表面脱乙酰化甲壳素颗粒固定溶菌酶及酶学性质研究

Study on Immobilized Lysozyme and Enzymatic Properties of Surface-deacetylated Chitin Granules

本研究以表面脱乙酰化甲壳素颗粒为载体,以戊二醛为交联剂,对溶菌酶进行固定化。本文利用正交试验设计,以蛋白利用率、活力回收率和保藏稳定性为指标,以半脱乙酰壳聚糖为底物,3-甲基-2-苯丙噻唑啉酮腙(MBTH)法为酶活力测定方法,优化了关键固定化参数,并考察了固定化对溶菌酶酶学性质的影响。结果表明:固定化过程中最佳脱乙酰时间(A)为25 min,戊二醛浓度(B)为5%,载体活化pH(C)为5.0,在此条件下固定化的溶菌酶与游离溶菌酶相比,其表观最适pH由4.0降至3.5;最适温度由75℃升高至80℃,且在70～90℃仍保持95%活力; Km为2.69 mg/mL,明显大于游离酶的1.75 mg/mL; 15 d稳定性保持66%。因此,固定化提高了溶菌酶的热稳定性和贮藏稳定性,具有广泛的应用前景。

In this study,lysozyme was immobilized by using surface-deacetylated chitin granules as a carrier and glutaraldehyde as a crosslinking agent.Orthogonal array design was used to optimize the key parameters of the immobilization,according to the comprehensive evaluation of protein utilization ratio,activity recovery and storage stability,on the basis of MBTH activity assay using half-deacetylated chitosan as a substrate. The effect of immobilization on the enzymatic properties of lysozyme was investigated. The results showed that the optimum immobilization conditions were as follows: deacetylation time 25 min,concentration of glutaraldehyde 5% and pH5.0 for the carrier activation. Compared with dissociative lysozyme,immobilized lysozyme in this condition reduced its apparent optimum pH from 4.0 to 3.5.The optimum temperature increased from 75 to 80 ℃ and maintained 95% or more of activity within 70~90 ℃.The kinetic parameters,Kmwas estimated to be 1.75 mg/mL for free and 2.69 mg/mL for immobilized lysozyme. The activity after storage for 15 d could still be maintained 66%. Therefore,immobilization increased the thermal stability and storage stability of lysozyme,which might confer it a wider range of applications.