Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

J Ind Microbiol Biotechnol. 2004 Jul;31(6):273-7. doi: 10.1007/s10295-004-0146-5. Epub 2004 Jul 10.

Abstract

Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Catalytic Domain / genetics
  • Enzyme Stability
  • Escherichia coli / genetics
  • Geobacillus stearothermophilus / enzymology*
  • Geobacillus stearothermophilus / genetics
  • Leucyl Aminopeptidase / genetics
  • Leucyl Aminopeptidase / isolation & purification
  • Leucyl Aminopeptidase / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Starch / metabolism
  • Temperature
  • alpha-Amylases / genetics
  • alpha-Amylases / metabolism*

Substances

  • Recombinant Fusion Proteins
  • Starch
  • alpha-Amylases
  • Leucyl Aminopeptidase