从土壤中分离出的依赖NAD⁺的氨基酸脱氢酶产生菌的初步报告

Preliminary report of NAD+-dependent amino acid dehydrogenase producing bacteria isolated from soil.

作者信息

Mohammadi Hamid Shahbaz, Omidinia Eskander, Lotfi Sahebghadam Abbas, Saghiri Reza

机构信息

Young Researcher Club, Dept. of Biochemistry, Science & Research Campus, Islamic Azad University, Tehran, Iran.

Dept. of Biochemistry, Pasteur Institute of Iran, Tehran 13164, Iran.

出版信息

Iran Biomed J. 2007 Apr;11(2):131-5.

PMID:18051956

Abstract

BACKGROUND

Amino acid dehydrogenases (L-amino acid: oxidoreductase deaminating; EC 1.4.1.X) are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD+, NADP+ or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria (HCY) and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteriadehydrogenases from soil bacteria.

METHODS

The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed.

RESULTS

In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD+-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat/Km (s(-1).mM(-1)) values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83.

CONCLUSION

This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria.

摘要

背景

氨基酸脱氢酶（L-氨基酸：氧化还原酶脱氨基；EC 1.4.1.X）是氧化还原酶更广泛超家族的成员，催化氨基酸可逆地氧化脱氨生成其酮酸和氨，同时还原NAD⁺、NADP⁺或FAD。作为生物催化剂，这些酶在生物传感器或诊断试剂盒中用于筛查氨基酸代谢紊乱，如苯丙酮尿症（PKU）、枫糖尿症（MSUD）、同型胱氨酸尿症（HCY）和高脯氨酸血症，受到了广泛关注。本研究旨在从土壤细菌中分离和筛选新型氨基酸脱氢酶。

方法

通过薄层色谱、活性染色从土壤中分离的L-甲硫氨酸和L-苯丙氨酸利用菌中筛选产酶细菌，并通过酶活性测定进行确认。通过表型和生化特征鉴定细菌菌株。还对酶进行了稳态动力学研究。

结果

在总共230个测试菌株中，有4个被鉴定为氨基酸脱氢酶产生菌，分别属于假单胞菌属、柠檬酸杆菌属和变形杆菌属。它们在氧化脱氨反应中对L-异亮氨酸、L-甲硫氨酸、L-半胱氨酸、L-丝氨酸和L-谷氨酰胺表现出所需的NAD⁺依赖性脱氢酶活性。L-异亮氨酸脱氢酶、L-甲硫氨酸脱氢酶和L-谷氨酰胺脱氢酶对L-异亮氨酸、L-甲硫氨酸和L-谷氨酰胺氧化脱氨的比活性分别为1.59、1.2和0.73 U/mg。这些菌株中的Kcat/Km（s⁻¹·mM⁻¹）值如下：L-异亮氨酸为113.6，L-甲硫氨酸为62.05，L-谷氨酰胺为95.83。