Cloning and characterization of a novel lipase from Vibrio harveyi strain AP6.
A lipolytic enzyme designated Vst, was cloned from Vibrio harveyi strain AP6. The vst gene was 1650 bp and was predicted to encode a 549 amino acid precursor with a molecular mass of about 61 kDa. The predicted polypeptide shared about 30% sequence identity with Bacillus esterases. Sequence alignment of Vst and related esterases revealed the presence of an active site serine located in the middle of the GESAG motif, at positions 212-216. This motif resembled the GXSXG consensus motif characteristic of lipolytic enzymes. Vst was expressed as a carboxy-terminal 6 x His tagged recombinant enzyme from E. coli TOP10 cells. SDS-PAGE and Western blot analysis using anti-His antibodies confirmed that the size of the mature protein was about 61 kDa. Substrate specificity of Vst was investigated using p -nitrophenyl (p NP) esters with varying carbon chain lengths, from C2 to C18. Vst showed the highest activity with the long chain p -nitrophenyl ester p NPC12 but was able to hydrolyze longer chain esters (p NPC14-p NPC16) as well as short and medium chain esters (p NPC4 and p NPC8).
Teo
Jeanette W P
JW
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117 597, Singapore.
Zhang
Lian-Hui
LH
Poh
Chit Laa
CL
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C477832:Vst protein, Vibrio harveyi; D008049:Lipase