To improve the inhibitory insensitivity towards Aspergillus usamii E001 GH11F xylanase(Auxyn11A)towards xylanase inhibitor protein(XIP-I),and Triticum aestivum xylanase inhibitor(TAXI-I)the site-directed mutagenesis w...To improve the inhibitory insensitivity towards Aspergillus usamii E001 GH11F xylanase(Auxyn11A)towards xylanase inhibitor protein(XIP-I),and Triticum aestivum xylanase inhibitor(TAXI-I)the site-directed mutagenesis was conducted based on the computer-aided redesign.Firstly,two xylanase inhibitory proteins-encoding xip-I and taxi-I were synthesized and expressed in Pichia pastoris SMD1168,namely SyXIP-I and SyTAXI-I.The highest inhibitory activities of SyXIP-I and SyTAXI-I on Auxyn11A occurred at molar ratio(xylanase:inhibitor)10:1,40℃ for 30 min,and 100:1,30℃ for 30 min,respectively.Secondly,nine variants of a Auxyn11A-encoding gene(Auxyn11A)were constructed as designed theoretically and expressed in E.coli BL21(DE3),respectively.One mutant,Auxyn11ASDSS exhibited the highest xylanase activity and showed 100%resistance to the inhibitor SyXIP-I,and Auxyn11A^(N2) showed 30%resistance.Secondly,to obtain both types of resistance,four variants,Auxyn11A^(N2−SDSSA),Auxyn11A^(SDSSA),AuxynM^(SDSS),and AuxynM^(SDSSA) was also constructed.Among them,AuxynM^(SDSSA) exhibited complete resistance to the SyXIP-I and SyTAXI-I.Furthermore,AuxynMSDSSA was purified.Its optimum temperature and pH were 60℃ and 5.0,and those of Auxyn11A were 50℃ and 5.0.The specific activity,K_(m),V_(max),and k_(cat)/K_(m) values of AuxynM^(SDSSA) were 1054.6 U/mg,4.0 mg/mL,6659.6μmol/min/mg,and 610.46 mL/mg/s,which were similia to those of Auxyn11A,1097.4 U/mg,4.23 mg/mL,6876.5μmol/min/mg,and 557.04 mL/mg/s,respectively.This study provided a novel thermophilic xylanase with complete resistance to the SyXIP-I and SyTAXI-I,therefore,making it a promising candidate for extensive applications in animal feed and food industry.展开更多
基金supported by the National Natural Science Foundation of China(21676117)the Natural Science Foundation of Jiangsu Province for Youth of China(No.BK20180622).
文摘To improve the inhibitory insensitivity towards Aspergillus usamii E001 GH11F xylanase(Auxyn11A)towards xylanase inhibitor protein(XIP-I),and Triticum aestivum xylanase inhibitor(TAXI-I)the site-directed mutagenesis was conducted based on the computer-aided redesign.Firstly,two xylanase inhibitory proteins-encoding xip-I and taxi-I were synthesized and expressed in Pichia pastoris SMD1168,namely SyXIP-I and SyTAXI-I.The highest inhibitory activities of SyXIP-I and SyTAXI-I on Auxyn11A occurred at molar ratio(xylanase:inhibitor)10:1,40℃ for 30 min,and 100:1,30℃ for 30 min,respectively.Secondly,nine variants of a Auxyn11A-encoding gene(Auxyn11A)were constructed as designed theoretically and expressed in E.coli BL21(DE3),respectively.One mutant,Auxyn11ASDSS exhibited the highest xylanase activity and showed 100%resistance to the inhibitor SyXIP-I,and Auxyn11A^(N2) showed 30%resistance.Secondly,to obtain both types of resistance,four variants,Auxyn11A^(N2−SDSSA),Auxyn11A^(SDSSA),AuxynM^(SDSS),and AuxynM^(SDSSA) was also constructed.Among them,AuxynM^(SDSSA) exhibited complete resistance to the SyXIP-I and SyTAXI-I.Furthermore,AuxynMSDSSA was purified.Its optimum temperature and pH were 60℃ and 5.0,and those of Auxyn11A were 50℃ and 5.0.The specific activity,K_(m),V_(max),and k_(cat)/K_(m) values of AuxynM^(SDSSA) were 1054.6 U/mg,4.0 mg/mL,6659.6μmol/min/mg,and 610.46 mL/mg/s,which were similia to those of Auxyn11A,1097.4 U/mg,4.23 mg/mL,6876.5μmol/min/mg,and 557.04 mL/mg/s,respectively.This study provided a novel thermophilic xylanase with complete resistance to the SyXIP-I and SyTAXI-I,therefore,making it a promising candidate for extensive applications in animal feed and food industry.
