k = -0.035 min-1
ln (v/v o)
0.6 -1.5
0.4 -2
Tm = -2.5
0.2 48ºC -3
0 -3.5
30 40 50 60 0 20 40 60 80 100
Temperature (ºC) time (min)
A. Bommarius
Ideal performance of biocat. Processes
dimensions of merit and thresholds for biocatalyst & process
• (Bio)catalyst Thresholds (pharma)

– catalytic rate constant: turnover frequency > 1 s-1
– biocatalyst stability: deactivation constant kd < 0.1 d-1
– product purity: eeP > 99.5%
• Process
– catalyst lifetime stability: total turnover number TTN > 10,000
(moles product/mole (bio)catalyst)
– volumetric productivity: space-time-yield STY > 0.1 kg/(L·d)
– process selectivity: enantiomeric ratio E [-] > 100
reviews: Rozzell, Chimica Oggi 1999 (6/7), 42-47

Bommarius et al., Chimia 2001, 55, 50-59
Enzyme activity
Volumetric activity: U/L of solution or culture broth
Specific activity: U/mg protein

 μmol/(minmg protein)
each at specific conditions of: T, pH, buffer/salts, solvent,

substrate type, [substrate],
[product], [inhibitor]
Enzyme performance requirements
J. Dong, E. Fernandez-Fueyo, F. Hollmann,* C. E. Paul, M. Pesic, S. Schmidt, Y. Wang, S. Younes, W.

Zhang, Angew. Chem. Int. Ed. 2018, 57, 9238 – 9261.
P. Tufvesson, J. Lima-Ramos, M. Nordblad, J. M. Woodley, Org. Process Res. Dev. 2011, 15, 266–274.
A biocatalyst can be…
• a pure enzyme
• a crude (cell free) enzyme preparation
• resting cells
• living/fermenting cells
• freeze dried cells
• immobilized (crude) enzyme
• immobilized cells
• …
The serine hydrolase mechanism
(Scheme 2.1, adapted)
Overall reaction:
Active site residues:

In detail:
1st half reaction:

2nd half reaction:
His
Asp Acyl-enzyme
Ser intermediate
H N N O O
O O H
Nu
R1
tetrahedral intermediate

(Scheme 2.1)
O O
R1 OH R1 HN-R3
H 2O R3-NH2
hydrolysis ester aminolysis
O
4 Enz R1 H2O2
R -OH
O Acyl-enzyme intermediate O
R1 OR4 4 3 R1 O-OH
Nu = H2O, R -OH, R -NH2, H2O2
acyl transfer peracid formation
R3 = H, alkyl, aryl, -NR52
R4 = alkyl, aryl, -N=CR52

Enantiotopos differentiation (prochiral substrates)
achiral precursor with
prochiral center
Better: Re/Si

Enantiotopos differentiation (prochiral substrates)
Double-step process Ph OAc

k1 * k3
OH
Ph OAc R Ph OH
OAc OH
k2 Ph OH k4
*
OAc
S

Desymmetrization of meso-substrates
(Scheme 2.4)

Single-step asymmetric synthesis
(Figure 2.1)
Enantiomer differentiation – kinetic resolution

(Scheme 2.6)

Example for kinetic resolution:
Hydrolysis of an ester in aqueous solution
O
OH
O O
+ HO
(R)-1-Phenylethanol
+ Novozym 435
O O
RT +
O puffer pH 7
O
rac-O-1-Acetyl-1-phenylethanol (S)-(-)-O-1-Acetyl-
1-phenylethanol
Novozym 435 = CAL B = Candida antarctica lipase B

Energy diagram of catalyzed versus uncatalyzed reaction
(Figure 1.7)
E = enzyme, S = substrate
[EnzS] = enzyme-substrate complex, P = product
K = equilibrium constant for [ES]-formation
kcat = reaction rate constant for [ES] → E + P
Ea = activation energy, ≠ denotes a transition
state, KM = Michaelis-Menten constant,
v = reaction velocity.

Energy diagram for an enzyme-catalyzed enantioselective reaction
(e.g. kinetic resolution) (Figure 1.8)
≠
≠
≠ ≠ ≠
E = enzyme; A and B = enantiomeric substrates, P and Q = enantiomeric products;

[EA] and [EB] = diastereomeric enzyme-substrate complexes; ≠ denotes a transition state;
Δ Δ G, Δ Δ H and Δ Δ S = free energy, enthalpy and entropy difference, resp.;
R = gas constant, T = temperature, vA and vB = reaction velocities of A and B, resp.

Time course of ee of kinetic resolution
Kinetic resolution
kR
A P
kS
B Q
substrate
product A+B
e.e. [%] substrate P+Q e.e. [%]
100 product A+B 100
P+Q
50 50
0 0
0 50 100 0 50 100
conversion [%] conversion [%]
kR/kS = 5 kR/kS = 20
E-value for kinetic resolution (irreversibel)
kR
E Mit kR >=k S
kS
E (Enantioselectivity) [E mainly used in biocatalysis, otherwise 's']
Requirements
No side reactions
Reaction irreversibel
No loss of catalyst activity
No substrate or product inhibition
E = f(c,eeP) = f(c,eeS) = f(eeS,eeP)

The dependence of the selectivity and the conversion of the
reaction is:
For the product: For the substrate:
c=conversion, e.e.=enantiomeric excess of substrate (S) or product (P),

E=Enantiomeric ratio

E-Value for kinetic resolution
Online tool:
http://biocatalysis.uni-graz.at/biocatalysis-tools/enantio

‚Kinetic Resolution‘ and ‚Dynamic Kinetic Resolution‘
Kinetic resolution Dynamic kinetic resolution
A P A P
50% 100%
B Q B Q
50%

Dynamic Kinetic Resolution DKR
Course of ee during conversion for DKR compared to KR

100
Product 82%
75
e.e. [%]
69%
50
25
0
0 25 50 75 100
conversion [%]
Kinetic resolution: E = 10, at c = 50%: e.e. = 69%

Dynamic kinetic resolution: at c = 50%: e.e. = 82% (= constant)

Dynamic Kinetic Resolution
kfast
A P
krac krac
kslow
B Q

Dynamic Kinetic Resolution E =10
100
kfast/krac
80
60
e.e. [%]
40
20
100:1
0
0 20 40 60 80 100
c [%]
100
kfast/krac
80
60
e.e. [%]
40 10:1
20
100:1
0
0 20 40 60 80 100
c [%]
100
kfast/krac
80
1:1
60
e.e. [%]
40 10:1
20
100:1
0
0 20 40 60 80 100
c [%]
100
kfast/krac
80 1:6
1:1
60
e.e. [%]
40 10:1
20
100:1
0
0 20 40 60 80 100
c [%]
Kinetic resolution with in-situ racemization
(Figure 2.9)
krac of the product should be negligible

(ideally also kspont)

World production of amino acids using biocatalytic processes
(1980-2004)
(Table 2.1 in 6th edition)

Competing synthesis routes for L-Phe

Important enzymatic routes to enantiomerically pure α-amino acids

Enzymatic resolution of amino acid esters via the esterase method
(Scheme 2.11)
COOR1 COOH COOR1

2 esterase or protease R2HN NHR2
NHR +
R buffer R R
DL L D
R = alkyl or aryl; R1 = short-chain alkyl; R2 = H or acyl

Resolution of N-acetyl amino acid esters by α-chymotrypsin (protease)

Dynamic resolution of amino acid esters
(Scheme 2.13)

Kinetic and dynamic resolution of amino acid amides via the amidase
method
(Scheme 2.14)
COOH L-amino acid CONH2 D-amino acid COOH

amidase amidase
H2 N NH2 NH2
Brevundimonas Ochrobactrum
R diminuta R anthropi SV3 R
L L D D
yield >99% yield >99%
-amino -caprolactam
racemase L19V/L78T mutant

Enzymatic resolution of N-acyl amino acids via the acylase-method
(Scheme 2.15)

Enzymatic resolution of hydantoins via the hydantoinase method
(Scheme 2.17) H
O N
D-hydantoinase L-hydantoinase
O
buffer buffer
R N
H
DL-hydantoin
CO2H CO2H
NH NH2 H2 N HN
R R
O O
D-N-carbamoyl L-N-carbamoyl
amino acid amino acid
H2O H2 O
carbamoylase carbamoylase
NH3 + NH3 +
CO2 CO2
CO2H CO2H
NH2 H2 N
R R
D L

Semi-Synthetic β-lactam Antibiotics
65% of the World Market for Antibiotics
~15 US Billion of Annual Dosage Sales

Worldwide
Chemical synthesis of semi-synthetic β-lactam antibiotics has

dominated industrial production since their discovery in the 1960s.
More recently, an enzymatic process using Penicillin G Acylase (PGA)

is conducted on an industrial scale by DSM (Heerlen, ND) for
Cephalexin, Ampicillin, and Amoxicillin allowing for cheaper, more
environmentally benign manufacture.
Utility of Pen G acylase– substrate specificity
•Used to prepare 6-APA, a key industrial intermediate for semi-synthetic

antibiotic production.
•Used to synthesize Amoxicillin and Ampicillin.
Van de Sandt E et al. (2000). Chimica Oggi. 17:65

De Vroom E et al. (1999) Chimica Oggi-Chemistry Today. 17: 65
Arroyo M et al. (2003) Appl Microbiol Biotechnol. 60:507.
Enzymatic vs Chemical Process for 6-APA
N
H S
O N
O
penicillin G CO2H
1. Me3SiCl pen acylase
2. PCl5/ H2 O
PhNMe2/ -40°C
37°C
CH2Cl2
N S H2N S
6-APA
Cl N N
O O
CO2SiMe3 CO2H
Process Chemical Enzymatic

Reagents Me3SiCl (0.6) Pen acylase (1-2)
(kg/ kg 6-APA) PCl5 (1.2) PhNMe2 (1.6) NH3 (0.09)
n-BuOH (8.4 ltr), NH3 (0.2)
Solvent CH2Cl2 (8.4) H2O (2)
(ltr/kg 6-APA)
Source: Roger Sheldon, TU Delft
Types of substrates for esterases and proteases
(Scheme 2.20)
O
O
R 1 R1
O R1 OR3
* OR3
R2
* 3
*
O R
H R2 3 OR3
OR *
Type I R1
O
O
O O
O O 3 R1
R 1
R1 R * O R3
R2 O R3 *
*
H R2
Type II O R3 R1 * O R3
O O
prochiral substrates meso-forms
R1, R2 = alkyl, aryl; R3 = Me, Et; * = center of (pro)chirality

Mild ester hydrolysis by porcine liver esterase
(Scheme 2.21)

Desymmetrization of cyclic meso-diacetates by porcine liver esterase
and acetylcholine esterase
(Scheme 2.29)
ACE = acetylcholin esterase VO Enzyme Technology and Biocatalysis, Kroutil

Resolution of α-chiral ester using the isoenzyme A-PLE
(Scheme 2.33)
A-PLE
buffer +
Cl OMe Cl OH Cl OMe
E >200 R S
rac O O O
MeO
MeO(CH2)3O NH2 O O
N NH2
HO H
Aliskiren
used against high blood pressure

Resolution of α-substituted propionates by carboxylesterase NP
(Scheme 2.35)
R = Aryl-
+
Aryl S* COOH Aryl COOMe
Carboxyl
esterase e.e. 95-100%
NP
R COOMe * switch in CIP-sequence priority
buffer
rac
+
Aryl-O S* COOH Aryl-O COOMe
R = Aryl-O- e.e. 87-93%
Anti-inflammatory agents: S CO2H S CO2H

MeO
Naproxen Ibuprofen
Cl
N
Herbicides: F3C O O Cl O O
R R
CO2H CO2H
Fluazifop Diclofop
Tuning stereoselectivity of
enzymatic reactions

Optimization of porcine liver esterase-catalyzed hydrolysis by substrate
modification (substrate engineering)
(Scheme 2.40) pro-R
COOMe COOMe COOH
crude PLE crude PLE
S NHX NHX R NHX
buffer buffer
COOH COOMe COOMe
pro-S

Selectivity enhancement of porcine liver esterase by addition of organic
cosolvents (medium engineering)
(Scheme 2.42)

Screening for Pseudomonas aeruginosa lipase variants showing
enhanced enantioselectivities using a chromogenic surrogate substrate
(Scheme 2.43)

Esterase- and lipase-kinetics
(Figure 2.12)

Palomo, J. M.; Fuentes, M.; Fernández-Lorente, G.; Mateo,C.; Guisán, J.M.; Fernández-Lafuente,R.
Biomacromolecules. 2003, 4, 1-6.
Substrate types for lipases
(Scheme 2.45)
O R1
R1 O
R2 * O R3 R2 *
H H O R3
Type III Type IV
O O
3 O H R3O H
R
R S
medium large medium
large
>90% of cases true

'Kazlauskas-rule': preferred enantiomer
sequence rule order of large>medium assumed
R1, R2 = alkyl, aryl; R3 = n-Pr or longer; * = center of (pro)chirality

Mirror-image orientation of the catalytic machinery of Candida rugosa
lipase and the protease subtilisin and enantiocomplementary ester
hydrolysis using Mucor sp. lipase and α-chymotrypsin
(Scheme 2.46)

Dynamic resolution of an allylic alcohol ester using Pseudomonas sp.
lipase and PdII catalysis
(Scheme 2.62, modified)
OAc Pseudomonas sp. OH
lipase
Ph buffer Ph
R
PdII cat. e.e. 96%, yield 81%
L2Pd
+OAc Ph
PdII cat.
OAc
Ph
S
Lipase-catalysed resolution of an epoxy-ester on industrial scale
(Scheme 2.66)
O O O
CO2Me Serratia marcescens CO2Me CO2H
lipase
+
buffer / toluene e.e. 99.9% 2S,3R
MeO rac-trans MeO 2R,3S MeO
steps spontaneous
decarboxylation
CO2
O NMe2
N
CH=O
AcO
S MeO
Diltiazem
MeO
treatment of hypertension,
angina pectoris, and some types
of arrhythmia.

Lipase-assisted chemoenzymatic synthesis of pregabalin on industrial
scale
(Scheme 2.67)
to treat e.g. epilepsy and

generalized anxiety disorder
Naturally occuring organic nitriles
(Scheme 2.95)
O
N C
Ph (CH2)n C N R C N O R1
C N
Acetylenic nitriles R2
(plants) n = 1,2 (plants)
(microorganisms) R1 = (un)saturated C13 to C21
R2 = H, -O-Acyl
Cyanolipids (plants)
NH2 OMe
C N
C N H C N
N
N Aryl O glucose
N N O
ribose
Toyocamycin Ricinine Cyanoglucosides
(microbial antibiotic) (plants) (fungi, algae, plants, insects)

General pathways of the enzymatic hydrolysis of nitriles
(Scheme 2.96)

Chemoselective microbial hydrolysis of acrylonitrile
(Scheme 2.99)
O
CN microorganism O
NH2
H2O OH
> 400 g/L
Rhodococcus rhodochrous
Pseudomonas chlororapis
Brevibacterium sp.
polymers pigments water treatment super absorber

nappies
Fotos: www.panalytical.com; www.sorbisches-gymnasium.de; olgsblog.de

Chemoselective microbial hydrolysis of aromatic and heteroaromatic
nitriles yielding carboxamides (product concentrations)
(Scheme 2.100)
Rhodococcus
O
rhodochrous
R C N
H2O R NH2
N
F H
489 g/L 306 g/L 1045 g/L
X N
N
X = O: 522 g/L ortho: 977 g/L 985 g/L
X = S: 210 g/L meta: 1465 g/L
para: 1099 g/L
Regioselective microbial hydrolysis of dinitriles
(Scheme 2.102)
C N Rhodococcus C N
rhodochrous
2 H 2O NH3
C N CO2H
C N CO2H CO2H
Acremonium sp. chemical
2 H2O NH3 reduction
C N C N NH2
3.25 g/l Tranexamic acid
Pseudomonas O
N C chlororaphis B23
C N
N C NH2
H2O NH3
93% yield, 96% selectivity

Stereocomplementary enantioselective hydrolysis of α-hydroxynitriles
(Scheme 2.104)

1 Enzyme Technology and Biocatalysis OH1

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

1 Enzyme Technology and Biocatalysis OH1

Uploaded by

Copyright:

Available Formats

Enzyme Technology and Biocatalysis

Enzymtechnologie und Biokatalyse

Major parts of the lecture are based

VO Enzyme Technology and Biocatalysis, Kroutil

Biocatalysis: An Industrial Perspective, 2018

Biocatalysis: Biochemical Fundamentals and Application

VO Enzyme Technology and Biocatalysis, Kroutil

Introduction and Background Information

 Enzyme Properties and Nomenclature:

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

 Formation of Carbon-Carbon Bonds:

 Addition and Elimination Reactions:

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

>2000: DNA synthesis improved, 2003: one gene: 15000 Euro

>2015: DNA synthesis: one gene: 100 Euro;

ADHs, Aldolases, BVMO, P450

HNL, nitrile hydratase, nitrilase

Seifenfabrikant, 40, 4, (1920)

Enantioselectivity of lipase: Z.physiol. Chemie, 140, 203, (1904)

D-glucose D-sorbitol L-sorbose

VO Enzyme Technology and Biocatalysis, Kroutil

• Very important monomer for non-ionic and ionic polyacrylamides

• Global annual production

 „Enzymes are sensitive”.

VO Enzyme Technology and Biocatalysis, Kroutil

 Enzymes are environmentally acceptable.

VO Enzyme Technology and Biocatalysis, Kroutil

Not valid anymore:

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

E. Fischer, Einfluβ der Konfiguration auf die Wirkung der Enzyme.

VO Enzyme Technology and Biocatalysis, Kroutil

No Induced Fit Inactive Enzyme Induced Fit Active Enzyme

Koshland DE. Application of a theory of enzyme specificity to protein synthesis.

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

Active site: gets the reagents close together

1) Gets the reagents close together

4) Stabilises transition state

5) May provide alternative mechanism

6) Employing metal ions for activation

VO Enzyme Technology and Biocatalysis, Kroutil

7. Translocases* movement of ions or molecules across

VO Enzyme Technology and Biocatalysis, Kroutil

VO Enzyme Technology and Biocatalysis, Kroutil

M. Dias Gomes, J. M. Woodley, Molecules 2019, 24, 3573

M. Dias Gomes, J. M. Woodley, Molecules 2019, 24, 3573

• Reversible Unfolding • Irreversible Deactivation

• (Bio)catalyst Thresholds (pharma)

reviews: Rozzell, Chimica Oggi 1999 (6/7), 42-47

Volumetric activity: U/L of solution or culture broth

Specific activity: U/mg protein

each at specific conditions of: T, pH, buffer/salts, solvent,

J. Dong, E. Fernandez-Fueyo, F. Hollmann,* C. E. Paul, M. Pesic, S. Schmidt, Y. Wang, S. Younes, W.

Active site residues:

VO Enzyme Technology and Biocatalysis, Kroutil

1st half reaction:

VO Enzyme Technology and Biocatalysis, Kroutil

2nd half reaction:

VO Enzyme Technology and Biocatalysis, Kroutil