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High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l(-1) ferulic acid could be added continually and 19.2 g l(-1) vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.

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2,3,5,6-Tetramethylpyrazine (TTMP) was produced using a newly isolated Bacillus mutant. Culture medium optimization studies showed that soytone, an enzyme-hydrolysate of soybean meal, with the supplementation of vitamins, can fully replace yeast extract plus peptone in supporting TTMP production from glucose. In a 5-l fermenter, using the optimized medium which contained 20% glucose, 5% soytone, 3% (NH(4))(2)HPO(4), and vitamin supplements, fermentations were carried out with stirring at 700 rpm, air flow at 1.0 vvm, controlled pH at 7.0, and temperature at 37 degrees C. TTMP reached 4.33 g l(-1) after 64.6 h cultivation. A product recovery method was described, which involved evaporation, crystallization, and lyophilization. The product purity was 99.88%, determined by GC with the normalization method. The main impurities were 2,3,5-trimethylpyrazine (0.09%) and 2-ethyl-3,5,6-trimethylpyrazine (0.02%), which were identified by GC/MS. (13)C NMR determination also gave a consistent result. Natural and high purity of the product and the utilization of cheap green renewable materials make this process promising to compete with TTMP chemical synthetic methods.

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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.

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