Cloning, sequence analysis and expression of bacterial lipase-coding DNA fragments from environment in Escherichia coli.
Thirteen pairs of primers were designed, synthesized and used to clone the whole coding sequences or mature peptide-coding sequences of lipases. Bacteria producing extracellular lipases were enriched for the extraction of total DNAs. Eight fragments with 500-1,200 bp in length were obtained by using touchdown PCR and sequenced. Five of them were found to be lipase-coding DNAs. One fragment called BL9 that was 95.9% similar to a coding sequence of putative lipase. This lipase contained a Gly-His-Ser-Met-Gly motif which is matched to the consensus Gly-X-Ser-X-Gly conserved among lipolytic enzymes. The BL9 DNA fragment was inserted into the expression vector pET32a(+) of Escherichia coli. A functional product was yielded in the supernatant and produced a hydrolyzed zone on the tributyrin agar.
Fan
Zhaoxin
Z
College of Life Science, Sichuan University, Sichuan Key Laboratory of Molecular Biology & Biotechnology, Chengdu, 610064, China. fanzhaoxin@sohu.com
Yue
Changwu
C
Tang
Yang
Y
Zhang
Yizheng
Y
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D017931:DNA Primers / Q000138:chemical synthesis; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008049:Lipase / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008049:Lipase
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