Cloning and expression of lipP, a gene encoding a cold-adapted lipase from Moritella sp.2-5-10-1.
A gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 isolated from Antarctic region was cloned and sequenced in this study. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of the lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine [Gly-Trp-Ser-Leu-Gly] and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. These sequences were involved in the lipase active site conformation. Structure factors that would allow proper enzyme flexibility at low temperatures were discussed. It was suggested that the changes in the primary structure of the psychrophilic lipases compared to the thermophilic ones could account for their ability to catalyze lipolysis at temperatures close to 0 degrees C. For expression, the sequence corresponding to the cold-adapted lipase of strain 2-5-10-1 was subcloned into the pET-28a expression vector to construct a recombinant lipase protein. Expression of the lipase by Escherichia coli BL21 (DE3) cells was observed as clear halos on 1% (vol/vol) tributyrin upon induction with IPTG at 25 degrees C.
Yang
Xiuxia
X
Department of Marine Biology, College of Marine Life Science, Ocean University of China, Qingdao 266003, PR China. xxyang@ouc.edu.cn
Lin
Xuezheng
X
Fan
Tingjun
T
Bian
Ji
J
Huang
Xiaohang
X
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016680:Genome, Bacterial; D015698:Genomic Library; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D034221:Moritella / Q000201:enzymology / Q000235:genetics* / Q000502:physiology
D001426:Bacterial Proteins; D008049:Lipase