A thermostable leucine aminopeptidase from Bacillus kaustophilus CCRC 11223.
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 degrees C, respectively, and 50% of its activity remained after incubation at 60 degrees C for 32 min. The enzyme preferentially hydrolyzed L-leucine- p-nitroanilide ( L-Leu- p-NA) followed by Cys derivative.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 60083, Chiayi, Taiwan.
Hsu
Wen-Hwei
WH
Wu
Cheng-Pu
CP
Chi
Meng-Chun
MC
Chou
Wei-Mou
WM
Hu
Hui-Yu
HY
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase