Directed Evolution Of Activity And Organic Acid Tolerance Of ?-glucosidase

?-glucosidase(BGL)is a rate-limiting enzyme of lignocellulose hydrolysis.However,organic acids produced during the process of lignocellulose pretreatment can inhibit BGL.In this study,we took BGL from Penicillium oxalicum 16 screened by our lab as the research object.The error-prone PCR method was used for directed evolution.The p GAPZa A and Pichia pastoris GS115 were used as the constitutive expression vector and host,respectively.We adopted high-throughput screening method including formate flat plate primary screening,96-well plate secondary screening and shake flask third screening method to obtain mutants with improved activity and organic acid tolerance.Using the single factor optimization method,we obtained the optimal error-prone PCR reaction condition:3 m M Mg²⁺and 0.2 m M Mn²⁺.Then 6 target mutants were obtained according to the above construction and screening methods,named as L1-1-D5,L1-3-H11,L2-1-C12,L2-2-C7,L3-F3 and L3-H4.According to enzymatic characterization results,the optimum temperature and optimum p H of L1-1-D5 were65°C and 4.0,respectively.The other 5 mutants were the same as the original enzyme,and their optimum temperature and optimum p H were both 70?and 5.0.The relative residual activities of L1-1-D5,L2-2-C7,and L3-H4 after incubating at 70?for 50minutes were 1.3 times,1.9 times,and 1.8 times compared with the original 16BGL,respectively.Compared with the original enzyme,the catalytic efficiency K_cat/K_m of L1-3-H11 increased to 1.2 times,and the specific activity increased by 3.0 times;the specific activities of L2-2-C7 and L3-H4 increased by 2.3 times and 0.7 times,respectively.The enzyme activities of L1-1-D5,L2-1-C12 and L3-F3 were increased by 0.5 times,0.9 times and 1.5 times,respectively.Under the conditions of 10 mg/m L formic acid at 50?for 60 min,the formic acid stability of L3-F3 increased by 1.7 times;acetic acid stability after 60 minutes in 25mg/m L acetic acid at 50°C is 1.2 times compared with the original enzyme.The acetic acid stability of L1-1-D5 and L2-1-C12 in 15 mg/m L acetic acid at 50?for 5 h is 1.1times as much as the original enzyme.The acetic acid tolerance(K_m/K_I)of L3-F3 is 1.4times that of the original enzyme.Compared with the original enzyme,the formic acid and acetic acid tolerance of L2-1-C12 were increased to 1.2 times and 2.2 times,respectively.In addition,by using a single-factor optimization method,we obtained the optimal reaction condition of DNA shuffling:digestion of 0.1?g DNA by 0.004 U DNase I at 37°C for 10 minutes.In this study,based on obtaining the mutants with beneficial properties and the DNA shuffling optimal reaction condition,it will lay a solid material foundation for the co-evolution and its mechanism of?-glucosidase activity and organic acid tolerance and also provide a method for the molecular modification and exploration of other enzymes if we combine the superposition principle of beneficial mutation.