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Catalysts 2017, 7(9), 257; doi:10.3390/catal7090257

Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

1
School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China
2
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
*
Author to whom correspondence should be addressed.
Received: 11 August 2017 / Revised: 26 August 2017 / Accepted: 26 August 2017 / Published: 31 August 2017
(This article belongs to the Special Issue Biocatalysis and Biotransformations)
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Abstract

A novel nicotine hydroxylase was isolated from Pseudomonas sp. ZZ-5 (HSPHZZ). The sequence encoding the enzyme was 1206 nucleotides long, and encoded a protein of 401 amino acids. Recombinant HSPHZZ was functionally overexpressed in Escherichia coli BL21-Codon Plus (DE3)-RIL cells and purified to homogeneity after Ni-NTA affinity chromatography. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that the enzyme could efficiently catalyze the conversion of 6-hydroxy-3-succinoylpyridine (HSP) into 2,5-dihydroxypyridine (2,5-DHP) and succinic acid in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The kinetic constants (Km, kcat, and kcat/Km) of HSPHZZ toward HSP were 0.18 mM, 2.1 s−1, and 11.7 s−1 mM−1, respectively. The optimum temperature, pH, and optimum concentrations of substrate and enzyme for 2,5-DHP production were 30 °C, 8.5, 1.0 mM, and 1.0 μM, respectively. Under optimum conditions, 85.3 mg/L 2,5-DHP was produced in 40 min with a conversion of 74.9%. These results demonstrated that HSPHZZ could be used for the enzymatic production of 2,5-DHP in biotechnology applications. View Full-Text
Keywords: HSP; 2,5-DHP; Pseudomonas; HSP hydroxylase; gene cloning HSP; 2,5-DHP; Pseudomonas; HSP hydroxylase; gene cloning
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Wei, T.; Zang, J.; Zheng, Y.; Tang, H.; Huang, S.; Mao, D. Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine. Catalysts 2017, 7, 257.

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