| Objective To establish the preparation method of the immobilized xylanase, enhance the stability and repeated utilization of Xylanase YS1069; establishing the preparation method of xylooligosaccharides by immobilized xylanase; evaluating the effect of xylooligosaccharides; providing bases for applications. Methods(1) In this study, the cross-linked enzyme aggregates(CLEAs) technology was combined with carrier immobilization technique. Screening proper resin for xylanase immobilization. Immobilized conditions of Xylanase were studied by using single factor experiment and the response surface design.(2) Studied the properties of immobilized xylanase CLEAs and free xylanase.(3) Useing immobilized xylanase to prepare xylooligosaccharides. Preparation conditions of xylooligosaccharides were studied by single factor experiment and the response surface design. Analyzing the content of the xylooligosaccharides by HPLC.(4) Copy oxidative damage model of human umbilical vein endothelial cells(HUVECs) with oxygenized low density lipoprotein(ox-LDL), to study antioxidant activity of xylooligosaccharides. Experiments were divided into fellow groups: normal group、model group made by ox-LDL、 the different concentration of xylooligosaccharides(5、10、20mg/m L). Detecting the activity of glutathione peroxidase(GSH-PX), superoxide dismutase(SOD) by the enzymatic chemical methods. Results(1) The study indicated that the best immobilized conditions were: amino resin LKZ-218 as carrier, p H 9.0 enzyme liquid precipitated by 2 m L isopropyl, precipitation 1h, cross-linked 2 h with the carrier LKZ-218 by 1.59 g/L genipin and immobilized 9.25 h in the environment of 20℃. The recovery rate was up to 65.63% and activity of enzyme was up to 190U/g.(2) By studying its enzymology properties we conclude that: the optimal temperature of immobilized xylanase CLEAs higher than free xylanase enzyme 10℃, up to 65℃; the optimum p H has not changed; comparing with the free enzyme, it has better p H stability and thermal stability. After repeated 10 times, the relative activity of the immobilized xylanase CLEAs was still 42.3%; the relative activity of free xylanase enzyme was 12% and the immobilized xylanase CLEAs was still 47% after storaged 12 weeks; immobilized xylanase CLEAs activity loss less than the free enzyme after disposed for some time in the organic reagents, surfactants, and metal ion solution, its stability increased significantly; studied the kinetics of the enzymic reaction conclude that the Km and Vmax of immobilized xylanase CLEAs were 9.6 g/L and 4.0mmol/L/min, the Km and Vmax of free enzyme were 9.1 g/L and 6.2 mmol/L/min.(3) The best conditions of substrate concentration、time、temperature、p H、enzyme amount for the preparation of xylooligosaccharide were: 52.5 g/L、3 h、57 ℃、p H 9、0.2 g/g; the HPLC results showed that the main components of enzymolysis products were xylose、xylibiose、xylotriose, most of which is xylibiose.(4) The enzymatic chemical analysis showed that: GSH-PX and SOD in model group was significantly lower than the normal group, but when we gived different concentrations of xylooligosaccharide, the GSH-PX and SOD activity increased with the increase of the xylooligosaccharide,s dose(compared with the model group,P<0.05). Conclusion The recovery rate and activity of the immobilized xylanase CLEAs were 65.63% and 190 U/g, which was preparation by crosslinked enzyme aggregates(CLEAs) technology combined with carrier immobilized technology.The stability increased significantly after the xylanase immobilized. The main components of xylooligosaccharide is xylibiose, which is preparated by the immobilized xylanase CLEAs, and have strong antioxidant activity. |
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