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

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 5.8、组氨酸添加量0.6%、甲醇添加量0.85%。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2.56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-activity and low-cost xylanase is really a hot topic in these fields. In this work, to further improve the expression level of xylanase by recombinant pPICZαA-QxynB in Pichia pastoirs, response surface methodology(RSM)was used to optimize the culture conditions. In the first place, Plackett-Burman design was used to investigate the significant factors affecting xylanase yield of recombinant pPICZαA-QxynB in P. pastoirs. The results showed that three significant factors played important 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 applied 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 concentration and 0.85% of methanol concentration was added into the culture medium every 24 h. Under the optimum conditions, 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 explain 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 with the average of 3 622 U/mL. Finally, the three sets of parallel experimental values were in conformity with the predicted values. These results suggested that the model could