木聚糖酶XynG1-1在毕赤酵母中的表达及发酵条件优化  

Expression of xylanase XynG1-1 in Pichia pastoris and optimization of its fermentation conditions

作　　者：王停停[1,2] 郑宏臣[2,3] 徐健勇[2,3] 彭梦[1,2] 刘逸寒[1] 路福平[1] 宋诙[2,3] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,工业酶国家工程实验室,天津300308 [3]中国科学院天津工业生物技术研究所,天津工业生物系统与过程重点实验室,天津300308

出　　处：《食品与发酵工业》2017年第1期37-43,共7页Food and Fermentation Industries

基　　金：国家“863”计划(2014AA021302、2014AA021303);中国科学院重点部署项目(KFZD-SW-201-2A);天津市科技计划项目(13ZCDZSY05000)

摘　　要：将来源于Paenibacillus campinasensis G1-1的木聚糖酶编码基因成功整合到毕赤酵母GS115基因组上,构建了高产木聚糖酶XynG1-1的毕赤酵母工程菌。采用响应面法对该工程菌的发酵条件进行优化。首先使用Design-Expert软件进行Plackett Burman实验设计筛选出影响产酶量的3个主要因素,即甲醇含量、生物素含量和培养时间。在此基础上使用Design-Expert软件进行Box-Behnken实验设计,通过响应面分析得出优化的发酵培养条件为:甲醇含量2.28%,培养时间37.29 h,生物素4 mg/L,酵母粉20 g/L,蛋白胨20 g/L,YNB 30 g/L,装液量100 m L/L,转速250 r/min、温度28℃、磷酸缓冲液pH 6.0。经实验验证,优化后的培养条件下胞外重组酶活达到707.2 IU/m L,与响应面预测结果一致,较优化前木聚糖酶酶活提高了7.9倍,较原始菌株产酶量提高了19.8倍。经10 L发酵罐扩大培养之后,重组木聚糖酶的酶活达到2 703 IU/m L。因此,该研究有效提高了木聚糖酶XynG1-1的发酵产量,并且,该重组酶保持了良好的酶学性质,可为工业化生产及应用奠定基础。The gene encoding xylanase from Paenibacillus campinasensis GI-1 was successfully integrated into the genome of Pichia pastoris GSllS, and engineered P. pastoris for high-yielding xylanase XynGl-1 was constructed. The fermentation conditions of the engineered bacteria were optimized by using the response surface method. Firstly, Plackett Burman design was carried out using Design-Expert software and three main factors influencing the yield of xylanase were screened out, which were content of methanol, content of biotin and incubation time. Then the Box-Be- hnken of Design-Expert software was used to design the experiment, the optimize culture conditions for fermentation were confirmed by the analysis of the response surface to be follows： yeast powder of 20 g/L, peptone of 20 g/L, YNB of 30 g/L, biotin of 4 mg/L, methanol content of 2.28% , liquid volume of 100 mL/L, incubation time of 37.29 h, rotational speed of 250 r/rain, temperature 28℃ , phosphate buffer pH 6.0. Verified by experiments, the extracellular recombined enzyme activity under the optimized cultivation condition was 707.2 IU/mL, which was con- sistent with the prediction of the response surface method and was 7.9-fold of the former xylanase enzyme activity and 19.8-fold of the enzyme activity produced by original strains. After enlarged culture in 10 L fermentation tank, the re- combined xylanase enzyme activity reached 2 703 IU/mL. Therefore, this study effectively improved the fermentation yield of xylanase XynGl-1, and the recombinant enzyme retained better enzymology properties. It established founda- tion for the further industrial production and application of xylanase XynGl-1.

关 键 词：毕赤酵母 木聚糖酶 响应面 发酵优化 酶学性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]