Hydrolysis Reaction Of Soybean Oil Catalyzed By Lipase In Ultrasonic Irradiation

Current mainstream hydrolysis technology of oils and fats needs high temperature/high operation pressure and conventional hydrolysis technology needs catalyzer, which would produce serious by-reactions and pollute entironment. Ultrasonic technology, the same as enzymatic technology, is a technology with the characters of investment-saving, warmer condition, lower energy cost, less by-reactions, and safety to environment. This thesis combined the potential advantages of the two technologies to investigate oils and fats hydrolysis reaction catalyzed by ultrasound-enzymatic technology.Solvent-free system is provide with the advantages of safety, low-cost, high volume concentration of substrates (or products) and easy to separate, which was selected to investigate hydrolysis of soybean oil catalyzed by C. lipolytica lipase in shaking bath. The reaction characters were obtained: optimum reaction temperature around 45℃, C. lipolytica is an alkali-lipase with wave-like changing of activity from pH 6.1-8.3, obvious enzyme concentration inhibition occurs when C. lipolytica concentration exceeds 1%(w/w), when water/oil ratio exceeds 0.675:1, raising of reaction rate tends to balance. Respond surface methodology (RSM) was used on the base of C. lipolytica reaction characters. A mathematics model was established to reflect influence of reaction conditions on the reaction course. Analysis of variance (ANOVA) and canonical analysis of experimental data showed the results that temperature and enzyme concentration are significant at 99% confidence level on the hydrolysis reaction; pH and water/oil ratio are non-significant on the reaction in the experimental range. The optimum reaction conditions are: temperature 42.2℃, pH 7.51, enzyme concentration 0.85(w/w) and water/oil ratio 1.24(w/w).Power of ultrasound is an important influence on the reaction catalyzed by enzyme. Enzyme inactivation did not present in the selected ultrasonic power range. Experimental results showed that reaction rate increases with the increasing of ultrasonic power, when ultrasonic intensity exceeds 1.20W/cm2, increasing of reaction rate gradually slows. But stability of reaction is improved by increasing ultrasonic intensity, which indicates that the fluctuation of power output would induce a remarkable variation in reaction rate in lower ultrasonic intensity. Selected 1.64W/cm2 of ultrasonic intensity for the following experiments.Effects of ultrasound on soybean oil hydrolysis reaction catalyzed by C. lipolytica lipase were investigated. Compared with that in shaking bath, heat-durability is enhanced about 5℃in ultrasonic bath; enzyme concentration inhibition does not present, which probably could attribute to agglomeration of enzyme on the oil-water interface is avoided or reduced by ultrasound; water is more"efficient"in ultrasonic reaction, in another word, the critical point is lower at which the kinetics could be considered as a pseudo-mono-substrate reaction; ultrasound causes little change to the ionization state of C. lipolytica, as similar degree-pH pattern were presented in shaking and ultrasonic conditions. RSM was also utilized in ultrasound bath and a mathematics model was established to reflect influence of reaction conditions on the reaction course. The optimum reaction conditions in ultrasonic bath are: temperature 47.0℃, pH 7.77, enzyme concentration 0.77(w/w) and water/oil ratio 0.71(w/w).Multiple-enzyme reaction is a technology of cooperating bi-enzyme or multi-enzyme to enhance the catalytic capability of enzymes. Selected five different source lipases of Sigma-3126, Bio-Eng PPL, CLL, Novo435 and Amano-G to investigate the complex effect. Combining CLL and Amano-G could give a better hydrolysis rate and yield. The optimum reaction condition was obtained: the concentration of CLL and Amano-G are 1%(w/w) and 0.1%(w/w), respectively; adding Amano-G after C. lipolytica acts 1h; temperature is 45℃; water-phase is de-ionized water, and water/oil ratio is 1:1(w/w). Under above condition, hydrolysis reaction could be accelerated by ultrasound, especially in the initial reaction stage. After 24h, reaction yield could be enhanced from 45.7% in conventional shaking bath to 94.2% catalyzed by ultrasonic-bi-enzyme.Kinetics of enzymatic reaction is an important approach to mechanism. Kinetics model can present the infection of influence factors on the reaction accurately. At present, in most kinetics models, the interfacial area of water-oil interface is hypothesized to be a constant, which is not suitable for solvent-free system. In the thesis, we established a new kinetics model in solvent-free system based on a model of active-substrate. And the model combined interfacial area of water-oil interface and active substrate.The applicability had been proved suitable in the range of volume fraction of oilΦ=0.1-0.9 by methylene blue dyeing-microscope technology, and the hypothesis had been also proved correct in the range ofΦ=0.1-0.9 by TLC. The kinetics model was used in hydrolysis reaction of soybean oil catalyzed by C. lipolytica lipase. Based on initial reaction rate and the principle of Sauter diameter changed with water/oil ratioΦ, parameters of the kinetics model were calculated using non-line regression fitting methodology in both shaking bath and ultrasonic bath. The influence of parameters Ke,k*cat and kd/kp on reaction rate were discussed, and explained the actual physical meaning of parameters.Substrate inhibition phenomenon was explained using the active-substrate model. It indicated that substrate inhibition phenomenon occurred in hydrolysis reaction is an"apparent"substrate inhibition. Reaction rate is controlled by active-substrate concentration. Ultrasound could reduce substrate inhibition and accelerate reaction rate, which essential reason is the effect of ultrasound on the active-substrate.Study on the thermodynamics can avoid the limitation of system structure and process mechanism, which is favored on the macroscopy characters of reaction. Based on the derivation of thermodynamics formula, activation energy Ea of soybean oil hydrolysis reaction catalyzed by C. lipolytica was determined, Ea in 200rpm shaking bath is 10.33 kJ/mol and in 240V ultrasonic bath is 10.24 kJ/mol, respectively. Additional, pre-exponential factor A was calculated, in 200rpm shaking bath is 2.85 and in 240V ultrasonic bath is 3.35, respectively. Parameters of thermodynamicsΔ~≠H,Δ~≠G, andΔ~≠S at 45℃, were also calculated and discussed in shaking bath and in ultrasonic bath, respectively.Based on the conclusions of others and our experimental results, the enzymatic hydrolysis reaction mechanism of oils and fats irradiated by ultrasound was discussed. We proposed a"localized O/W emulsion"function for the enzymatic hydrolysis reaction of oils and fats accelerated by ultrasound.