Significantly enhanced thermostability and inhibitory resistance of <i>Aspergillus usamii</i> xylanase by modifying its N-terminus and thumb regions

Dong Zhang; Zhu Zhu; Yuqing Lei; Xiaoya Su; Liangze Ma; Minchen Wu

doi:10.1007/s43393-024-00263-x

Systems Microbiology and Biomanufacturing ›› 2024, Vol. 4 ›› Issue (3) : 928-940. DOI: 10.1007/s43393-024-00263-x

Original Article

Significantly enhanced thermostability and inhibitory resistance of Aspergillus usamii xylanase by modifying its N-terminus and thumb regions

Dong Zhang¹^,² ,
Zhu Zhu³ ,
Yuqing Lei² ,
Xiaoya Su² ,
Liangze Ma⁴ ,
Minchen Wu¹^,²^,^f

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Abstract

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 °C for 30 min, and 100:1, 30 °C 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, Auxyn11A^SDSS 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, AuxynM^SDSSA was purified. Its optimum temperature and pH were 60 °C and 5.0, and those of Auxyn11A were 50 °C 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, 6659.6 µmol/min/mg, and 610.46 mL/mg/s, which were similia to those of Auxyn11A, 1097.4 U/mg, 4.23 mg/mL, 6876.5 µmol/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.

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Dong Zhang, Zhu Zhu, Yuqing Lei, Xiaoya Su, Liangze Ma, Minchen Wu. Significantly enhanced thermostability and inhibitory resistance of Aspergillus usamii xylanase by modifying its N-terminus and thumb regions. Systems Microbiology and Biomanufacturing, 2024, 4(3): 928‒940 https://doi.org/10.1007/s43393-024-00263-x