Cloning And Expression Of The Encoding Gene Of Thermostable Lipase From Thermomyces Lanuginosus

Lipase(triacylglycerol acylhydrolases, EC 3.1.1.3), which are widely existed in organisms, catalyze both hydrolysis and synthesis of the ester bonds of triglycerides at oil water interface. Lipase are thus of great importance in detergents, food ingredients and many other industrial applications. Lipase produced by microoraganisms are much more attractive in industrial field because of their easy getting, various use and great output .Thermomyces lanuginosus is a thermophilic fungus with higher growth temperature, which is shown to produce considerable thermostable enzymes such as lipase, cellucose , endocellulase, xylanase, laccase and so on. However the high culture temperature needed by T.lanuginosus to produce lipase greatly is difficult in industrial applications. In recent years most of studies focus on the cloning and expression of gene in different expression systems by gene engineering methods.During growth in a induced medium containing olive oil, T. lanuginosus produced lipase. Based on AF054513, a reported lipase gene from T. lanuginosus, and other published lipase gene in database, we designed special primers and cloned the full length genomic DNA and cDNA of two lipase gene using PCR,RT-PCR and TAIL-PCR methods. We named the two lipase gene as lgy and ln, respectively. DNA sequencing revealed that genomic DNA of the T.lanuginosus lipase gene lgy is 1071bp, contains three introns. And the cDNA of gene lgy we cloned is identical to the registered lipase gene AF054513 who contains an ORF of 873bp encoding 291 amino acids. The DNA and cDNA sequence has been registered in Genbank with accession number EU022703 and EU370914, respectively. The genomic DNA of ln is 1209bp, contains three introns too. The full length of ln cDNA is 1095bp, which contains an ORF of 873bp encoding 291 amino acids. Registered in GenBank , the accession number is EU004197 and EU004196.The alignment results of putative amino acids sequence shows both the lipase gene, lgy and ln, has the sequence"G-H-S-L-G", which is the conserved domain of mould lipase primary structure. Sequence alignment also shows that the two amino acids sequence are homology with the number of 78.69%.The coding region of lgy and ln without their signal sequences , first 17 amino acid residues, and expression vector pPIC9K were digested with SnaBⅠand BlnⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/lgy and pPIC9K/ln was constructed and sequenced to confirm the correct reading frame. The construct plasmid pPIC9K/lgy and pPIC9K/ln was linearized with a restriction enzyme SacI (insertion at 5'AOX1), and transformed into Pichia pastoris GS115 competent cell by electroporation methods, and screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of P. pastoris integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels of interest protein every 24 hours. The engineering strains with high expression level, named as GS-LGY-3 and GS-LN-6, respectively, were tested and characterized.Streaked culture for single colonies of His+ transformants GS-LGY-3 and GS-LN-6 on YPD plate for 10 generations, and then PCR analysis showed the interest gene was integrated in P. pastoris genome. The expression level was also kept stable. The expression lipase LGY exhibited optimum catalytic activity at pH 8.0 and 60℃. It was stable at 60℃within 60min. The expression lipase LN exhibited optimum catalytic activity at pH 9.0 and 60℃. The enzyme was stable at 50℃but much more thermostable at high temperature than LGY. Ca2+ enhanced both the enzyme activity greatly, whereas Ag+, Fe2+, K+, Zn2+ caused obvious inhibition.The T.lanuginosus lipase was secreted into the culture medium by the yeast P. pastoris in an active form, and some specific properties of expressed lipase was the same with lipase from the native strain T.lanuginosus(It exhibited optimum catalytic activity at 60℃and was stable at 60℃within 60min.). It implied the expressed lipase could be widely applied in the biological fields and chemical industry. Therefore, we hope to reconstruct the lipase gene and optimize the expression condition to obtain the yeast engineering strains suitable for industrial applications.