Bioconversion Of L-Phenylalanine Into Natural 2-Phenylethanol By Yeast Cells

2-phenylethanol is a higher aromatic alcohol with a rose-like odor,and takes on extensive applications in food,daily chemical and light industries as flavors or fragrances. The worldwide demand for natural products impels the research and development on production of 2-phenylethanol by biotechnology.The most efficient biotechnological approach used to obtain natural 2-phenylethanol is the bioconversion of L-phenylalanine to 2-phenylethanol by yeast cells.Since the precursor L-phenylalanine is now produced microbiologically,2-phenylethanol produced in this way can undoubtedly be labeled "natural",and it is considered to be the best substitute for the product extracted from essential oils of roses or other plants.The future for development and utilization of this technology is therefore bright.Aimed at improving the concentration and productivity of 2-phenylethanol,the thesis comprehensively studied the bioconversion process of 2-phenylethanol from L-phenylalanine by yeast cells.Some remarkable results were obtained finally,in which the 2-phenylethanol concentration and productivity were improved significantly,and provided an available foundation for the industrial application.A 2-phenylethanol-tolerant strain,Saccharomyces cerevisiae BD,capable of producing high concentration of 2-phenylethanol from L-phenylalanine,was screened out from fifteen yeast strains collected.After separation and ultraviolet radiation of the strain BD,BD18 was selected as a desirable strain for 2-phenylethanol production with the 2-phenylethanol concentration of 2.04 g/L in initial medium.The experiments of single factor and orthogonal design were carried out to optimize the compositions of bioconversion and seed medium,as well as cultivation conditions for 2-phenylethanol production.The best operations process was as following:The strain maintained in slant medium was inoculated into 40 mL of preculturing medium containing 40 g/L glucose,20 g/L peptone and 10 g/L yeast extract,and incubated at 30℃and 200 rpm for 24 h.Then a 3 mL cell suspension was transferred to 250 mL Erlenmeyer flasks containing 30 mL of biotransformation medium consisting of 120 g/L sucrose,5 g/L yeast extract,7.5 g/L KH₂PO₄,9.6 g/L K₂HPO₄ and 0.5 g/L MgSO₄·7H₂O. After inoculation,10 g/L L-phenylalanine,without prior sterilization,were added to the bioconversion medium and incubated at 30℃and 200 rpm.This process resulted in a improvement of 2-phenylethanol concentration,up to 4.64 g/L with a molar yield of 62.7%and productivity of 0.26 g/(L·h).A model of polynomial regressive equation between the 2-phenylethanol concentration and sucrose,yeast extract and L-phenylalanine in the medium was established after the Box-Behnken central composite design and the response surface analysis were put into practice.The model exhibited fine exactitude and provided a predictive model for optimizing the medium in the production of 2-phenylethanol by bioconversion.In situ product recovery techniques were applied to production of 2-phenylethanol by bioconversion,which was performed in aqueous/organic solvent and aqueous/solid absorbent two-phase system respectively.The selection of organic solvents and absorbents favorable for establishing two-phase system was conducted,and the bioconversion conditions in two-phase system for production of 2-phenylethanol were investigated too.When oleic acid with 1/3 volume of medium was added into medium, and shaking speed,temperature and concentration of substrate were set at 250 r/min, 30℃,14 g/L respectively,the 2-phenylethanol concentration could reach 14.9 g/L and 1.74 g/L in oleic acid phase and aqueous phase respectively after 18 h cultivation,with a molar yield of 64.7%.The productivity of 2-phenylethanol achieved at 0.37 g/(L·h). When polypropylene glycol 2000 with 1/2 volume of medium was added into medium, the concentration of substrate were set at 12 g/L,the 2-phenylethanol concentration could reach 11.1 g/L in polypropylene glycol 2000 phase after 18 h cultivation,with a molar yield of 68.1%.The productivity of 2-phenylethanol achieved at 0.31 g/(L·h).When 2g macroporous resin D101 used as adsorbent and 12 g/L L-phenylalanine were added into 30 mL medium,the total 2-phenylethanol concentration achieved was 6.17 g/L,of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin.After 24 h cultivation,the molar yield and productivity of 2-phenylethanol reached 69.5%and 0.26 g/(L·h)respectively.Production of 2-phenylethanol by conventional bioconversion process and with organic solvent extraction or macroporous resin adsorption were scaled up in 5 L bioreactor,and the results were almost same as in flask scale.It means that scale-up of the bioconversion from flask scale achieved success easily for production of 2-phenylethanol.The kinetic models for cell growth,2-phenylethanol production and sucrose consumption of Saccharomyces cerevisiae were established based on the data of bioconversion process in 5 L bioreactor.Three kinetic models were proposed by the Logistic equation for the cell growth,the Luedeking-Piret equation for 2-phenylethanol production and the Luedeking-Piret-like equation for sucrose consumption respectively. The models exhibiting high precision for simulation could exactly describe and predict 2-phenylethanol production by biotransformation in practice.The selection of organic solvent favorable for extraction separation of 2-phenylethanol was performed,and it was found that acetic ether is the best extract for 2-phenylethanol separation from bioconversion liquid.In extracting separation,if phase ratio of acetic ether to bioconversion liquid was set at 0.5,the recovery and purity of 2-phenylethanol could reach 93.6%and 90.7%respectively.Through static adsorption experiments,the macroporous resin D101 was also found to be the best absorbent for 2-phenylethanol separation.If separation was conducted on column packed with D101 the optimal loading volume was 5.5 BV(bed volume),and the flow rate was 10 BV/h when the concentration of 2-phenylethanol in bioconversion liquid was about 4.5 g/L. After sample loading,D 101 was firstly leached by distilled water with the same volume of loaded sample,and then eluted by 95%ethanol with 3 BV.By this process,the product with recovery of 84.4%and purity of 85.2%was obtained after the ethanol was evaporated.