Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (5): 269-278.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0892
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Received:
2021-07-12
Online:
2022-05-26
Published:
2022-06-10
Contact:
DUAN Xu-guo
E-mail:870129814@qq.com;xgduan@njfu.edu.cn
ZHU Qiu-yu, DUAN Xu-guo. Recombinant Expression and Site-directed Mutagenesis of L-aspartate-α-decarboxylase,and the Establishment of High-throughput Assay Method[J]. Biotechnology Bulletin, 2022, 38(5): 269-278.
引物Primer | 引物序列Primer sequence(5'-3') |
---|---|
BspanD-F | GAAGGAGATATACATATGTATCGAACAATGATGAGCGG |
BspanD-R | GCTTGTCGACGGAGCTCCTACAAAATTGTACGGGCTGGT |
I88M-F | GGTCATTATTATGTCCCACAAAATGATGTC |
I88M-R | GACATCATTTTGTGGGACATAATAATGACC |
I46V-F | GAAAAAGTACAAGTTGTGAATAATAATAATGGAG |
I46V-R | CTCCATTATTATTATTCACAACTTGTACTTTTTC |
K104S-F | CCATGAGCCGAGTGTGGCTGTTCT |
K104S-R | AGAACAGCCACACTCGGCTCATGG |
I126*-F | GAACCAGCCCGTACATAATTGTAGGAGC |
I126*-R | GCTCCTACAATTATGTACGGGCTGGTTC |
Table 1 Primers used in this study
引物Primer | 引物序列Primer sequence(5'-3') |
---|---|
BspanD-F | GAAGGAGATATACATATGTATCGAACAATGATGAGCGG |
BspanD-R | GCTTGTCGACGGAGCTCCTACAAAATTGTACGGGCTGGT |
I88M-F | GGTCATTATTATGTCCCACAAAATGATGTC |
I88M-R | GACATCATTTTGTGGGACATAATAATGACC |
I46V-F | GAAAAAGTACAAGTTGTGAATAATAATAATGGAG |
I46V-R | CTCCATTATTATTATTCACAACTTGTACTTTTTC |
K104S-F | CCATGAGCCGAGTGTGGCTGTTCT |
K104S-R | AGAACAGCCACACTCGGCTCATGG |
I126*-F | GAACCAGCCCGTACATAATTGTAGGAGC |
I126*-R | GCTCCTACAATTATGTACGGGCTGGTTC |
Fig. 1 Agarose gel electropherogram and structural sch-ematic diagram of expression vector A:PCR product of panDBs and plasmid enzyme digestion product(M:DNA marker;1:PCR product of panDBs;2:double restriction enzyme digestion product of plasmid pET24a(+)). B:Structural schematic diagram of recombinant expression vector pET24a(+)-panDBs
Fig. 2 Electropherogram of recombinant protein panDBs and its mutant proteins M:Protein marker. 1:Supernatant of panDBs. 2:Supernatant of mutant panDBs-1. 3:Supernatant of mutant panDBs-2
Fig. 4 Development of a colorimetric high-throughput scr-eening method for PanD assay A:Absorption spectra of protonated and deprotonated forms of CPR. B:Difference in absorption spectra between the two forms of CPR. C:Absorbance at 567 nm as a function of the concentration of CPR
Mutation sites | Vmax/ (μmol·min-1·mg-1) | Km/ (mmol·L-1) | |
---|---|---|---|
panDBs | - | 0.61±0.06 | 2.78±0.09 |
panDBs-1 | I46V/I88M/K104S/I126* | 0.68±0.03 | 2.98±0.16 |
panDBs-2 | I88M | 1.11±0.11 | 1.49±0.05 |
Table 2 Partial kinetic parameters of recombinant enzyme and its mutants
Mutation sites | Vmax/ (μmol·min-1·mg-1) | Km/ (mmol·L-1) | |
---|---|---|---|
panDBs | - | 0.61±0.06 | 2.78±0.09 |
panDBs-1 | I46V/I88M/K104S/I126* | 0.68±0.03 | 2.98±0.16 |
panDBs-2 | I88M | 1.11±0.11 | 1.49±0.05 |
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