纳米SiO_2改性壳聚糖载体的制备及固定化壳聚糖酶的研究

Study on Nano-SiO_2 modified chitosan carrier and immobilization of chitosanase

利用壳聚糖与硅酸乙酯(TEOS)形成的溶胶-凝胶制备杂化载体,经戊二醛交联后固定化壳聚糖酶,结合扫描电镜观察、X射线衍射及红外光谱分析对杂化载体进行了表征,初步比较了固定化酶与游离酶的酶学特性。结果表明,以5%的戊二醛浓度交联载体时,固定化酶的活性最高;最大结合酶量为2mL(U=2.5);固定化酶的最适pH7.0,酶活曲线向碱性偏移;固定化酶的最适酶促反应温度比游离酶升高5℃,在更宽的温度范围内具有活性。通过Lineweaver-Burk作图得出固定化酶的K'm为10.64g/L,较游离酶1.29g/L有所升高。该固定化酶具有较强的热稳定性,重复使用10次后,酶活力损失小于30%;在4℃低温条件下,稳定性较好,半衰期约为28d。

Chitosanase was immobilized on hybrid carrier by cross-linking reaction with glutaraldehyde.The carrier was formed by the sol-gel of TEOS which doped with chitosan. Characterization of carrier was verified with Scanning Electron Microscopy （SEM）, Infrared spectrum and X-ray diffraction while characteristics of the immobilized chitosanase and free chitosanase were compared. Results showed that the optimal conditions for immobilization were as follows.5% solution of glutaraldehyde and 2m （U = 2..5）chitosanase immobilized on the carrier possessed the highest chitosanolytic activity.Optimal pH for the immobilized enzyme was 7.0 so that the enzyme activity curve of the immobilized chitosanase moved to the alkaline relative to free chitosanase.After the chitosanase immobilized,optimal temperature was increased by 5℃.There was wider temperature in the scope of enzyme activity.Through the Lineweaver-Burk mapping,the Km which was 10.64g/L for immobilized enzyme was rised significantly than the Km which was 1.29g/L for free enzyme.The immobilized enzyme showed better operation and storage stability than the free enzyme.Reused 10times,the loss of enzyme activity was tess than 30%.At 4℃, the stability of immobilized enzyme was good,and the half-life was approximately 28d.