N-乙酰神经氨酸醛缩酶的固定化及固定化酶性质研究

使用LX-1000HFA氨基树脂对N-乙酰神经氨酸醛缩酶(NAL)进行固定化,并对游离酶与固定化酶的酶学性质及稳定性进行了对比研究。结果显示,最佳固定化条件为载体投放量5.0 g,固定化时间12 h,缓冲液浓度1.0 mol/L,pH7.5,温度25℃。在此条件下制备的固定化NAL活力最高,比酶活可达200 U/g湿载体。与游离酶相比,最适反应温度提高了5℃,最适反应pH没有变化,温度和pH耐受性明显提升。同时固定化酶储存稳定性和操作稳定性也显著增强,在4℃条件下储存10 d后其酶活仅损失6%,重复使用10次后仍保持初始酶活的80%。因此,该固定化酶具有良好的温度稳定性、pH稳定性、储存稳定性和操作稳定性,为酶法工业化生产N-乙酰神经氨酸研究提供了理论依据。

Immobilization of N-acetylneuraminic Acid Aldolaseand Properties of the Immobilized Enzyme

N-acetylneuraminic acid aldolase(NAL)was immobilized using LX-1000HFA amino resin.The immobilized conditions were optimized,and the enzymatic properties and stabilitiesbetweenfree and immobilized enzymes were compared.The optimized conditions of immobilization were asthat the quantity of carrier was 5.0 g,the immobilization time was 12 h,the buffer concentration was 1.0 mol/L,the pH was 7.5,and the temperature was 25℃.The prepared immobilized NAL had the highest viability and the enzyme activity,reached 200 U/g wet resin.Compared with the free enzyme,the optimal reaction temperature increased by 5℃,the optimal reaction pH remainedunchanged,and thermal-and pH-tolerancewere significantly improved.Meanwhile,the storage stability and operational stability of the immobilized enzyme were also greatly enhanced.Only 6%of the enzyme activity was lost after storage for 10 d at 4℃,and the immobilized enzyme retained 80%itsactivity after repeated usage for 10 times.Therefore,the immobilized enzyme hasfine thermal stability,pH stability,storage stability,and operational stability,which provides a theoretical basis for the industrial production of N-acetylneuraminic acid via enzymatic method.