A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223 |
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| Authors: | Long-Liu?Lin Wen-Hwei?Hsu Cheng-Pu?Wu Meng-Chun?Chi Wei-Mou?Chou Email author" target="_blank">Hui-Yu?HuEmail author |
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| Institution: | (1) Department of Applied Chemistry, National Chiayi University, 60083 Chiayi, Taiwan;(2) Institute of Molecular Biology, National Chung Hsing University, 402 Taichung, Taiwan;(3) Graduate Institute of Biotechnology, National Chiayi University, 60083 Chiayi, Taiwan;(4) Department of Food and Nutrition, Hungkuang University, 433 Taichung, Taiwan |
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| Abstract: | Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His6-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian |
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| Keywords: | Bacillus kaustophilus Gene cloning Leucine aminopeptidase Phylogeny |
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