转氨酶的固定化及酶学性质研究

Immobilization of transaminase and characterization of its enzymatic properties

利用环氧树脂载体进行固定化转氨酶的研究,通过单因素优化确定了较优的酶固定化条件:ES-103b树脂载体、转氨酶和辅酶磷酸吡哆醛最佳质量比为50∶6∶5,在p H为7.0的1mol/L磷酸钾缓冲液介质中吸附25 h。将得到的固定化颗粒重新置于pH为9.0的100 mmol/L磷酸钾缓冲液中,30℃保温30 h。取出后置于pH为8.5的3 mol/L甘氨酸-NaOH缓冲液中,25℃保温12 h,制得固定化转氨酶,其比活力为52.7 U/g(湿载体),酶活回收率达到49.3%。酶学性质研究表明:固定化转氨酶催化反应最适pH、温度分别为7.0、50℃;固定化酶热稳定性及p H稳定性明显提高,50℃条件下的半衰期为20.2 d;在pH 8.0的三乙醇胺缓冲液中(4℃),10 d后酶活仍保持初始酶活的82.3%。

This study foc used on the transaminase immobilization using epoxy-activated resin as support. Optimal conditions for immobilization were as follows： Resin ES-103 b, lyophilized enzyme and PLP were added into 1 mol/L potassium phosphate solution（pH 7.0） at the ratio of 50∶6∶5 and incubated at room temperature for 25 h. The obtained immobilized enzyme was placed into 100mmol/L potassium phosphate buffer（p H 9.0） for 30 h at 30 ℃, followed by its incubation in 3 mol/L Glycine-NaOH buffer（pH 8.5） at 25 ℃ for 12 h. The activity of immobilized enzyme was determined as 52.7 U/g（wet support） with activity recovery of 49.3%. The enzymatic characteristics of the immobilized transaminase were investigated. The optimal reaction temperature and pH were 50℃ and 8.0, respectively. The immobilized transaminase possessed an improved p H and thermal stability. The half-time of the immobilized enzyme at 50 ℃ was 20.2 d. The immobilized transaminase remained 82.3% of the original activity after 10 d of storage in TEA buffer（pH 8.0） at 4 ℃.