Research On Synthesis Of Ethyl Lactic With Nonaqueous Enzyme-catalysis And The Effect Of Ultrasound On The Reaction System

Ethyl lactate is a kind of edible flavors on the approved list of GB 2760-86 standards of China, has the characteristics of no corrosive, no pollution, non-volatile, easily recyclable, meeting the sanitary standards for food additives. In recent years, as the gradual increase of demand of ethyl lactate in market and more requirements for the environment protection, the traditional production process of ethyl lactate can not meet industry needs any longer.This paper based on the technologic condition of non-aqueous enzyme catalytic synthesis of ethyl lactate, then study the reaction efficiency of non-aqueous enzyme catalytic synthesis of ethyl lactate, and the effection of surface microstructure or characteristic of Novozym435, which is effected by the ultrasonic wave. the main results listed as followed:Firstly, tertiary butyl alcohol and Novozym435 were adopted as the solvent and lipase in the process of the synthesis of ethyl lactate respectively. The optimal experimental conditions were obtained by single factor experiments, the temperature was 50℃, the mole ratio of acid to alcohol was 1:8, lactic acid concentration was 0.3mol / L, enzyme concentration was 35g/mol and the rotational speed was 200r/min.Secondly, on the basis of single factor experiments, response surface methodology analysis was carried out to determine the optimum reaction parameters for the synthesis of ethyl lactate, which listed as followed: lactic acid concentration was 0.42mol/L, the mole ratio of acid to alcohol was 1:6, enzyme concentration was 42g/mol, the temperature is 50℃and the rotational speed was 200r/min. Under this condition, the yield of ethyl lactate was 70.21%±0.2%, and could still reach above 60% after the enzyme being reused 6 times.Thirdly, under the optimal conditions of the synthesis of ethyl lactate, it was investigated the effects of three different ultrasonic modes (Normal, Sweep, and Degas) on the non-aqueous enzymatic reaction. The optimal conditions were obtained: ultrasonic mode was Sweep, frequency was 130 kHz, ultrasonic intensity was 60%. The effects of ultrasonic on the Secondary protein structure and microstructure of immobilized Novozym435 were also studied.Fourthly, the effects of ultrasonic on the non-aqueous enzymatic reaction for the synthesis of ethyl lactate were investigated. It was obtained that the maximum reaction rate under ultrasonic was 18.2μmol / (min*g), the Michaelis constant of lactic acid and ethanol, namely KA and KB were 0.0116mol / L, 0.0329mol / L respectively. Vmax increased with the aid of ultrasound, which agreed with the fact that ultrasound improved the reaction rate significantly. As the KA and KB were both decreased, indicating the affinity between the reaction substrate and the lipase were strengthened, consistent with the fact that ultrasound improved the yield of ethyl lactateFifth, compared with the immobilized Novozym435 without ultrasound treatment, Ultrasound treatment increased the enzyme activity. There is no significant change in the enzyme stability, the nature of the immobilized enzyme surface structure (SEM test) and the enzyme protein secondary structure (FT-IR test).