毕赤酵母产重组木聚糖酶发酵条件的优化及其酶学性质

Optimization of condition for fermentation of recombi nant Pichia pastoris and enzymatic properties of xylanase produced

目的优化毕赤酵母工程菌GS115/xyn11A产重组木聚糖酶的发酵条件,并检测其酶学性质。方法采用单因素试验和L(934)正交试验考察摇瓶发酵条件下培养基起始pH值、诱导剂甲醇添加量、诱导温度及诱导时间对产酶活性的影响;并分析重组木聚糖酶的酶学性质。结果影响重组毕赤酵母产酶的因素重要性依次为:培养基起始pH值>诱导时间>诱导温度>甲醇添加量,重组酵母产酶最佳条件为:起始pH值7.5,甲醇添加量1.5%,32℃诱导96 h,在此条件下进行诱导表达重组木聚糖酶的酶活性可达228.35 IU/ml;酶的最适反应温度为50℃,最适反应pH值为5.5,在低于40℃和pH 4.5～7.5的范围内较稳定。结论优化了毕赤酵母产重组木聚糖酶的发酵条件,为木聚糖酶的工业化生产及应用提供了依据。

Objective To optimize the condition for fermentation of recombinant Pichia pastoris GSl15 /xynllA and deter- mine the enzymatic properties of produced xylanase. Methods The effects of the initial pH value, methanol concentration as well as temperature and time for induction on expression of xylanase in recombinant P. pastoris GSll5/xynllA were investigated by single factor test and L9 （34） orthogonal test, and the enzymaticproperties of recombinant xylanase were analyzed. Results In the order of importance to expression of xylanase in recombinant P. pastoris, the influencing factors were initial pH value of medium, time for in- duction, temperature for induction and methanol concentration. The optimal initial pH value, methanol concentration as well as tem- perature and time for induction were 7. 5, 1. 5%, 32 ~C and 96 h respectively. Under the optimal condition, the recombinant xy- lanase reached an activity of 228.35 IU/ml, of which the optimal working temperature and pH value were 50 ~C and 5. 5 respective- ly, and showed high stability at 40 ℃ or below and pH 4. 5 - 7. 5. Conclusion The condition for fermentation of recombinant P. p astoris GS 115/xynl 1A was optimized, which provided a basis for industrial production and application of xylanase.