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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (1): 63-70    DOI: 10.13523/j.cb.20190108
技术与方法     
重组Humicola insolens角质酶的高密度发酵优化 *
黄燕1,2,3,孙益荣1,2,3,吴敬1,2,3,宿玲恰1,2,3,**()
1 江南大学食品科学与技术国家重点实验室 无锡 214122
2 江南大学生物工程学院工业生物技术教育部重点实验室 无锡 214122
3 江南大学 教育部食品安全国际合作联合实验室 无锡 214122
Optimization of High Density Fermentation of Recombinant Humicola insolens Cutinase
Yan HUANG1,2,3,Yi-rong SUN1,2,3,Jing WU1,2,3,Ling-qia SU1,2,3,**()
1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,China
2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122,China
3 Joint Laboratory for International Cooperation in Food Safety by the Ministry of Education, Jiangnan University, Wuxi 21412,China
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摘要：

在采用前期已构建的重组菌E.coli BL21(DE3)/pET20b(+)-hic进行高密度发酵制备角质酶时发现,在高诱导强度发酵时,菌体浓度下降明显。同时,通过测定纯化重组角质酶的磷脂水解活性,考察验证了重组酶对宿主细胞的损伤作用。重组酶的磷脂酰乙醇胺活性为9.8U/mg(NPB水解比活力为1 047.6 U/mg),在卵黄平板出现了明显的反应圈现象。在此基础上尝试了高菌体浓度结合高诱导强度的发酵策略,以进一步提高重组酶在3L罐中的表达水平。优化后的最佳条件及结果为:OD600为75时,恒速流加0.8g/(L ·h)的乳糖溶液,发酵24h后,酶活达到最大值4 788.0U/ml,约为摇瓶发酵酶活的28倍,与OD600为50时、流加0.2 g/(L ·h)进行诱导的发酵策略(酶活2 233.0 U/ml)相比,提高幅度约为114.0%,发酵时间缩短40.0%。
关键词： Humicola insolens;  角质酶;  磷脂酶活性;  高密度发酵    
Abstract:

During the high-density fermentation of cutinase using E.coli BL21(DE3)/pET20b(+)-hic, it was found that the concentration of bacteria decreased obviously under high induction intensity fermentation. The phospholipid activity of the purified recombinant cutinase was determined in order to verify the effect of recombinant enzyme on E.coli. The phosphatidyl ethanolamine activity of the recombinant enzyme was 9.8U/mg (the specific activity of NPB hydrolysis was 1 047.6U/mg). Then, the strategy of high cell concentration and high induction intensity was used to further improve the expression level of recombinant enzyme in 3L fermentor. The optimizing fermentation conditions was: the induce phases started when the cell concentration OD600 reached 75 and the temperature was adjusted to 30℃, lactose solution were added with constant flow rate of 0.8g/(L ·h) for 24h. The highest activity of enzyme reached 4 788.0U/ml, which is 28 times higher than the enzyme activity in shake flask. Comparing with other fermentation strategy which induced at OD600 of 50 with 0.2g/(L ·h) lactose solution with enzyme activity 2 233.0U/ml, the enzyme activity increased about 114.0% and the fermentation time was shortened by 40.0%.
Key words: Humicola insolens    Cutinase    Phospholipase activity    High density fermentation
收稿日期: 2018-09-18 出版日期: 2019-02-28
ZTFLH:  Q814  
基金资助: * 国家杰出青年基金(31425020);高等学校学科创新引智计划资助项目(B170210)
通讯作者: 宿玲恰     E-mail: sulingqia@jiangnan.edu.cn
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引用本文:

黄燕,孙益荣,吴敬,宿玲恰. 重组Humicola insolens角质酶的高密度发酵优化 *[J]. 中国生物工程杂志, 2019, 39(1): 63-70.

Yan HUANG,Yi-rong SUN,Jing WU,Ling-qia SU. Optimization of High Density Fermentation of Recombinant Humicola insolens Cutinase. China Biotechnology, 2019, 39(1): 63-70.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190108    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I1/63

图1  在OD600为50条件下诱导强度对重组菌生长 (a) 和产酶 (b) 的影响
图2  在OD600 50,乳糖流速0.2g/(L ·h)诱导条件下发酵上清液SDS-PAGE分析
图3  纯化的重组角质酶SDS-PAGE分析
纯化步骤 总蛋白质含量 (mg) 总酶活 (U) 比活力(U/mg) 得率 (%) 纯化倍数 (倍)
粗酶液 5.65 4.42×103 7.83×102 100 1.00
硫酸铵沉淀 2.46 2.20×103 8.94×102 49.7 1.14
MonoQ阴离子交换柱 0.89 9.41×102 1.05×103 21.2 1.33
表1  重组角质酶的纯化工艺
图4  重组角质酶的磷脂水解活性检测
图5  在OD600为75条件下诱导强度对重组菌生长 (a) 和产酶 (b) 的影响
图6  在OD600 75、乳糖流速0.8g/(L ·h)诱导条件下发酵上清液SDS-PAGE分析; 箭头所指处为重组表达的角质酶
图7  在OD600为95条件下诱导强度对重组菌生长 (a) 和产酶 (b) 的影响
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