Cloning,Expression And Characterization Of Novel Thermophilic Lipolytic Enzymes

Lipolytic enzymes,including esterases and lipases,catalyze the formation and cleavage of ester bonds.Thermostable lipolytic enzymes,having high catalytic temperature,good thermal stability,pH stability,tolerance to organic solvent and so on,have a wide range of applications in food,cosmetics,feed and pharmaceutical industry.The traditional screening methods of microbial strains have limitations due to its wide range of sources and numerous species.With the rapid development of biotechnology,bioinformatics technology plays more and more important roles in the discovery of novel thermostable lipolytic enzymes.In this paper,two thermophilic esterase genes derived from hyperthermophile,Aquiex aeolicus VF5,were first identified,and the cloning,expression and characteristics of two esterases were also studied,which laid a foundation for its further reform and industrial applications.(1)Two predicted thermophilic esterase genes,named Aaeo1 and Aaeo2,derived from hyperthermophile,A.aeolicus VF5,were screened from Genebank database using bioinformatics methods such as Blast,multiple sequence alignments and structural prediction.The two sequences include a pentapeptide consensus sequence,Gly-Xaa-Ser-Xaa-Gly,and a catalytic triad,Ser-Asp-His,which is highly conserved in lipolytic enzymes of the ?/? hydrolase superfamily.Protein blast search showed that Aaeo1 and Aaeo2 have some identity with other thermostable lipolytic enzymes.The two esterases sequences,Aaeo1 and Aaeo2,were classified into family VIII and family V by constructing a phylogenetic tree according to bacterial lipolytic enzymes classification,respectively.(2)Two esterase genes were optimized and artificially synthesized according to the codon preference of P.pastoris,and the recombinant expression stains were constructed,named as GS115/pPIC9K-Aaeo1 and GS115/pPIC9K-Aaeo2.The esterase activity was detected in the fermentation supernatant,which indicates that Aaeo1 and Aaeo2 may be esterase.The confirmation was carried by site-directed mutagenesis of three hypothetical catalytic sites in Aaeo1 and Aaeo2 sequences,and the mutant strains have little catalytic activity compared to their wild-type enzymes.These results indicated that Aaeo1 and Aaeo2 were true lipolytic enzymes.(3)The enzymatic properties of both esterases were determined.Aaeo1 has a broad substrate profile but the optimum substrate for Aaeo2 is p NPC4(p-nitrophenyl butyrate).The optimum temperatures of Aaeo1 and Aaeo2 were 80 oC and 85 oC,respectively.Both showed higher thermostable in 70 °C and 80 °C,and had over 70% residual activity after incubation for 120 min.The denaturation temperature(Tm)of Aaeo1 and Aaeo2 were calculated to be 85.3±0.4 oC and 86.3±0.3 oC,respectively.Aaeo1 and Aaeo2 both have the optimum pH at 8.0.Aaeo1 retained more than 60% residual activity after incubation for 240 min in pH8.0 and 9.0,while Aaeo2 had low pH stability in pH7.0-9.0.Different kinds and concentrations metal ions have different effects on Aaeo1 and Aaeo2.The two esterases exhibited certain tolerance to various organic solvents and some detergents.The activities of both esterases were inhibited by PMSF,DEPC,and EDTA,but were activated by DTT.The Km and kcat values of p NPC4 for Aaeo1 were 5.11±0.31 mM and 16.12±2.32 s-1,and the Km and kcat values for Aaeo2 were 0.79±0.03 mM and 3.59±1.15 s-1,respectively.(4)Given that Aaeo1 and Aaeo2 are derived from prokaryotic A.aeolicus,the recombinant strains BL21/p MBP3-Aaeo1 and BL21/pMBP3-Aaeo2,Origami2/pMBP3-Aaeo1 and Origami2/p MBP3-Aaeo2 were constructed using the fusion expression vector p MBP3.The level of protein expression and the specific activity of fusion protein are low.Comparison of P.pastoris and E.coli expression systems,the protein concentration and specific activity of Aaeo1 and Aaeo2 expressed in P.pastoris system were higher than those in E.coli.