Haloalkane Dehalogenase - Biochemistry - Lecture Slides, Slides of Biochemistry

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Module 10:

White Biotechnology

Lecture39:

White Biotechnology

(Contd.)

Haloalkane dehalogenase

Cl

OH

OH

Cl

OH

OH

E Cl

g

OH

OH

O

-Cl+NAD

-NADH

subs conc: 100g/L; pH = 6.8; temp: 30

o

C

reaction type: kinetic resolution coupled with dehalogenation/oxidationcatalyst: immobilized whole cellsstrain:

Alcaligenes

sp. or

Pseudomonas

sp. depending on substrate

developer: Daiso Co. Ltd; yield = 50%; ee = 99.5%

Some related optically active halohydrins can also be resolved

Cl

OH

CN

Cl

OH

CO

2

R

R

OH

OH

O

H

O

E

Halohydrin dehalogenase

Cl

OH

O

OEt

OH

O

OEt

NC

E

NaCN, H

2

SO

4

-NaCl

NaCl

subs conc: 80g/L; pH = 7.3; temp: 40

o

C

reaction type: cyanationcatalyst: soluble recombinant enzymescatalyst: soluble recombinant enzymesenzyme: mutant halohydrin dehaloggenasedeveloper: Codexis Inc.; ee = 99.9%

The product is a key chiral intermediate for atrovastatin

C

H

O

O

H

O

Pyruvate decarboxylase (lyase)

O

CHO

O

H

E

acetaldehydeacetaldehyde

benzaldehyde

phenylacetylcarbinol (PAC)

subs conc: 3.3 g/L; medium: waterreaction type: Asymmetric C-C bond formationreaction type: Asymmetric C C bond formationcatalyst: whole cellsstrain:

Saccharomyces cerevisiae

developer: Krebs Biochemicals & Industries Ltd.

O

• Mechanism similar like crossed benzoin condensation reaction, ThDP acts as a cofactor* PAC can be converted chemically in a two step process to D-pseudoephedrine

O

H

O

O

H

NHMe

O

H

NHMe

MeNH

2

Ac

O 2

Pt/H

2

Hydroxynitrile lyase/ oxynitrilase

O

CHO

O

OH

CN

E

y

y

y

y

HCN

b

/^

l

subs conc: 198.22 g/molmedium: biphasic solvent system: aq buffer/tert-butyl methyl etherreaction type: nucleophilic addition of cyanideenzyme: hydroxynitrile lyasestrain:

Hevea brasiliensis

(rubber tree)

strain:

Hevea

brasiliensis

(rubber

tree)

developer: DSM, ee = 98%; yield = 98%

* the product is an intermediate for the manufactute of pyrethroids insecticides

cypermethrin

deltamethrin

Flucythrinate

Fumarase

CO

2

H

HO

2

C

OH

HO

2

C

CO

2

H

E

+H O

Fumarase

2

+H

2

O

Fumaric acid

L-Malic acid

subs conc: 150 g/L; pH

= 8 0; temp: 25

o

C

subs conc: 150 g/L; pH

8.0; temp: 25 C

reaction type: C-O bond formationcatalyst: suspended whole cellsenzyme: (S)-malate hydrolasestrain:

Corynebacterium clutamicum

d

l^

A

i^

G

bH

i ld

d

eveloper: Amino GmbH; yield = 85%

* About 40,00 ton of malic acid are used worldwide annually as a food supplement,cosmetics and pharmaceutical industry* L-malic acid is used as a component of amino acid infusions for parenteral nutrion.

O

H

O

T

B

S

O

T

B

S

(R)-3-hydroxy-butyric acid is used for the synthesis of a carbapenem intermediate O

H

CO

2

Me

OTBS

CHO

OTBS

OSiR

3

ClSO

2

NCO

OTBS

ClSO

2

NCO

reduction NH

OAc

OO

Carbapenem intermediate

E

Enoyl-CoA hydratase

CO

2

H

O H

CO

2

H

E H

2

O

pH = 7 2-7 5; temp: 30-

oC

(β

)-hydroxy-isobutyric acid

pH

= 7.2-7.5; temp: 30-33 C

reaction type: hydrationcatalyst: whole cellsenzyme: acyl-CoA dehydrogenase (E1), enoyl-CoA hydratase (E2)strain:

Candida rugosa

IFO 0750 M

developer: Kanegafuchi; ee = 98%

p

g

;

The biotransformation occurs in three steps. First the aliphatic acid is dehydrogenated to α,β

-unsaturated acid. In a subsequent step, enantioselective hydration and hydrolysis takes place

CO

H 2

SCoA

O

SCoA

O

E

O H

SC A

O

O H

CO H

E

O H

SCoA

C

O

2

H