来源于瘤胃厌氧真菌Neocallimastix frontalis木聚糖酶在毕赤酵母中的表达

Expression of a Xylanase Gene Originated from Rumen Anaerobic Fungi Neocallimastix frontalis in Pichia pastoris

厌氧真菌Neocallimastix frontalis是瘤胃中降解木聚糖和纤维素的主要微生物之一,其木聚糖酶具有潜在的应用价值。对来源于Neocallimastix frontalis木聚糖酶基因Xyn11B进行密码子优化;通过全基因合成优化后的木聚糖酶基因Xyn11Bm,构建该基因的酵母表达载体p PIC9K-Xyn11Bm,并在毕赤酵母GS115中诱导表达。摇瓶水平时,重组Xyn11Bm酶活性最高为4 874.8U/m L。在10 L发酵罐中诱导96 h后,重组Xyn11Bm的酶活性为5 139.7 U/m L,菌体湿重和干重达到216.7 g/L和117.3 g/L。酶学性质分析表明,重组Xyn11Bm的最适反应温度为50℃,最适反应p H为5.0。在p H5.0-8.0时该酶具有较好的稳定性,但温度稳定性较差。底物特异性分析表明,重组Xyn11Bm可水解燕麦木聚糖、桦木木聚糖和可溶性木聚糖4-O-Me-D-glucurono-D-xylan,但不降解地衣多糖和大麦β-葡聚糖。结果表明重组Xyn11Bm具有潜在的应用价值。

The anaerobic fungus Neocallimastixfrontalis 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 Xyn1 1Bm 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 Xyn 11Bm 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 Xyn1 1Bm had potential application value.