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Literature summary extracted from

  • 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 (1995), Eur. J. Biochem., 230, 773-778.
    View publication on PubMed

Application

EC Number Application Comment Organism
3.5.1.93 synthesis mutant enzyme M269Y may be a promising enzyme in the one-step enzymic production of 7-amino-cephalosporanic acid from cephalosporin C Pseudomonas sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.93 expression in Escherichia coli. Semi-large-scale fermentation of recombinant enzyme Pseudomonas sp.

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.93 A271F 118% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 56.2% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 A271L 104% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 100% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 A271Y 101% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 122% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K100Q 81% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 106% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K114Q 86% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 101% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K170Q 130% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 95.6% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K187Q 113% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 91.1% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K255Q 107% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 97% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K301Q 101% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate Pseudomonas sp.
3.5.1.93 K44Q 102% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 111% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K507Q 102% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 113.9% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 K629Q 94.2% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate Pseudomonas sp.
3.5.1.93 K73Q 46.9% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, 47% of wild-type activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 S293C 1.21% of wild-type activity with glutaryl-7-aminocephalosporanic acid as substrate, no activity with cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 Y270E no activity with glutaryl-7-aminocephalosporanic acid or cephalosporin C as substrate Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.
3.5.1.93 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 Pseudomonas sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.93 21.3
-
 cephalosporin C 37°C, mutant enzyme Y269F Pseudomonas sp.
3.5.1.93 21.6
-
 cephalosporin C 37°C, wild-type enzyme Pseudomonas sp.
3.5.1.93 22.1
-
 cephalosporin C 37°C, mutant enzyme Y269Y Pseudomonas sp.
3.5.1.93 27.8
-
 cephalosporin C 37°C, mutant enzyme Y270F Pseudomonas sp.

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.93 Pseudomonas sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.93
-
 Pseudomonas sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.93 cephalosporin C + H2O
-
 Pseudomonas sp. 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate
-
 ?
3.5.1.93 glutaryl-7-aminocephalosporanic acid + H2O
-
 Pseudomonas sp. 7-aminocephalosporanic acid + glutarate
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.93 2.91
-
 cephalosporin C 37°C, mutant enzyme Y270F Pseudomonas sp.
3.5.1.93 5.93
-
 cephalosporin C 37°C, wild-type enzyme Pseudomonas sp.
3.5.1.93 9.4
-
 cephalosporin C 37°C, mutant enzyme Y269Y Pseudomonas sp.
3.5.1.93 10.4
-
 cephalosporin C 37°C, mutant enzyme Y269F Pseudomonas sp.