可视化高通量检测天冬氨酸转氨甲酰酶活性的方法

doi:10.16085/j.issn.1000-6613.2021-0750

摘要/Abstract

摘要：

天冬氨酸转氨甲酰酶（ATCase）是嘧啶生物合成途径的第一个酶，其活性的反馈调节机制在控制嘌呤和嘧啶合成途径的平衡中起重要作用。目前，检测该酶活性的方法是基于安替比林和2,3-丁二酮肟的显色法。然而，该方法需先避光反应16h，再在45℃水浴30min并均匀光照，操作比较繁琐。本研究以对二甲氨基苯甲醛（PDAB）盐酸溶液为显色试剂，建立了一种检测ATCase活性的方法。该方法的原理是ATCase催化产生的N-氨甲酰基-L-天冬氨酸（N-CP-L-Asp）与PDAB在室温下反应15min，可生成黄色物质并能通过比色法定量检测。研究表明，在0.1~5mmol/L N-CP-DL-Asp的浓度范围内，黄色随化合物浓度的增加而加深，且在438nm的吸光度具有良好的线性关系，精密度RSD为0.87%~1.52%，加标回收率为96.6%~101.9%。该方法成功用于重组表达ATCase的活性测试，测得比酶活为56.83U/mg。该方法可以高效、快速和可视化地检测ATCase的活性，并且可以通过酶标仪实现高通量检测。

关键词: 生物催化, 酶, 天冬氨酸转氨甲酰酶, 活性, 对二甲氨基苯甲醛, 可视化, 高通量检测

Abstract:

Aspartate transcarbamylase (ATCase) is the first enzyme in the pyrimidine biosynthesis pathway, and the feedback regulation mechanism of its activity plays an important role in controlling the balance of purine and pyrimidine metabolism. Currently, the activity of ATCase is detected through a spectrophotometric method with antipyrine and 2,3-butanedione oxime as the color reagent. However, this method requires a reaction in the darkness for 16h, and then a water bath at 45℃ for 30min with uniform illumination, which is complicated to operate. In this study, p-dimethylaminobenzaldehyde (PDAB) hydrochloric acid solution was used as the color reagent to establish a method for detecting the activity of ATCase. The principle of this method is that N-carbamoyl-L-aspartic acid (the product of ATCase) reacts with PDAB at room temperature for 15min, which can produce a yellow substance and can be quantitatively detected by colorimetry. In the range of 0.1—5mmol/L, the yellow color deepened with the increase of the N-CP-DL-Asp concentration, and the absorbance at 438nm had a good linear relationship. The precision RSD was 0.87%—1.52%, and the recovery rate of standard addition was 96.6%—101.9%. This method was successfully used to determine the activity of recombinantly expressed ATCase, and the specific activity was 56.83U/mg. This method realizes the efficient, rapid and visual detection of the activity of ATCase, and can achieve high-throughput detection by using a microplate reader.

Key words: biocatalysis, enzyme, aspartate transcarbamylase, reactivity, p-dimethylaminobenzaldehyde, visualization, high-throughput detection

中图分类号:

Q555⁺.6

高博, 冯旭东, 李春. 可视化高通量检测天冬氨酸转氨甲酰酶活性的方法[J]. 化工进展, 2022, 41(4): 2054-2059.

GAO Bo, FENG Xudong, LI Chun. Visual and high-throughput method for detecting the activity of aspartate transcarbamylase[J]. Chemical Industry and Engineering Progress, 2022, 41(4): 2054-2059.

图/表 11

参考文献 12

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N-CP-DL-Asp浓度/mmol·L^-1	平均吸光度	标准偏差	RSD/%
2	0.691	0.006	0.87
3	1.028	0.012	1.17
4	1.379	0.021	1.52

N-CP-DL-Asp浓度/mmol·L^-1	平均吸光度	标准偏差	RSD/%
2	0.691	0.006	0.87
3	1.028	0.012	1.17
4	1.379	0.021	1.52

上清液体积/μL	Abs₄₃₈对照组	Abs₄₃₈实验组
10	0.242	1.081
20	0.289	1.026
30	0.274	1.179
50	0.287	1.051

上清液体积/μL	Abs₄₃₈对照组	Abs₄₃₈实验组
10	0.242	1.081
20	0.289	1.026
30	0.274	1.179
50	0.287	1.051

序号	吸光度	比酶活/U·mg^-1	平均比酶活/U·mg^-1
1	1.095	57.46	56.83
2	1.044	54.79
3	1.110	58.24