Study On Yarrowia Lipolytica Extracellmlar Lipases

Lipases (EC3.1.1.3) are serine hydrolases that catalyze esterification, transesterification, hydrolysis and alcoholysis reaction, are widely used in the field of biofuels, food processing, nutrition, cosmetics, pharmaceutical. Yarrowia lipolytica is one of the non-conventional yeasts used as a model to produce lipases, the mixed lipase has been successfully used in the synthesis of biodiesel, polyol ester and other products. To refine the understanding of the lipase family of Y.lipolytica, expand the scope of its application. In this paper, the study focuses on the extracellular lipase of the Y.lipolytica. It is known that Lip2?Lip7 and Lip8 are extracellular lipases, in this paper, Lip2?Lip7 and Lip8 were studied by heterologous expression and basic theory of enzymatic properties, which lay the foundation for the subsequent application of the lipase family of Y.lipolytica. For the characterization of three extracellular lipases, some researches have been done as follows:(1) Successful construction of three recombinant plasmids pPICZaA-lip2, pPICZaA-lip7 and pPICZaA-lip8. The secretory expression of Lip2,Lip7 and Lip8 was successfully achieved in Pichia pastoris GS115. Optimization of shake flask fermentation conditions, the optimal induction temperature of 25 ?, the most suitable induction time is 72 h, the hydrolytic activity were 3.38 U/mL,0.18 U/mL,0.05 U/mL(2) In this paper, we systematically compared the catalytic ability of three extracellular lipase in P.pastoris, the 5L fermentor fermentation liquid hydrolytic activity were 4000 U/mL,4.12 U/mL,1.71 U/mL, hydrolytic activity of Lip2 was better than that of Lip7 and Lip8. The enzymatic properties of basic research shows:the optimum pH of Lip2 and Lip7 were 8.0; the optimum temperature of Lip2, Lip7 and Lip8 were respectively 40 ?,40 ? and 50?; different metal ions on three lipase activity inhibition or promote different levels, which Ni2+ inhibition is most obvious; Lip2 biased in favor of the hydrolysis of long chain esters, while Lip7 and Lip8 biased medium chain ester, and the differences of lipase substrate specificity is interpreted from the space structure. These differences in enzymatic properties, lay the foundation for further research and application of the three lipase.(3) Because Escherichia coli has the advantages of short fermentation period and low cost, this paper makes a preliminary study on the heterologous expression of lipase in E.coli. pET22b-lip7 recombinant plasmid was successfully constructed, and the heterologous expression of Lip7 in E.coli was achieved. Optimization of shake flask fermentation conditions, the induction time of 12 h, the expression of host Rosetta, induced by temperature is 20?, Lip7 hydrolytic activity is 0.29 U/mL(4) As a kind of lipase with extensive application value, Lip2, which is widely used, has a poor thermal stability, which is an obstacle to its application. This paper attempts to further improve the thermal stability of Lip2 in the early stage of the laboratory. Three mutant T117G/C244A, F237C/C244A, T117G/F237C/C244A were successfully constructed by site directed mutagenesis technique, and secretory expression was successfully achieved in P.pastoris GS115. The half-life of T117G/F237C/C244A at 50? was greater than 100 min. The thermal stability of T117G/F237C/C244A was much higher than that of the previous mutant (14.4 min). In this study, further enhances the thermal stability of Lip2. Through the analysis of the spatial structure of lipase, the mechanism of improving the thermal stability of the mutant was discussed, which provided a theoretical basis for understanding the relationship between the structure and function of the lipase family.