Gene Resources Exploitation And Molecular Improvement Of Corn Bran Xylan Degrading Enzymes

Xylanases can hydrolyze xylan backbone of hemicellulose into monosaccharides and oligosaccharides and have been widely used in many fields.Corn bran,a byproduct of corn processing,is an important feed ingredient.It is rich in xylans,but the structure of xylan obtained from corn bran is extremely complex and difficult to degrade by traditional xylanase enzymes used in feed.In this study,the fungus Aspergillus niger MJ5 stored in the laboratory and eight Fusarium sp.strains purchased from the China Center of Industrial Culture Collection?CICC?were used as materials to screen for corn bran xylan-degrading enzyme producing strains.Salt ion medium with corn bran was used as the induction medium and xylan from corn bran was used as a substrate to detect xylanase activity of the culture supernatant.The results indicated that A.niger MJ5 and Fusarium graminearum 2697 had higher corn bran xylanase activities.From the genome sequencing results of the two strains,15 possible GH5 and GH10 family xylanase genes were found and expressed in Pichia pastoris expression system.Eleven possible GH5 or GH10 family xylanase genes were cloned from A.niger MJ5 and expressed in P.pastoris.Among them,one of the recombinant proteins,AnXYN10A,had corn bran xylanase activity.The optimal pH for AnXYN10A activity was 5.0,and the optimal temperature was60°C.When wheat arabinoxylan was used as a substrate,the specific activity,K_m,and V_max values of the enzyme,were 2507 U/mg,5.67 mg/mL,and 2835?mol/min·mg,respectively.When xylan from corn bran was used as a substrate,the specific activity of the enzyme was 22 U/mg,and K_m and V_maxax were 12.7 mg/mL and 36?mol/min·mg,respectively.Four possible GH10 family xylanase genes were cloned from F.graminearum 2697 and expressed in P.pastoris.Recombinant FgXYN10A and FgXYN10B had corn bran xylanase activity.The optimum temperature and pH for activity of FgXYN10A were 50°C and 5.0,respectively,and the corresponding values for FgXYN10B were 60°C and pH 5.5,respectively.The specific activities of FgXYN10A and FgXYN10B with wheat arabinoxylan as substrate were 1105 U/mg and 451 U/mg,respectively.When xylan from corn bran was used as the substrate,the specific activities of FgXYN10A and FgXYN10B were 19 U/mg and 14 U/mg,respectively.The corn bran xylanase activity of AnXYN10A from A.niger MJ5 was better than that of F.graminearum 2697.In order to further improve the stability and catalytic activity of AnXYN10A,two pairs of disulfide bonds were introduced into AnXYN10A at G89-I130 and Y177-G210,respectively.The optimal pH,temperature,and specific activity of the two disulfide bond mutants were similar to those of the wild type,and the thermal stability at 60°C for 10 min was approximately 5%higher than that of the wild type.Using sequence and structure analysis,three mutants,T302Y,L24Y,and R161A,were designed.Their optimal temperatures and pH were consistent with those of the wild type.The thermal stability of T302Y was improved.When T302Y was cultivated at 60°C for 10 min,the residual activity of T302Y was 80%,and that of the wild type was 55%.The specific activity was 19%lower than that of the wild type.The specific activities of L24Y and R161A were 12%and 11%higher than that of the wild type.We combined the disulfide bond mutation with T302Y and R161A to obtain AnXYN10A-M.Both the wild type and AnXYN10A-M had the highest activity at pH 5.0 and 60°C.While the stability of AnXYN10A-M was higher than that of the wild type,after cultivation at 60°C for30 min,the residual activity of wild type and AnXYN10A-M was 30%and 67%,respectively.The half-life of AnXYN10A-M at 60°C was 60 min,which was increased 3-fold compared to that of the wild type?15 min?.T_m of AnXYN10A-M was 63.3°C,which was 6.1°C higher than that of the wild type?57.2°C?.When wheat arabinoxylan and corn bran xylan were used as substrates,the specific activities were 2251 U/mg and 2507 U/mg,respectively,which were equivalent to that of the wild type.In this study,15 possible xylanase genes were successfully cloned from A.niger MJ5 and F.graminearum 2697,and then expressed in a P.pastoris expression system.The recombinant xylanases,AnXYN10A,FgXYN10A,FgXYN10B,and FgXYN10C,are active against the xylan obtained from corn bran.To summarize,mutational research of AnXYN10A was carried out,and the thermal stability of the combined mutant AnXYN10A-M was improved.It has potential for applications in the animal feed industry.