南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化

doi:10.13560/j.cnki.biotech.bull.1985.2020-0529

摘要/Abstract

摘要：

旨在利用大肠杆菌实现南极假丝酵母脂肪酶B（CALB）基因的高效可溶性表达,并降低生产成本。构建带有不同信号肽的CALB基因表达质粒,转化至不同大肠杆菌宿主中,在摇瓶中进行基础培养基、诱导条件、培养基组成成分和进程曲线的优化。结果显示,带有PelB信号肽的重组菌pET25b-CALB-1/Rosetta（DE3）在20℃下使用0.5%（W/V）乳糖在TY培养基中诱导表达效果最好,优化后的合成培养基成分为1.75%（W/V）山梨醇、2.25%（W/V）鱼蛋白胨、1%（W/V）安琪酵母提取物、0.75%（W/V）Na₂HPO₄。在摇瓶中诱导60 h后,CALB酶活力最高达到35.67 U/mL,相比于初始发酵酶活力提高了17.77倍,是目前大肠杆菌生产CALB的最高水平。成功地构建了大肠杆菌表达系统,经过系统优化后,CALB的高水平可溶性表达得以实现。

关键词: 南极假丝酵母脂肪酶B, 大肠杆菌, 可溶性表达, 发酵优化

Abstract:

In order to achieve efficient soluble expression of Candida antarctica lipase B（CALB）in Escherichia coli at lower cost,CALB gene expression plasmids with different signal peptides were constructed and transformed into different E. coli hosts,and then a series of fermentation experiments were employed in shaking flasks to optimize the culture medium types,inducing conditions,components of medium and time course of cultivation. The results showed that the recombinant strain pET25b-CALB-1/Rosetta（DE3）with PelB signal peptide was induced to have the best expression in TY medium at 20℃ employing 0.5%（W/V）lactose. The optimal composition of fermentation medium was 1.75%（W/V）sorbitol,2.25%（W/V）fish peptone,1%（W/V）Angel yeast extract and 0.75%（W/V）Na₂HPO₄. The recombination system achieved a maximum CALB activity of 35.67 U/mL after induction for 60 h,which was 17.77 times higher than that before optimization,i.e.,the highest one of CALB produced by E. coli at present. In conclusion,the expression system in E. coli was successfully established,and the high-level soluble expression of CALB was obtained after system optimization.

Key words: Candida antarctica lipase B, Escherichia coli, soluble expression, fermentation optimization

吴蓉, 曹佳睿, 曹君, 刘飞翔, 杨猛, 苏二正. 南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化[J]. 生物技术通报, 2021, 37(2): 138-148.

WU Rong, CAO Jia-rui, CAO Jun, LIU Fei-xiang, YANG Meng, SU Er-zheng. Expression and Fermentation Optimization of Candida antarctica Lipase B in Escherichia coli[J]. Biotechnology Bulletin, 2021, 37(2): 138-148.

图/表 12

图1 重组质粒的构建和双酶切验证 A：含有PelB信号肽的重组质粒;B：含有DsbA信号肽的重组质粒;C：双酶切验证结果;M：Marker;1：质粒pET25b-CALB-1;2：质粒pET25b-CALB-2

图2 重组菌的SDS-PAGE分析图 A：发酵液;B：菌体上清液;C：菌体沉淀;M：标准蛋白Marker;1：pET25b/BL21（DE3）空载体对照;2：pET25b/Rosetta（DE3）空载体对照;3：诱导后的重组菌pET25b-CALB-1/BL（DE3）;4：诱导后的重组菌pET25b-CALB-1/Rosetta（DE3）;5：诱导后的重组菌pET25b-CALB-2/BL（DE3）;6：诱导后的重组菌pET25b-CALB-2/Rosetta（DE3）

图3 不同基础培养基对CALB可溶性表达的影响

图4 不同浓度的IPTG（A）和乳糖（B）对CALB表达的影响

图5 不同诱导温度对CALB表达的影响

表1 不同碳源对重组大肠杆菌的生长和CALB表达的影响

Carbon sources （1.0%,W/V）	OD₆₀₀	Enzyme activity（U/mL）	Specific activity（U/mg）
Sucrose	4.72±0.12	1.59±0.02	2.83±0.05
Glucose	2.84±0.09	0.78±0.01	5.77±0.07
Glycerol	10.10±0.24	5.93±0.06	5.84±0.08
Sorbitol	13.20±0.21	12.12±0.11	7.61±0.08
Starch	8.82±0.17	2.45±0.02	2.58±0.04
Dextrin	12.70±0.43	7.43±0.07	6.07±0.05

图6 不同浓度的糊精对CALB表达的影响

表2 不同氮源对重组大肠杆菌的生长和CALB表达的影响

Nitrogen sources （2.0%,W/V）	OD₆₀₀	Enzyme activity （U/mL）	Specific activity （U/mg）
Tryptone	12.69±0.32	19.41±0.20	12.15±0.09
SM	15.16±0.37	9.50±0.07	4.22±0.06
BBP	12.88±0.25	20.99±0.19	6.66±0.07
FP	16.14±0.40	22.70±0.17	6.97±0.06
PP	12.55±0.11	1.32±0.03	0.66±00.05
CSL	11.59±0.31	5.32±0.06	2.48±0.02

图7 不同浓度的鱼蛋白胨对CALB表达的影响

图8 不同浓度的安琪酵母提取物对CALB表达的影响

图9 不同浓度的Na2HPO4对CALB表达的影响

图10 最优条件下的发酵进程曲线

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