Recombinant Expression and Site-directed Mutagenesis of L-aspartate-α-decarboxylase,and the Establishment of High-throughput Assay Method

doi:10.13560/j.cnki.biotech.bull.1985.2021-0892

Abstract

Abstract:

The L-aspartate-α-decarboxylase-encoded gene from Bacillus subtilis was cloned and heterologously expressed and two variants were constructed by site-directed mutagenesis. Regarding the issues of low detection throughput,long period,and high cost in the detection of enzyme activity,a simple and efficient high-throughput assay method was established. Having chlorophenol red（CPR）indicator and 4-morpholineethanesulfonic acid（MES）buffer system,the detection conditions were optimized to improve the accuracy and sensitivity of assay method,and a high-throughput assay method based on the microplate was established. The L-aspartate-α-decarboxylase and its variants were used as model enzymes to verify the high-throughput assay method. The optimized conditions of detecting L-aspartate-α-decarboxylase enzyme activity were：MES buffer 2 mmol/L,CPR indicator 75 μmol/L,L-aspartic acid 75 mmol/L,pH 6.5,temperature 37℃,reaction time 10 min,and the detection wavelength was 567 nm. The 3 model enzymes were used to verify the high-throughput assay method,and the results presented consistent with the results obtained by HPLC method. In conclusion,this high-throughput assay method is simple and rapid,and can be used in the rapid detection of L-aspartate-α-decarboxylase. Thus the establishment of this method lays the foundation for the site-directed evolution of L-aspartate-α-decarboxylase and its variants

Key words: L-aspartate-α-decarboxylase, site-directed mutation, indicator, microplate method, high-throughput assay

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.

Figures/Tables 9

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. 3 Principle for the determination of panD activity by colorimetric method

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

Fig. 5 Effects of buffer concentrations on sensing pH change using CPR as indicator

Fig. 6 Effects of indicator concentration on sensing pH change using CPR as indicator

Fig. 7 Determination of PanD activity A：Validation curve of CPR-MES system in a microplate. B：Enzymatic reaction rate curve

Table 2 Partial kinetic parameters of recombinant enzyme and its mutants

	Mutation sites	V_max/ （μmol·min^-1·mg^-1）	K_m/ （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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