High-efficiency Expression And Immobilization Of Monoacylglycerol Lipase GMGL

Monoglyceride lipases are hydrolytic enzymes that specifically catalyze the hydrolysis of monoglyceride substrates.Due to the high substrate specificity,monoglyceride lipases have great application value in industrial applications.At the same time,monoglyceride lipases play an important physiological regulatory role in different organisms.Based on a large number of studies on the relationship between structure and function of monoglyceride lipases,their catalytic and physiological functions have gradually become clear.However,the research on their high-efficiency expression and preparation of enzyme preparations have not been studied deeply,which limits the industrial application prospect of them.This study took the monoglyceride lipase from Geobacillus sp.12AMOR1(GMGL)of Bacillus licheniformis as the research object.We optimized the high-efficiency expression and fermentation conditions of GMGL in E.coli.In addition,the immobilized preparation conditions,characterization and preliminary application of enzymes were studied.The main research results are as follows:(1)Optimization of fermentation process of engineered strain GMGL/E.coli BL21(DE3).The effects of inducer concentration,induction time,induction temperature,induction pH and glucose addition on the preparation of GMGL/E.coli BL21(DE3)fermentation were studied in shaking flask,5 L fermenter and 50 L fermenter.The results showed that the optimum IPTG concentration was 1.0 mmol/L,the optimum induction time was late logarithmic growth phase(OD₆₀₀ was 20),the optimum induction temperature was 25?,the optimum pH was 7.0,and the optimum glucose flow was variable speed flow.Under the optimum condition,the activity of GMGL reached 1985 U/m L.It was 67.50%higher than that before horizontal fermentation in fermentation tank and 16.54 times of that before horizontal fermentation in shaking flask.In 50 L fermenter,the highest GMGL activity was2335 U/m L at 27 h,which was 17.63%higher than that in 5 L fermenter.(2)Immobilization of recombinant monoglyceride lipase GMGL.Seven resins with different carrier materials were selected to immobilize GMGL.Among them,the lipase GMGL immobilized with epoxy resin ECR8285M showed the highest hydrolysis activity(i.e.,2595 U/g).The immobilization process was further optimized by single-factor test and response surface methodology:the optimum enzyme loading was 84mg/g,the salt ion strength of phosphate buffer was 0.54 mol/L,and the buffer pH was 8.70.Under the optimum condition,the enzyme activity of immobilized GMGL was 4908 U/g,which was 197.45%higher than that before optimization.(3)Characterization and enzymatic properties of immobilized GMGL.Before and after immobilization,GMGL was characterized by scanning electron microscopy(SEM)and Fourier transform infrared(FTIR)spectroscopy,which confirmed that GMGL was successfully immobilized on ECR8285M resin.Studies on enzymatic properties showed that the optimum reaction temperature was 60?.After incubation at 55?for 1 h,the hydrolysis activity of ECR8285M-GMGL remained above 80%,showing better thermal stability than free GMGL.The optimum reaction pH was 7.0,showing excellent stability in neutral solution environment.ECR8285M-GMGL had a strong tolerance to n-hexane and the residual hydrolytic activity was still 82.30%after 3 h treatment.After sealed and stored at 4?for 120 days,the hydrolytic activity of ECR8285M-GMGL was still over 92%.In addition,after continuous stirring for 6 days,no crack was observed,which indicated that ECR8285M-GMGL had good storage stability,high mechanical strength and attractive industrial application potential.