Study On Biotransformation Of γ-Decalactone By Yarrowia Lipolytica

Gamma-decalactone (GDL) is the lactone of4-hydroxydecanoic acid (4HDA), found inthe fruits and the fermentation products naturally. This compound exhibits an oily-peachyaroma and has been widely used in food additives, pharmaceutical and chemical synthesisindustries. People’s demands for the product are increasing, but it’s not found occuring widelyin natural, and it synthetised by chemical method is racemic molecule. GDL synthetised bybiological method has the characteristics of natural equivalent, and the optical activity GDLproduct has not supplied in the current international market. Therefore, the biological methodhas broad prospects.In this paper, the strain of Yarrowia lipolytica was screened for GDL production. Asystematic and complete research was carried on in order to increase GDL production. Firstly,the genome shuffling method was employed to improve the yield of GDL. And the cultureconditions were optimized and the models for cell growth kinetics and GDL productiondynamics were established. Then an organic-inorganic composite material was prepared andused for immobilizing cells. The composite material could not only improve the mechanicalstrength and permeability of the cells but also prolong their service life. The bioconversion ofionic liquid system was established to increase the material exchange between the inside andoutside of cells. The analysis results of influence factors on the GDL production and ACOisozyme activity indicated that pyridine ring showed a preferable function of promotion andstimulation. Finally the GDL extraction efficiency of the supercritical carbon dioxide methodwas compared with the traditional solvent method. The details are as follows:(1) Under alkaline conditions, GDL reacts with hydrochloric acid hydroxylamine to formhydroxamic acid, which can reaction with ferric iron and further generate a colored complex.The spectrophotometric assay of GDL was established with good accuracy andreproducibility with a detection range from0.125to8.14g L-1. In order to improve thescreening efficiency of GDL producing strains, a high-throughput screening method wasperformed on the96-well microculture plates. Cells were cultured on96-well microplates.And GDL in the fermented broth was determined by using this newly rapidspectrophotometric quantitative determination method. As a result of screening,215GDL-producing strains were selected from4327samples. The highest production of thesestrains was179mg L-1. This strain was identified to be Yarrowia lipolytica according to thecell morphology, culture characteristics and the sequence of26S rRNA.(2) Ultraviolet radiation was performed to establish the positive mutant library. Theoptimal conditions for protoplast formation and fusion were obtained. By using recursivefusion method and after three rounds of fusion, a mutant strain, named G3-3.21, was obtainedand showed the best genetic stability and highest GDL yield with3.75g L-1, which was6.58times higher than that of parental strain. The results of comparative analysis on the mutantstrains indicated that GDL production required the Acyl-CoA oxidase (ACO) isozymes havinghigher ability of degrading the long chain substrates and lower ability of degrading the middleor short chain substrates. This was in favor of GDL accumulating and maintaining the highest yield in a certain period and avoiding the consequent β-oxidization before its formationthrough intramolecular cyclization.(3) In order to improve GDL production, the eight variables were optimized by using theTaguchi designed method. The wheat bran, complex nitrogen source (yeast extract and(NH4)2HPO4) and initial pH were determined to be the major effect factors. The centralcomposite design in response surface methodology was used to further optimize these mainfactors. As a result, the optimal medium composition was determined, and GDL productionwas4.767g L-1. The analysis of wheat bran components showed that the nicotinic acid couldimprove the yield of GDL greatly due to the promoting effect of pyridine ring in the nicotinicacid. By analysis on the characteristics of the cell growth and GDL production, the models forcells growth kinetics and GDL production dynamics were established. The growth kineticsmodel for Z703and G3-3.21strains weredX0.309(XdXdt=115.2)Xandd=0.438(1X,t16.7)Xrespectively. The GDL production dynamics model for the Z703and G3-3.21strain weredPdt=0.00105Xand dPdt=0.00784X, respectively.(4) In order to reduce the toxicity of substrate and product for cells, Organic alginatesodium compouned by inorganic attapulgite clay was used for cell immobilization. GDLproduction of immobilized cells was up to6.15g L-1,1.32times higher than that of free cells.In addition, the cells could be used for3times. In addition, ionic liquid with better biologicalcompatibility was introduced in this study to increase the material exchange between theinside and outside of the cells. In this ionic liquid biotransformation system, GDL productionwas7.85g·L-1. Compared with the same anion, when the cation was pyridine, GDLproduction and ACO activity were generally higher than those of imidazole ion. when theanions were bromine ion and nitrate ion, GDL production and the ACO activity were lowerthan those of tetra-fluoride phosphate ion and hexa-fluoride phosphate ion.(5) The wall-breaking conditions of the free cells were studied, total GDL produciton(both inside and outside the cells) was5.11g L-1, which was increased by14.06%comparedwith the produciton outside the cells. Acidification and heating experiments showed that theyeast adopted in this study had lactonization ability itself and could catalyze4-hydroxyl-decanoic lactone into GDL without heating and acidification. The extractiontechnology of GDL of immobilized cell in ionic liquid biotransformation system wasresearched and extraction effects of the two methods were compared. The extraction rate was86.2%by the butyl acetate extraction method, while the extraction rate was91.7%bysupercritical CO2extraction method.