High-level Expression,Characterization And Application Of Acidic Lipases

Acidic lipases,which exhibit high and stable hydrolytic activity towards oils and fats in acid environment,pocess good application prospects in food,chemical,pharmaceutical,feed and other industries.In our laboratory,two acidic lipases ANL from Aspergillus niger NICM1207 and ATL from Aspergillus Terreus were successfully heterologous expressed in Pichia pastoris GS115.However,the low expression level of acidic lipases from Aspergillus sp.remains a major obstacle for industrial application.Methods such as fusion expression and site-directed mutagenesis were carried out to improve the expression level.And then,the recombintant acidic lipases were purified and characterized,and the application in gastrointestinal digestion in vitro of the acidic lipases were studied.The main works and innovative results are listed as follows:?1?Fusion expression with cellulose binding domain?CBD?,maltose binding protein?MBP?and small ubiquitin-related modifier?SUMO?was investigated to enhance the expression level of A.niger lipase?ANL?in P.pastoris.When fused with SUMO,named SANL,the total protein concentration and enzyme activity in the shake flask fermentation were 4.31-and 1.60-fold higher than the parent ANL,respectively.The highest activity reached 960 U·m L^-1 in 3-L bioreactor,which was 1.85-fold higher than the parent ANL.SANL exhibited the maximum activity at pH2.5 and high stability in a broad range of pH?4.0-10.0?.SANL showed the K_m values towards olive oil,triolein and p-nitrophenyl palmitate were 0.42-,0.69-and 0.15-fold lower and the k_cat/K_m values were 1.18-,1.79-and 5.51-fold higher than ANL,respectively.?2?The catalytic activity of A.terreus lipase?ATL?was improved by rational design.According to the sequence analysis and homologous modeling,several amino acids involved in the lid domain and substrate binding pocket domains of the acidic lipase ATL were mutated by site-directed mutagenesis,and eight mutants were constructed.These mutants and the wild type lipase ATL were expressed in P.pastoris and the enzymatic properties were characterized.The mutants ATLLid and ATLV218W exhibited higher hydrolytic activity than ATL towards p-nitrophenyl laurate.The k_cat values of ATLLid and ATLV218W towards p-nitrophenyl laurate were 39.37-and 50.79-fold higher,and the k_cat/K_m values were 2.85-and 8.48-fold higher than the parent ATL,respectively.Although thermostability of these mutants decreased slightly,ATLLid and ATLV218W still exhibited the maximum activity at pH5.0 and high stability in a broad range of pH?4.0-8.0?,which were similar to the parent.Using homologous modeling and molecular docking technology the mechanism for the improvement of catalytic activity was analyzed.?3?In the gastrointestinal digestion experiments,ANL and SANL exhibited higher tolerance to pepsin and higher hydrolysis activity towards triolein from pH3.0 to 6.0 than A.terreus lipases ATL,ATLLid and ATLV218W,which exhibited the superiority of A.niger lipase in gastric digestion.However,all of these Aspergillus.sp lipases were significantly inhibited by NaTDC,which may limit their application in intestinal digestion.Combined with the Rhizopus chinensis CCTCC M201021 lipase?RCL?and ANL/SANL the oil hydrolysis rate can be acccelarated in the gastrointestinal digestion in vitro.