Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68239
標題: Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp strain TS-23 alpha-amylase
作者: Huang, H.B.
Chi, M.C.
Hsu, W.H.
Liang, W.C.
Lin, L.L.
關鍵字: Bacillus kaustophilus;leucine aminopeptidase;Bacillus sp strain TS-23;amylase;starch-binding domain;adsorption-elution purification;x-ray crystallography;escherichia-coli;bovine lens;cyclodextrin;glycosyltransferase;cyclomaltodextrin glucanotransferase;aspergillus-niger;hybrid enzymes;fusion gene;glucoamylase;adsorption
Project: Bioprocess and Biosystems Engineering
期刊/報告no:: Bioprocess and Biosystems Engineering, Volume 27, Issue 6, Page(s) 389-398.
摘要: 
The starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70 degrees C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K-m value. Starch-binding assays showed that the K-d and B-max values for the fusion enzyme were 2.3 mu M and 0.35 mu mol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris-HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.
URI: http://hdl.handle.net/11455/68239
ISSN: 1615-7591
DOI: 10.1007/s00449-005-0001-8
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