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A. V. Stepashkina, S. S. Savin, O. E. Skirgello, V. I. Tishkov
Expression
and characterization of mutant forms of penicillin acylase
from Alcaligenes faecalis
Abstract
Sequencing
of six plasmids with gene of penicillin acylase from Alcaligenes faecalis
VKM B1518 (AfPA) revealed presence of random
nucleotide mutations resulted aroused after polymerase chain reaction. Six
mutant AfPAs as well as wild-type enzyme were
expressed in E.coli cells. Data obtained
showed that some amino acid changes influenced on enzyme activity and level of expression
of the mutants as well as on speed of cell growth. Four mutant AfPAs were purified and characterized. It was found that
studied amino acid replacements did not change catalytic efficiency Amino acid
changes βQ133R and βK184E (mutant AfPAM2) are not essential for thermal stability while in the case of
mutants AfPAM4 (βY90H), M5 (αD132G, βR97C) and M6 (αV5E, αN183S and βE439G) inactivation rate
constant in comparison with wild-type enzyme increased 2.4, 2.75 and 8.3 fold,
respectively.
Key words: penicillin acylase, Alcaligenes faecalis,
random mutagenesis, expression, catalytic properties, thermal stability.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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