title: Reaction kinetics for the biocatalytic conversion of phenazine-1-carboxylic acid to 2-hydroxyphenazine.
abstract: The phenazine derivative 2-hydroxyphenazine (2-OH-PHZ) plays an important role in the biocontrol of plant diseases, and exhibits stronger bacteriostatic and fungistatic activity than phenazine-1-carboxylic acid (PCA) toward some pathogens. PhzO has been shown to be responsible for the conversion of PCA to 2-OH-PHZ, however the kinetics of the reaction have not been systematically studied. Further, the yield of 2-OH-PHZ in fermentation culture is quite low and enhancement in our understanding of the reaction kinetics may contribute to improvements in large-scale, high-yield production of 2-OH-PHZ for biological control and other applications. In this study we confirmed previous reports that free PCA is converted to 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the action of a single enzyme PhzO, and particularly demonstrate that this reaction is dependent on NADP(H) and Fe3+. Fe3+ enhanced the conversion from PCA to 2-OH-PHZ and 28°C was a optimum temperature for the conversion. However, PCA added in excess to the culture inhibited the production of 2-OH-PHZ. 2-OH-PCA was extracted and purified from the broth, and it was confirmed that the decarboxylation of 2-OH-PCA could occur without the involvement of any enzyme. A kinetic analysis of the conversion of 2-OH-PCA to 2-OH-PHZ in the absence of enzyme and under different temperatures and pHs in vitro, revealed that the conversion followed first-order reaction kinetics. In the fermentation, the concentration of 2-OH-PCA increased to about 90 mg/L within a red precipitate fraction, as compared to 37 mg/L within the supernatant. The results of this study elucidate the reaction kinetics involved in the biosynthesis of 2-OH-PHZ and provide insights into in vitro methods to enhance yields of 2-OH-PHZ.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Mingmin
authors.initials: M
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Huasong
authors.initials: H
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Xuehong
authors.initials: X
authors.lastname: Zhang
mesh_terms: D055162:Biocatalysis; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D009243:NAD / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry; D010619:Phenazines / Q000737:chemistry
chemical_list: D010619:Phenazines; D009243:NAD; C037165:1-phenazinecarboxylic acid; C088013:2-hydroxyphenazine; D007501:Iron; D010105:Oxygenases