谷氨酰胺转氨酶在Pichia pastoris GS115中的表达及其发酵条件响应面法优化  

Expression of transglutaminase in Pichia pastoris GS115 and optimization of its fermentation conditions using response surface methodology

作　　者：于凡 刘双 陈海清 王红静 马雯[1] 石楠[2] 檀建新[1] YU Fan;LIU Shuang;CHEN Haiqing;WANG Hongjing;MA Wen;SHI Nan;TAN Jianxin(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China;School of Life Sciences/Key Laboratory of Microbial Diversity Research and Application of Hebei Province,Hebei University,Baoding 071002,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]河北大学生命科学学院/河北省微生物多样性研究与应用重点实验室,河北保定071002

出　　处：《河北农业大学学报》2020年第6期83-89,107,共8页Journal of Hebei Agricultural University

基　　金：河北省重点研发计划项目(16275505D);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18).

摘　　要：为了将筛选得到的吸水链霉菌107.3的谷氨酰胺转氨酶基因在毕赤酵母GS115中表达,并通过优化发酵条件来提高重组菌株酶的表达量,本试验构建了由吸水链霉菌前肽pro、kex2酶切位点、成熟酶mTGase 3部分组成的融合基因,电转至毕赤酵母GS115中进行表达;采用Plackett-Burman(PB)设计和响应面法(RSM)在摇瓶水平研究了重组毕赤酵母的发酵条件(接种量、诱导温度和时间、初始pH值和甲醇浓度)。结果表明:吸水链霉菌的TGase基因在毕赤酵母GS115中得获得成功表达,酶活力为0.3 U/mL;通过PB试验设计确定了影响产酶的显著性因素为诱导初始pH、甲醇浓度和接种量。进一步选择该3个显著因素进行RSM中心组合试验,确定了最优的产酶条件为:接种量OD600为6、pH 7、甲醇浓度2.0%、诱导温度23℃、诱导时间72 h。优化发酵条件后,重组菌株产TGase酶活力可达1.1 U/mL,是初始酶活的3.6倍。本研究构建的MTG异源表达体系为MTG生产和分子改造提供了平台。In order to express the transglutaminase(TGase)gene of Streptomyces hygroscopicus 107.3 in Pichia pastoris GS115 and improve the expression of recombinant enzyme by optimizing fermentation conditions,a fusion gene containing TGase propeptide(pro),kex2 protease cutting site and mature TGase(mTGase)of S.hygroscopicus was constructed in this experiment and transferred to P.pastoris GS115 for expression.Plackett Burman(PB)design and response surface methodology(RSM)were used to study the expression conditions(inoculum size,induction temperature and time,initial pH value and methanol concentration)at a level of cultivation in shake flask.The results indicated that the TGase gene of S.hygroscopicus was successfully expressed in P.pastoris GS115,and the enzyme activity was 0.3 U/mL.Three significant factors including initial pH,methanol concentration and inoculation amount,which affected the enzyme production,were screened out by PB design and further subjected to RSM Box-Benhnken test to determine the optimal enzyme production conditions.The results showed that the optimal conditions for a production were as follows:inoculum OD600=6,pH=7,methanol concentration 2.0%,induction temperature 23℃and induction time 72 h.After optimizing the fermentation conditions,the TGase activity of the recombinant strain was up to 1.1 U/mL,which was 3.6 times of the initial enzyme activity.The heterologous expression system of MTG constructed in this study provides a platform for MTG production and molecular modification.

关 键 词：谷氨酰胺转氨酶 毕赤酵母 Plackett-Burman(PB)设计 响应面法 吸水链霉菌 

分 类 号：Q786[生物学—分子生物学]