实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

一种快速定量检测氨基酸脱羧酶活力的比色方法及其应用

  • 陈庆菊 ,
  • 邓欢 ,
  • 胡红 ,
  • 卢昌文 ,
  • 唐志如
展开
  • 西南大学动物科技学院, 生物饲料与分子营养实验室, 重庆 400715
陈庆菊(1995-),女,贵州龙里人,硕士研究生,研究方向为动物营养与饲料科学。E-mail:1054082657@qq.com

收稿日期: 2019-07-04

  网络出版日期: 2020-01-19

基金资助

国家自然科学基金(31772610);国家重点研发计划(2018YFD0501000);重庆市留学人才创新计划重点项目(cx2017024)

A Colorimetric Method for Rapid and Quantitative Determination of Amino Acid Decarboxylase Activity and Its Application

  • CHEN Qingju ,
  • DENG Huan ,
  • HU Hong ,
  • LU Changwen ,
  • TANG Zhiru
Expand
  • Key Laboratory for Bio-Feed and Animal Nutrition, College of Animal Science and Technology, Southwest University, Chongqing 400715, China

Received date: 2019-07-04

  Online published: 2020-01-19

Supported by

 

摘要

本试验旨在探究一种快速定量检测氨基酸脱羧酶活力的比色方法。试验采用脱羧酶标准品进行标准曲线的制备,再用待测样品的吸光度值和标准曲线计算样品氨基酸脱羧酶活力。采用本试验建立的检测氨基酸脱羧酶活力的比色方法,对猪结肠微生物和脑组织样品进行氨基酸脱羧酶活力的测定;通过对未知样品的氨基酸脱羧酶活力进行测定,验证该方法的准确性。结果显示:以酪氨酸脱羧酶标准品建立了线性关系较好的氨基酸脱羧酶活力标准曲线:y=0.478 9x+0.112 6,R2=0.998 0。不同氨基酸脱羧酶活力与构建的酪氨酸脱羧酶活力标准曲线相似度高,说明本方法对多种氨基酸脱羧酶活力有广泛的适用性。采用本试验建立的比色方法快速地测定出了猪结肠微生物和脑组织中氨基酸脱羧酶活力;并且,使用该方法测得的未知样品的氨基酸脱羧酶活力落入已构建的标准曲线范围中,说明本方法的准确性较高。由结果可知,本试验建立的比色方法是检测氨基酸脱羧酶活力的一种快速方法,成本低廉,操作简单,对仪器要求不高,方法可靠,适用于多种样品中氨基酸脱羧酶活力的测定,可为研究动物组织、微生物培养液、细胞培养以及血液样本中的氨基酸脱羧酶活力提供有效的测定方法。

本文引用格式

陈庆菊 , 邓欢 , 胡红 , 卢昌文 , 唐志如 . 一种快速定量检测氨基酸脱羧酶活力的比色方法及其应用[J]. 动物营养学报, 2020 , 32(1) : 390 -396 . DOI: 10.3969/j.issn.1006-267x.2020.01.045

Abstract

This study was designed to investigate a colorimetric method for rapid and quantitative detection of amino acid decarboxylase activity. The experiment used the decarboxylase standard to prepare the standard curve, and then used the absorbance value and the standard curve of the sample to calculate the amino acid decarboxylase activity of the sample. The amino acid decarboxylase activity of pig colonic microorganism and brain tissue samples was determined by the colorimetric method for detecting amino acid decarboxylase activity established in this experiment. The accuracy of the method was verified by measuring the amino acid decarboxylase activity of unknown samples. The results showed as follows:the standard curve with good linear relationship of the amino acid decarboxylase activity established by the tyrosine decarboxylase standard was:y=0.478 9x+0.112 6, R2=0.998 0. The standard curves of different amino acid decarboxylase activities had a high similarity to the standard curve of tyrosine decarboxylase activity, indicating that the method had wide applicability to various amino acid decarboxylase activities. The amino acid decarboxylase activity in pig colonic microorganism and brain tissue was rapidly determined by the colorimetric method established in this experiment. Moreover, the amino acid decarboxylase activity of the unknown sample measured by this method fell within the range of the established standard curve, indicating that the method had high accuracy. The results indicate that the method established in this experiment is a rapid method for detecting the activity of amino acid decarboxylase. It is lower in cost, simpler in operation, lower in requirements on instruments, reliable in method, and suitable for determination of amino acid decarboxylase activity in various samples. It can provide an effective assay for studying the activity of amino acid decarboxylase in animal tissues, microbial culture fluids, cell cultures and blood samples.

参考文献

[1] LI T F,HUO L,PULLEY C,et al.Decarboxylation mechanisms in biological system[J].Bioorganic Chemistry,2012,43:2-14.
[2] JIANG H,XIA X X,FENG Y,et al.Development of a robust system for high-throughput colorimetric assay of diverse amino acid decarboxylases[J].Process Biochemistry,2017,60:27-34.
[3] GOTO T,TIBA K,SAKURADA Y,et al.Determination of hepatic cysteinesulfinate decarboxylase activity in fish by means of OPA-prelabeling and reverse-phase high-performance liquid chromatographic separation[J].Fisheries Science,2010,67(3):553-555.
[4] VIENOŽINSKIENE J,JANUŠEVI ČIVT R,PAULIUKONIS A,et al.Lysine decarboxylase assay by the pH-stat method[J].Analytical Biochemistry,1985,146(1):180-183.  
[5] ROSENBERG R M,HERREID R M,PIAZZA G J,et al.Indicator assay for amino acid decarboxylases[J].Analytical Biochemistry,1989,181(1):59-65.  
[6] KIM Y H,SATHIYANARAYANAN G,KIM H J,et al.A liquid-based colorimetric assay of lysine decarboxylase and its application to enzymatic assay[J].Journal of Microbiology and Biotechnology,2015,25(12):2110-2115.  
[7] MÉDICI R,DE MARÍA P D,OTTEN L G,et al.A high-throughput screening assay for amino acid decarboxylase activity[J].Advanced Synthesis & Catalysis,2011,353(13):2369-2376.  
[8] HENNIG A,BAKIRCI H,NAU W M.Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes[J].Nature Methods,2007,4(8):629-632.  
[9] YU K,HU S,HUANG J,et al.A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase[J].Enzyme and Microbial Technology,2011,49(3):272-276.  
[10] ALCÁZAR R,TIBURCIO A F.Determination of arginine and ornithine decarboxylase activities in plants[M]//ALCÁZAR R,TIBURCIO A.Methods in molecular biology.New York,NY:Humana Press,2018.
[11] SCRIVEN F,WLASICHUK K B,PALCIC M M.A continual spectrophotometric assay for amino acid decarboxylases[J].Analytical Biochemistry,1988,170(2):367-371.  
[12] 李金龙,王瑶,马娅君,等.一种猪胃肠道内容物与粪便微生物梯度离心分离方法[J].动物营养学报,2018,30(5):1837-1844.
[13] PESSIONE E,CIRRINCIONE S.Bioactive molecules released in food by lactic acid bacteria:encrypted peptides and biogenic amines[J].Frontiers in Microbiology,2016,7:876.
[14] HUSSAIN H,FUAT A R M,VIMALA B,et al.Screening method for detection of immediate amino acid decarboxylases-producing bacteria implicated in food poisoning[J].Tropical Biomedicine,2011,28(2):351-361.
[15] SHI F,XIE Y L,JIANG J J,et al.Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH[J].Enzyme and Microbial Technology,2014,61/62:35-43.
[16] BOVER-CID S,HUGAS M,IZQUIERDO-PULIDO M,et al.Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages[J].International Journal of Food Microbiology,2001,66(3):185-189.  
文章导航

/