| Mono-and diacylglycerol lipase is a highly selective lipase for lipid substrates that specifically reacts with mono-and diglycerides and has no hydrolytic activity with triglycerides substrate.Its unique substrate specificity makes it valuable in the field of lipid modification.Until now a few mono-and diacylglycerol lipases have been reported,but their basic theory and applications need to be further studied.In this study,a novel mono-and diacylglycerol lipase Pr Lip was mined from the genome of Penicillium roqueforti.It was studied including its expression and purification,enzymatic properties,crystallography and application in deacidifying high-acid lipids,which aims to provide structural basis for elucidation of the catalytic mechanism and give scientific guidance for industrial application.The detailed results are as follows:(1)Preparation and enzymatic properties of lipase Pr Lip.An engineering strain of Pichia pastoris X33 was constructed to screen a positive clone with high expression of target protein.After fermented for 60 h at 30℃,Pr Lip was purified by anion exchange chromatography with the purity above 90%.SDS-PAGE showed that the molecular weight of target band was 31 k Da.The enzymatic characterization results showed that the optimal reaction temperature and p H were 45℃ and 7.0,respectively,and its thermostable half-life under 40℃ was 6 h.It was more stable in alkaline environment,and had a good tolerance against temperature and most surfactants.Pr Lip was characterized as a mono-and diacylglycerol lipase through hydrolysis and esterification.(2)Crystallization and structure analysis of lipase Pr Lip.The homogeneity of Pr Lip was further improved by gel filtration chromatography and then it was used for protein crystals by vapor diffusion method.The final crystallization conditions were as followed: protein concentration 9 mg/m L,temperature 4℃,0.1 M sodium acetate(p H 4.0),26%(w/v)PEG 1500.Through molecular replacement and structural modification,the three-dimensional structure with resolution of 1.8 ?(PDB ID: 6L7N)was obtained.Pr Lip is a typical α/β hydrolytic folding enzyme consisting of 8 parallel β-sheets and 12 α-helices.The lid region is comprised of Gly82 to Val96,which is a typical amphiphilic α-helix through structural comparison.The catalytic triad is Ser145-Asp199-His259,and the oxygen anion hole is Ser83 and Leu146.(3)Immobilization of lipase Pr Lip and its application in deacidifying high-acid rice bran oil.After screening 8 kinds of resins and optimizing immobilization conditions,the esterification activity of immobilized Pr Lip reached 879 U/g under the optimal conditions of ECR8806 resin,p H7.0(20 m M)buffer and 20 mg/g enzyme-carrier ratio.Then it was applied to deacidify high-acid rice bran oil.After optimizing reaction parameters,the final reaction conditions were as followed: using ethanol as acyl receptors,when the molar ratio of ethanol to free fatty acids was 2:1,reaction temperature of 35℃,the enzyme dosage of 60 U/g and 4 m L/g(with respect to oil)n-hexane,the free fatty acids in rice bran oil decreased from 39.16% to 1.02% and the deacidification efficiency reached 97.4% with the catalysis of immobilized Pr Lip after 8 h. |
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