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中国精品科技期刊2020
周晨妍, 刘振华, 王丹丹, 李同彪, 高启禹. 产木聚糖酶毕赤酵母工程菌株构建及表达条件优化研究[J]. 华体会体育, 2016, (13): 162-167. DOI: 10.13386/j.issn1002-0306.2016.13.024
引用本文: 周晨妍, 刘振华, 王丹丹, 李同彪, 高启禹. 产木聚糖酶毕赤酵母工程菌株构建及表达条件优化研究[J]. 华体会体育, 2016, (13): 162-167. DOI: 10.13386/j.issn1002-0306.2016.13.024
ZHOU Chen- yan, LIU Zhen-hua, WANG Dan-dan, LI Tong-biao, GAO Qi-yu. Study on the construction and expression of the engineered strain of Pichia pastoris for xylanase production[J]. Science and Technology of Food Industry, 2016, (13): 162-167. DOI: 10.13386/j.issn1002-0306.2016.13.024
Citation: ZHOU Chen- yan, LIU Zhen-hua, WANG Dan-dan, LI Tong-biao, GAO Qi-yu. Study on the construction and expression of the engineered strain of Pichia pastoris for xylanase production[J]. Science and Technology of Food Industry, 2016, (13): 162-167. DOI: 10.13386/j.issn1002-0306.2016.13.024

产木聚糖酶毕赤酵母工程菌株构建及表达条件优化研究

Study on the construction and expression of the engineered strain of Pichia pastoris for xylanase production

  • 摘要: 将黑曲霉XZ-3S木聚糖酶Xyn43A成熟肽基因插入表达载体p PIC9K,重组质粒SalⅠ线性化后分别电击转化2种毕赤酵母GS115和KM71,转化液经MD平板、G418浓度梯度平板和摇瓶复筛,获得两株重组菌GS115/Xyn43A(Mut+)和KM71/Xyn43A(Muts)。其中GS115/Xyn43A菌株最优表达条件为:甲醇浓度2.0%,接种时间24 h,诱导时间108 h,诱导温度30℃、诱导培养基初始p H6.3;KM71/Xyn43A菌株最优表达条件为:甲醇浓度1.75%,接种时间26 h,诱导时间132 h,诱导温度30℃、诱导培养基初始p H6.5。在最优表达条件下两重组菌GS115/Xyn43A和KM71/Xyn43A比酶活力分别可达139.36、143.29 U/mg。 

     

    Abstract: A gene encoding xylanase Xyn43 A from Aspergillus niger XZ- 3S was cloned into the Pichia pastoris expression vector,p PIC9 K. The recombinant plasmid was linearized with Sal I and then transformed into Pichia pastoris GS115 and KM71 by electroporation,respectively. After screening by MD medium,G418 concentration plate and shake bottle selection,two recombinant strains GS115 / Xyn43 A and KM71 / Xyn43 A were obtained. The best optimization schemes were obtained by single factor experiment and L9( 3~4) experiment. The optimal expression conditions of GS115 / Xyn43 A were methanol concentration of 2.0%,inoculation time of 24 h,induction time of 108 h,induction temperature of 30 ℃,and initial p H6.3.The optimal expression conditions of KM71 / Xyn43 A were methanol concentration of 1.75%,inoculation time of of26 h,induction time of 132 h,induction temperature of30 ℃,and initial p H6.5. Under the optimum conditions,the xylanase activity of the two recombinant strains were139.36 U / mg and 143.29 U / mg,respectively.

     

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