| Lipase(EC 3.1.1.3) is also known as triglyceride hydrolysis enzyme. Owing to the advantages of the efficiency of production, high selectivity and non-pollution to the environment, lipases are widely used in light industry, medicine, food, environment, energy and other industries. Rhizopus sp. are the important producing strains of the microbial lipases. The gene sequence similarity of lipases from Rhizopus oryzae and Rhizopus chinensis are more than 80%, consisting of signal peptide, prosequence and mature peptide. In our previous studies, we found the secretion level of R. oryzae was much less than that of R. chinensis in Pichia pastoris. Through the analysis of lipase gene sequence, the significant difference between them are the difference of prosequence. Three potential N-glycosylation sites are found in the propeptide of RCL, while ROL possesses only one potential N-glycosylation site in its prosequence. Therefore, in view of the lipase prosequence and its N-glycosylation, this paper conducted the mechanism study of the expression and secretion of lipases in The research contents are as follows:(1) The conservative sites Cys28 in the prosequence of R. chinensis lipase promotes the expression and secretion of lipase in P. pastoris. The recombinant genetic engineering strain expressing Cys28 Ser lipase mutant was constructed. Compared with the wild type, the enzyme activity and secretion levels of the mutant decrease significantly, suggesting that the Cys28 in the prosequence may work as intra-molecular disulfide agent, promoting the secretion and expression of lipase.(2) Identify the N-glycosylation of R. chinensis lipase. The mutant R. chinensis lipase pro RCLCNQ and the N-glycosylation sites mutant pro RCLCN14 Q, pro RCLCN48 Q and pro RCLCN60 Q were constructed and compared with lipase r27 RCLC excision of part of prosequence. Using glycosidase, MADLI-TOF-MS and LC-MS/MS methods to determine the the N-glycosylation of R. chinensis lipase expressed in P. pastoris, results showed that about 90% of N-14 and N-60 sites produce N-glycan, while only 10% of N-48 sites occurres N-glycosylation.(3) The N-glycosylation in the prosequence of R. chinensis lipase is the key role of lipase expression and secretion in P. pastoris and the N-glycan of lipase has an important effect on enzyme properties. The secretion levesl, extracellular enzyme activities and extracellular total protein concentration of r27 RCLC, pro RCLCNQ and N-glycosylation sites mutants in P. pastoris were studied. At the same time, the enzyme properties of lipases including the optimum reaction temperature and p H stability, thermal stability, the optimum p H, organic solvent tolerance and enzymatic reaction kinetics were compared. Results showed that the mutation of the glycosylation site N-60 cause the mutant pro RCLCN60 Q unable to be secreted into the supernatant, suggesting that the N-60 plays a key role in the expression of lipase. However, the mutations of N-14 and N-48 have no effect on enzyme secretion and expression, but the mutation of N-14 causes the enzyme catalytic activity decrease significantly, indicating that the N-glycan on N-14 site can promote the catalytic efficiency of lipase; Besides, the N-glycosylation enhances the thermal stability and the organic solvent tolerance of R. chinensis lipase.(4) The complete prosequence and N-glycans are the obstructive factors of R. chinensis lipase in vitro refolding. Aimed at r27 RCLC removed part of prosequence, pro RCLCN14 Q containing a N-glycan, pro RCLCNQ containing two of N-glycan and pro RCL without N-glycan expressed in E. coli, lipases spontaneously folding efficiency were studied in vitro study. Research results showed that the renaturation of r27 RCLC is the most efficient; and the renaturation rate of pro RCL is higher than pro RCLCNQ and pro RCLCN14 Q, indicating that the existence of the complete prosequence and N-glycan have great negative effect on lipase in vitro refolding.(5) The rational design of the introduction of N-glycosylation sites in R. oryzae achieve the high-efficiency expression of lipase in P. pastoris. One and two N-glycosylation sites were introducted in the prosequence of R. oryzae lipase respectively. The recombinant genetic engineering strains expressing lipase pro Ro LA, pro Ro LB and pro Ro LAB were constructed. Results showed that after the introduction of N-glycosylation sites in R. oryzae lipases, the expression level increase from almost no secretion to relative high level(200 U?m L-1). The secretion of pro ROLAB introducing two N-glycosylation sites is obvious higher than that of introducing a N-glycosylation sites mutant pro ROLA and pro ROLB, and the production of pro ROLA is higher than pro ROLB. The results showed that the introduction of N-glycosylation sites is likely to produce the additional degree of glycosylation, significantly increasing the secretion of lipase, and the design of the different N-glycosylation sites in R. oryzae lipase have different impact on lipase ssecretion level.Based on the above results, it showed that R. chinensis lipase occured complicated N-glycosylation modification in the production process in P. pastoris. The N-glycosylation modification has an important effect on the expression level, correctly fold and enzymology properties such as catalytic activity and stability of lipase. N-glycosylation modification on lipase has a significant role in promoting secretion. Based on this, this study provides a rational design of N-glycosylation, a very effective strategy to improve enzyme expression level. On the other hand, N-glycosylation significantly improves lipase heat resistance and organic solvent resistance lipase, providing a new train of thought for the lipase molecular modification to improve the adaptability of biocatalysis in nonaqueous phase. Keywords: Pichia pastoris, Lipase, Rhizopus sp, N-glycosylation, prosequence... |
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