中国生物工程杂志

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 2.58mmol/L compared to 3.31mmol/L of the free YADH, it would reduce its appetency for the substrate.