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

摘要

A novel nicotine hydroxylase was isolated from Pseudomonas sp. ZZ-5 (HSPH ZZ ). The sequence encoding the enzyme was 1206 nucleotides long, and encoded a protein of 401 amino acids. Recombinant HSPH ZZ 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 (K m , k cat , and k cat /K m ) of HSPH ZZ 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 HSPH ZZ could be used for the enzymatic production of 2,5-DHP in biotechnology applications.