Cloning And Heterologous Expression Of Cold-active Lipases From Antarctic Psychrotrophic Bacterium Psychrobacter Sp. G

Antarctic environment is characterized by various challenging conditions for the survival of cold-evolved microorganisms, which have successfully developed adaptation mechanisms enabling them to thrive at low temperatures. These adaptations include changes in membrane chemistry and modifications of protein structure and function. Antarctic psychrotrophic/psychrophilic microorganisms produce cold-active enzymes, which have unique low-temperature physiological and biochemical characteristics, very high catalytic constant (K_cat) values and lower and more stable Michaelis constant (K_m) values. The main features of cold active lipases are lower activation energy and higher catalytic activity at low temperature, and they have significant advantages in some areas in comparison to the lipases from mesophiles.The genomic DNA of Antarctic psychrotrophic bacterium Psychrobacter sp. G was used to construct its plasmid genomic library, and to screen the cold-active lipolytic enzyme genes. Two genes encoding for cold-active lipolytic enzymes, Lip-1452 (with an open reading frame of 1452 bp in length) and Lip-948 (with an open reading frame of 948 bp in length), were screened. The primary structure of the two lipases deduced from the nucleotide sequence showed a consensus pentapeptide containing the active serine (Lip-1452, GDSAG, and Lip-948, GNSMG) and a conserved His-Gly dipeptide in the N-terminal part of the two enzymes, respectively. Protein sequence alignment and conserved regions analysis indicated that the two lipases probably belonged to family IV and family V of the bacterial lipolytic enzymes, respectively.The cold-active lipase gene Lip-948 and Lip-1452 were both ligated into plasmid pColdâ…¢and transformed into E. coli BL21(DE3). The lipase activity assay showed that the specific activity of LIP-948 heterologous expressed was higher than that of LIP-1452. Then Lip-948 was ligated into different expression vectors such as pColdâ… , pMAL-c4x and pET32a, and transformed into E. coli BL21(DE3). SDS–PAGE analysis showed that there was substantive expression of lipase LIP-948 in E. coli. The soluble proportion of total LIP-948 heterologously expressed in different expression vectors mentioned above were 4.5%, 18.0% and 55.6%; the corresponding activity were 5.33 U/mL, 5.83 U/mL and 5.83 U/mL, respectively. The expression system of pColdâ…¢+ Lip-948 in E. coli was optimized showing that the most effective concentration of IPTG was 0.5 mmol/L, the OD600 of bacteria density ranged from 0.4 to 0.6, the best induction temperature and time was 15℃and 24 h.Co-expression of molecular chaperones with the pColdâ… + Lip-948 was also carried out. The results showed that co-expression of different chaperones led to an increase or decrease in the formation of soluble LIP-948 in varying degrees. Co-expression of pColdâ… + Lip-948 with chaperone pTf16 and pGro7 decreased the amount of soluble LIP-948, while the soluble expression was enhanced when pColdâ… + Lip-948 was co-expressed with"chaperone team"plasmids (pKJE7, pG-Tf2, pG-KJE8), respectively. LIP-948 was most efficiently expressed in soluble form when it was co-expressed with pG-KJE8, which was up to 19.8% of intracellular soluble proteins and with a specific activity of 108.77 U/mg.The soluble LIP-948 was purified with amylase affinity chromatography and its enzymatic characters were studied. The optimal temperature and pH of LIP-948 was 35℃and 8, respectively. The activity of LIP-948 dropped dramatically after incubation at 50℃for 15 min, and the activity was enhanced by Sr²⁺, Ca²⁺. It preferentially hydrolyzed 4-nitrophenyl esters with the shorter carbon chain.