Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme.
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 alpha-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 alpha-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.
Yamashita
M
M
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University.
Kinoshita
T
T
Ihara
M
M
Mikawa
T
T
Murooka
Y
Y
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006361:Heating; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase