重组大肠杆菌产卤代烷脱卤酶的发酵条件优化  

Optimization of fermentation conditions of recombinant Escherichia coli expressing haloalkane dehalogenase

作　　者：名晓东 郭自涛 陈剑雄 顾正华 辛瑜 孙海彦[2] 关彦明 张梁 MING Xiaodong;GUO Zitao;CHEN Jianxiong;GU Zhenghua;XIN Yu;SUN Haiyan;GUAN Yanming;ZHANG Liang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China;China National Research Institute of Food and Fermentation Industries,Beijing,100015,China)

机构地区：[1]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [2]中国热带农业科学院热带生物技术研究所,海南海口571101 [3]中国食品发酵工业研究院有限公司,北京100015

出　　处：《食品与发酵工业》2022年第13期16-24,共9页Food and Fermentation Industries

基　　金：国家重点研发计划绿色生物制造专项(2021YFC2100300);国家现代农业产业技术体系项目(CARS-11-HNSHY);中国热带农业科学院基本科研业务费项目(1630052021012,1630052020023,1630052020024)。

摘　　要：对产卤代烷脱卤酶的重组大肠杆菌BL21(DE3)/pET28a-DhaA进行发酵优化以提高其表达量。该研究从TB培养基出发,通过单因素实验与响应面实验在摇瓶水平上对培养基各成分进行优化;其次,在最优培养基的基础上对发酵的工艺条件进行优化;最后,在5 L发酵罐中进行了初步放大验证。结果表明,最优的培养基组成为:甘油10 g/L,酵母粉23 g/L,蛋白胨14 g/L,MgSO_( 4)·7H_(2)O 1.3 g/L,ZnSO_( 4)·7H_(2)O 0.1 g/L,酶活力可达到(1182.94±10.86)U/L,较初始培养基提高了94%;最优的发酵工艺条件为:接种量5%,装液量40 mL/250 mL,37℃培养至OD_(600)为1.8时加入终浓度为0.4 mmol/L的异丙基-β-D-硫代半乳糖苷于20℃诱导22 h,酶活力可达到(3585.83±15.02)U/L,提高至未进行优化前的5.87倍;5 L发酵罐初步放大验证实验中,酶活力最高达到(9682.62±191.16)U/L,提高至摇瓶水平的2.7倍。通过在摇瓶水平对发酵培养基与工艺进行优化,以及在5 L发酵罐中进行放大验证,极大地提高了卤代烷脱卤酶的酶活力,为放大生产奠定了基础。In this study,the fermentation of a recombinant Escherichia coli BL21(DE3)/pET28a-DhaA was optimized to improve its production of haloalkane dehalogenase.Firstly,based on TB medium,the components of culture medium were optimized at shaking flask level by single factor experiment and response surface experiment.Secondly,the fermentation conditions were optimized base on the optimal culture medium.Finally,the preliminary amplification was verified in a 5 L fermentation tank.The results showed that the optimal medium composition was glycerol 10 g/L,yeast powder 23 g/L,peptone 14 g/L,MgSO_(4)·7H_(2)O 1.3 g/L,ZnSO_( 4)·7H_(2)O 0.1 g/L.The enzyme activity can reach(1182.94±10.86)U/L,which was 94%higher than that of the initial medium.The optimal fermentation conditions were:the inoculum volume 5%,the loading volume 40 mL/250 mL,0.4 mmol/L IPTG was added to induce 22 h at 20℃,when the OD_(600) reached 1.8 at 37℃.The enzyme activity was achieved at(3585.83±15.02)U/L,which was 5.87 times higher than the initial conditions.At last,the enzyme activity reached(9682.62±191.16)U/L in a 5 L fermentation tank,which was 2.7 times higher than that of a shaking flask.After optimization,the enzyme activity of haloalkane dehalogenase was greatly improved,and our study provides a foundation for scale-up production.

关 键 词：卤代烷脱卤酶 大肠杆菌 发酵优化 响应面法 

分 类 号：TQ925[轻工技术与工程—发酵工程]