毕赤酵母重组菌高密度发酵产疏绵状嗜热丝孢菌脂肪酶  

Producing of Thermomyces lanuginosus Lipase Using High Density Fermentation of Recombinant Pichia pastoris

作　　者：蔡海莺[1,2] Dumba Trish 张婷[1] 来佳 赵敏洁[2] 李杨[2] 于林凯 张辉[2] 冯凤琴[2] Cai Haiying;Dumba Trish;Zhang Ting;Lai Jia;Zhao Minjie;Li Yang;Yu Linkai;Zhang Hui;Feng Fengqin(Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing,Zhejiang Provincial Key Lab for Chem ＆ Bio Processing Technology of Farm Product,School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023;College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058)

机构地区：[1]浙江省农业生物资源生化制造协同创新中心浙江省农产品化学与生物加工技术重点实验室浙江科技学院生物与化学工程学院,杭州310023 [2]浙江大学生物系统工程与食品科学学院,杭州310058

出　　处：《中国食品学报》2018年第7期99-107,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：浙江省重大科技专项(2012C12005-2);浙江省重点研发计划项目(2017C02009);浙江省教育厅科研一般项目(Y201737161)

摘　　要：疏绵状嗜热丝孢菌脂肪酶TLL是一种热稳定、具有位置专一性等良好属性和应用前景的脂肪酶,已成功在多种宿主中实现重组表达。本研究构建含前导肽的TLL脂肪酶毕赤酵母重组工程菌,在摇瓶水平对该重组工程菌的培养和诱导条件进行优化,通过单因素试验,得到的最佳发酵条件:1.5%(体积分数)甲醇,pH 7.0,质量分数0.5%玉米油和0.1%阿拉伯胶,该条件下摇瓶发酵最高酶活为2 265.3 U/mL。以甲醇为唯一碳源进行高密度发酵,202 h后菌体OD600值达到最高282,对应发酵上清液的最高酶活为22 561.4 U/mL。参考摇瓶优化结果,采用辅助碳源与甲醇共诱导高密度发酵,培养和诱导208 h后,最终菌体OD600值达到362,对应发酵上清液的最高酶活为24 522.6 U/mL,较以甲醇为唯一碳源的对应值分别提高了28.37%和10.87%。另外,最高酶活较摇瓶水平发酵的最高酶活2 265.3 U/mL提高10.83倍,表明高密度发酵和共诱导是大幅提高酵母表达重组TLL蛋白的重要手段。Thermomyces lanuginosus lipase（TLL） is a promising industrial enzyme with many favorable properties including high thermos-stability and regio-specificity, which has been successfully produced in several heterologous expression systems. In this study, recombinant Pichia pastoris strains with coding gene of TLL and its propeptides have been constructed. The fermentation conditions of strains in flask were optimized, and the optimized conditions are as follows：1.5% ethanol, pH 7, 0.5% maize oil and 0.1% Arabic gum supplement, respectively. Through the fermentation of recombinant strain using the combined optimized factors, the lipase activity of supernatant liquid could reach 2 265.3 U/mL.Through high density fermentation in a 5 liter tank, the recombinant P. pastoris strain showed the highest cell density（OD600） and lipase activity after 202 h fermentation, 282 and 22 561.4 U/mL respectively. According to the optimized fermentation condition in flask level, a co-feeding induction strategy using assistant carbon resource and methanol was performed. The result showed that, after 208 h fermentation, the cell density（OD600） and lipase activity reached 362 and24 522.6 U/mL respectively, which were 28.37% and 10.87% higher than those induced by sole methanol. Furthermore,the lipase activity increased to 10.83 times than the highest level 2 265.3 U/mL fermented in flask, indicating that high density fermentation and co-feeding induction strategies were useful measures for increasing the heterologous expression of lipase TLL in recombinant P. pastoris.

关 键 词：疏绵状嗜热丝孢菌脂肪酶 高密度发酵 毕赤酵母 重组表达 共诱导 

分 类 号：TQ925.6[轻工技术与工程—发酵工程]