Ethyl acetate, one of the esters, has pleasant, fruity and flower-like aroma, which is produced by several microorganisms. H. anomala has been known to produce ethyl acetate most abundantly under aerobic condition. For an exact quantitative analysis of ethyl acetate, direct injection of culture broth or esterase (in whole cells) reaction mixture into gas chromatography was employed. Ethyl acetate is very volatile and its volatility to ethanol is about 20 times greater. Therefore, evaporated ethyl acetate during fermentation was recovered by trapping system.
Ethanol and ethyl acetate were identified as main volatile components and the rest were acetic acid, n-propyl acetate and isobutyl acetate. Ethyl acetate formation during cultivation was increased with a rapid cell growth and decreased with a shortage of glucose and ethanol in a culture broth.
Esterase activity in whole cells was highest in 21 hr aged cells with a broad range of optimum, which were in an early stationary phase. Esterase in whole cells was most active at pH 7.0, 30℃ and its activation energy was 25.9 Kcal/mole in the range of 10℃-30℃. Esterase in whole cells hydrolyzed ethyl acetate gradually and slightly inhibited by 10 mM - 100 mM or above ethyl acetate. Ethyl acetate was also produced from ethanol and acetic acid anaerobically by reverse reaction of esterase in whole cells.