Abstract
Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent K m, k cat, and V max values to be 0.35 mM, 67.7 s−1, and 78.2 U mg−1, respectively. The catalytic efficiency (k cat/K m) value of Fcs was 193.4 mM−1 s−1 for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation.
Highlights
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavoring agents used in the world
As for the localization of fcs and ech, it was reported that the translational start codon GTG of fcs overlapped with the stop codon TAG of ech in Amycolatopsis sp. strain HR167 [10]
It was found that the ech and fcs genes were located just in the same way as that reported in Amycolatopsis sp. strain HR167 [10]
Summary
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavoring agents used in the world. It is widely used in foods, beverages, perfumes, and pharmaceuticals owing to its unique and irreplaceable flavor [1]. Natural vanillin extracted from vanilla pods provides only about 0.25% (40 tons of 16,000 tons) of vanillin sold in the market, whereas the remainder is mostly produced through the chemical synthesis of lignin or fossil hydrocarbons [3]. Vanillin produced by biotechnological approaches in microbial systems is classified as natural vanillin by the European and US food legislations [5]. Because of the increasing concerns for ‘‘natural’’ and ‘‘healthy’’ products among consumers worldwide, the microbial production of natural vanillin has become one of the major points of research in this field
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