牛瘤胃宏基因组文库来源的新型木聚糖酶基因的克隆、表达及酶学性质

Clone,Expression and Enzymological Properties of a Novel Xylanase from Cattle Rumen Macrogenomic Library

为了从牛瘤胃的微生物中获取新型的木聚糖酶,采用直接法提取牛瘤胃内容物微生物总DNA,构建宏基因组文库,通过功能驱动法筛选得到1个木聚糖酶基因(yh-1),该基因大小为987 bp,编码329个氨基酸;SDS-PAGE电泳分析YH-1的相对分子量为36.2 kDa,该酶的最适反应温度和最适反应pH是50℃和8.0~8.5;该酶有较强的温度稳定性,50℃条件下保温60 min,仍保留90%以上的剩余酶活性,65~75℃保留80%以上的剩余酶活性;2 mmol/L Zn^(2+)、Na^+、Co^(2+)和Ni^(2+)对该酶具有一定的激活作用;经过24 h的处理后,该酶可以从玉米芯、稻草和甘蔗叶中分别获得0.014、0.381和0.19 mg的还原糖。上述特征使得该酶在烘焙体系以及其他领域具有广泛的应用前景。

In order to obtain novel xylanase gene from microorganisms in cattle rumen,the cattle rumen microbial total DNA was extracted through direct method and a cattle rumen macrogenomic library was constructed. A gene encoding xylanase gene（ yh-1） was isolated and obtained through functionally driving and screening method. The gene size was at 987 bp encoding 329 amino acids; the relative molecular mass of yh-1 was 36. 2 kDa by SDS-PAGE electrophoresis analysis. The most suitable reaction temperature and pH of the enzyme were 50 ℃ and 8. 0-8. 5,respectively.The enzyme showed a strong thermostability,the residual enzyme activity still retained over 90% when keeping warm at 50 ℃ for 60 min,and retained over 80% of the activity when keeping warm at 65-75 ℃ for 60 min. 2 mmol/L of Zn^2＋,Na^＋,Co^2＋,and Ni^2＋possessed a certain activation effects on the enzyme. The enzyme could respectively obtained reducing sugar at 0. 014 mg,0. 381 mg,and 0. 19 mg from corn cobs,rice straw and sugarcane leaves after incubated for 24 h. The above mentioned properties made it a broad application potential in baking system as well as in other fields.