Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (10): 93-106.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0486
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LIU Jin-sheng(), CHEN Zhen-ya(), HUO Yi-xin, GUO Shu-yuan()
Received:
2023-05-23
Online:
2023-10-26
Published:
2023-11-28
Contact:
CHEN Zhen-ya, GUO Shu-yuan
E-mail:ljs15536837748@163.com;chenzhenya@bit.edu.cn;guosy@bit.edu.cn
LIU Jin-sheng, CHEN Zhen-ya, HUO Yi-xin, GUO Shu-yuan. Application of FACS Technology in the Directed Evolution of Enzyme[J]. Biotechnology Bulletin, 2023, 39(10): 93-106.
筛选方法 Screening method | 通量 Throughput | 突变文库 Mutant library | 特点 Characteristic | 参考文献 References |
---|---|---|---|---|
平板 | 105-106 | 操作简便;半定量分析;通量低;精度低 | [ | |
微孔板 | 1/s | 105 | 操作简便;定量分析;通量低;精度低 | [ |
FACS | 104/s | >108 | 通量高;消耗少;精度高;单细胞定量分析;设备昂贵;不便携 | [ |
FADS | <103/s | 107 | 通量高;消耗少;精度高;单细胞定量分析;工艺复杂度高;搭建难 | [ |
Table 1 Comparison of screening methods
筛选方法 Screening method | 通量 Throughput | 突变文库 Mutant library | 特点 Characteristic | 参考文献 References |
---|---|---|---|---|
平板 | 105-106 | 操作简便;半定量分析;通量低;精度低 | [ | |
微孔板 | 1/s | 105 | 操作简便;定量分析;通量低;精度低 | [ |
FACS | 104/s | >108 | 通量高;消耗少;精度高;单细胞定量分析;设备昂贵;不便携 | [ |
FADS | <103/s | 107 | 通量高;消耗少;精度高;单细胞定量分析;工艺复杂度高;搭建难 | [ |
类别 Classification | 目标酶 Target enzyme | 结果 Result | 宿主细胞 Host cell | 参考文献 Reference |
---|---|---|---|---|
细胞表面酶 | 丝氨酸蛋白酶OmpT | kcat/Km值提高60倍 | 大肠杆菌E. coli | [ |
分选酶A | kcat/Km值提高140倍 | 酿酒酵母S. cerevisiae | [ | |
半乳糖氧化酶 | Km值降低4.4倍 | 大肠杆菌E. coli | [ | |
D-氨基酸氧化酶 | kcat值提高4.2倍 | 大肠杆菌E. coli | [ | |
胞内酶 | β-1,3-半乳糖基转移酶 | 底物耐受性提高300倍 | 大肠杆菌E. coli | [ |
α-1,3-岩藻糖基转移酶 | 合成Lewis X和3'岩藻糖基乳糖的kcat/Km值分别提高6倍和14倍 | 幽门螺旋杆菌Helicobacter pylori | [ | |
α-1,2-岩藻糖基转移酶 | 2'岩藻糖基乳糖的滴度和产量分别提高1.72倍和1.51倍 | 大肠杆菌E. coli | [ | |
多聚唾液酸转移酶 | 比酶活和热稳定性分别提高 1.5-2.1倍和9倍 | 奈瑟菌Neisseria | [ | |
葡萄糖氧化酶 | Vmax值分别提高1.3和2.3倍 | 酿酒酵母S. cerevisiae | [ | |
ATP磷酸核糖转移酶 | L-组氨酸产量为0.1 mmol/L,野生型未生产L-组氨酸 | 谷氨酸棒状杆菌Corynebacterium glutamicum | [ | |
丝氨酸乙酰转移酶 | L-半胱氨酸产量提高2.36倍 | 大肠杆菌E. coli | [ | |
3C蛋白酶 | 蛋白水解活性提高4倍 | 大肠杆菌E. coli | [ | |
咖啡因去甲基化酶 | Vmax/Km值和产物选择性分别提高33和22倍 | 酿酒酵母S. cerevisiae | [ | |
丙酮酸羧化酶 | 赖氨酸滴度分别增加9%和19% | 谷氨酸棒状杆菌C. glutamicum | [ | |
莽草酸生产菌株 | 莽草酸滴度达到(3.72±0.35)mmol/L,比野生型提高2.4倍 | 谷氨酸棒状杆菌C. glutamicum | [ | |
丝氨酸羟甲基转移酶 | L-丝氨酸产量达到34.78 g/L,比野生型菌株提高35.9% | 谷氨酸棒状杆菌C. glutamicum | [ |
Table 2 Application of FACS in non-secretory enzymes
类别 Classification | 目标酶 Target enzyme | 结果 Result | 宿主细胞 Host cell | 参考文献 Reference |
---|---|---|---|---|
细胞表面酶 | 丝氨酸蛋白酶OmpT | kcat/Km值提高60倍 | 大肠杆菌E. coli | [ |
分选酶A | kcat/Km值提高140倍 | 酿酒酵母S. cerevisiae | [ | |
半乳糖氧化酶 | Km值降低4.4倍 | 大肠杆菌E. coli | [ | |
D-氨基酸氧化酶 | kcat值提高4.2倍 | 大肠杆菌E. coli | [ | |
胞内酶 | β-1,3-半乳糖基转移酶 | 底物耐受性提高300倍 | 大肠杆菌E. coli | [ |
α-1,3-岩藻糖基转移酶 | 合成Lewis X和3'岩藻糖基乳糖的kcat/Km值分别提高6倍和14倍 | 幽门螺旋杆菌Helicobacter pylori | [ | |
α-1,2-岩藻糖基转移酶 | 2'岩藻糖基乳糖的滴度和产量分别提高1.72倍和1.51倍 | 大肠杆菌E. coli | [ | |
多聚唾液酸转移酶 | 比酶活和热稳定性分别提高 1.5-2.1倍和9倍 | 奈瑟菌Neisseria | [ | |
葡萄糖氧化酶 | Vmax值分别提高1.3和2.3倍 | 酿酒酵母S. cerevisiae | [ | |
ATP磷酸核糖转移酶 | L-组氨酸产量为0.1 mmol/L,野生型未生产L-组氨酸 | 谷氨酸棒状杆菌Corynebacterium glutamicum | [ | |
丝氨酸乙酰转移酶 | L-半胱氨酸产量提高2.36倍 | 大肠杆菌E. coli | [ | |
3C蛋白酶 | 蛋白水解活性提高4倍 | 大肠杆菌E. coli | [ | |
咖啡因去甲基化酶 | Vmax/Km值和产物选择性分别提高33和22倍 | 酿酒酵母S. cerevisiae | [ | |
丙酮酸羧化酶 | 赖氨酸滴度分别增加9%和19% | 谷氨酸棒状杆菌C. glutamicum | [ | |
莽草酸生产菌株 | 莽草酸滴度达到(3.72±0.35)mmol/L,比野生型提高2.4倍 | 谷氨酸棒状杆菌C. glutamicum | [ | |
丝氨酸羟甲基转移酶 | L-丝氨酸产量达到34.78 g/L,比野生型菌株提高35.9% | 谷氨酸棒状杆菌C. glutamicum | [ |
Fig. 2 FACS screening of non-secretory enzymes a: Screening of cell surface enzymes with fluorescent-labeled substrates. b: Screening of intracellular enzymes with fluorescent-labeled substrates
类别 Classification | 目标酶 Target enzyme | 结果 Result | 宿主细胞 Host cell | 参考文献 Reference |
---|---|---|---|---|
双乳化液滴法 | 血清对氧磷酶 | kcat/Km值提高100倍 | 大肠杆菌E. coli | [ |
碱性磷酸酶 | 实现活性碱性磷酸酶的100万倍富集 | 大肠杆菌E. coli | [ | |
α-L-苏糖核酸聚合酶 | 获得一个不依赖于锰的α-L-苏氨酸核糖核酸(TNA)聚合酶突变体 | 大肠杆菌E. coli | [ | |
几丁质酶A | 比酶活提高2倍 | 大肠杆菌E. coli | [ | |
人工金属酶 | 建立FACS筛选双乳化液滴中人工金属酶的方法 | 大肠杆菌E. coli | [ | |
水凝胶微珠法 | RNA聚合酶 | 获得一个对利福霉素有耐药性的突变体 | 大肠杆菌E. coli | [ |
植酸酶 | kcat值提高31% | 大肠杆菌E. coli | [ | |
木聚糖酶 | 比酶活提高1.3倍 | 毕赤酵母Pichia pastoris | [ | |
转肽酶 | kcat/Km值提高114倍 | 大肠杆菌E. coli | [ |
Table 3 Application of FACS in secretory enzymes
类别 Classification | 目标酶 Target enzyme | 结果 Result | 宿主细胞 Host cell | 参考文献 Reference |
---|---|---|---|---|
双乳化液滴法 | 血清对氧磷酶 | kcat/Km值提高100倍 | 大肠杆菌E. coli | [ |
碱性磷酸酶 | 实现活性碱性磷酸酶的100万倍富集 | 大肠杆菌E. coli | [ | |
α-L-苏糖核酸聚合酶 | 获得一个不依赖于锰的α-L-苏氨酸核糖核酸(TNA)聚合酶突变体 | 大肠杆菌E. coli | [ | |
几丁质酶A | 比酶活提高2倍 | 大肠杆菌E. coli | [ | |
人工金属酶 | 建立FACS筛选双乳化液滴中人工金属酶的方法 | 大肠杆菌E. coli | [ | |
水凝胶微珠法 | RNA聚合酶 | 获得一个对利福霉素有耐药性的突变体 | 大肠杆菌E. coli | [ |
植酸酶 | kcat值提高31% | 大肠杆菌E. coli | [ | |
木聚糖酶 | 比酶活提高1.3倍 | 毕赤酵母Pichia pastoris | [ | |
转肽酶 | kcat/Km值提高114倍 | 大肠杆菌E. coli | [ |
类别 Classification | 材料 Material | 特点 Characteristic | 参考文献 References |
---|---|---|---|
天然水凝胶 | 纤维蛋白;胶原蛋白;海藻酸盐;透明质酸;壳聚糖;琼脂糖 | 通过物理或离子相互作用力形成凝胶;良好的生物相容性 | [ |
合成水凝胶 | 聚乙二醇;聚丙烯酸;聚乙烯醇 | 通过共价键形成聚合体;可再生的;易获得 |
Table 4 Types of hydrogels
类别 Classification | 材料 Material | 特点 Characteristic | 参考文献 References |
---|---|---|---|
天然水凝胶 | 纤维蛋白;胶原蛋白;海藻酸盐;透明质酸;壳聚糖;琼脂糖 | 通过物理或离子相互作用力形成凝胶;良好的生物相容性 | [ |
合成水凝胶 | 聚乙二醇;聚丙烯酸;聚乙烯醇 | 通过共价键形成聚合体;可再生的;易获得 |
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