Cloning And Expression Of The Lipase Gene From Marine Microorganisms

Ocean ecosystem is the largest ecosystem on the earth, where there are enormous varieties of microorganisms living in. Marine microorganisms produce different enzymes to fit in its living environment which is differ from that of terra firma. As ester hydrolase, lipase and esterase of marine microorganisms has wide industrial application prospect. The main subject of this study is to screen speciality lipase or esterase gene from marine microorganisms, and characterize those protein.By analysis of 16S rDNA sequence, a Burkholderia sp. S30 and Acinetobacter sp. S9 showing extracellular lipolytic activity towards tributyrin were isolated from the marine eel and abalone gastrointestinal contents.Second, marine lipase from Burkholderia sp. S30 were investigated. A lipase gene(Lip) encoding 364 amino acids was obtained by PCR, and 1-41 amino acids of the whole amino acids were predicted to be the signal peptide. The sequence has been submitted to GenBank under the accession number KJ631420. The Lip gene was amplified and integrated into the genome of P. pastor is X33 via the pPICZa-C vector, and the recombinant lipase was expressed into the culture medium. After induced with methanol, recombinant lipase reached 118.8 U/L with weight of 36.6 kDa. The optimum pH and temperature of recombinant lipase were 8.5 and 55℃, respectively. Furthermore, this lipase exhibited remarkable stability at pH between 8.0-10.0 and under 60℃.Third, a esterase gene(Est) was cloned from Acinetobacter sp. S9. The gene Est encoding 303 amino acids was obtained by PCR from Acinetobacter sp. S9, and 1-19 amino acids of the whole amino acids were predicted to be the signal peptide. The sequence has been submitted to GenBank under the accession number KJ019019. The Est gene was amplified and integrated into the genome of P. pastoris X33 via the pPICZa-C vector, and the recombinant esterase was expressed into the culture medium. After induced with methanol, recombinant esterase reached 1683.1 U/L with weight of 33.7 kDa, and was 2.6 times higher than the original strain. The specific activities of the recombinant esterase were 27.28 U/mg after ammonium sulfate precipitation, ion-exchange column chromatography and gel filtration chromatography. The optimum pH and temperature of recombinant esterase were 8.0 and 40℃, respectively. And this lipase exhibited remarkable stability at pH between 8.0-10.0 and under 70℃. The apparent Km and Vmax values for p-nitrophenyl acetate were 0.804 umol/mL and 13.587 μmol/mL-min, respectively.Compared with the terrestrial, the recombinant proteins possess the advantages of low reaction temperature and high pH resistance. The properties make them very attractive for industrial applications.