Study On Cyclization Mechanism Of Biotransformation γ-Decalactone By Yeast Of MF-Y11

γ-Decalactone is a lactone compound having a peach and apricot flavor.It can be bio-synthesized by yeast,using castor oil,ricinoleic acid or methyl ricinoleate as a substrate.Ric-inoleic acid is biotransformed in the yeast cell byβ-oxidation to obtain 4-hydroxydecanoic acid,an important precursor ofγ-decalactone,which is further cyclized intoγ-decalactone un-der the cyclase.In this paper,the direct precursor ofγ-decalactone,4-hydroxydecanoic acid,was prepared by chemical synthesis,and the synthesized 4-hydroxy decanoic acid was used as a substrate for yeast MF-Y11 fermentation.The cyclization mechanism of lactone cyclase converting 4-hydroxy decanoic acid intoγ-decalactone was preliminarily elucidated by stud-ying the changes of productγ-decalactone,substrate 4-hydroxydecanoic acid and medium pH during fermentation under different factors.The specific content has the following aspects:Firstly,γ-decalactone was saponified by sodium hydroxide,and acidification was carried out by ion exchange using a D113 weak acid cation resin to obtain 4-hydroxydecanoic acid having a purity of 98.7%and a yield of 32.0%.Based on the characterization of derivatization mass spectrometry,infrared spectroscopy and nuclear magnetic resonance spectroscopy,the chemical structure was determined and some physicochemical properties of 4-hydroxyl were supplemented.It is concluded that the upper limit pH value of 4-hydroxydecanoic acid start-ing spontaneous cyclization at room temperature is 5.03,and the lower limit pH value is 1.93;the lower limit temperature value of 4-hydroxydecanoic acid starting spontaneous cyclization under heating conditions is 42°C（Melting point）,the lower limit temperature is 150℃,and on this basis,an analytical method for 4-hydroxydecanoic acid is established.Secondly,by optimizing the fermentation parameters of yeast MF-Y11 to 4-hydroxy decanoic acid during fermentation,the optimal transformation conditions were as follows:initial pH of the medium 7.50,conversion temperature 30°C,the best substrate was added.the optimal substrate addition amount is 2.00g/L.Under this condition,the concentration ofγ-decalactone reached a maximum value of 0.50 g/L,and it was found that the cyclase in yeast MF-Y11 was no active at a pH of 4.82.The maximum thermal half-life of4-hydroxydecanoate cyclase was 4.41 h,and the maximum heat inactivation rate constant was0.16 h^-1.By studying the reaction kinetics curve of yeast MF-Y11 intracellular cyclase under optimal conditions,it was found that the enzyme might be an allosteric enzyme,and the fer-mentation kinetic parameter Vmax=0.48mmoL·L^-1h^-1(0.060 g·L^-1 h^-1),Km=5.05 mmoL·L^-1(0.91 g L^-1)was calculated.Thirdly,through the analysis of fermentation kinetics and metabolic flow of4-hydroxycarboxylic acid under the influence of different factors in yeast,it is concluded that low concentration of 4-hydroxycarboxylic acid has higher conversion efficiency to gam-ma-decanolide.Among them,the metal ions which promoted the cyclization ability of4-hydroxycarboxylic acid ranked Mg²⁺>Ca²⁺>Zn²⁺,from large to small.The inhibition con-centration of gamma-decanolide on yeast was about 0.50g/L under the optimum conditions.Fourthly,by applying the results of the study,under the optimum conditions of the above4-hydroxydecanoate cyclase,the yeast MF-Y11 was converted to 12-hydroxystearic acid by using the water-insoluble organic solvent DC345 which is less toxic to yeast.When the yeast bioconverts 12-hydroxystearic acid of 15.00/L,the concentration ofγ-decalactone in the 72h transformation can reach 2.25g/l.In particular,the yeast conversion of 3.00 g/L of12-hydroxystearic acid in the 24 hγ-decalactone molar conversion can reach 81%,it is 3.63times higher than that without adding DC345.,In this paper,the cyclization mechanism of yeast MF-Y11 transforming gγ-decalactone was studied,which provided a theoretical basis for the industrialization ofγ-decalactone and a new method for the preparation ofγ-decalactone,making it possible to industrialize.