Amidase - Biochemistry - Lecture Slides, Slides of Biochemistry

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Module 10:

White Biotechnology

Lecture37:

White Biotechnology

(Contd.)

Amidase

H N N

NH

2

HN N

NH

2

HN NH

OH

E

H

O 2

NH

O

H

O

H

O

H

O 2

subs conc: 20g/LpH = 8 0; temp: 40 47

o^ C

pH = 8.0; temp: 40-

o^ C

reaction type: amide hydrolysis (kinetic resolution)catalyst: suspended whole cellsstrain:

Klebsiella terrigena

DSM 9174

Developer: Lonza AG, yield = 41%; ee = 99.4%

The product is used in the synthesis of orally active HIV protease inhibitor crixivan (Merck)

N

N

N

OH

HN

OH

O

O

NHtBu

Crixivan

H

N

H

2

S

Penicillin amidase

O

HN

N

S

O

CO H

O

OH

N

H

2

N

S

O

CO

H 2

E

CO

H 2

Penicillin G

6-amino penicillianic acid (6-APA)

subs conc: 100g/Lsubs

conc: 100g/L

pH = 8.0, temp: 37

o^ C

reaction type: carboxylic acid amide hydrolysisenzyme: penicillin amidohydrolasestrain:

Arthobacter viscosus

De eloper: Dr Vig Medicaments IndiaDe

veloper: Dr. Vig Medicaments, India yield = 99%

6-APA is used as an intermediate for the manufacture of semi-synthetic penicillins

Penicillin amidase

H N

S

OH

N

H

2

S

E

O

N

O

CO

H 2

O

N

O

CO

H 2

Penicillin G

6 amino penicillianic acid (6 APA)6-amino penicillianic acid (6-APA)

subs conc: 100g/LpH = 8.4, temp: 30-

o C

pH

8.4, temp: 30 36 C

reaction type: carboxylic acid amide hydrolysisenzyme: penicillin amidohydrolasestrain:

Bacillus megaterium

Developer: Asahi Kasei Chemicals Corporation

i ld

yield = 86%%

6-APA is used as an intermediate for the manufacture of semi-synthetic penicillins

O

O

Penicillin amidase

O

NH

O

OEt

O

N

O

OEt

N

H

O

OEt

E

H

SiMe

3

H

SiMe

3

N

H

2

SiMe

3

subs conc: 100g/L; pH = 5.4-5.6; temp: 25

o^ C

reaction type: amide hydrolysisreaction type: amide hydrolysiscatalyst: immobilized enzymestrain: Penicillin G amidohydrolase expressed in E. colideveloper: Pfizer Inc. yield = 43-46% (amine); ee = 99.5%

The product

β

-amino acid is a chiral synthon for the synthesis of

xemilofiban hydrochloride, an anti-platelet agent.

Xemilofiban

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Aminoacylase

N

N

H

A CO

H 2

N

CO

H 2

N

CO NHAc

H 2

E

Aminoacylase

N

S

NHAc

N

S

NH

2

S

NHAc

H

O 2

subs conc:150 g/L; pH = 7.0reaction type: carboxylic acid amide hydrolysiscatalyst: immobilized enzyme

N

t l L

i^

id

id h d

l

enzyme: N-acetyl-L-aminoacid amidohydrolasestrain:

Aspergillus niger

developer: Celltech Group plc, ee = 99%

Th

d

t (L thi

l l l

i^

) i

d

t^

f^

tih

t^

i

The product (L-thiazolylalanine) is used as a component of antihypertensiveinhibitors of the enzyme renin, where it acts as a mimic of histidine.

N

CO

H 2

O

N

O

N

CO NHAc

H 2

Ac

O, OH 2

N

S

NHAc

N

O

S

Me

S

NHAc

The unwanted enantiomer can be racemized via an oxazolinone that tautomerizes to the enol

OH

N

O

N

S

Me

CO

H 2

CO

H 2

The process can be applied to other unnatural alanine derivatives such as;

NH

2

NH

2

NC

CO H

N

NH

CO 2

H 2

NH

CO 2

H 2

Aminoacylase

S

NHAc

CO

H 2

S

NHA

CO

H 2

S

NH

CO

H 2

E Co

2+

NHAc

NHAc

NH

2

Co

subs conc: 98 g/L[Co

2+

]: 0.029 g/L (activator)

[^

]^

g

(^

)

pH = 7.0; temp: 37

o C

strain:

Aspergillus oryzae

developer: Degussa AG; yield = 80%; ee = 99.5%

*** Co**

2+

is added to increase the operational stability of the acylase.

*** L aminoacids are used for parenteral nutrition feed and food additives cosmetics pesticides* L-aminoacids are used for parenteral nutrition, feed and food additives, cosmetics, pesticidesand intermediates for pharmaceuticals and chiral synthons for organic synthesis.* Several proteinogenic and non-proteinogenic amino acids are produced in the same way.**

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