3.5.5.1: nitrilase

4.6fold increased specific activity compared to the wild type enzyme

F168K

F168L

2.4fold improved specific activity

F168M

3fold increased specific activity compared to the wild type enzyme

F168T

1.7fold increased specific activity compared to the wild type enzyme

F168T/L201Q

3.1fold increased specific activity compared to the wild type enzyme

F168V

F168V/L201N

15.3fold increased specific activity compared to the wild type enzyme

F168V/T201N/S192F/M191T/F192S

Q1W2L4

the mutant enzyme shows 136% improvement in specific activity. Vmax and kcat are enhanced 1.23fold and 1.23fold, while the Km is decreased 1.53fold

756946

F174I

1.8fold improved specific activity

L201A

1.9fold increased specific activity compared to the wild type enzyme

L201G

1.5fold increased specific activity compared to the wild type enzyme

L201H

2.7fold increased specific activity compared to the wild type enzyme

L201K

3.5fold increased specific activity compared to the wild type enzyme

L201N

5.5fold increased specific activity compared to the wild type enzyme

L201Q

4.9fold increased specific activity compared to the wild type enzyme

L201S

2.2fold increased specific activity compared to the wild type enzyme

L201T

2fold increased specific activity compared to the wild type enzyme

Q339K

t1/2 at 45°C is 16.4 min, compared to 12.5 min for the wild-type enzyme

Q343K

t1/2 at 45°C is 22.8 min, compared to 12.5 min for the wild-type enzyme

T201F

t1/2 at 45°C is 169 min, compared to 12.5 min for the wild-type enzyme

T201F/Q339K/Q343K

t1/2 at 45°C is 180 min, compared to 12.5 min for the wild-type enzyme. The mutant enzyme exhibits about 14fold longer half-life at 45°C compared to the wild-type enzyme

T201I

t1/2 at 45°C is 55 min, compared to 12.5 min for the wild-type enzyme

T201L

t1/2 at 45°C is 119 min, compared to 12.5 min for the wild-type enzyme

T201W

t1/2 at 45°C is 135 min, compared to 12.5 min for the wild-type enzyme

T210A

7.3fold improved specific activity

T210I

1.8fold improved specific activity

Y65C

1.8fold improved specific activity

F168K

F168M

3fold increased specific activity compared to the wild type enzyme

F168T

1.7fold increased specific activity compared to the wild type enzyme

F168V

4.1fold increased specific activity compared to the wild type enzyme

F174I

1.8fold improved specific activity

L201A

1.9fold increased specific activity compared to the wild type enzyme

T210A

7.3fold improved specific activity

T210I

1.8fold improved specific activity

Y65C

1.8fold improved specific activity

Q339K

Acidovorax facilis ZJB09122

t1/2 at 45°C is 16.4 min, compared to 12.5 min for the wild-type enzyme

T201F

Acidovorax facilis ZJB09122

t1/2 at 45°C is 169 min, compared to 12.5 min for the wild-type enzyme

T201I

Acidovorax facilis ZJB09122

t1/2 at 45°C is 55 min, compared to 12.5 min for the wild-type enzyme

T201L

Acidovorax facilis ZJB09122

t1/2 at 45°C is 119 min, compared to 12.5 min for the wild-type enzyme

T201W

Acidovorax facilis ZJB09122

t1/2 at 45°C is 135 min, compared to 12.5 min for the wild-type enzyme

A198V

the mutant enzyme shows 2.4fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/I290F

the mutant enzyme shows 1.2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 140% activity at pH 4.5 compared to the wild type enzyme

A198V/I290F/H135Y

the mutant enzyme shows 1.7fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 150% activity at pH 4.5 compared to the wild type enzyme

A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T

the mutant enzyme shows 2.8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 740% activity at pH 4.5 compared to the wild type enzyme

A198V/I290F/Y177C/A285T

the mutant enzyme shows 0.9fold conversion rate of 2-phenylpropionitrile at pH 7.5 and 240% activity at pH 4.5 compared to the wild type enzyme

A198V/I290F/Y213H/T350S

the mutant enzyme shows 2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 200% activity at pH 4.5 compared to the wild type enzyme

A198V/Q197H

the mutant enzyme shows 2.9fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/Q197H/L176M

the mutant enzyme shows 6.5fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/Q197H/L176M/V306I

the mutant enzyme shows 8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

I290F/Q3L

the mutant enzyme shows 2.3fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 100% activity at pH 4.5 compared to the wild type enzyme

A198V

the mutant enzyme shows 2.4fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/Q197H

the mutant enzyme shows 2.9fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/Q197H/L176M

the mutant enzyme shows 6.5fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

A198V/Q197H/L176M/V306I

the mutant enzyme shows 8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme

I290F/Q3L

the mutant enzyme shows 2.3fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 100% activity at pH 4.5 compared to the wild type enzyme

A200G

37% increased activity compared to the wild type enzyme

C179A

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C179N

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C180A

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C180N

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C186A

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C186N

site-directed mutagenesis, completely inactive mutant, determined with substrate indole-3-acetonitrile

C236S

mutant has a nearly 3fold increase in reactivity

H101Q/C239S/D246E

47% increased activity compared to the wild type enzyme

I29L

113% increased activity compared to the wild type enzyme

P172S/C236S/V291I

84% increased activity compared to the wild type enzyme

R89S/G179G

89% increased activity compared to the wild type enzyme

T28S

30% increased activity compared to the wild type enzyme

E327G/Q86R/E96G/D254E/E35K/Q322R/E327G/Q86R/E6G/D254E/E327G

Bacillus pumilus

B3GNT7

mutamt enzyme shows activity at pH 10. The wild-type enzyme exhibits the optimum activity at pH 8 and is not able to hydrolyze HCN at pH higher than 9. However, cyanide wastes should be maintained at alkaline pH to prevent HCN release. Therefore, the CynD from Bacillus pumilus was engineered for an increased activity under these conditions (pH 9-10), and several mutants were shown to meet this requirement

C182A

inactive mutant

F80H

a single amino acid residue in position 80 (H80 in isoenzyme CrNIT1 and F80 in isoenzyme CrNIT2) is identified, that when exchanged, leads to an almost complete switch in substrate preference ratio. Position 80 exerts an influence on the helical twist

H80F

C191A

decrease/loss of labeling for activity-based proteomics probes

F154A

decrease/loss of labeling for activity-based proteomics probes

F164A

decrease/loss of labeling for activity-based proteomics probes

F192A

decrease/loss of labeling for activity-based proteomics probes

F217A

decrease/loss of labeling for activity-based proteomics probes

Y192R

increase in labeling with proteomics probe Leu-Glu-alpha-CA probe

Y54A

site-directed mutagenesis, the mutant enzyme converts 2-hydroxy-2-phenylpropionitrile with about the same activity as that of the wild-type enzyme, but forms significantly reduced amounts of amides from mandelonitrile and acetophenone cyanohydrin, it shows different kinetics of acetophenone cyanohydrin conversion and product formation compared to the wild-type

Y54F

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54M

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54P

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54V

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54A

Y54F

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54M

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54P

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

Y54V

site-directed mutagenesis, altered substrate specificity and enantioselectivity compared to the wild-type enzyme, overview

C165A

C165S

G103A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

I104A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

K9A

increased specific activities

L79A

increased specific activities

M114A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

N164A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

Q116A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

R129A

R129H

the mutant enzyme is active only for meta-substituted benzonitriles

R129K

the mutant enzyme is active only for meta-substituted benzonitriles

R130A

the mutant exhibits increased specific activity with 3-methylbenzonitrile and benzonitrile compared to the wild type enzyme

S188A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

T115A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

V49A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

Y142A

Y142F

mutant shows slightly lower kcat/Km values compared to the wild type enzyme

Y142I

low specific activities

Y142L

low specific activities

Y142S

mutant exhibits slightly higher kcat/Km values for aromatic nitriles and shows no activity toward aliphatic nitriles

Y142V

decreased activity

Y175A

increased specific activities

Q116A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

R129A

the mutant exhibits no activity with 3-methylbenzonitrile and benzonitrile

R130A

the mutant exhibits increased specific activity with 3-methylbenzonitrile and benzonitrile compared to the wild type enzyme

S188A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

T115A

the mutant exhibits decreased specific activity with 3-methylbenzonitrile and increased specific activity with benzonitrile compared to the wild type enzyme

K96R

E142A

mutant enzyme shows almost no activity

F193A

mutant enzyme shows almost no activity

F202V

the mutant enzyme shows 6.25fold improvement in activity towards 3-(4-chlorophenyl) glutaronitrile relative to that of wild-type enzyme

F64A

lower activity as compared to wild-type enzyme

H141A

fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity

I201A

fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity

M197A

fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity

P194A

fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity

P194A/I201A/F202V

the mutant enzyme with a larger substrate-binding pocket displays significantly enhanced catalytic activity and enantioselectivity (S, 99% ee) toward 3-(4-chlorophenyl) glutaronitrile and other 3-substituted glutaronitriles

P202A

fold increase in activity as compared to wild-type enzyme. No tradeoff occurred in stereoselectivity

Q205A

lower activity as compared to wild-type enzyme

T139A

mutant enzyme shows almost no activity

V198A

lower activity as compared to wild-type enzyme. The enantiomeric excess value of mutant enzyme V198A is 75% (S)

W170A

mutant enzyme shows almost no activity

Y140A

lower activity as compared to wild-type enzyme

Y59A

mutant enzyme shows almost no activity