Biotransformation Of L-phenylalanine To 2-phenylethanol By Microorganism

As one of the most important components of natural flavors, 2-phenylethanol is widely used in foods, beverages and perfumes industries. Natural 2-phenylethanol extracted from rose features both a high price and a limited supply. The increasing concern for health nutrition and the limit of chemistry food additive in United states and European Union stimulate a worldwide demand for natural 2-phenylethanol. Biotransformation is an alternative or equivalent technology to get natural 2-phenylethanol. Natural L-phenylalanine is available and economical substrate for the 2-phenylethanol production. Therefore, the bioconversion of L-phenylalanine to 2-phenylethanol becomes more and more attractive.The paper focused on the biotransformation of L-phenylalanine to 2-phenylethanol by microorganisms. The optimization of fermentation with free and immobilized cells is studied. Then the production of 2-phenylalcohol from L-phenylalanine by microbial conversion in aqueous/organic solvent biphasic system was studied.Saccharomyces cerevisiae, Kluyveromyces marxianus and Kluyveromyces sinensis were reported to be three of the best strains which could produced more 2-phenylethanol than others. The results showed that Saccharomyces cerevisiae was the best strain which had bigger capacity to transform L-phenylalanine to 2-phenylethanol among the three stains tested. The conditions of liquid fermentation of 2-phenylethanol were studied. It showed that the effect of single factors, such as carbon source, nitrogen source, growth factors and inorganic salts on the production of 2-phenylethanol. Through orthogonal test, the optimum medium composition had been obtained. It was 40g/L sucrose, 7g/L L-phenylalanine, 0.5g/L Na2HPO4, 1.8 g/L Yeast Nitrogen Base( without amino Acid and Ammonium Sulfate). The optimum shaking culture condition was as follows: 24 hours strain age, 6% inoculation quality, initial pH 4.0, 50mL medium in 250mL shaking flask, 30℃, 100r/min shaking speed, 48hours incubation. Under this condition, productivity of 2-phenylethanol was 2.0g/L, which increased about 33.3% than that in original medium.The cells of Saccharomyces cerevisiae were entrapped into calcium alginate. The immobilized conditions were alginate 4.0%, cells 20%, Ca2+ 0.1mol/L .The optimum pH for free and immobilized cells were 4.0 and 3.5, respectively. The optimum reaction temperature for free and immobilized cells was 30℃. The pH stabilities of free cells and immobilized cells were good at all pH levels. For storage stability, the half-life of free cells was less than 5 days and the half-life of immobilized cells was more than 10 days.Microbial conversion of L-phenylalanine to 2-phenylalcohol with yeast was performed in an aqueous/organic solvent biphasic system. Saccharomyces cerevisiae had more potential to transform L-phenylalanine to 2-phenylalcohol than Kluyveromyces marxianus. The results of the solvent screening indicated that oleic acid was the best solvent which can extract 2-phenylalcohol efficiently. The optimum values of volume ratio of organic phase to aqueous phase, reaction temperature, pH and liquid volume were 1:1, 30℃, 4.0 and 20mL/250mL, respectively. Under above optimum conditions, the content of 2-phenylethanol reached 3.0g/L 48h later, which increased about 50% than that in monophasic aqueous system.