Investigation On The Spectrum Characteristics Of Enzymes In Microwave Irradiation Assisted Enzymatic Catalysis

Microwave radiation can be used to dispel protein, and also can be used to accelerate enzymatic catalysis reactions. The proper microwave irradiation doesn't damage the primary structure of protein, while excessive microwave radiation would dispel, or deactive protein. Applying microwave irradiation-enzyme coupling catalysis (MIECC) can result in many unique findings, which were quite different from those observed from conventional heated enzymatic reactions. In this paper, the enzymatic esterification of caprylic acid and butanol, caprylic acid and glycerol were studied. The fluorescence change of LRI and Novozym 435 were recorded to investigate the formation change of the enzyme protein during the reactions. In addition,the effect of microwave irradiation time on the activity of the lipase and proteinase, along with their UV and fluorescence spectrum change were investigated. The main contents and results are as follows:1. We studied the fluorescence and ultraviolet spectrum change of immobilized lipase from Mucor miehei in the microwave assisted enzymatic esterification of caprylic acid and butanol in organic medium, by investigating the fluorescence spectra in solvent or aqueous buffer after incubated the lipase with the solvent, caprylic acid and butanol under microwave irradiation, respectively. The comparison was made with the conventional heated enzymatic esterification in the solvents.It was found that both of the heating modes, the microwave irradiation and conventional heating, can enhance the fluorescence intensity without shifting of the emission wavelength of the lipase. At the circumstance that the irradiation can accelerate the esterification, the irradiation enhanced the exposure of the lipase protein molecules in the aqueous environment after incubating the lipase with solvents or the substrates. The effect of the reaction mixture on the fluorescence intensity was driven by the solvents. The trend of the plot of log P versus the initial reaction rate was similar with that of the log P versus the fluorescence intensity of lipase in aqueous buffer after the esterification; but different with that of the log P versus the fluorescence intensity of lipase in organic medium.2. We studied the fluorescence and ultraviolet spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol, by investigating the fluorescence spectra in aqueous buffer after incubated the lipase with the caprylic acid and glycerol under microwave irradiation, respectively.The comparison between the microwave irradiated and conventional heated reactions was made. The microwave irradiation can enhance the fluorescence intensity more without shifting of the emission wavelength of the lipase protein. The trend of the plot of temperature, or the ratio caprylic acid to glycerol, or the water dosage versus the initial reaction rate, all of them were similar with that of the temperature, or the ratio caprylic acid to glycerol, or water dosage versus the fluorescence intensity of lipase in aqueous buffer after the esterification, respectively. Additionally, the fluorescence spectrum change of Novozym 435 in the microwave assisted enzymatic esterification of caprylic acid and glycerol under different reaction conditions was studied. The fluorescence spectra in aqueous buffer were recorded after incubating the lipase with the caprylic acid or glycerol or the reaction mixture at the 40% caprylic acid conversion. The trend of the plot of reaction parameters versus the ratio 1- monoglyceride to 2- monoglyceride and the ratio 1,3-diglyceride to 1,2-diglyceride was similar with that of different reaction conditon versus the fluorescence intensity of lipase in aqueous buffer after the esterification,respectively.3. The activity change of lipase(Lipex 100 T,Lipolase 100 T) and proteinase(Properase 1000 E,Purafect 2000 E) induced by microwave irradiation were investigated. We also studied the fluorescence or ultraviolet spectrum with different irradiation time. The activity of lipase and proteinase increased firstly and then decreased with rise microwave irradiation. The fluorescence and ultraviolet intensity enhanced firstly, and reduced with the rise irradiation time later without shifting of the emission wavelength of the lipase and proteinase. The trend of the plot of irradiation time versus the fluorescence or ultraviolet intensity of the lipase after storaged the lipase for two months was coincident with that of irradiation time versus the fluorescence or ultraviolet intensity of lipase irradiated after 30s. But the fluorescence or ultraviolet intensity of lipase after storged two months stronger than that of lipase irradiated after 30s.