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L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

ABSTRACT: An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.

SUBMITTER: Sonke T

PROVIDER: S-EPMC1317364 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

Sonke Theo T Ernste Sandra S Tandler Renate F RF Kaptein Bernard B Peeters Wilco P H WP van Assema Friso B J FB Wubbolts Marcel G MG Schoemaker Hans E HE

Applied and environmental microbiology 20051201 12

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). S ...[more]

PMID: 16332774

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Project description:Azoxystrobin is one of the most popular strobilurin fungicides, widely used in agricultural fields for decades.Extensive use of azoxystrobin poses a major threat to ecosystems. However, little is known about the kinetics and mechanism of azoxystrobin biodegradation. The present study reports a newly isolated bacterial strain, Ochrobactrum anthropi SH14, utilizing azoxystrobin as a sole carbon source, was isolated from contaminated soils. Strain SH14 degraded 86.3% of azoxystrobin (50 μg·mL-1) in a mineral salt medium within five days. Maximum specific degradation rate (qmax), half-saturation constant (Ks), and inhibition constant (Ki) were noted as 0.6122 d-1, 6.8291 μg·mL-1, and 188.4680 μg·mL-1, respectively.Conditions for strain SH14 based azoxystrobin degradation were optimized by response surface methodology. Optimum degradation was determined to be 30.2 °C, pH 7.9, and 1.1 × 107 CFU·mL-1 of inoculum. Strain SH14 degraded azoxystrobin via a novel metabolic pathway with the formation of N-(4,6-dimethoxypyrimidin-2-yl)-acetamide,2-amino-4-(4-chlorophenyl)-3-cyano-5,6-dimethyl-pyridine, and 3-quinolinecarboxylic acid,6,8-difluoro-4-hydroxy-ethyl ester as the main intermediate products, which were further transformed without any persistent accumulative product. This is the first report of azoxystrobin degradation pathway in a microorganism. Strain SH14 also degraded other strobilurin fungicides, including kresoxim-methyl (89.4%), pyraclostrobin (88.5%), trifloxystrobin (78.7%), picoxystrobin (76.6%), and fluoxastrobin (57.2%) by following first-order kinetic model. Bioaugmentation of azoxystrobin-contaminated soils with strain SH14 remarkably enhanced the degradation of azoxystrobin, and its half-life was substantially reduced by 95.7 and 65.6 days in sterile and non-sterile soils, respectively, in comparison with the controls without strain SH14. The study presents O. anthropi SH14 for enhanced biodegradation of azoxystrobin and elaborates on the metabolic pathways to eliminate its residual toxicity from the environment.

Dataset Information

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

Publications

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

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