响应面法优化重组巴斯德毕赤酵母合成ECH42的培养基组分及其重组酶降解几丁质的研究

采用响应面法在摇瓶水平对重组巴斯德毕赤酵母合成内切几丁质酶的培养基组分进行优化,并探讨重组内切几丁质酶降解几丁质的最佳反应条件。首先对培养基中显著影响内切几丁质酶活力的关键组分通过Plackett-Burman试验设计进行筛选;然后通过Box-Behnken试验设计和响应面法确定关键组分的最佳浓度。结果筛选出3个具有显著效应的关键组分为酵母膏、油酸和吐温-80,最佳浓度分别为：2.45%、0.17%和0.62%。优化后的最佳培养基组成为：2.45%酵母膏、2.00%蛋白胨、0.50%酵母氮碱（YNB）、0.50%甲醇、0.17%油酸、0.62%吐温-80和0.40% PTM₁。在该培养基中,重组巴斯德毕赤酵母在摇瓶水平（25mL/250mL）发酵生产内切几丁质酶的活力高达92.26U/mL。重组内切几丁质酶催化几丁质降解的最佳反应条件为：粉末几丁质浓度为4%,pH和温度分别为7.0和30℃,反应时间为10h。研究结果为后期在发酵罐中大规模生产内切几丁质酶和几丁寡糖提供了基础。

Medium component optimization via response surface methodology for endochitinase ECH42 biosynthesis and investigation of chitin degradation by ECH42 of recombinant Pichia pastoris

The optimization of medium components for the endochitinase (ECH42) biosynthesis by recombinant Pichia pastoris was investigated at shake flask level by response surface analysis. The critical medium components that had obvious influence on the activity of endochitinase was first screened by Plackett-Burman design. The optimal concentrations were further determined by Box-Behnken design and response surface methodology. Yeast extract, oleic acid and Tween-80 were found to play an important role in the biosynthesis of the recombinant endochitinase, and their optimal concentrations were 2.45%, 0.17% and 0.62%, respectively. The optimized medium consisted of 0.50% methanol, 0.50% yeast nitrogen base (YNB), 2.00% tryptone, 0.17% oleic acid, 2.45% yeast extract, 0.4% PTM₁ and 0.62% Tween-80. The activity of the recombinant endochitinase was up to 92.26U/mL in a 250mL shake flask under the optimized condition. The optimal reaction condition of chitin degradation catalyzed by ECH42 included powder chitin concentration 4%, pH 7.0, temperature 30°C and reaction time 10h. These results provide a basis for the large-scale production of the endochitinase and chito-oligosaccharide in the fermentor.