纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

Study of Immobilization of Yeast Alcohol Dehydrogenase Cross-linked Magnetic Chitosan Microspheres

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres,M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS呈规则的圆球形,粒径在30nm左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50mg磁性壳聚糖微球,加入20ml0.25mg/ml酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05mol/L,pH7.0),在4℃固定2h。M-CS容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。

Magnetic chitosan microspheres （M-CS） were prepared and used for yeast alcohol dehydrogenase （YADH） immobilization. The optimum technology and the properties of immobilized YADH were studied. The optimal immobilization conditions for YADH were： 20ml of 0.25mg/ml of YADH in phosphate buffer （0.05mol/ L, pH 7.0） reacted with 50mg of magnetic M-CS at 4℃ for 2h. The microspheres were characterized by transmission electron microscopy, the results showed that M-CS were regular sphere with an average size of 30nm and had magnetic response characteristic. The M-CS suspended in H2O solution was easily precipitated and separated by magnetic field. Mechanical strength and crosslinking degree of M-CS were influenced by the ratio of carrier and immobilized YADH, ion concentration in phosphate buffer and pH of the solution. The immobilization was slightly influenced by the reaction temperature. The immobilization would improve its thermal, basic resistant and acid resistant stability. After the immobilized enzyme was kept between 35 ℃ to 75 ℃ for one hour , it still had 70 % of initial enzyme activity , when it was kept pH between 5.0 to 7.4 for one hour , it still had 80% of initial enzyme activity. The optimal reaction temperature of the immobilized enzyme was 40℃ compared to 30℃ of the free YADH, the optimal reaction pH of the immobilized enzyme was 6.8 as same as one of the free enzyme. Storaged at the temperature of 4℃ for 30 days without any protection by reagent,the free enzyme only kept 26% of the original activity but the immobilized enzyme still retained 60% of the activity . The immobilized enzyme maintained 70% of the activity after circular use 5 times. The Km value of immobilized YADH for Pyruvate was 2.58 mmol/L compared to 3.31 mmol/L of the free YADH, it would reduce its appetency for the substrate.