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Literature summary for 3.5.5.1 extracted from

  • Schreiner, U.; Hecher, B.; Obrowsky, S.; Waich, K.; Klempier, N.; Steinkellner, G.; Gruber, K.; Rozzell, J.; Glieder, A.; Winkler, M.
    Directed evolution of Alcaligenes faecalis nitrilase (2010), Enzyme Microb. Technol., 47, 140-146.
No PubMed abstract available
Search for more literature for 3.5.5.1

Protein Variants

Protein Variants Comment Organism
A198V the mutant enzyme shows 2.4fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/I290F the mutant enzyme shows 1.2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 140% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/I290F/H135Y the mutant enzyme shows 1.7fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 150% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T the mutant enzyme shows 2.8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 740% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/I290F/Y177C/A285T the mutant enzyme shows 0.9fold conversion rate of 2-phenylpropionitrile at pH 7.5 and 240% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/I290F/Y213H/T350S the mutant enzyme shows 2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 200% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/Q197H the mutant enzyme shows 2.9fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/Q197H/L176M the mutant enzyme shows 6.5fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme Alcaligenes faecalis
A198V/Q197H/L176M/V306I the mutant enzyme shows 8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme Alcaligenes faecalis
I290F/Q3L the mutant enzyme shows 2.3fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 100% activity at pH 4.5 compared to the wild type enzyme Alcaligenes faecalis

Organism

Organism UniProt Comment Textmining
Alcaligenes faecalis P20960
-
-
Alcaligenes faecalis JM3 P20960
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni Sepharose column chromatography Alcaligenes faecalis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.1
-
 the specific activity is less than 0.1 micromol/min/mg, wild type enzyme, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 4.5 and 30°C Alcaligenes faecalis
0.14
-
 mutant enzyme A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 7.5 and 30°C Alcaligenes faecalis
0.52
-
 mutant enzyme A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 4.5 and 30°C Alcaligenes faecalis
0.62
-
 wild type enzyme, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 7.5 and 30°C Alcaligenes faecalis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-2-chloromandelonitrile + H2O
-
 Alcaligenes faecalis (R)-2-chloromandelic acid + (S)-2-chloromandelic acid + NH3 the wild type enzyme yields 76% (R)-2-chloromandelic acid and 9% (S)-2-chloromandelic acid at pH 7.5, as well as 12% (R)-2-chloromandelic acid and no (S)-2-chloromandelic acid at pH 4.5 ?
(R)-2-chloromandelonitrile + H2O
-
 Alcaligenes faecalis JM3 (R)-2-chloromandelic acid + (S)-2-chloromandelic acid + NH3 the wild type enzyme yields 76% (R)-2-chloromandelic acid and 9% (S)-2-chloromandelic acid at pH 7.5, as well as 12% (R)-2-chloromandelic acid and no (S)-2-chloromandelic acid at pH 4.5 ?
2-phenylpropionitrile + 2 H2O
-
 Alcaligenes faecalis 2-phenylpropionic acid + NH3
-
 ?
2-phenylpropionitrile + 2 H2O
-
 Alcaligenes faecalis JM3 2-phenylpropionic acid + NH3
-
 ?

Synonyms

Synonyms Comment Organism
NIT
-
 Alcaligenes faecalis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 wild type enzyme Alcaligenes faecalis

pH Stability

pH Stability pH Stability Maximum Comment Organism
4 7.5 the wild type enzyme shows no activity after 30 min incubation at pH 4.5, about 10% activity after 30 min incubation at pH 5.0, about 20% activity after 30 min incubation at pH 5.5, about 60% activity after 30 min incubation at pH 6.0, and 100% activity after 30 min incubation at pH 6.5-7.5 Alcaligenes faecalis