Effects Of Microwave Radiation On Glucose Isomerase

Glucose isomerase (D-glucose isomerase, EC5.3.1.5, GI) is a a key enzyme for large-scale industrial preparation of high fructose corn syrup from starch (high fructose corn syrup, HFCS). In addition, this enzyme is also able to convert xylan into xylulose, and then produce ethanol by microbial fermentation. This biotransformation techno logy as a sustainable energy has good prospects sooner or later. Microwave has become a hot research aspects, because it is a new physical processing methods, with no chemical residue and safe. Microwave inactivation technique not only can effectively inactivate the enzyme in food, but also has a positive impact on food texture, color, flavor, nutritional value, microbial stability and rehydration under a certain conditions.In order to find a convenient regeneration method for glucose isomerase, the research of microwave treatment on changes properties and structure of glucose isomerase is of great significance. Not providing experimental data and reference value, but microwave irradiation assisted can also be applied to glucose isomerase catalytic. This paper is to study the effects of microwave radiation treatment on properties and structure of glucose isomerase, and microwave treatment on immobilized glucose isomerase catalytic processes. The main concludes are as follows:(1) In a fixed treatment temperature of70℃and5min, when microwave power was300W,400W, the relative activity of glucose isomerase were increased by14.23%,8.42%; And when the microwave power was600W,800W, the relative activity of glucose isomerase reduced to95.69%and95.69%. When microwave processing temperature was greater than80℃, the relative activity of glucose isomerase decreased significantly(P＜0.05). After microwave treatment of300W, glucose isomerase activity within the range of65～90℃were over85%; microwave treatment significantly decreased the stability of the glucose isomerase under high temperature and low temperature (P＜0.05), but there was no significant change in the stability of the optimal temperature (75℃)(P＞0.05). Different conditions of microwave treatment had a different effect on Km and Vmax of glucose isomerase. After treatment of300,400W, Km has shrunk, indicating that the affinity of GI for substrate became larger; after600,800W microwave treatment, Km increased.(2) After SDS-PAGE, it showed that microwave radiation had no effect on glucose isomerase primary structure. The UV spectra showed that the different microwave power and time led to the amino acid residues and microenvironment changes of the glucose isomerase, and some chromophore group exposed. After high power (800W) and long time (10,15,20min) microwave treatment, the UV absorption reduced. The conformation changes of the enzyme might be due to the molecule unfolding. Fluorescence spectrum showed the tertiary structure location of Tyr and Trp residue changed, and the micro environmental of Tyr and Trp residue changed; More Trp residues exposed to the molecular surface, a more polar environment. The changes of glucose isomerase protein molecules after microwave treatment might make the Trp residue more close to the Tyr residues. With the energy transferred, the conformational change of tryptophan residues or more away from the disulfide bond fluorescence quenching would cause the increasing of fluorescence intensity. Raman spectroscopy showed that α-helical secondary structure lost its stability; orderly structure gradually disappeared, and disordered structures continued to show. The changes of active site and substrate binding amino acid residues associated microenvironment reflected the changes of microwave radiation on the regional structure of glucose isomerase activity which affected enzyme substrate binding, resulting in changes of glucose isomerase activity.(3) Under water bath heating (70℃), the activity of immobilized enzyme was125.41U/g; And under microwave radiation heating (400W,70℃), the activity of immobilized enzyme was156.13U/g,24.50%higher than the water bath condition(P＜0.05). Under water bath heating conditions, immobilized glucose isomerase optimum reaction temperature was80℃; optimum pH was7.0; optimal concentration of metal ions was Mg2+0.05mol/L, Co2+0.001mol/L; optimum stirring speed was160r/min; the buffer concentration was200mmol/L. Under microwave radiation heating condition, immobilized glucose isomerase optimum reaction temperature was75℃; optimum pH was7.0; optimal concentration of metal ions was Mg2+0.08mol/L, Co2+0.001mol/L; the optimum stirring speed was160r/min, the buffer concentratton was200mmol/L. Under water bath heating, the Km and Vmax of immobilized enzyme was1.02mol/L and8.2×10-4mol/(L·min); and under the condition of microwave radiation heating, the the Km and Vmax was0.98mol/L and9.8 ×10-4mol/(L·min), respectively. The stability performance was stronger under the condition of water bath heating. Under microwave radiation heating, relative activity was only30.38%after15times; but slightly less than under the condition of water bath heating. When temperature was60℃,70℃, under microwave radiation heating residual enzyme activity were higher than the former, larger than90%; high temperature microwave radiation heating had a significant effect on the stability of immobilized glucose isomerase(P＜0.05).