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Information on EC 3.5.1.93 - glutaryl-7-aminocephalosporanic-acid acylase and Organism(s) Pseudomonas sp. and UniProt Accession P07662
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3.5.1.93 glutaryl-7-aminocephalosporanic-acid acylaseIUBMB Comments
Forms 7-aminocephalosporanic acid, a key intermediate in the synthesis of cephem antibiotics. It reacts only weakly with cephalosporin C.
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Word Map
- 3.5.1.93
- penicillin
-
semisynthetic
- acylases
-
deacylation
-
d-amino
- synthesis
-
autoproteolytic
- beta-lactam
- diminuta
- acremonium
-
7-adca
-
alphabeta2
- industry
- biotechnology
- pharmacology
The taxonomic range for the selected organisms is: Pseudomonas sp.
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
Synonyms
cephalosporin acylase, gl-7-aca acylase, cephalosporin c acylase, glutaryl-7-aminocephalosporanic acid acylase, glutaryl acylase, cpc acylase, scpcacy, gl-7aca acylase, glutaryl-7-aca acylase, j1 acylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CCA
cephalosporin acylase
cephalosporin C acylase
CPC acylase
GL-7-ACA acylase
GLA
glutaryl-7-ACA acylase
glutaryl-7-ACA acylase
-
glutaryl-7-ACA acylases and cephalosporin acylases have been collectively referred to as cephalosporin acylases because they all have either glutaryl-7-aminocephalosporanic acid or cephalosporin C as their substrates
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PATHWAY SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
(7R)-7-(4-carboxybutanamido)cephalosporanate amidohydrolase
Forms 7-aminocephalosporanic acid, a key intermediate in the synthesis of cephem antibiotics. It reacts only weakly with cephalosporin C.
CAS REGISTRY NUMBER
COMMENTARY
56645-46-6
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
adipyl-7-aminodesacetoxycephalosporanic acid + H2O
deacetoxycephalosporanic acid + adipate
-
-
-
?
adipyl-aminopenicillanic acid + H2O
adipate + aminopenicillanic acid
wild-type enzyme shows 70.6% of the activity with glutaryl-7-aminocephalosporanic acid
-
-
?
cephalosporin C + H2O
cephalosporanic acid + 2-amino-5-hydroxypentanoate
wild-type enzyme shows 2.3% of the activity with glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
-
-
?
glutaryl-aminopenicillanic acid + H2O
glutarate + aminopenicillanic acid
wild-type enzyme shows 90.6% of the activity with glutaryl-7-aminocephalosporanic acid
-
-
?
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
adipyl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + adipate
-
activity is 0.56% of the activity with glutaryl-7-aminocephalosporanic acid
-
-
?
adipyl-7-aminocephalosporanic acid + H2O
adipate + 7-aminocephalosporanate
-
-
-
-
?
adipyl-7-aminodesacetoxycephalosporanic acid + H2O
adipate + 7-aminodesacetoxycephalosporanate
-
57.4% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
cephalosporin C + H2O
7-aminocephalosporanate + 2-amino-5-hydroxypentanoate
-
-
-
-
?
D-glutamyl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + D-glutamate
-
activity is 0.85% of the activity with glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl deacetoxy 7-aminocephalosporanic acid + H2O
deacetoxycephalosporanic acid + glutarate
-
-
-
-
?
glutaryl deacetyl-7-aminocephalosporanic acid + H2O
deacetyl-7-aminocephalosporanic acid + glutarate
-
-
-
-
?
glutaryl-3-chloro-7-aminodesacetoxycephalosporanic acid + H2O
glutarate + 3-chloro-7-aminodesacetoxycephalosporanate
-
81.9% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-7-amino cephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
-
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
glutarate + 7-aminocephalosporanate
-
9.9% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-7-aminodesacetoxycephalosporanic acid + H2O
glutarate + 7-aminodesacetoxycephalosporanate
-
71.5% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-D-Phe-Gly + H2O
glutarate + D-Phe-Gly
-
about 25% activity with 50 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-GDPP + H2O
glutarate + GDPP
-
substrate with highest catalytic efficiency
-
-
?
glutaryl-L-Ala + H2O
glutarate + L-Ala
-
about 25% activity with 50 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-L-Phe + H2O
glutarate + L-Phe
-
about 10% activity with 50 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-L-Phe-Gly + H2O
glutarate + L-Phe-Gly
-
about 50% activity with 50 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
pentanoyl-7-aminocephalosporanic acid + H2O
pentanoate + 7-aminocephalosporanate
-
about 2.4% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
succinyl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + succinate
-
-
-
-
?
succinyl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + succinate
-
activity is 9.9fold higher than with glutaryl-7-aminocephalosporanic acid
-
-
?
succinyl-7-aminocephalosporanic acid + H2O
succinate + 7-aminocephalosporanate
-
25.8% activity with 5 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
-
-
-
-
?
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
-
-
-
?
cephalosporin C + H2O
(7R)-7-aminocephalosporanate + 2-aminoadipate
-
-
-
-
?
cephalosporin C + H2O
(7R)-7-aminocephalosporanate + 2-aminoadipate
-
-
-
?
cephalosporin C + H2O
(7R)-7-aminocephalosporanate + 2-aminoadipate
-
the wild-type enzyme shows weak activity with cephalosporin C, but some enzyme mutants show increased activity
-
-
?
cephalosporin C + H2O
(7R)-7-aminocephalosporanate + 2-aminoadipate
-
the wild-type enzyme shows weak activity with cephalosporin C, while the activity of double mutant H57betaS/H70betaS with this substrate is enhanced
-
-
?
cephalosporin C + H2O
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate
-
-
-
-
?
cephalosporin C + H2O
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate
-
-
-
?
cephalosporin C + H2O
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate
-
activity determined using a biological and colorimetic method. For the spectrophotometric assay p-dimethylaminobenzaldehyde is added to produce a yellow condensation product which can be measured at a wavelength of 414 nm
-
-
?
cephalosporin C + H2O
cephalosporamic acid + 2-amino-5-hydroxypentanoate
-
-
-
-
?
cephalosporin C + H2O
cephalosporamic acid + 2-amino-5-hydroxypentanoate
the recombinant enzyme exhibits 2.3times more cephalosporin C specific deacylation activity compared to glutaryl-7-aminocephalosporanic acid
-
-
?
cephalosporin C + H2O
cephalosporanic acid + 2-amino-5-hydroxypentanoate
-
-
-
-
?
cephalosporin C + H2O
cephalosporanic acid + 2-amino-5-hydroxypentanoate
-
weak activity
-
-
?
cephalosporin C + H2O
cephalosporanic acid + 2-amino-5-hydroxypentanoate
-
low activity
-
-
?
cephalosporin C + H2O
cephalosporanic acid + 2-amino-5-hydroxypentanoate
-
double mutant H296S/H309S exhibits 22fold enhanced specificity and reactivity for cephalosporin C over the natural substrate glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
-
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
100% activity
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanate + glutarate
-
rate of synthesis is slower than rate of hydrolysis
-
-
r
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + glutarate
-
-
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + glutarate
-
activity assay
-
-
?
glutaryl-7-aminocephalosporanic acid + H2O
7-aminocephalosporanic acid + glutarate
-
double mutant H296S/H309S exhibits 22fold enhanced specificity and reactivity for cephalosporin C over the natural substrate glutaryl-7-aminocephalosporanic acid
-
-
?
glutaryl-glycine + H2O
glutarate + glycine
-
less than 5% activity with 50 mM substrate compared to glutaryl-7-aminocephalosporanic acid
-
-
?
additional information
?
-
-
purified enzyme exhibits also gamma-glutamyltranspeptidase activity with L-gamma-glutamyl-p-nitroanilide
-
-
?
additional information
?
-
-
the enzyme has transacylase activity 10times that of its hydrolytic activity
-
-
?
additional information
?
-
-
less than 1% activity with TLGEGDPP compared to EGDPPDLADQG, no activity with adipyl-7-aminocephalosporanic acid
-
-
?
additional information
?
-
-
the enzyme also removes N-terminal Glu or Asp from peptides. Cephalosporin acylase is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibits exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction
-
-
?
additional information
?
-
-
the precursor cephalosporin acylase (without signal peptide) is activated by two peptide bond cleavages, which excise an internal spacer. The first cleavage occurs between Gly169 and Ser170 and the second cleavage occurs between Glu159 and Gly160
-
-
?
additional information
?
-
-
the recombinant enzyme shows no activity towards cephalosporin C, butanoyl-7-aminodesacetoxycephalosporanic acid and pentanoyl-7-aminodesacetoxycephalosporanic acid
-
-
?
additional information
?
-
-
the amino acid moiety of cephalosporin C that are potentially involved in the enzyme substrate selectivity are Met165alpha, His23beta, Arg24beta, Val25beta, Tyr32beta, Phe55beta, Phe58beta, Phe72beta, Leu154beta, Leu175beta, Ile176beta and His178beta, substrate docking in the active site, overview
-
-
?
additional information
?
-
substrate chephalosporin has a similar negative charge in pH 8.5 solution
-
-
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
cephalosporin C + H2O
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate
-
-
-
?
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
-
-
-
-
?
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
(7R)-7-aminocephalosporanate + glutarate
-
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
inhibition of mutant M165alphaS/H57betaS/H70betaS, no inhibition of wild-type enzyme and mutant H57betaS/H70betaS
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4.5
4-(glutarylamino)benzoic acid
-
at 37°C, pH not specified in the publication
6.4
glutaryl-gamma-Glu-Cys-Gly
-
at 37°C, pH not specified in the publication
0.14
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266H
0.42
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266S
0.52
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme F229L
0.7
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S
0.82
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme F375L
0.96
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, wild-type enzyme
0.016
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266H
0.033
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, wild-type enzyme
0.043
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S
0.048
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme F229L
0.094
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266S
0.27
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme F375L
11.7
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme K198A
12.2
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme R121A
12.7
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme D286A
13.4
glutaryl-7-aminocephalosporanic acid
mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit
14.4
glutaryl-7-aminocephalosporanic acid
alpha-subunit mutant enzyme Y151F
17.8
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme Q50N/K198A
17.9
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme Q50N
0.041
(7R)-7-(4-carboxybutanamido)cephalosporanate
pH 8.5, 37°C, free mutant enzyme
0.043
(7R)-7-(4-carboxybutanamido)cephalosporanate
pH 8.5, 37°C, free wild-type enzyme
0.0438
(7R)-7-(4-carboxybutanamido)cephalosporanate
pH 8.5, 37°C, immobilized mutant enzyme
0.0484
(7R)-7-(4-carboxybutanamido)cephalosporanate
pH 8.5, 37°C, immobilized wild-type enzyme
0.4
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/F58betaD, pH 8.0, 37°C
0.41
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/M165alphaS/I176betaS, pH 8.0, 37°C
0.6
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L176betaK, pH 8.0, 37°C
1.5
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant wild-type enzyme, pH 8.0, 25°C
1.5
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant wild-type enzyme, pH 8.0, 25°C
1.8
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L176betaT, pH 8.0, 37°C
2
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 37°C
2
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L154betaY, pH 8.0, 37°C
3.2
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L176betaQ, pH 8.0, 37°C
3.6
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/V25betaR, pH 8.0, 37°C
3.8
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L175betaT, pH 8.0, 37°C
6.3
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/F58betaI, pH 8.0, 37°C
6.6
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 37°C
6.9
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C
9.2
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 37°C
21.9
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/M165alphaS, pH 8.0, 37°C
21.9
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C
4.3
adipyl-7-aminocephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
4.4
adipyl-7-aminocephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
7.5
adipyl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
0.17
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme N266Q/F375L
0.17
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme Y178H/N266M
0.19
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme Y178H/N266H
0.28
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme Y178H/N266Q/F375L
0.7
adipyl-7-aminodesacetoxycephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
3.8
adipyl-7-aminodesacetoxycephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
7.9
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
1.5
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/F58betaD, pH 8.0, 37°C
1.5
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L176betaC, pH 8.0, 37°C
1.8
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/M165alphaS/I176betaS, pH 8.0, 37°C
2
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 37°C
2.3
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/V25betaR, pH 8.0, 37°C
3.1
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 37°C
3.8
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L175betaT, pH 8.0, 37°C
4.5
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L154betaY, pH 8.0, 37°C
5.4
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L176betaT, pH 8.0, 37°C
7.5
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L176betaK, pH 8.0, 37°C
12.2
cephalosporin C
-
recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C
16.4
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 37°C
27.7
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/M165alphaS, pH 8.0, 37°C
27.7
cephalosporin C
-
recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C
33.7
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/F58betaI, pH 8.0, 37°C
37
cephalosporin C
mutant enzyme L677F, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication
43
cephalosporin C
wild type enzyme, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication
86
cephalosporin C
mutant enzyme P295A, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication
105
cephalosporin C
mutant enzyme A675G, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication
121
cephalosporin C
mutant enzyme H309V, in 100 mM Tris-HCl buffer, pH 8.0, temperature not specified in the publication
1
glutaryl-3-chloro-7-aminodesacetoxycephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
2.2
glutaryl-3-chloro-7-aminodesacetoxycephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
8.7
glutaryl-3-chloro-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
0.7
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H296S
0.9
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215F
0.9
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215Y/H309S
1.1
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme E89A/A215Y
1.2
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215V
1.2
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme Y271F
1.7
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215L
1.7
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215Y
1.7
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme D416Y
1.9
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H296F
2
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme Y215Y/F270S
2.5
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215Y/H296S/H309S
2.7
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme F270M
2.8
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H296N
4
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme A215Y/H296S
4.3
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H417Y
4.6
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H309S
5.5
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H296S/H417M
6.9
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme H296S/H309S
9
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme D416Y/H417Y
25
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme I44V/E49stop/D416Y/H417Y
37.2
glutaryl-7-amino cephalosporanic acid
-
pH 8.0, 37°C, mutant enzyme S22P/T394P/D416Y/H417Y
0.5
glutaryl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
1.5
glutaryl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
2.5
glutaryl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
3
glutaryl-7-aminocephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
3.3
glutaryl-7-aminocephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
5.4
glutaryl-7-aminocephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
6.9
glutaryl-7-aminocephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
15.4
glutaryl-7-aminocephalosporanic acid
-
pH 8.0, 37°C, enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane
1.3
glutaryl-7-aminodesacetoxycephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
1.5
glutaryl-7-aminodesacetoxycephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
14.1
glutaryl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
16.1
pentanoyl-7-aminocephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
17.4
pentanoyl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
2.8
succinyl-7-aminocephalosporanic acid
-
recombinant enzyme, at pH 8.0 and 25°C
2.8
succinyl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
3.2
succinyl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
5.4
succinyl-7-aminocephalosporanic acid
-
mutant enzyme H296S/H309S, at pH 8.0 and 25°C
additional information
additional information
-
kinetics of wild-type and mutant enzymes with cephalosporin C, overview
-
additional information
additional information
Michaelis-Menten kinetics of free and immobilized wild-type and mutant enzymes, overview
-
additional information
additional information
Michaelis constant (Km) and maximum reaction velocity (Vmax) for the free and immobilized enzyme are calculated according to Lineweaver-Burk plots
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.16
4-(glutarylamino)benzoic acid
-
at 37°C, pH not specified in the publication
3.2
glutaryl-gamma-Glu-Cys-Gly
-
at 37°C, pH not specified in the publication
1.28
succinyl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
0.34
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266S
0.46
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, wild-type enzyme
0.47
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme F229L
0.55
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266H
0.61
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S
0.67
adipyl-7-aminodesacetoxycephalosporanic acid
pH 7.5, 37°C, mutant enzyme F375L
0.21
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme Q50N/K198A
0.22
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme Q50N
0.24
glutaryl-7-aminocephalosporanic acid
alpha-subunit mutant enzyme Y151F
0.38
glutaryl-7-aminocephalosporanic acid
mutant enzyme with mutation Y151F in the alpha-subunit and Q50N in the beta-subunit
0.41
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme D286A
0.45
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme R121A
0.47
glutaryl-7-aminocephalosporanic acid
beta-subunit mutant enzyme K198betaA
1.1
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme F375L
2.2
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266S
3.1
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme F229L
3.3
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme N266H
3.5
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S
4.3
glutaryl-7-aminocephalosporanic acid
pH 7.5, 37°C, wild-type enzyme
0.48
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme N266Q/F375L
0.66
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme Y178H/N266Q/F375L
0.95
adipyl-7-aminodesacetoxycephalosporanic acid
-
mutant enzyme Y178H/N266H
0.022
cephalosporin C
-
wild-type enzyme and deletion mutant D4(212-ADLA-215), pH 8.5, 37°C
7.1
glutaryl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.04
4-(glutarylamino)benzoic acid
-
at 37°C, pH not specified in the publication
14.2
glutaryl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
0.5
glutaryl-gamma-Glu-Cys-Gly
-
at 37°C, pH not specified in the publication
0.4
succinyl-7-aminocephalosporanic acid
-
at 37°C, pH not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
21
glutaryl-7-aminocephalosporanic acid
-
wild type enzyme, at pH 8.0 and 25°C
1
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C
6.3
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 25°C
21
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
wild-type enzyme, pH 8.0, 25°C
21
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C
33
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/I176betaQ, pH 8.0, 25°C
65
(7R)-7-(4-carboxybutanamido)cephalosporanate
-
recombinant mutant H57betaS/H70betaS/I176betaK, pH 8.0, 25°C
1
(7R)-7-aminocephalosporanate
-
recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C
2
(7R)-7-aminocephalosporanate
-
recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 25°C
6.9
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 25°C
77
cephalosporin C
-
recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
12.3
mutant enzyme with mutation Y151F in alpha-subunit and mutation Q50N in beta-subunit
additional information
-
double mutant H296S/H309S exhibits 22fold enhanced specificity and reactivity for cephalosporin C over the natural substrate glutaryl-7-aminocephalosporanic acid
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8 - 8.5
-
soluble enzyme and enzyme immobilized on silica gel modified by epoxide silanization
8.3
-
enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane
8.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 10
7 - 9
-
activity decrease from pH 7 to pH 9, synthesis of glutaryl-7-aminocephalosporanic acid
8 - 10
additional information
-
pH-dependent activity profile for the enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane is considerably expanded
6 - 10
-
pH 6.0: soluble enzyme shows 35% of maximal activity, enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane shows 60% of maximal activity, pH 10.0: soluble enzyme shows 67% of maximal activity, enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane shows 75% of maximal activity
6 - 10
-
pH 6.0: soluble enzyme shows about 35% of maximal activity, enzyme immobilized on silica gel modified by epoxide silanization shows about 65% of maximal activity, pH 10.0: soluble enzyme shows about 70% of maximal activity, enzyme immobilized on silica gel modified by epoxide silanization shows about 85% of maximal activity
8 - 10
-
pH 8: about 50% of maximal activity, pH 9-10: maximal activity, hydrolysis of glutaryl-7-aminocephalosporanic acid
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
50
50
-
soluble enzyme and enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane
50
-
soluble enzyme and enzyme immobilized on silica gel modified by epoxide silanization
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 60
-
20°C: soluble enzyme shows 35% of maximal activity, enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane shows 45% of maximal activity, 60°C: soluble enzyme shows 20% of maximal activity, enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane shows 40% of maximal activity
30 - 55
30 - 55
-
30°C: about 45% of maximal activity, 55°C: about 70% of maximal activity
30 - 55
-
30°C: soluble enzyme shows about 45% of maximal activity, enzyme immobilized on silica gel modified by epoxide silanization shows about 75% of maximal activity, 55°C: soluble enzyme shows about 40% of maximal activity, enzyme immobilized on silica gel modified by epoxide silanization shows about 55% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
additional information
evolution
-
phylogenetic analysis and active site evolution concerning cephalosporin C binding, overview
evolution
-
the enzyme is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, in which the precursor gene is translated into a single polypeptide chain and then folded into a self-activating pre-protein activated by intramolecular double cleavages
additional information
-
a deep cavity constitutes the active site, structure overview. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity, ligand covalently binds to the catalytic serine residue forming a tetrahedral adduct and mimickingc the transition state of the enzyme for both the maturation step and the catalysis of the substrates
additional information
-
substrate binding, molecular modeling study, overview
additional information
the protein structure of Pseudomonas SE83 CCA is obtained through the computational modeling with X-ray crystal structure of Pseudomonas N176 CCA (PDB accession code 4HST) as the template, three-dimensional structure, homology modeling, overview. The key residue of the enzyme, 1Ser of beta-subunit, is located in the active-site cavity, which is on the opposite side to the C-terminal of beta-subunit
additional information
-
three-dimensional structure model of wild-type enzyme, overview
additional information
three-dimensional structure model of wild-type enzyme, overview
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
28000
54000
58000
61000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
heterodimer
additional information
-
a deep cavity constitutes the active site. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity
heterodimer
alphabeta structure, but not linked via disulfide bridge and not separable by SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
proteolytic modification
the enzyme is processed in two sequential steps of intramolecular autoproteolysis involving two distinct proteolytic mechanisms, the first mediatzed by a serine residue and the second by a glutamate
proteolytic modification
-
a first autoproteolytic cleavage site of the pro-enzyme is G239-S240, a second cleavage site is A232-S233, determined by tandem MS/MS analysis of the purified alpha-subunit C-terminus
proteolytic modification
-
the enzyme is synthesized as a single folded precursor protein made of 782 amino acid residues that, once folded, undergoes an autocatalytic processing event to produce the mature alpha/beta heterodimer, an internal linker segment of ten residues (GDASDAAGGG) is removed from the pre-enzyme between the final alpha- and beta-chains, maturation process, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour diffusion method, mutant enzymes Y202L, R226K, S170C and E159Q
polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and have unit-cell parameters a = 141.41, b = 192.0, c = 80.75 A. They belong to a space group P2(1)2(1)2(1) and diffract to at least 2.7 A resolution
-
preliminary crystals of the double mutant can be grown from 4% PEG 8000, 100 mM Tris-HCl, pH 8.5, carried out by vapour diffusion using hanging-drop method at 17.85°C, 1.57 A resolution
-
recombinant wild-type enzyme, and unaltered mutant and selenomethionine-labeled mutant H57betaS/H70betaS enzyme, hanging drop vapour diffusion method, reservoir solutions containing 30% PEG, 20% glycerol, and 100 mM Tris, pH 8.0, for 2-4 h, the crystals are cryoprotected using paratone, X-ray diffraction structure determination and analysis at 1.57-2.48 A resolution, modeling
-
vapour-diffusion method. A bipyramidal crystal form is obtained from a solution containing polyethylene glycol and CaCl2. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit cell parameters are a = b = 73.5 A, c = 380.3 A
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
F375L
1.5fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 1.2fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 3.9fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 8.2fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme. Improved activity ratio for adipyl-7-aminodesacetoxycephalosporanic acid to glutaryl-7-aminocephalosporanic acid of the mutant enzyme is a consequence of a decreased catalytic efficiency towards glutaryl-7-aminocephalosporanic acid
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
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
N266H
1.2fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 6.9fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.3fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 2fold decrease in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme. Nearly 10fold improved catalytic efficiency on adipyl-7-aminodesacetoxycephalosporanic acid, resulting from a 50% increase in turnover-number and a 6fold decrease in KM-value without decreasing the catalytic efficiency on glutaryl-7-aminocephalosporanic acid
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
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
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
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
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
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
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
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
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
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
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
P295A
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
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
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
additional information
A675G
the mutant exhibits 112.8% activity with cephalosporin C compared to the wild type enzyme
A675G
the mutant exhibits significantly enhanced specific enzymatic activity compared to the wild type enzyme (35% increase)
A675G
site-directed mutagenesis, the mutant shows 13% increased activity compared to the wild-type enzyme
H296S/H309S
-
Vmax/Km for cephalosporin C is 4fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 21.6fold lower than wild-type value
H296S/H309S
-
the mutant also shows activity with cephalosporin C (3fold increase compared to the wild type enzyme)
H309S
-
Vmax/Km for cephalosporin C is 1.5fold higher than wild-type value, Vmax/Km for glutaryl-7-amino cephalosporanic acid is 8.9fold lower than wild-type value
H309S
site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme
H309V
the mutant exhibits 88.9% activity with cephalosporin C compared to the wild type enzyme
H309V
site-directed mutagenesis, the mutant shows 11.1% decreased activity compared to the wild-type enzyme
H57betaS/H70betaS
-
site-directed mutagenesis, interest in designing a single step enzymatic conversion of cephalosporin C to (7R)-7-aminocephalosporanate catalyzed by a true cephalosporin C acylase, the engineered mutant displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid compared to the wild-type enzyme. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity
H57betaS/H70betaS
-
site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme
H57betaS/H70betaS/L154betaY
-
random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme
H57betaS/H70betaS/L154betaY
-
site-directed mutagenesis, interest in designing a single step enzymatic conversion of cephalosporin C to (7R)-7-aminocephalosporanate catalyzed by a true cephalosporin C acylase, the engineered mutant displays highly enhanced catalytic efficiency, with a 11000fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, on cephalosporin C over glutaryl-7-aminocephalosporanic acid compared to the wild-type enzyme under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects
L666F
the mutant exhibits significantly reduced specific enzymatic activity (75.4%) with cephalosporin C compared to the wild type enzyme
L666F
site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme
P295A
the mutant exhibits 20% activity with cephalosporin C compared to the wild type enzyme
P295A
site-directed mutagenesis, the mutant shows 80% decreased activity compared to the wild-type enzyme
additional information
-
construction of 14 mutants with deletion in the alpha-C-terminal region, the majority of the fragment-deleted mutant proteins completely lose their activity due to failure of the first autocleavage, while mutant proteins D2 (227-AM-228 deletion) and D4 (212-ADLA-215 deletion) formally activate into mature enzyme with high activity. The Kcat/Kmvalues of mutant proteins D2 and D4 are 46% and 102% higher than that of wild-type control, respectively
additional information
-
mutant screening of H57betaS/H70betaS triple mutants generated by site-saturation mutagenesis, introduction of multiple mutations, overview
additional information
the enzyme is mutated by PCR method to add a 3G3K tag to the C-terminal of the beta-subunit, the wild-type and mutated enzyme have nearly equal specific activity. The mutant enzyme has a 20% higher expressed activity than its wild-type counterpart. Immobilization of mutated and wild-type enzyme on glyoxyl agarose support via surface lysine or the poly-lysine tag, respectively
additional information
-
three-dimensional structure model of the wild-type enzyme for semi-rational determination of mutation residues, overview
additional information
three-dimensional structure model of the wild-type enzyme for semi-rational determination of mutation residues, overview
additional information
proton-producing enzyme cephalosporin C acylase (CCA) is covalently bound on an epoxy-activated porous support. The microenvironmental pH change in immobilized CCA during the reaction is detected using pH-sensitive fluorescein labeling (pH-sensitive fluorescein molecule co-immobilized on the carrier). The high catalytic velocity of the initial stage of conversion results in a sharp intraparticle pH gradient, which is likely the key factor relating to low operational stability. Mass transfer in the immobilized CCA is relatively severe during CPC hydrolysis. Reducing the intraparticle pH gradient, i.e. diminishing the diffusion limitation, is important for the catalysis of CCA. Accordingly, another strategy for a two-stage catalytic process is developed to reduce the reaction rate of stage I at a low temperature to preserve enzymatic activity and to shorten the duration of catalysis at a high reaction temperature in stage II. The reaction using the two-stage catalytic process (10-37°C shift at 30 min) shows significantly improved stability compared with that of the single-temperature reaction at 37°C (29 batches versus five batches, respectively) and a shorter catalytic period than the reaction at 10°C (40 min versus 70 min, respectively)
additional information
the purified recombinant His-tagged enzyme is immobilized on different supports, i.e. an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface, validation and optimization. The CCA immobilized on EP-PEI has the best stability and its activity declines very slowly at 45°C. The enzyme immobilized on the cationic support EP-EDA has a half-life of 100 min at 45°C and pH 8.0, a value much lower than on EP-PEI. Stabilization factors of enzyme immobilized on EP-PEI and EP-EDA are 35.6fold and 5.5fold, respectively. The Km values of CCA immobilized on EP-PEI and EP-EDA are even lower than that of the free enzyme, indicating that a higher substrate affinity is obtained after the immobilization on the cationic supports. The reusability of the EP-PEI support significantly reduces the cost of the biocatalyst preparation
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10
37°C, 125 min, 35% loss of wild-type activity, 10% loss of activity of mutant enzyme K198betaA
660767
7
37°C, 125 min, 10% loss of wild-type activity, 7% loss of activity of mutant enzyme K198betaA
660767
8
37°C, 125 min, 13% loss of wild-type activity, 7% loss of activity of mutant enzyme K198betaA
660767
9
37°C, 125 min, 20% loss of wild-type activity, 3% loss of activity of mutant enzyme K198betaA
660767
5.5
the enzyme activities of both free and immobilized enzymes are greatly reduced at pH 5.5
733139
6 - 8.5
the purified wild-type enzyme is not stable, stability is increased by immobilization
756210
8 - 9.5
-
LX-1000EP-immobilized enzyme shows good stability in solutions at pH 8.0-9.5. after 2 h of incubation
713590
additional information
-
the stability of the enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane is significantly improved over its free form at lower pH values
660933
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
half-life: 68.1 h (wild-type enzyme), 88.3 h (mutant enzyme R121betaA), 107.5 h (mutant enzyme K198betaA), 53.9 h (mutant enzyme D286betA)
10 - 37
purified recombinant enzyme, the immobilized enzyme CCA is more labile at 37°C than at 20°C or 10°C
15 - 40
-
the LX-1000EP-immobilized enzyme is stable at temperatures up to 40°C (8% loss of activity after 2 h of incubation at 40°C)
25
-
there is no fatal decrease of the enzyme stability up to 25°C. Above 25°C, the stability of the free enzyme decays sharply
37
45
50
55
-
1 h, soluble enzyme loses 77% of activity. t1/2 is 17.03 min for the soluble enzyme and 34.74 min for the immobilized enzyme
60
-
pH 8.0, 6 h, 25 mM Tris-HCl, 22% inactivation of wild-type enzyme, 26% inactivation of mutant enzyme M164A, 29% inactivation of mutant enzyme M164G, 20% inactivation of mutant enzyme M164L, 29% inactivation of mutant enzyme M164N, 10% inactivation of mutant enzyme M164Q
additional information
-
thermal stability of the enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane is increased significantly compared to that of the soluble enzyme
37
-
1 h, soluble enzyme and enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane are stable, t1/2 is 307.7 min for the soluble enzyme and 733.7 min for the immobilized enzyme
37
-
60 min, soluble enzyme and enzyme immobilized on silica gel modified by epoxide silanization lose about 10% of its activity
45
-
t1/2 is 49.97 min for the soluble enzyme and 200 min for the immobilized enzyme
45
free wild-type and mutant enzymes: pH 8.5, inactivation after 1 h, immobilized wild-type and mutant enzymes: pH 8.5, 30% and 10% activity remaing after 4 h
50
-
1 h, less than 10% loss of activity at pH 6.0, about 60% less of activity at pH 5, about 35% loss of activity at pH 7, about 50% loss of activity at pH 8, about 90% loss of activity at pH 9
50
-
t1/2 is 29.72 min for the soluble enzyme and 65.9 min for the immobilized enzyme
50
-
60 min, soluble enzyme loses 77% of its activity, enzyme immobilized on silica gel modified by epoxide silanization loses 60% of its activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
FP-IDA-Ni2+ resins-immobilized GLA is recycled 21times without significant loss of activity
immobilized mutant enzyme M269Y/C305S is effective in more than 60 cycles for the production of 7-aminocephalosporanate
-
the enzyme is thermostabilized by immobilization using epoxide silanization
-
the stability of the enzyme immobilized on silica gel modified with 3-aminopropyltriethoxysilane is significantly improved over its free form at lower pH values
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
25°C, 4 weeks, mutant enzyme C305S loses 25% of its activity, mutant enzyme M269Y/C305S loses 25% of its activity
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni2+-HiTrap chelating column chromatography and Cu2+-immobilized iminodiacetic acid affinity chromatography
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)pLysS
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning in Escherichia coli strain DH5alpha, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strains JM109(DE3) and BL21(DE3), optimization of the culture conditions for Escherichia coli JM109(DE3)/pET28-sCPCAcyA675G, overview
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli. Semi-large-scale fermentation of recombinant enzyme
-
full-length wild-type enzyme or the double mutant variant H296S/H309S are transformed in Escherichia coli BL21
-
into the vector pET24 for expression in Escherichia coli BL21DE3 pLysS cells
-
phylogenetic analysis, recombinant exxpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)pLysS
-
recombinant expression in in Escherichia coli JM109(DE3)/pET28, the enzyme performs self-activation
-
recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant expression of wild-type enzyme and of unaltered mutant and selenomethionine-labeled mutant H57betaS/H70betaS in Escherichia coli strain BL21(DE3)pLysS
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
enzymatic production of 7-aminocephalosporanate. Construction of site-directed mutants with enhanced activity and stability
biotechnology
-
a biological and colometric method is evaluated for determination of cephalosporin acylase product in bacteria
industry
pharmacology
-
the product of the reaction 7-aminocephalosporanic acid is a starting material for semisynthetic cephalosporin antibiotics. High thermal stability of the enzyme immobilized on silica gels indicates that it can be successfully used for the production of 7-aminocephalosporanic acid on an industrial scale
synthesis
industry
-
the huge production of engineered GLA produced in Escherichia coli under optimized conditions makes this enzyme an economic tool to be used in bioconversion processes at the industrial level, e.g., in 7-ACA production
industry
cephalosporin C acylase (CCA), a proton-forming enzyme, is an important industrial enzyme that can directly catalyze the substrate cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA), which is an intermediate in many types of synthetic cephalosporins. Immobilized CCA on porous carriers is applied in industry for the production of 7-ACA because of its high efficiency and environmentally friendly nature compared with the chemical process and two-step enzymatic process involving two enzymes. Mutational improvement of catalytic efficiency and operational stability
synthesis
-
immobilized mutant enzyme M269Y/C305S may be a promising enzyme in one-step enzymatic production of 7-aminocephalosporanate from cephalosporin C
synthesis
-
mutant enzyme M269Y may be a promising enzyme in the one-step enzymic production of 7-amino-cephalosporanic acid from cephalosporin C
synthesis
-
production of 7-aminocephalosporanate on a large scale at 25°C using enzyme immobilized by epoxide silanization
synthesis
-
the enzyme mutant may be suitable for industrial application of the mono-step process for cephalosporin C conversion to (7R)-7-aminocephalosporanate, which is the precursor for production of semisynthetic cephalosporins
synthesis
cephalosporin C acylase (CCA), a proton-forming enzyme, is an important industrial enzyme that can directly catalyze the substrate cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA), which is an intermediate in many types of synthetic cephalosporins. Immobilized CCA on porous carriers is applied in industry for the production of 7-ACA because of its high efficiency and environmentally friendly nature compared with the chemical process and two-step enzymatic process involving two enzymes
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kinoshita, T.; Tada, T.; Saito, Y.; Ishii, Y.; Sato, A.; Murata, M.
Crystallization and preliminary X-ray analysis of cephalosporin C acylase from Pseudomonas sp. strain N176
Acta Crystallogr. Sect. D
56
458-459
2000
Pseudomonas sp.
Yamada, H.; Ishii, Y.; Noguchi, Y.; Miura, T.; Mori, T.; Saito, Y.
Protein engineering of a cephalosporin C acylase
Ann. N. Y. Acad. Sci.
799
74-81
1996
Pseudomonas sp.
Binder, R.; Brown, J.; Romancik, G.
Biochemical characterization of a glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas strain BL072
Appl. Environ. Microbiol.
60
1805-1809
1994
Pseudomonas sp., Pseudomonas sp. BL072
Saito, Y.; Fujimura, T.; Ishii, Y.; Noguchi, Y.; Miura, T.; Niwa, M.; Shimomura, K.
Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene
Appl. Environ. Microbiol.
62
2919-2925
1996
Pseudomonas sp.
Zhang, W.; Liu, Y.; Zheng, H.; Yang, S.; Jiang, W.
Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis
Appl. Environ. Microbiol.
71
5290-5296
2005
Pseudomonas sp. (P07662), Pseudomonas sp.
Park, S.W.; Choi, S.Y.; Chung, K.H.; H43ong, S.I.; Kim, S.W.
Characteristics of GL-7-ACA acylase immobilized on silica gel through silanization
Biochem. Eng. J.
11
87-93
2002
Pseudomonas sp., Pseudomonas sp. KAC-1
-
Kim, J.K.; Yang, I.S.; Rhee, S.; Dauter, Z.; Lee, Y.S.; Park, S.S.; Kim, K.H.
Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation
Biochemistry
42
4084-4093
2003
Pseudomonas sp. (A4ZVL3), Pseudomonas sp.
Ishiye, M.; Niwa, M.
Nucleotide sequence and expression in Escherichia coli of the cephalosporin acylase gene of a Pseudomonas strain
Biochim. Biophys. Acta
1132
233-239
1992
Pseudomonas sp., Pseudomonas sp. V22
Ishii, Y.; Saito, Y.; Fujimura, T.; Sasaki, H.; Noguchi, Y.; Yamada, H.; Niwa, M.; Shimomura, K.
High-level production, chemical modification and site-directed mutagenesis of a cephalosporin C acylase from Pseudomonas strain N176
Eur. J. Biochem.
230
773-778
1995
Pseudomonas sp.
Otten, L.G.; Sio, C.F.; Vrielink, J.; Cool, R.H.; Quax, W.J.
Altering the substrate specificity of cephalosporin acylase by directed evolution of the beta-subunit
J. Biol. Chem.
277
42121-42127
2002
Pseudomonas sp. (P07662), Pseudomonas sp. SY-77 (P07662)
Aramori, I.; Fukagawa, M.; Tsumura, M.; Iwami, M.; Ono, H.; Ishitani, Y.; Koji, H.; Kohsaka, M.; Ueda, Y.; Imanaka, H.
Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases
J. Ferment. Bioeng.
73
185-192
1992
Brevibacillus laterosporus, Brevundimonas diminuta, Pseudomonas sp., Brevundimonas diminuta N176, Brevibacillus laterosporus J1
-
Kwon, T.H.; Rhee, S.; Lee, Y.S.; Park, S.S.; Kim, K.H.
Crystallization and preliminary X-Ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16
J. Struct. Biol.
131
79-81
2000
Pseudomonas sp.
Kim, J.K.; Yang, I.S.; Shin, H.J.; Cho, K.J.; Ryu, E.K.; Kim, S.H.; Park, S.S.; Kim, K.H.
Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries
Proc. Natl. Acad. Sci. USA
103
1732-1737
2006
Pseudomonas sp. (A4ZVL3)
Pollegioni, L.; Lorenzi, S.; Rosini, E.; Marcone, G.L.; Molla, G.; Verga, R.; Cabri, W.; Pilone, M.S.
Evolution of an acylase active on cephalosporin C
Protein Sci.
14
3064-3076
2005
Pseudomonas sp.
Lim, J.S.; Park, S.W.; Kim, S.W.
Thermal and operational characteristics of glutaryl-7-aminocephalosporanic acid acylase immobilized on silica gel modified by epoxide silanization
World J. Microbiol. Biotechnol.
22
39-44
2006
Pseudomonas sp., Pseudomonas sp. KAC-1
Sonawane, V.C.
Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes
Crit. Rev. Biotechnol.
26
95-120
2006
Aeromonas sp., Achromobacter xylosoxidans, Arthrobacter sp., Rhizobium viscosum, Brevibacillus laterosporus, Bacillus sp. (in: Bacteria), Brevundimonas diminuta, Burkholderia cepacia, Sphingomonas paucimobilis, Flavobacterium sp., Paecilomyces sp. (in: Eurotiomycetes), Pseudomonas sp., Pseudomonas putida, Pseudomonas nitroreducens, Aeromonas sp. ACY 95, Burkholderia cepacia BY21, Arthrobacter sp. 5-8A, Flavobacterium sp. 650, Pseudomonas sp. A14, Brevibacillus laterosporus J1, Pseudomonas nitroreducens CCRC 11041, Bacillus sp. (in: Bacteria) SV12, Pseudomonas sp. BLO 72, Brevundimonas diminuta N 176
Zhou, H.; Yu, H.; Luo, H.; Shi, Y.; Ma, X.; Shen, Z.
Inducible and constitutive expression of glutaryl-7-aminocephalosporanic acid acylase by fusion to maltose-binding protein
Enzyme Microb. Technol.
40
555-562
2007
Pseudomonas sp.
-
Zheng, H.; Chen, J.; Su, L.; Zhao, Y.; Yang, Y.; Zeng, H.Y.; Xu, G.; Yang, S.; Jiang, W.
One-step purification and immobilization of his-tagged GL-7-ACA acylase
Enzyme Microb. Technol.
41
474-479
2007
Pseudomonas sp. (O86089)
-
Otten, L.G.; Sio, C.F.; Reis, C.R.; Koch, G.; Cool, R.H.; Quax, W.J.
A highly active adipyl-cephalosporin acylase obtained via rational randomization
FEBS J.
274
5600-5610
2007
Pseudomonas sp., Pseudomonas sp. SY-77
Zheng, H.; Zhu, T.; Chen, J.; Zhao, Y.; Jiang, W.; Zhao, G.; Yang, S.; Yang, Y.
Construction of recombinant Escherichia coli D11/pMSTO and its use in enzymatic preparation of 7-aminocephalosporanic acid in one pot
J. Biotechnol.
129
400-405
2007
Pseudomonas sp.
Volonte, F.; Marinelli, F.; Gastaldo, L.; Sacchi, S.; Pilone, M.S.; Pollegioni, L.; Molla, G.
Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli
Protein Expr. Purif.
61
131-137
2008
Pseudomonas sp.
Anandan, A.; Vallet, C.; Coyle, T.; Moustafa, I.M.; Vrielink, A.
Crystallization and preliminary diffraction analysis of an engineered cephalosporin acylase
Acta Crystallogr. Sect. F
66
808-810
2010
Pseudomonas sp.
Tanomand, A.; Abeshov, R.; Farajnia, S.
Determination of cephalosporin acylase activity by biological and colorimetric method in bacteria
Afr. J. Biotechnol.
8
6697-6699
2009
Pseudomonas sp.
-
Zhu, X.; Luo, H.; Chang, Y.; Su, H.; Li, Q.; Yu, H.; Shen, Z.
Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid
World J. Microbiol. Biotechnol.
27
823-829
2011
Pseudomonas sp., Pseudomonas sp. SE83
Yin, J.; Deng, Z.; Zhao, G.; Huang, X.
The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis
J. Biol. Chem.
286
24476-24486
2011
Pseudomonas sp.
Wang, Y.; Yu, H.; Song, W.; An, M.; Zhang, J.; Luo, H.; Shen, Z.
Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel
J. Biosci. Bioeng.
113
36-41
2012
Pseudomonas sp., Pseudomonas sp. (P15558)
Rosini, E.; Monelli, C.; Pollegioni, L.; Riva, S.; Monti, D.
On the substrate preference of glutaryl acylases
J. Mol. Catal. B
76
52-58
2012
Pseudomonas sp.
-
Luo, H.; Zhao, H.; Chang, Y.; Wang, Q.; Yu, H.; Shen, Z.
Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support
Appl. Biochem. Biotechnol.
175
2114-2123
2014
Pseudomonas sp. (P15557)
Golden, E.; Paterson, R.; Tie, W.; Anandan, A.; Flematti, G.; Molla, G.; Rosini, E.; Pollegioni, L.; Vrielink, A.
Structure of a class III engineered cephalosporin acylase: Comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity
Biochem. J.
451
217-226
2013
Pseudomonas sp.
Conti, G.; Pollegioni, L.; Molla, G.; Rosini, E.
Strategic manipulation of an industrial biocatalyst - evolution of a cephalosporin C acylase
FEBS J.
281
2443-2455
2014
Pseudomonas sp.
Zhang, J.; Yu, H.; Wang, Y.; Luo, H.; Shen, Z.
Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect of deleting its flanking residues in the alpha-C-terminal region
J. Biotechnol.
184
138-145
2014
Pseudomonas sp.
Wei, Y.; Luo, H.; Chang, Y.; Yu, H.; Shen, Z.
Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine
Biocatal. Biotransform.
33
250-259
2015
Pseudomonas sp. (A0A1D8GRD5)
-
Luo, H.; Zhu, L.; Chang, Y.; Liu, X.; Liu, Z.; Sun, H.; Li, X.; Yu, H.; Shen, Z.
Microenvironmental pH changes in immobilized cephalosporin C acylase during a proton-producing reaction and regulation by a two-stage catalytic process
Biores. Technol.
223
157-165
2017
Pseudomonas sp. (A0A1D8GRD5)
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