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標題: A thermostable leucine aminopeptidase from Bacillus kaustophilus CCRC 11223
作者: Lin, L.L.
Hsu, W.H.
Wu, C.P.
Chi, M.C.
Chou, W.M.
Hu, H.Y.
關鍵字: Bacillus kaustophilus;gene cloning;leucine aminopeptidase;phylogeny;escherichia-coli;bovine lens;crystal-structure;genus bacillus;purification;cloning;enzyme;identification;thermozymes;expression
Project: Extremophiles
期刊/報告no:: Extremophiles, Volume 8, Issue 1, Page(s) 79-87.
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. His(6)-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 65degreesC, respectively, and 50% of its activity remained after incubation at 60degreesC for 32 min. The enzyme preferentially hydrolyzed L-leucine-p-nitroanilide (L-Leu-p-NA) followed by Cys derivative.
ISSN: 1431-0651
DOI: 10.1007/s00792-003-0364-1
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