Purification, Gene Cloning And Expression Of Lipase From Aspergillus Oryzae And Its Application In The Synthesis Of Vitamin A

Lipase?EC 3.1.1.3?is a kind of hydrolase widely used as biocatalyst for organic synthesis.Lipase-catalyzed reactions in organic media have become increasingly important in biocatalysis since the lipases need no co-enzyme and have demonstrated high catalytic efficiency,high selectivity?chemoselectivity,regioselectivity and stereoselectivity?and robust stability.In the study,the lipase from Aspergillus oryzae WZ007?AOL?was purified and characterized in details.Furthermore,the gene encoding AOL was successfully cloned and then heterologously expressed in E.coli BL21?DE3?.In addition,AOL's applications in the synthesis of vitamin A were investigated.A High-throughputassayforscreeninglipase-catalyzed transesterification activity was established,in which lipase-catalyzed transesterification with vinyl acetate and n-butanol in organic media was employed as model reaction and the content of acetaldehyde was detected by phenol reagent.The results showed there was a good linear relationship between the value of optical density and the concentration of acetaldehyde within the range of 0.001-0.150 mM.The strain of WZ007with high transesterification activity was screened using the high-throughput screening assay.Then,the strain of WZ007 was identified to be A.oryzae relying on electron microscope observation of spores,physiological and biochemical properties,ITS sequence analysis and detection of aflatoxin content.Particularly,the results showed that the strain did not produce aflatoxin and thus had a good bio-safety.After the cells of A.oryzae WZ007 were disrupted by ultrasonic cell disruption,the intracellular AOL was purified using a four-step procedure:acetone precipitation,DEAE ion exchange chromatography,octyl hydrophobicchromatographyandhigh-Qanionexchange chromatography.The purity of the purified AOL was verified through the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Enzymatic properties of AOL that the enzyme had a“lid”structure and could hydrolyze various triglycerides as substrate.The optimal values of temperature and pH value were 50 and pH 7.5,respectively.The enzyme showed excellent thermo-stability and tolerance to organic solvents and surfactant,while the enzyme activity was severely inhibited by Cu²⁺,Zn²⁺and Ag⁺.The addition of 5 mM dithiothreitol resulted in 1.8-fold increase of the lipase activity,suggesting that the free sulfhydryl groups of enzyme were pertinent to the lipase activity.The sequences of peptide fragments of AOL obtained by MALDI-TOF-MS had a high identity with a triacylglycerol lipase from A.oryzae?GenBank accession number:BAA92327.1?.The gene encoding AOL which designated as aol was amplified by PCR using the cDNA of A.oryzae WZ007 as template and the primers was designed according to the known the gene tglA.The gene aol contained a 765-bp open reading frame encoding a protein of 254 amino acids?GenBank accession number:KP975533?.SignalP 4.1 analysis showed that there was no signal peptide in the sequence of AOL.Tmpred software analysis indicated that the enzyme AOL had no transmembrane segments and thus was an intracellular enzyme.Prediction of secondary structure of AOL was performed by PSIPRED and the three-dimensional structure of lipase was modelled through SWISS-MODEL homology modeling server.The catalytic triad of AOL was Ser170-His235-Asp222and the lid structure of AOL was constituted by ¹³³VAGYLAG¹³⁹ with alpha helix structure.Furthermore,the gene aol was successfully cloned into the pEASY-E1 vector through AT-cloning strategy and subsequently heterologously expressed in E.coli BL21?DE3?.The recombinant E.coli showed stereoselective hydrolytic activity towards ethyl R,S-2-phenoxy propionate,demonstrating that the targeted recombiant AOL was functional expressed in E.coli.Regioselective acetylation of vitamin A intermediate diols for the synthesis of monoacetate was catalyzed by whole-cells of A.oryzae WZ007,and the influence factors on biocatalysis were investigated and optimized.The optimized biocatalytic conditions were listed as following:MTBE as solvent,vinyl acetate as acylating agent,1:4 molar ratio of substrate towards acylating agent,35?reaction temperature and lyophilized cells at phosphate buffer pH 7.5.After the lipase-catalyzed reaction beening conducted in 1 L reaction system for 10 h under the optimized reaction conditions?500 mM diols as substrate,50 g/L cells of A.oryzae WZ007 as biocatalyst?,the conversion rate and the yield of monoacetate reached 100%and 98.2%,respectively.Moreover,the product from the AOL-catalyzed reaction was identified as vitamin A intermediates monoacetate by the analyses of nuclear magnetic resonance and gas chromatography-mass spectrometer.