Study On The Xylanase Mutagenesis Of Resistance To Inhibit Xylanase Activity

Xylanase inhibitor proteins have been recently found existing in wheat,which exclusively inhibitor exogenous xylanases.It has been proved that these xylanase inhibitor proteins have negative effect on function of xylanases used in feed industy.So we plan to obtain the insensitive xylanase mutants to solve this problem.Aspergillus niger xylanase p ET-20b-xyn X4 has expressed in E.coli successfully by our laboratory.The inhibition rate is 87.61%.The study is based on structure of the complex between XIP-I and Aspergillus nidulans,and revealed 22 extensive interactions between the two proteins.The mutagenesis named A1,A1,A2,A3,A4,A5,No.1~17.The amino acid sequence alignment between Aspergillus aculeatus xylanase and Aspergillus niger xylanase showed that three differences amino acid.The mutagenesis named B1?B2?B3.According to enzyme activity and inhibition rate of the obtained mutagenesis,we designed 10 mutagenesis named 18~28.Than we used Quik Change Site-Directed Mutagenesis to obtain the mutant gene make the amino acids mutanted A.The correct sequencing of the mutant strains for a large number of induced expressions,and then to use Co2+ chelating agarose gel affinity chromatography and dextran gel purified,and ultimately obtained the pure enzyme.The results showed that:(1)A1,A2,A3,No.6,No.13,No.15,No.16,No.19,No.20,No.22,No.25 were not detected enzyme activity.On the condition of mole ratio between the two proteins,A4 and NO.1 inhibition rate are 75% and 73.13%,decrease 12.61% and 14.48% compared with the wild-type.(2)The optimum p H of XYN7,NO.1,B1 were 4.5,showed that the mutant No.1 improve the acid-resistance of the enzyme.XYN7 enzyme optimum temperatures for 45?,No.4 and A4 mutant enzyme optimum temperatures for 50?,B1 mutant enzyme optimum temperatures for 40?.The pure enzyme half inactivation times of four mutant enzymes were 19.553/19.154/36.517/31.111 min.The wild-type enzyme half inactivation time was 130.304 min.(3)After the enzymatic kinetic analysis showed that four mutant enzymes Vmax values were 0.006/0.002/0.002/0.003?mol·m L·s-1and compared 0.018?mol·m L·s-1of the wild-type compared to more significantly reduce,the mutant enzyme reaction rate decreases.The Km values of the four mutant enzymes were 3.56/0.82/0.69/0.81mg·m L-1 respectively,than that of the wild-type enzyme of 8.16 mg·m L-1,and the mutant enzyme and substrate affinity and capacity increased.Kcat value of the mutant enzymes are obvious 2.88/1.21/0.91/1.29 s-1compared to the wild-type enzyme 9.14 reduced very significantly,indicating that the binding efficiency of the mutant enzyme and the substrate is lower than compared to wild-type.Based on the crystal structure,amino acid sequence alignment and Quik Change Site-Directed Mutagenesis,the present obtain the mutant enzymes.Researching on the molecular level of xylanases and inhibition mechanism could provide a theoretical basis to improve the insensitive xylanases of inhibitors from all sorts of microbial xylanases.