响应面法优化毕赤酵母基因工程菌表达木聚糖酶条件研究  

Optimization of fermentation conditions of recombinant pPICZαA-QxynB(GS115) for xylanase production by response surface methodology

作　　者：杨诗逸 李琦[1] 奚丽娅 陈明真[1] 赵林果[1] 

机构地区：[1]南京林业大学南方现代林业协同创新中心,江苏省生物质绿色燃料与化学品重点实验室,南京210037

出　　处：《林业工程学报》2016年第6期88-94,共7页Journal of Forestry Engineering

基　　金：江苏省高校自然科学研究重大项目(13KJA220004);江苏省“333高层次人才培养工程”专项资助(BRA2015317);江苏省“六大人才高峰”资助项目(2014-JY-011);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：木聚糖酶作为降解木聚糖的关键酶之一,在制药、纸浆造纸和食品等领域有着广泛的应用。为了进一步提高重组毕赤酵母基因工程菌pPICZαA-QxynB(GS115)表达产木聚糖酶的能力,采用响应面法对其发酵条件进行优化。先通过Plackett-Burman法筛选出3个对产酶有显著影响的重要因素,分别为初始pH、组氨酸添加量和甲醇添加量;再利用单因素法逼近最大响应区域,并结合Box-Behnken试验获得最优的发酵条件为初始pH 58、组氨酸添加量06%、甲醇添加量085%。在最优的发酵条件下,重组菌pPICZαA-QxynB(GS115)表达木聚糖酶酶活达到3 604 U/mL,较未优化前提高了256倍,其3组平行试验值与模型的预测值基本相符,说明预测模型的可信度高,研究结果能为建立重组菌pPICZαA-QxynB(GS115)表达木聚糖酶的制备工艺提供依据。Xylan, as the major component of hemicellulose, is widely distributed in agricultural by-products including corncob, rice husk and wheat bran. Among xylan-degrading enzymes, xylanase is the key enzyme for random cleavage of the xylan backbone. It shows great potential in development of industry processes, such as medicine, paper and pulping industry, food industry and bioconversion of lignocellulosic waste to ethanol. Therefore, the topic of producing high-activ- ity and low-cost xylanase is really a hot topic in these fields. In this work, to further improve the expression level of -yla- nase by recombinant pPICZc-A-QxynB in Pichia pastoirs, response surface methodology （RSM） was used to optimize the cuhure conditions. In the first place, Plackett-Burman design was used to investigate the significant factors affecting xy- lanase yield of recombinant pPICZαA-QxynB in P. pastoirs. The results showed that three significant factors played im- portant roles in the fermentation conditions, including initial pH value, His concentration and methanol concentration. Then, the single factor method was used to approach the optimal level of the three factors, Box-Behnken design was ap- plied to obtain the optimal fermentation conditions. Based on the model, the optimal culture conditions for recombinant xylanase production in P. pastoirs were obtained as following：medium optimal initial pH value 5.8, 0. 6% of His concen- tration and 0. 85% of methanol concentration was added into the culture medium every 24 h. Under the optimum condi- tions, the maximum activity of xylanase was 3 604 U/mL after 13-day cultivation at a temperature of 28 ℃ in shake flasks, which was nearly 2. 56 times higher than the native conditions. Here, the R2 value was 0. 933 9, which could ex- plain 93.39% of the variability of the response. To confirm these results, a validation experiment was performed under these optimized conditions in triplicate, which the observed experimental values of xylanase activity were 3 624, 3 706 and 3 538 U/mL w

关 键 词：木聚糖酶 响应面法 发酵条件优化 基因工程菌pPICZαA-QxynB(GS115) 

分 类 号：Q812[生物学—生物工程]