Abstract:

In this study, engineering Pichia pastoris containing double copies of Aspergillus sulphureus xylanase gene xynA was constructed in order to earn a high expression level and low production cost. In a 10 L fermentor, double\|copy recombinant strain yielded the enzyme activity of 3 574 U/mL. The recombinant xylanase exhibited optimal activity at 50℃ and pH 2.2~3.4. Residual activity of the raw recombinant xylanase was over 70%, when fermentation broth was pretreated with Na2HPO4\|citric acid buffer of pH 1.7 for 100 min. And the recombinant xylanase exhibited strong resistance to pepsin and trypsin. High level of catalytic activity in low pH and acid resistance suggested that the recombinant xylanase has a prospective application in feed industry as an additive. Then the recombinant enzymes action effect was rapidly evaluated by in vitro simulating swine gastrointestinal environment. The experiment was conducted to determine the optimal supplementation level of recombinant enzyme in wheat\|based diet preliminarily. In this experiment, wheat\|based diet was divided into 4 treats containing 0, 2 000 U/kg, 4 000 U/kg or 6 000 U/kg recombinant enzyme, with 5 repetitions per treat. Then SDS\|Ⅱ monogastric animal bionic digestive system was used to digest experimental diet by imitating gastrointestinal tract environment of swine. Increasing the level of recombinant acidic β\|1, 4\|xylanase in the diet increased the apparent digestibility of gross energy (GE), crude protein (CP) (linear and quadratic effect; P<0.01) and neutral detergent fibre (NDF) (linear and quadratic effect; P<0.05). The optimal supplementation level of recombinant enzyme was determined to be 4 000 U/kg.

Key words: acidic xylanase, double\, copy strain, pichia pastoris, in vitro method

摘要：

通过构建硫色曲霉酸性木聚糖酶基因xynA串联双拷贝工程菌，提高酶的发酵活力。10 L发酵罐中，双拷贝菌株发酵活力达3 574 U/mL。重组木聚糖酶的最适催化温度为50℃，最适反应pH为2.2~3.4，在pH 1.7的酸性缓冲液下保持100 min，酶活残留70%以上。重组木聚糖酶对胃蛋白酶和胰蛋白酶也具有较好的耐受性。该木聚糖酶在低pH条件下的高催化活性、耐酸性及蛋白酶耐受性，使之具有较好的应用前景。通过体外模拟猪胃肠道环境以快速评价重组酶的作用效果。试验以小麦型日粮作为基础日粮，设计4个处理组，重组酶的添加量分别为0 U/kg、2 000 U/kg、4 000 U/kg和6 000 U/kg，每个处理5个重复，使用SDS\|Ⅱ单胃动物仿生消化系统模拟猪的胃肠道环境消化试验日粮，消化后的残渣烘干后测定其总能、粗蛋白质、中性洗涤纤维含量以计算其消化率。结果表明，添加重组酸性木聚糖酶显著提高了总能、粗蛋白质（线性和二次；P<0.01）和中性洗涤纤维的消化率（线性和二次；P<0.05）。重组酶的最适宜添加量为4 000 U/kg。

关键词: 酸性木聚糖酶；双拷贝菌株；毕赤酵母；体外法