The Key Enzymes From Apple Wine Yeasts For Esters Synthesis

Esters are the major and important determinant of apple wine flavor, enzymatically synthesizedby yeast during the fermentation process. Two pathways of the synthesis of esters by yeast : onewas synthesized from alcohols and carboxylic acids by esterase ,another was synthesized by thecondensation of acyl-Coenzyme A(CoA) and alcohols under the action of alcohol acyltransf -erase(EC 2.3.1).Study on the key enzyme which enzymatically synthesized the esters during the applewine fermentation process, instructed the fermentation of alcoholic beverages in theoretics andpractice, improving flavors of the alcoholic beverages, increasing the qualities of apple wine.A method for determination of aroma compounds for apple wine and enzyme activities wasdeveloped in the base of work formerly. The analysis was performed with headspace solid-phasemicroextraction coupled with gas chromatography-mass spectrometry. The optimum parameters ofthis method were: 75 μm Carboxen-PDMS fiber, extraction time: 40min,extraction temperature:45℃,NaCl concentration: 25%.The standard deviations for qualitative analysis of the main alcoholsand esters were from 0.011 to 0.249,the recoveries for all this anayltes ranged from 90.65% to103.50% with the regression coefficients above 0.9560.During different fermentation phases, the amount of the main alcohols and esters fermentedrespectively by three yeasts which shown good enological characterization of apple wine wereanalyzed. The amounts of ethyl acetate and phenylethyl acetate which imparted apple wine specialaroma synthesized by H. valbyensis were higher than those by M.pulcherrima and S. cerevisiae.Phenylethyl acetate was synthesized from phenylethyl alcohol and acetyl-CoA through analyzingactivities of esterase and alcohol acetyltransferase (EC 2.3.1.84, AATFase) in each yeast andanalyzing the amount of the substrates.AATFase which was the key enzyme of esters synthesized was purified from H. valbyensis .Firstly, intact cells were collected when the yeast had been cultured for 36h by centrifugation anddisrupted. The cell membrane fractions were collected by high speed centrifugation. Secondly, thecrude enzyme was solved from the cell membrane fractions of H. valbyensis by 1% Triton and thenpurified by three steps of chromatographic separations: DEAE Sepharose ,Sephadex G-75,OctylSepharose. Finally, the purified enzyme had a single band on an SDS polyacrylamide gel, and itsmolecular mass was estimated to be about 37KD. Specific activities of purified enzyme was 195times than crude enzyme and yield of activity was about 12%.The optimal temperature and pH for the purified AATase activity were 30℃ and pH7.0,respectively. It was stable from pH7.0 to pH8.0,but was unstable at temperatures above 10℃ . Theactivity was strongly inhibited by heavy metal ions such as Hg²⁺,Zn²⁺,Pb²⁺, slightly stimulated byMg²⁺ and slightly inhibited by EDTA,Mn²⁺. The enzyme catalyzes the esterification of otheralcohols by acetyl-COA to acetate ester, but had specificity to phenethyl acetate.