Abstract
To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50, 000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in α-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of α-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.
| Original language | English |
|---|---|
| Pages (from-to) | 1233-1246 |
| Number of pages | 14 |
| Journal | Journal of Biochemistry |
| Volume | 116 |
| Issue number | 6 |
| Publication status | Published - 1994 Dec 1 |
| Externally published | Yes |
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Keywords
- Pullulanase
- Random mutagenesis
ASJC Scopus subject areas
- Statistics, Probability and Uncertainty
- Applied Mathematics
- Physiology (medical)
- Radiology Nuclear Medicine and imaging
- Molecular Biology
- Biochemistry
Cite this
Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme. / Yamashita, Mitsuo; Kinoshita, Takuya; Ihara, Michiko; Mikawa, Tomomi; Murooka, Yoshikatu.
In: Journal of Biochemistry, Vol. 116, No. 6, 01.12.1994, p. 1233-1246.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme
AU - Yamashita, Mitsuo
AU - Kinoshita, Takuya
AU - Ihara, Michiko
AU - Mikawa, Tomomi
AU - Murooka, Yoshikatu
PY - 1994/12/1
Y1 - 1994/12/1
N2 - To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50, 000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in α-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of α-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.
AB - To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50, 000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in α-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of α-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.
KW - Pullulanase
KW - Random mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=0028596968&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028596968&partnerID=8YFLogxK
M3 - Article
C2 - 7706211
AN - SCOPUS:0028596968
VL - 116
SP - 1233
EP - 1246
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 6
ER -