嗜热脂肪地芽孢杆菌木聚糖酶XynA的异源表达、酶学表征及低聚木糖的抗氧化性研究

Heterologous expression and characterization of xylanase XynA from Geobacillus stearothermophilus and antioxidant activity of Xylo-oligosaccharides

试验旨在开发木聚糖酶XynA,为饲用木聚糖酶提供新的资源。研究毕赤酵母中异源表达嗜热脂肪地芽孢杆菌XynA的最佳发酵条件、酶学性质及水解产物低聚木糖(xylo-oligosaccharide,XOS)的抗氧化性。结果显示,1.0%的甲醇诱导96 h,即可产生酶活约为33.7 U/mL的XynA粗酶液。XynA最适反应pH值为9.0,最适反应温度为65℃,在pH值5.0~10.5、45~75℃时能发挥80%以上活性。薄层层析(TLC)分析发现,XynA水解甘蔗渣木聚糖的主要产物为木三糖-木六糖,1000 mg/L的XOS对1,1-二苯基-2-三硝基苯肼(DPPH)自由基的清除率达到72.04%,对羟自由基的清除率达到99.16%。研究表明,异源表达后的XynA具有较好的酶活和稳定性,能够高效水解木聚糖产生XOS,有助于其在饲料工业中的应用。

The experiment aims to develop xylanase XynA and provide a new resource for feeding xylanase.The XynA of Geobacillus stearothermophilus was heterologous expressed in Pichia pastoris,the optimal fermentation conditions,enzymatic properties and antioxidant properties of the hydrolysis product Xylo-oligosaccharide(XOS)were studied.The results showed that the crude XynA with enzyme activity of about 33.7 U/mL could be produced after 96 h induced by 1.0%methanol concentration.The optimum pH value of XynA is 9.0,the optimum temperature is 65℃,moreover,XynA remains above 80%of the activity at pH value ranging from 5.0 to 10.5 and temperatures between 45 and 75℃.Thinlayer chromatography(TLC)analysis revealed that the main products of the hydrolysis of bagasse xylan by XynA were xylotriose and xylohexaose.The scavenging rate of DPPH free radical reached 72.04%and the scavenging rate of hydroxylradical reached 99.16%with 1000 mg/L XOS.The study indicates that XynA exhibits robust activity and stability,efficiently hydrolyzing xylan to produce XOS,thereby facilitating its application in the feed industry.