Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/71627
標題: Stabilization of a truncated Bacillus sp strain TS-23 alpha-amylase by replacing histidine-436 with aspartate
作者: Lo, H.F.
Chen, Y.H.
Hsiao, N.W.
Chen, H.L.
Hu, H.Y.
Hsu, W.H.
Lin, L.L.
關鍵字: Bacillus sp strain TS-23;alpha-amylase;site-directed mutagenesis;histidine;thermostability;meningosepticum glycerol kinase;sequence-based classification;crystal-structure;escherichia-coli;cyclodextrin glycosyltransferase;enzymatic-properties;glycosyl hydrolases;angstrom resolution;amylolytic enzymes;protein stability
Project: World Journal of Microbiology & Biotechnology
期刊/報告no:: World Journal of Microbiology & Biotechnology, Volume 21, Issue 4, Page(s) 411-416.
摘要: 
Histidine-436 of a truncated Bacillus sp. strain TS-23 alpha-amylase (His(6)-tagged Delta NC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His(6)-tagged Delta NC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54 kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the K-m value, more than 66% increase in the value of catalytic efficiency (k(cat)/K-m) was observed in H436D, H436K, and H436Y. At 70 degrees C, H436D exhibited an increased half-life with respect to the wild-type enzyme.
URI: http://hdl.handle.net/11455/71627
ISSN: 0959-3993
DOI: 10.1007/s11274-004-1764-9
Appears in Collections:期刊論文

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