3.5.1.93: glutaryl-7-aminocephalosporanic-acid acylase

no hydrolysis of glutaryl-7-aminocephalosporanic acid

H309A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

S125A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

W173A

kcat/KM is 2.1fold lower than wild-type value

Y126A

kcat/KM is 9.2 fold lower than wild-type value

Y126F

kcat/KM is identical to wild-type value

Y57A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

Y57F

no hydrolysis of glutaryl-7-aminocephalosporanic acid

D264A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

H309A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

S125A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

Y57A

no hydrolysis of glutaryl-7-aminocephalosporanic acid

Y57F

no hydrolysis of glutaryl-7-aminocephalosporanic acid

F177H

F177P

F177T

L24F

beta-subunit mutant, no activity with glutaryl-7-aminocephalosporanic acid, activity with cephalosporin C is 25.9% of the wild-type activity

L24G

beta-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 27% of the wild-type activity, no activity with cephalosporin C

L24K

beta-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 1.1% of the wild-type activity, no activity with cephalosporin C

L24R

beta-subunit mutant, no activity with glutaryl-7-aminocephalosporanic acid, activity with cephalosporin C is 87.7% of the wild-type activity

L24W

beta-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 11.3% of the wild-type activity, activity with cephalosporin C is 98.8% of wild-type activity

Q50L

Q50M

Q50M/F58A

beta-subunit mutation, activity with cephalosporin C is 180% of wild-type activity

Q50M/F58D

beta-subunit mutation, activity with cephalosporin C is 130% of wild-type activity

Q50M/F58G

beta-subunit mutation, activity with cephalosporin C is 290% of wild-type activity

Q50M/F58H

beta-subunit mutation, activity with cephalosporin C is 160% of wild-type activity

Q50M/F58I

beta-subunit mutation, activity with cephalosporin C is 150% of wild-type activity

Q50M/F58L

beta-subunit mutation, activity with cephalosporin C is 120% of wild-type activity

Q50M/F58P

beta-subunit mutation, activity with cephalosporin C is 240% of wild-type activity

Q50M/F58S

beta-subunit mutation, activity with cephalosporin C is 260% of wild-type activity

Q50M/F58T

beta-subunit mutation, activity with cephalosporin C is 230% of wild-type activity

Q50M/Y149F

Q50M mutation in beta-subunit, Y149F mutation in alpha-subunit, activity with cephalosporin C is 157% of wild-type activity

Q50M/Y149K

Q50M mutation in beta-subunit, Y149K mutation in alpha-subunit, activity with cephalosporin C is 600% of wild-type activity

Q50M/Y149K/F177G

beta-subunit mutation, activity with cephalosporin C is 787% of wild-type activity

Q50M/Y149K/F177P

beta-subunit mutation, activity with cephalosporin C is 301% of wild-type activity

Q50M/Y149K/F177S

beta-subunit mutation, activity with cephalosporin C is 456% of wild-type activity

Q50M/Y149K/M145A

Q50M and Y149K are mutation in the beta-subunit, M145A is a mutation in the alpha-subunit, activity with cephalosporin C is 335% of wild-type activity

Q50M/Y149K/M145L

Q50M and Y149K are mutations in the beta-subunit, M145L is a mutation in the alpha-subunit, activity with cephalosporin C is 704% of wild-type activity

Q50M/Y149K/M145P

Q50M and Y149K are mutations in the beta-subunit, M145P is a mutation in the alpha-subunit, activity with cephalosporin C is 442% of wild-type activity

Q50M/Y149K/M145T

Q50M and Y149K are mutation in the beta-subunit, M145T is a mutation in the alpha-subunit, activity with cephalosporin C is 433% of wild-type activity

Q50M/Y149K/Y33N

beta-subunit mutation, activity with cephalosporin C is 460% of wild-type activity

Q50M/Y149K/Y33S

beta-subunit mutation, activity with cephalosporin C is 568% of wild-type activity

Q50M/Y149K/Y33T

beta-subunit mutation, activity with cephalosporin C is 424% of wild-type activity

Q50M/Y33D

beta-subunit mutant, activity with cephalosporin C is 450% of wild-type activity

Q50M/Y33G

beta-subunit mutant, activity with cephalosporin C is 70% of wild-type activity

Q50M/Y33H

beta-subunit mutant, activity with cephalosporin C is 101% of wild-type activity

Q50M/Y33L

beta-subunit mutant, activity with cephalosporin C is 202% of wild-type activity

Q50M/Y33N

beta-subunit mutant, activity with cephalosporin C is 350% of wild-type activity

Q50M/Y33P

beta-subunit mutant, activity with cephalosporin C is 99% of wild-type activity

Q50M/Y33S

beta-subunit mutant, activity with cephalosporin C is 60% of wild-type activity

Q50M/Y33T

beta-subunit mutant, activity with cephalosporin C is 109% of wild-type activity

Q50M/Y33V

beta-subunit mutant, activity with cephalosporin C is 135% of wild-type activity

Q50R

Q50T

Q50Y

R155G

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 88% of the wild-type activity

R57C

R57I

R57K

R57S

beta-subunit mutant, no activity with glutaryl-7-aminocephalosporanic acid and cephalosporin C

S152G

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 75.4% of the wild-type activity, activity with cephalosporin C is 38.6% of wild-type activity

S152H

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 63.7% of the wild-type activity, activity with cephalosporin C is 11.9% of wild-type activity

S152N

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 78% of the wild-type activity, activity with cephalosporin C is 24.8% of wild-type activity

S152P

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 76.2% of the wild-type activity, activity with cephalosporin C is 60.4% of wild-type activity

S152T

alpha-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 81.4% of the wild-type activity, activity with cephalosporin C is 34.7% of wild-type activity

S1A

S1betaC

no activity with glutaryl-7-aminocephalosporanic acid and cephalosporin C

S1C

Q9L5D6

beta-subunit mutant, no intermolecular cleavage in posttranslational modification, no activity with glutaryl-7-aminocephalosporanic acid

662111

V70G

beta-subunit mutant, no activity with glutaryl-7-aminocephalosporanic acid, activity with cephalosporin C is 32.1% of the wild-type activity

V70S

beta-subunit mutant, activity with glutaryl-7-aminocephalosporanic acid is 28.8% of the wild-type activity, no activity with cephalosporin C

Y149C

Y149G

Y149L

Y149N

Y149P

Y149R

Y33F

Y33I

Y33S

A215E

Vmax/Km for cephalosporin C is 1.2fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 7.6fold lower than wild-type value

A215F

Vmax/Km for cephalosporin C is 2fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 3.9fold lower than wild-type value

A215L

Vmax/Km for cephalosporin C is 1.3fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 4.2fold lower than wild-type value

A215V

Vmax/Km for cephalosporin C is 1.5fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 1.5fold lower than wild-type value

A215Y

Vmax/Km for cephalosporin C is 4.3fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 1.6fold lower than wild-type value

A215Y/H296S

Vmax/Km for cephalosporin C is 1.2fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 25.1fold lower than wild-type value

A215Y/H296S/H309S

Vmax/Km for cephalosporin C is 3.5fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 13.7fold lower than wild-type value

A215Y/H309S

Vmax/Km for cephalosporin C is 3.8fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 2.9fold higher than wild-type value

A271F

118% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 56.2% of wild-type activity with cephalosporin C as substrate

A271L

104% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 100% of wild-type activity with cephalosporin C as substrate

A271Y

101% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 122% of wild-type activity with cephalosporin C as substrate

A675G

A677A

site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme

A677F

site-directed mutagenesis, the mutant shows 24.6% decreased activity compared to the wild-type enzyme

C102S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C199S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C277S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C305S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C. Expression in Escherichia coli is 2-3fold higher than that of the wild-type enzyme

C391S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C493S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C496S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

C748S

mutant enzyme retains activity towards glutaryl-7-aminocephalosporanic acid and cephalosporin C

D286betaA

beta-subunit mutant enzyme, KM-value is 1.1fold lower than the wild-type value, turnover-number is 1.03fold higher than the wild-type value. Half-life at 37°C is 53.9 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 9.0 compared to pH 7.0 for wild-type enzyme

D416Y

Vmax/Km for cephalosporin C is 1.5fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 5.2fold lower than wild-type value

D416Y/H417Y

Vmax/Km for cephalosporin C is 5.3fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 151fold lower than wild-type value

E159D

the mutant is still active, but significantly retarded in the second autocleavage compared to the wild type enzyme

E89A/A215Y

Vmax/Km for cephalosporin C is 6fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 25.2fold lower than wild-type value

F229L

turnover number for adipyl-7-aminodesacetoxycephalosporanic acid is nealy identical to wild-type value, 1.8fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.4fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 1.45fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme

662118

F270M

Vmax/Km for cephalosporin C is 1.8fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 1.7fold lower than wild-type value

F375L

F375M

decreased activity compared to the wild type enzyme

G160A

the mutant performs the second autocleavage more slowly than the wild type enzyme

G160L

the mutant performs the second autocleavage more slowly than the wild type enzyme

H296A

site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme

H296F

Vmax/Km for cephalosporin C is 6fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 50.3fold lower than wild-type value

H296N

Vmax/Km for cephalosporin C is 1.2fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is fold lower than wild-type value

H296S

Vmax/Km for cephalosporin C is 1.7fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 10.1fold lower than wild-type value

H296S/H309S

H296S/H417M

Vmax/Km for cephalosporin C is 2fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 151fold lower than wild-type value

H296T

Vmax/Km for cephalosporin C is 1.5fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 5.2fold lower than wild-type value

H309A

site-directed mutagenesis

H309L

site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme

H309S

H309V

H309Y

Vmax/Km for cephalosporin C is 1.5fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 16.8fold lower than wild-type value

H417Y

Vmax/Km for cephalosporin C is more than 6fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 4.4fold lower than wild-type value

H57betaS/H70betaS

H57betaS/H70betaS/A215alphaY

site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme

H57betaS/H70betaS/F58betaD

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/F58betaI

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/F72betaR

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/I176betaK

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/I176betaQ

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/I176betaT

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/L154betaY

H57betaS/H70betaS/L154betaY/M165alphaS

site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme

H57betaS/H70betaS/L175betaT

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/M165alphaS

site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme

H57betaS/H70betaS/M165alphaS/I176betaS

site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme

H57betaS/H70betaS/R24betaP

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

H57betaS/H70betaS/V25betaR

random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme

I44V/E49stop/D416Y/H417Y

Vmax/Km for cephalosporin C is more than 6fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is more than 1510fold lower than wild-type value

K100Q

81% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 106% of wild-type activity with cephalosporin C as substrate

K114Q

86% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 101% of wild-type activity with cephalosporin C as substrate

K170Q

130% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 95.6% of wild-type activity with cephalosporin C as substrate

K187Q

113% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 91.1% of wild-type activity with cephalosporin C as substrate

K198betaA

beta-subunit mutant enzyme, KM-value is 1.04fold higher than the wild-type value, turnover-number is 1.1fold lower than the wild-type value. Half-life at 37°C is 107.5 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 8.0 compared to pH 7.0 for wild-type enzyme. Mutant enzyme shows higher stability at alkaline pH than wild-type enzyme

K255Q

107% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 97% of wild-type activity with cephalosporin C as substrate

K301Q

101% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate

K44Q

102% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 111% of wild-type activity with cephalosporin C as substrate

K507Q

102% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 113.9% of wild-type activity with cephalosporin C as substrate

K629Q

94.2% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate

K73Q

46.9% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 47% of wild-type activity with cephalosporin C as substrate

L666F

L677A

the mutant exhibits significantly reduced specific enzymatic activity compared to the wild type enzyme

M116A

at pH 7.5, 76.9% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 95.8% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 108% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate

M157A

at pH 7.5, 70.9% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 58% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 65% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate. Stability after treatment with H2O2 is lower than that of wild-type enzyme

M164A

at pH 7.5, 167% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 104% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 86.6% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate. Stability after treatment with H2O2 is higher than that of wild-type enzyme. The ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.2fold lower than the wild-type ratio. The ratio of turnover number to KM-value for glutaryl-7-aminocephalosporanic acid is 2.16fold higher than the wild-type ratio

M164F

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.3fold lower than the wild-type ratio

M164G

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.3fold lower than the wild-type ratio. The ratio of turnover number to KM-value for glutaryl-7-aminocephalosporanic acid is 2.23fold higher than the wild-type ratio

M164L

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 2.1fold lower than the wild-type ratio

M164N

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.4fold lower than the wild-type ratio. The ratio of turnover number to KM-value for glutaryl-7-aminocephalosporanic acid is 1.81fold higher than the wild-type ratio

M164P

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.3fold lower than the wild-type ratio

M164Q

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 5.3fold lower than the wild-type ratio. The ratio of turnover number to KM-value for glutaryl-7-aminocephalosporanic acid is nearly identical to the wild-type ratio

M164S

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.5fold lower than the wild-type ratio

M164T

the ratio of specific activity with cephalosporin C as substrate to that with glutaryl-7-aminocephalosporanic acid as substrate is 1.4fold lower than the wild-type ratio

M165alphaS/H57betaS/H70betaS

site-directed mutagenesis, the engineered mutant shows higher activity on both cephalosporin C and (7R)-7-(4-carboxybutanamido)cephalosporanate compared to the double H57betaS/H70betaS mutant

733289

M174A

at pH 7.5, 96.5% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 109% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 122% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate

M227A

at pH 7.5, 106% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 74.2% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 105% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate. Stability after treatment with H2O2 is lower than that of wild-type enzyme

M269F

98.2% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 165% of wild-type activity with cephalosporin C as substrate. The ratio of turnover number to Km-value with cephalosporin C as substrate is 1.8fold higher than the wild-type ratio

M269L

92.5% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 107.9% of wild-type activity with cephalosporin C as substrate

M269Y

91.5% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 155% of wild-type activity with cephalosporin C as substrate. The ratio of turnover number to Km-value with cephalosporin C as substrate is 1.6fold higher than the wild-type ratio

M269Y/C305S

1.6fold higher activity with cephalosporin C than wild-type enzyme

M270F

site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme

M271V/Q291K/T374S

1.3fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 1.4fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.2fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 1.3fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme

662118

M98A

at pH 7.5, 126% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 83.1% of the specific activity measured with wild-type enzyme with glutaryl-7-aminocephalosporanic acid as substrate. At pH 8.7, 91.2% of the specific activity measured with wild-type enzyme with cephalosporin C as substrate

N266H

N266M

decreased activity compared to the wild type enzyme

N266Q

decreased activity compared to the wild type enzyme

N266Q/F375L

decreased activity compared to the wild type enzyme

N266S

1.4fold decrease in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 2.3fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.95fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 2.8fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme

P295A

Q50betaN/K198betaA

beta-subunit mutant enzyme, KM-value is 2.14fold lower than the wild-type value, turnover-number is 1.45fold higher than the wild-type value, immobilized mutant enzyme shows 34.2% increase in specific activity compared to immobilized wild-type enzyme. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme

Q50N

beta-subunit mutant enzyme, KM-value is 2.05fold lower than the wild-type value, turnover-number is 1.45fold higher than the wild-type value. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme

R121A

beta-subunit mutant enzyme, KM-value is identical to the wild-type value, turnover-number is nearly identical to wild-type value. Half-life at 37°C is 88.3 h compared to 68.1 h for wild-type enzyme. Optimal pH is pH 6.0 compared to pH 7.0 for wild-type enzyme

R263A

site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme

R263L

site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme

S170C

the mutant simultaneously loses the activities for both the second autocleavage and substrate hydrolysis

S22P/T394P/D416Y/H417Y

Vmax/Km for cephalosporin C is more than 6fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is more than 1510fold lower than wild-type value

S293C

1.21% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, no activity with cephalosporin C as substrate

Y151F

alpha-subunit mutant enzyme, KM-value is 1.9fold lower than the wild-type value, turnover-number is 1.17fold higher than the wild-type value. Ability of the mutant enzyme to hydrolyze adipoyl 6-aminopenicillinic acid is improved, compared to activity of wild-type enzyme

Y151F/Q50N

mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit, KM-value is 1.2fold lower than the wild-type value, turnover-number is 1.09fold higher than the wild-type value

Y178F/F375H

the mutation synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36fold, The activity of this double mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid

Y178H

decreased activity compared to the wild type enzyme

Y178H/N266H

decreased activity compared to the wild type enzyme

Y178H/N266M

decreased activity compared to the wild type enzyme

Y178H/N266Q/F375L

decreased activity compared to the wild type enzyme

Y215Y/F270S

Vmax/Km for cephalosporin C is 2.5fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 1.2fold lower than wild-type value

Y270A

70.6% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 24.3% of wild-type activity with cephalosporin C as substrate

Y270E

no activity with glutaryl-7-aminocephalosporanic acid or cephalosporin C as substrate

Y270F

50.4% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 46.1% of wild-type activity with cephalosporin C as substrate. The ratio of turnover number to Km-value with cephalosporin C as substrate is 2.6fold lower than the wild-type ratio

Y270L

28.1% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 32.3% of wild-type activity with cephalosporin C as substrate

Y270S

61.7% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 28.3% of wild-type activity with cephalosporin C as substrate

Y271F

Vmax/Km for cephalosporin C is 1.5fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 5.4fold lower than wild-type value

Y27V

Vmax/Km for cephalosporin C is 1.5fold lower than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 2.1fold lower than wild-type value

E455D

the ratio of turnover number to Km-value of the beta-subunit mutant enzyme is 4.5fold lower than the wild-type ratio

E455L

the ratio of turnover number to Km-value of the beta-subunit mutant enzyme is 34fold lower than the wild-type ratio

E455Q

the ratio of turnover number to Km-value of the beta-subunit mutant enzyme is 6.1fold lower than the wild-type ratio

H23D

the ratio of turnover number to Km-value of the beta-subunit mutant enzyme is 9.7fold lower than to the wild-type ratio

H23Q

the ratio of turnover number to Km-value of the beta-subunit mutant enzyme is 4.3fold lower than the wild-type ratio

W4A

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.6fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 2.2fold lower than wild-type value, t1/2 at 37°C is 9.8fold lower than wild-type value

W4F

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.8fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 1.6fold lower than wild-type value, t1/2 at 37°C is 37.5fold lower than wild-type value

W4H

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.9fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 1.4fold lower than wild-type value, t1/2 at 37°C is 1.9fold lower than wild-type value

W4L

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.8fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 2.1fold lower than wild-type value, t1/2 at 37°C is 6.1fold lower than wild-type value

W4T

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.7fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 2.4fold lower than wild-type value, t1/2 at 37°C is 50fold lower than wild-type value

W4Y

beta-subunit mutant, KM-value for glutaryl-7-aminocephalosporanic acid is 1.6fold higher than wild-type value, turnover number for glutaryl-7-aminocephalosporanic acid is 1.3fold lower than wild-type value, t1/2 at 37°C is 12fold lower than wild-type value

F58betaN/H70betaS/I176betaT

site-directed mutagenesis, mutation of wild-type variant M31betaF, comparison of substrate binding structures and abilities with the wild-type, molecular dynamics simulations and modeling, overview

758446

H57betaS/H70betaS

H57betaS/H70betaS/F72betaR

site-directed mutagenesis, the mutant enzyme shows increased activity on cephalosporin compared to the wild-type enzyme, this mutant is the most active acylase on glutaryl-7-aminocephalosporanic acid

756357

H57betaS/H70betaS/L154betaY

site-directed mutagenesis, the mutant enzyme shows increased activity on cephalosporin compared to the wild-type enzyme

756357

L154betaF

site-directed mutagenesis, mutant M8, the mutant displays improved stability and activity compared to the wild-type

L154betaF/L180betaF

site-directed mutagenesis, mutant M10, the mutant displays improved stability and activity compared to the wild-type

L154betaF/Y167betaF/L180betaF

site-directed mutagenesis, the mutant displays improved stability and activity compared to the wild-type

L180betaF

site-directed mutagenesis, mutant M9, the mutant displays improved stability and activity compared to the wild-type

M31betaF/H57betaS/H70betaS

site-directed mutagenesis, mutant M1, the mutant displays improved stability and activity compared to the mutant M2

M31betaF/H57betaS/V68betaA/H70betaS

site-directed mutagenesis, mutant M2, the mutant shows a decrease in stability compared to wild-type, which is largely owing to the mutation V68betaA at the active site

M31betaF/H57betaS/V68betaA/H70betaS/L154betaF/L180betaF

site-directed mutagenesis

Y167betaF

site-directed mutagenesis, the mutant displays improved stability and activity compared to the wild-type

C471betaS

site-directed mutagenesis, mutant M3, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif

C471betaS/A114alphaS

site-directed mutagenesis, mutant M10, the mutant shows altered catalytic efficiency compared to wild-type

C471betaS/A114alphaS/A38betaS

site-directed mutagenesis, mutant M12

C471betaS/A114alphaS/A38betaS/L180betaF

site-directed mutagenesis, mutant M13, the mutant shows altered catalytic efficiency compared to wild-type

C471betaS/A38betaS/L154betaF

site-directed mutagenesis, mutant M14, the mutant shows altered catalytic efficiency compared to wild-type

C471betaS/A38betaS/L154betaF/L180betaF

site-directed mutagenesis, mutant M15, the mutant shows altered catalytic efficiency compared to wild-type

C47C471betaS/A38betaS

site-directed mutagenesis, mutant M11, the mutant shows altered catalytic efficiency compared to wild-type

L154betaF

site-directed mutagenesis, mutant M4, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif

L180betaF

site-directed mutagenesis, mutant M5, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif

N3betaT

site-directed mutagenesis, mutant M1, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif

N3betaV

site-directed mutagenesis, mutant M2, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif

F229L

F375L

F375M

decreased activity compared to the wild type enzyme

M271V/Q291K/T374S

N266H

N266M

decreased activity compared to the wild type enzyme

N266Q

decreased activity compared to the wild type enzyme

N266S

F177betaG

A4ZVL3

site-directed mutagenesis

758270

F177betaG/M145alphaA

F177betaG/M145alphaA/Y149alphaV

A4ZVL3

site-directed mutagenesis, mutant CAAC2, the mutant has an altered substrate binding site with increased affinity for binding of long acyl chains, it shows altered substrate specificity, lower enzymatic activity with cephalosporin but higher activity with aculeacin A, in comparison with the wild-type enzyme

758270

F375A

turnover number is 2.45fold lower than wild-type value, KM-value is 1.9fold lower than wild-type value, turnover number is 1.7fold lower than wild-type value, KM-value is 2.2fold higher than wild-type value

F375C

turnover number is 1.1fold higher than wild-type value, KM-value is 5.9fold lower than wild-type value, turnover number is 1.7fold lower than wild-type value, KM-value is 1.2fold higher than wild-type value

F375D

turnover number is 32.7fold lower than wild-type value, KM-value is 11fold higher than wild-type value, turnover number is 400fold lower than wild-type value, KM-value is 7fold higher than wild-type value

F375E

turnover number is 6.1fold lower than wild-type value, KM-value is 12fold higher than wild-type value, turnover number is 500fold lower than wild-type value, KM-value is 6.7fold higher than wild-type value

F375G

turnover number is 6.7fold lower than wild-type value, KM-value is 6.25fold lower than wild-type value, turnover number is 1.6fold lower than wild-type value, KM-value is 2.1fold higher than wild-type value

F375H

turnover number is 2.4fold higher than wild-type value, KM-value is 1.1fold lower than wild-type value, turnover number is 3fold lower than wild-type value, KM-value is 3.5fold higher than wild-type value

F375I

turnover number is 5fold lower than wild-type value, KM-value is 2.1fold higher than wild-type value, turnover number is 6fold lower than wild-type value, KM-value is 3.2fold higher than wild-type value

F375K

turnover number is 13.6fold lower than wild-type value, KM-value is 2.2fold higher than wild-type value, turnover number is 1.8fold lower than wild-type value, KM-value is 22.6fold higher than wild-type value

F375L

turnover number is 1.4fold higher than wild-type value, KM-value is 1.4fold lower than wild-type value, turnover number is 3.6fold lower than wild-type value, KM-value is 8.4fold higher than wild-type value

F375M

turnover number is 1.3fold higher than wild-type value, KM-value is 1.5fold lower than wild-type value, turnover number is 2fold lower than wild-type value, KM-value is 5.5fold higher than wild-type value

F375N

turnover number is 1.8fold higher than wild-type value, KM-value is 1.2fold lower than wild-type value, turnover number is 2fold lower than wild-type value, KM-value is 5.8fold higher than wild-type value

F375Q

turnover number is 1.4fold higher than wild-type value, KM-value is 2.1fold higher than wild-type value, turnover number is 5fold lower than wild-type value, KM-value is 22.6fold higher than wild-type value

F375R

turnover number is 27.2fold lower than wild-type value, KM-value is 10fold higher than wild-type value, turnover number is 19fold lower than wild-type value, KM-value is 19fold higher than wild-type value

F375S

turnover number is 4.5fold lower than wild-type value, KM-value is nearly identical to wild-type value, turnover number is 1.3fold lower than wild-type value, KM-value is 2.3fold higher than wild-type value

F375T

turnover number is 2fold lower than wild-type value, KM-value is 1.1fold lower than wild-type value, turnover number is 2.2fold lower than wild-type value, KM-value is 2.9fold higher than wild-type value

F375V

turnover number is 5.4fold lower than wild-type value, KM-value is 1.4fold higher than wild-type value, turnover number is 3.9fold lower than wild-type value, KM-value is 3.2fold higher than wild-type value

F375W

turnover number is 18.8fold lower than wild-type value, KM-value is 1.5fold higher than wild-type value, turnover number is 8.5fold lower than wild-type value, KM-value is 25.8fold higher than wild-type value

F375Y

turnover number is 1.14fold lower than wild-type value, KM-value is 2.4fold lower than wild-type value, turnover number is 1.1fold lower than wild-type value, KM-value is 2.6fold higher than wild-type value

N266A

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 2.3fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.8fold higher than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 2.9fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 8.4fold higher than wild-type value

N266C

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.3fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.5fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 2fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 5.2fold higher than wild-type value

N266D

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 205fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.8fold higher than wild-type value. Less efficient cleavage of the spacer from the alpha-subunit compared to wild-type enzyme

N266E

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 17.8fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 11.5fold higher than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 160fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 9.4fold higher than wild-type value. Less efficient cleavage of the spacer from the alpha-subunit compared to wild-type enzyme

N266F

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.1fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 2fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 4.4fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 3.2fold higher than wild-type value

N266G

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.8fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.2fold higher than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 2fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 17.4fold higher than wild-type value

N266H

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.15fold higher than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 8.6fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 1.3fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 1.4fold higher than wild-type value. 100% improved conversion of cephalosporin C compared to wild type enzyme

N266L

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.64fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.2fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 48.8fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 13.9fold higher than wild-type value

N266M

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 4.6fold higher than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 3.6fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 2.2fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 5.4fold higher than wild-type value

N266P

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 6.7fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 3.7fold higher than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 30.8fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 41.9fold higher than wild-type value

N266Q

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.12fold higher than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.9fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 1.7fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 3.9fold higher wild-type value. 100% improved conversion of cephalosporin C compared to wild type enzyme

N266S

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.7fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.2fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 2.5fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 2.4fold higher than wild-type value

N266T

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 2.7fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 4.75fold higher than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 8.2fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 48.4fold higher than wild-type value

N266W

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.2fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 2fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 3fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 5.8fold higher than wild-type value. 30% improved conversion of cephalosporin C compared to wild type enzyme

N266Y

turnover number for adipyl-7-aminodeacetoxycephalosporanic acid is 1.2fold lower than wild-type value, KM-value for adipyl-7-aminodeacetoxycephalosporanic acid is 1.8fold lower than wild-type value, turnover-number for glutaryl-7-aminocephalosporanic acid is 6.6fold lower than wild-type value, KM-value for glutaryl-7-aminocephalosporanic acid is 3.5fold higher than wild-type value