| It is not ideal that the activity and stability of xylanase in a strongly acidic environment in the production of animal feed and fruit juice drinks,so we want to find a new xylanase of high activity and acid resistance necessarily.A.niger xylanase xyn? has expressed in E.coli Successfully by our laboratory,the optimum pH of zhe xynlanase is 3.8,and the stability in an acidic environment is not high.This study is based G/11 family xylanase optimum pH of two conjoined formula speculated that the optimum pH with the YS bivalent lanes are negatively correlated,so we design change to the back of the Y amino acid mutations into S,expectations optimum pH of new xynlanase will lower than before.According to the sequence of xylanase from Aspergillus niger,we found 9 mutation sites,than,we using fusion PCR method to obtain the mutant gene make the amino acids back of Y mutated to S,in accordance with the location of the mutation were named No.1 to No.9.Make gene connected with an expression vector p ET-20 b after restricted by enzyme,than make the gene transferred into E.coli BL21 to for expressed.I got four point mutations mutants of 7,8,9 and 3-7 double mutants final.8 and 3-7 double mutant enzyme is not detected activity by confirmed,so follow study we select7,9 and 2 come from the laboratory.The correct sequencing of the mutant strains for a large number of induced expressions,the cells were disrupted to obtain a crude enzyme solution,and then to use Co2 chelating agarose gel affinity chromatography and dextran gel purified,and ultimately obtained the pure enzyme.G/11 family xylanase optimum pH of two conjoined formula speculate optimum pH with the YS bivalent takes the shape of a negative correlation,so the design mutation sites in the back of the Y amino acid mutations into S,in order to explore the new xylanase.The results showed that:(1)No.2,No.7,No.9,three mutants' Optimum pH compared to the wild-type enzyme 23 decreased by 0.2 units,and forecast match.(2): 7 mutant enzyme optimum temperatures for 48?,high 2?compared with the wild-type 23,extend the half-life nearly 10 min,proved the bit 7 point mutations to improve the temperature stability of the enzyme.(3): After the Enzymatic Kinetic analysis showed that three mutant enzymes Vmax values were 0.013/0.030/0.025 ?mol/ml/min and 0.063 mmol of the wild-type enzyme/m L/min compared to more significantly reduce,the mutant enzyme reaction rate decreases.The Km values of the three mutant enzymes were 3.14/4.02/9.38 mg/m L respectively,than that of the wild-type enzyme of 13.56 mg/m L,and the mutant enzyme and substrate affinity and capacity increased.Kcat value of the mutant enzymes are obvious 37.60/39.40/22.05 compared to the wild-type enzyme 277.37 reduced very significantly,indicating that the binding efficiency of the mutant enzyme and the substrate is lower than compared to wild-type.In this study,the pH diad formula based on mutant gene fusion PCR method for fixed-point,and ultimately gets the new mutant enzyme.Research from the molecular level of xylanase acid resistance mechanism prove YS diad can reduce the optimum pH of xylanase provides a theoretical basis to improve the acid resistance of xylanase. |
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