重组毕赤酵母高产木聚糖酶菌株的筛选及诱导条件优化

Screening for High-yield Strain of Xylanase Produced by Engineered Pichia pastoris and Optimization of the Methanol Induced Conditions

[目的]筛选重组毕赤酵母高产木聚糖酶菌株并优化其甲醇诱导条件。[方法]用不同浓度的G418 YPD平板筛选高拷贝转化子,不同时间间隔添加不同浓度的甲醇进行木聚糖酶诱导表达。[结果]在G418浓度为10.0 mg/m l时,筛选得到产酶活力最高的16号菌株,以0.5%甲醇于30℃诱导产酶,第6天时酶活力达到最高,为826.2 IU/m l,木聚糖酶比活力达5 229.1 IU/mg;该菌在诱导时间间隔为12 h、诱导浓度为0.8%时,产酶量高且稳定。[结论]该菌是甲醇依赖型的高拷贝高酶活菌株。

[ Objective ] The aim of the study was to screen high-yield strain of xylanase by engineered Pichia pastoris and to optimize the methanol induced condition. [ Method] Different concentrations of G418 on YPD plates were used to screen high-copy transformant, and then different concentrations of methanol were added into the medium at different time intervals to find the point of highest enzyme production. [ Re- suit] A high-yield strain No. 16 was obtained which could bear 10.0 mg/ml of G418. When it was induced by 0.5% methanol at 30 %, the xylanase activity could reach the highest point of 826.2 IU/ml. And its specific activity reached 5 229.1 IU/mg. The enzyme production was high and stable when the methanol induced time interval was 12 h and the methanol concentration was 0.8%. [ Conclusion] This strain was a methanol-dependent and high-yield one.