Gene Cloning, Expression and Enzymatic Characterization of Acetyl Xylan Esterase

Abstract:

Acetyl xylan esterase（Axe） can hydrolyze O-acetyl located in acetylated xylan. Thus，the steric hindrance effects of O-acetyl can be eliminated when xylan is hydroyzed by xylanase. In this study，a mature peptide gene，Auaxe，encoding an acetyl xylan esterase（AuAXE），was amplified by RT-PCR technique using the total RNA from Aspergillus usamii E001 as template. The recombinant expressing plasmid pPIC9KM-Auaxe was constructed and linearized with SalⅠ，followed by transforming it into Pichia pastoris GS115 by electroporation. The recombinant P. pastoris was screened by G418，induced by methanol for 72 h，and then the recombinant acetyl xylan esterase（reAuAxe） was obtained. The activity of crude reAuAxe reached 35.6 U/mL. After purification，the reAuAxe was purified to electrophoretic homogeneity with the activity of 390.5 U/mg. SDS-PAGE analysis showed that the apparent molecular mass of purified reAuAxe was about 34.0 kDa. Additionally，the enzymatic properties of purified reAuAxe were also analyzed. Its optimal temperature and pH were 50 ℃ and 6.0，respectively. It was stable at a temperature of 45 ℃ or below，and at a pH range of 4.5~7.0. Its enzymatic acvitity was not signficantly affected by an array of tested metal ions and EDTA. This paper laid a solid theoretical foundation for this enzyme's study in depth and application.