Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase.
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 microM and 0.35 micromol/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.
Huang
Hsien-Bin
HB
Department of Life Sciences and Institute of Molecular Biology, National Chung Cheng University, Chiayi, 621, Taiwan.
Chi
Meng-Chun
MC
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, 621, Taiwan.
Hsu
Wen-Hwei
WH
Institute of Molecular Biology, National Chung Hsing University, 402-27, Taichung, Taiwan.
Liang
Wan-Chi
WC
Department of Applied Chemistry, National Chaiyi University, 300 University Road, Chiayi, 60083, Taiwan.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chaiyi University, 300 University Road, Chiayi, 60083, Taiwan. llin@mail.ncyu.edu.tw.
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D002845:Chromatography / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007931:Leucyl Aminopeptidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013045:Species Specificity; D013213:Starch / Q000737:chemistry; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D013213:Starch; D000516:alpha-Amylases; D007931:Leucyl Aminopeptidase