关键词: Neocallimastix frontalis, 木聚糖酶, 毕赤酵母, 密码子优化, 酶学性质

Abstract: The anaerobic fungus Neocallimastix frontalis is one of main microorganisms in the rumen degrading xylan and cellulose and its xylanase has the potential application value. In this study, a xylanase gene Xyn11B originated from N. frontalis was codon optimized, and the optimized gene Xyn11Bm was synthesized. Based on the gene engineering technology, the yeast expression vector pPIC9K-Xyn11Bm was constructed, and the xylanase was induced and expressed in Pichia pastoris GS115. In shake flask level, enzyme activity of the recombinant Xyn11Bm reached the maximum up to 4874.8 U/mL. In 10 L fermentor, at 96 h after induction, the activity of recombinant enzyme was 5139.7 U/mL, cell wet weight and dry weight were 216.7 g/L and 117.3 g/L. Enzymatic properties analysis showed that the optimum reaction temperature and pH of Xyn11Bm were 50℃ and 5.0. In pH5.0-8.0, the enzyme had sound stability, but poor temperature stability. Substrate specificity analysis showed that recombinant Xyn11Bm could hydrolyze oat spelt xylan, birch xylan and soluble xylan 4-O-Me-D-glucurono-D-xylan, but not degrade lichenin and barley β-glucan. This indicated that the recombinant Xyn11Bm had potential application value.

Key words: Neocallimastix frontalis, xylanase, Pichia pastoris, codon optimization, enzymatic properties