生物技术通报 ›› 2021, Vol. 37 ›› Issue (2): 138-148.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0529
吴蓉1(), 曹佳睿1, 曹君1, 刘飞翔1, 杨猛1, 苏二正1,2()
收稿日期:
2020-05-25
出版日期:
2021-02-26
发布日期:
2021-02-26
作者简介:
吴蓉,女,博士研究生,研究方向:酶与生物催化;E-mail: 基金资助:
WU Rong1(), CAO Jia-rui1, CAO Jun1, LIU Fei-xiang1, YANG Meng1, SU Er-zheng1,2()
Received:
2020-05-25
Published:
2021-02-26
Online:
2021-02-26
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。
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)
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 |
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 |
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 |
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 |
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