The Expression,Enzymatic Characterization And Molecular Modification Of Aspergillus Oryzae Lipase

Lipase?EC 3.1.1.3?,which can specifically catalyze the hydrolysis of triacylglycerol,is widely found in nature.As an important source of lipase production,microorganisms have been studied,such as yeast,Aspergillus,Pseudomonas,Penicillium,Rhizopus and other microbial strains with industrial application prospects.Therefore,in this study,we selected eight Aspergillus oryzae lipase genes from the NCBI database?Genbank?to been expressed in Escherichia coli BL21?DE3?.Subsequently,the lipase gene with the highest enzyme activity was selected for molecular modification to further enhance the activity.The enzymatic properties of the mutant lipase were studied.The results of research are shown as follows:1.Eight Aspergillus oryzae lipase genes which optimized for E.coli codon preference were constructed in pET-28a?+?and expressed in E.coli BL21?DE3?successfully,and their enzymatic properties were preliminary studied.The hydrolysis activity of the eight lipases was measured,and the highest lipase activity of AOL-3was 521.12 U/g.The optimum temperature,pH and substrate specificity were studied.It was found that the optimum temperature was between 30? and 40?,the optimum pH was between 6.5 and 7.5.The substrate specificity of lipases was determined by using different carbon chain length ethyl esters as substrates,and it was found that there were different catalytic preferences.2.Molecular modification of AOL-3.Random mutations were performed using error-prone PCR and combined with site-directed saturation mutations.Efficient and rapid screening was performed with the plates using tributyrin as a substrate.After the first round of random mutation,the mutant strain numbered AOL-3?38/93?was screened,in which the 38th phenylalanine?F?of AOL-3 was mutated to asparagine?N?.The mutant lipase was named AOL-3^F38N.The specific enzyme activity of the crude mutant lipase AOL-3^F38N was 1770.9 U/g,which was 3.4 times of the enzyme AOL-3.A second round of random mutations was performed on the first round of the screened mutants,and a mutant strain numbered AOL-3?38/93-230/6?was screened,of which the 230th proline?V?of AOL-3^F38N38N was mutated to arginine?R?.The mutant lipase was named AOL-3^F38N/V230R38N/V230R which the specific enzyme activity was 2094.48U/mg,which was 4.0 times that of the enzyme AOL-3.3.The mutant lipase AOL-3^F38N/V230R and the AOL-3 were purified,and the enzymatic properties of AOL-3^F38N/V230R were studied.The specific enzyme activities of AOL-3^F38N/V230R and AOL-3 after nickel ion affinity chromatography were 188.61U/mg and 46.64 U/mg,respectively.The optimum temperature and pH of the mutant lipase were 40? and 7.5,respectively.It had good temperature stability in the range of 35?-65?.The pH stability was poor and the enzyme activity dropped sharply under alkaline conditions.The substrate specificity of the mutant lipase for fatty acid ethyl esters with different carbon chain lengths changed from C₄ to C₆-C₁₀,which increased the hydrolysis activity of the medium and long-chain ethyl esters.In the low concentration?1 mM?metal ion solution,the experimental metal ions had no obvious inhibitory effect on the mutant lipase AOL-3^F38N/V230R,and Mn²⁺had an activation effect.However,under relative high concentration?5 mM?metal ion conditions,its effect was changed.Zn²⁺,Fe³⁺,Ni⁺completely inhibited the activity of mutant lipase,and Mg²⁺and Mn²⁺had obvious activation effects.The Kinetic parameter K_m and V_maxax of AOL-3 were 2.40 mM and 7.89 mM/min,and AOL-3^F38N/V230R38N/V230R were determinated as 1.92 mM and 12.22 mM/min?...