Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (1): 289-298.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0516
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ZHANG Xue(), TAN Yu-meng, JIANG Hai-xia, YANG Guang-yu()
Received:
2021-04-15
Online:
2022-01-26
Published:
2022-02-22
Contact:
YANG Guang-yu
E-mail:yukimura0214@sjtu.edu.cn;yanggy@sjtu.edu.cn
ZHANG Xue, TAN Yu-meng, JIANG Hai-xia, YANG Guang-yu. Directed Evolution of α-1,2-fucosyltransferase by a Single-cell Ultra-high-throughput Screening Method[J]. Biotechnology Bulletin, 2022, 38(1): 289-298.
Fig. 1 Fluorescent substrate structure and the schematic diagram of FACS screening strategy A:The fluorescent substrates. B:Schematic diagram of single cell intracellular fluorescence capture
引物Primer | 序列Sequence(5'-3') |
---|---|
FutC-ep -F | CGAGCTCGACGATGACGATAAAATGGCCTTTAA |
FutC-ep- R | CCCAAGCTTGGGTCAATCTAAAGCGTTATAC |
Table 1 Primers for random mutation library construction
引物Primer | 序列Sequence(5'-3') |
---|---|
FutC-ep -F | CGAGCTCGACGATGACGATAAAATGGCCTTTAA |
FutC-ep- R | CCCAAGCTTGGGTCAATCTAAAGCGTTATAC |
Fig. 2 TLC analysis of fluorescent products and intra-cellular fluorescence enrichment reaction analysis of negative and positive strains under UV light irradiation A:TLC analysis of the two fluorescent substrates(Negtive)and the fluorescent products catalyzed by FutC(FutC). B:Comparison of the intracellular fluorescence enrichment reaction of the negative strain and the positive strain under UV light irradiation,the negative strain(Negtive,JM107 Nan- LacZ- pUCKC18-FKP/pUC18)and positive strains(FutC,JM107 Nan- LacZ- pUCKC18-FKP/pUC18-FutC)
分选前阳性比例 Proportion of positive cells before sorting/% | 分选后阳性比例 Proportion of positive cells after sorting/% | 富集倍数 Enrichment factor |
---|---|---|
50 | 100 | 2 |
10 | 100 | 10 |
1 | 90 | 90 |
0.1 | 73.3 | 733 |
Table 2 Model screening of FutC
分选前阳性比例 Proportion of positive cells before sorting/% | 分选后阳性比例 Proportion of positive cells after sorting/% | 富集倍数 Enrichment factor |
---|---|---|
50 | 100 | 2 |
10 | 100 | 10 |
1 | 90 | 90 |
0.1 | 73.3 | 733 |
Fig. 5 Construction of the FutC random mutation library A:Error-prone PCR products of futC gene with different Mn2+ concentration,1, 2, 3, 4 are:PCR products with 0, 0.04, 0.06, and 0.1 mmol/L Mn2+. B:Ligation efficiency of random mutation library,and 5 is a negative control
Fig. 8 Establishment of HPLC enzyme activity determin-ation method and protein purification of FutC WT and mutant A:HPLC analysis of substrate lactose-bodipy and product 2'-fucosyllactose-bodipy. B:Standard curve for the quantification of product 2'-fucosyllactose-bodipy. C:SDS-PAGE analysis of purified FutC wild-type and mutant proteins,WT:wild type,1:K102E, 2:R105C,3:K282E
酶Enzyme | 突变位点Mutation | 比活力Specific activity/(mU·mg-1) |
---|---|---|
WT | None | 57.6±3.6 |
1 | K102E | 156.8±2.9 |
2 | R105C | 175.7±11.0 |
3 | K282E | 151.9±7.9 |
Table 3 Specific activities of WT FutC and selected mutants using lactose-bodipy as acceptor
酶Enzyme | 突变位点Mutation | 比活力Specific activity/(mU·mg-1) |
---|---|---|
WT | None | 57.6±3.6 |
1 | K102E | 156.8±2.9 |
2 | R105C | 175.7±11.0 |
3 | K282E | 151.9±7.9 |
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