L-天冬氨酸-α-脱羧酶的重组表达、定点突变及高通量检测方法的建立

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

摘要/Abstract

摘要：

将枯草芽孢杆菌L-天冬氨酸-α-脱羧酶基因进行了克隆和异源表达,并通过定点突变构建了2个突变体。针对该酶活力检测时存在的检测通量低、周期长和成本高等缺点,旨在建立一种简单高效的酶活力高通量检测方法。采用氯酚红（CPR）指示剂和4-吗啉乙磺酸（MES）缓冲液体系,并对检测条件进行优化,提高检测的准确性和灵敏性,建立了基于比色法的微孔板高通量检测方法,然后以L-天冬氨酸-α-脱羧酶及其突变体作为模型酶,对高通量检测方法进行了验证。优化后的酶活检测条件为MES缓冲液2 mmol/L,CPR指示剂75 μmol/L,L-天冬氨酸75 mmol/L,pH 6.5,温度37℃,反应时间10 min,检测波长为567 nm。采用3种模型酶对微孔板高通量检测方法进行了验证,结果显示该方法与HPLC法测得的结果一致。高通量检测方法具有操作简便易行、灵敏度高等优点,能够用于L-天冬氨酸-α-脱羧酶的快速检测。该方法的建立将为L-天冬氨酸-α-脱羧酶进行定向进化及突变体的高通量筛选奠定基础。

关键词: L-天冬氨酸-α-脱羧酶, 定点突变, 指示剂, 微孔板法, 高通量检测

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

朱秋雨, 段绪果. L-天冬氨酸-α-脱羧酶的重组表达、定点突变及高通量检测方法的建立[J]. 生物技术通报, 2022, 38(5): 269-278.

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.

图/表 9

表1 本研究中使用的引物

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

图1 琼脂糖凝胶电泳图及表达载体结构示意图 A：panDBs 基因PCR产物和质粒酶切产物（M：标准DNA分子量;1：panDBs PCR产物;2：质粒pET24a（+）双酶切产物）;B：重组表达载体pET24a（+）-panDBs示意图

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

图2 重组酶panDBs及突变体蛋白电泳图 M：标准蛋白分子量;1：panDBs上清;2：突变体panDBs-1上清;3：突变体panDBs-2上清

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

图3 比色法测定L-天冬氨酸-α-脱羧酶酶活的原理

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

图4 基于比色法的PanD高通量筛选方法的建立 A：质子化和去质子化的CPR吸收光谱;B：质子化和去质子化的吸收光谱差异;C：567 nm处的吸光度与氯酚红（CPR）浓度的关系

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

图5 CPR为指示剂时缓冲液浓度对测定体系pH敏感性的影响

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

图6 CPR为指示剂时指示剂浓度对测定体系pH敏感性的影响

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

图7 PanD活性的测定 A：微孔板中CPR-MES体系校准曲线;B：酶反应速率曲线

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

表2 重组酶及其突变体的部分动力学参数

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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