Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.
Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.
Yamamoto
Takuo
T
Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase Minami-machi, Okayama 700-0834, Japan. amaseken@hayashibara.co.jp
Mukai
Kazuhisa
K
Yamashita
Hiroshi
H
Kubota
Michio
M
Fukuda
Shigeharu
S
Kurimoto
Masashi
M
Tsujisaka
Yoshio
Y
D001483:Base Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D046968:Thermoanaerobacter / Q000201:enzymology*
D017931:DNA Primers; D005964:Glucosyltransferases; C506185:kojibiose phosphorylase, Thermoanaerobacter brockii