新型耐热耐酸普鲁兰酶的分离纯化与酶学特性分析

Purification and Enzymatic Characterization of a Novel Acid-resistant and Thermostable Pullulanase

从泡盛曲霉(Aspergillus awamori) XF发酵液中分离纯化普鲁兰酶(PulXF)并研究其酶学性质。通过硫酸铵沉淀、阴离子交换层析、凝胶过滤层析和疏水层析,纯化得到一种电泳纯的PulXF。纯化倍数8.16倍,比活力为309.28 U/mg。SDS-PAGE检测得单条带,表明PulXF为单亚基蛋白,分子量63.7 kDa。在50-80℃,pH4.5~8.0范围内,酶能保持高活性,最适反应温度60℃,最适pH5.0。在较广的pH范围内(pH3.0~8.0),28℃下,经过24 h酶活能保持80%以上活性。K_m值和V_(max)分别为0.92 mg/mL和11.90μmol/min。酶活测定及TLC分析显示PulXF对普鲁兰多糖有强水解活性,终产物麦芽三糖;对支链淀粉和可溶性淀粉有较弱活性,对直链淀粉、α-环糊精、β-环糊精和糖原无活性。表明从泡盛曲霉XF分离的PulXF属于Ⅰ型普鲁兰酶。Ca^(2+)、Zn^(2+)和Mg^(2+)能够促进酶的活性,其中Ca^(2+)激活作用最强。而PulXF在5%浓度的SDS、CTAB、Tween 80和30%的乙醇中保持高稳定性。基于PulXF在苛刻环境下的高稳定性及高活性,很有希望在淀粉加工、食品饮料等领域以及需要在高温、高酸环境下使用的生物技术工业中使用。

This study reports on the purification and characterization of the pullulanase from a newly isolated Aspergillus awamori XF( PulXF). The enzyme was purified to apparent electrophoretic homogeneity through ammonium sulphate precipitation,anion exchange chromatography,gel chromatography and hydrophobicity chromatography. The crude enzyme was purified 8.16 times with 309.28 U/mg specific activity. SDS-PAGE revealed that the enzyme is monomeric,with a molecular weight of 63.7 kDa.PulXF was highly active in the temperature range of 50 80 ℃,with an optimum activity at 60 ℃. It was highly active in the pH range of 4.58.0,with optimum pH at 5.0,and retained more than 80% of its original activity in a broad pH range of 38 for 24 h at 28 ℃.The enzyme displayed Kmand Vmaxvalues,for pullulan of 0.92 mg/m L and 11.90 μmol/min,respectively,demonstrate its greater affinity.PulXF hydrolyzed pullulan,producing maltotriose,and low hydrolytic activities were also detected with amylopectin and soluable starch,but almost not with amylose,α-cyclodextrine,β-cyclodextrine and glycogen.This proved that the purified PulXF from Aspergillus awamori XF was classified under pullulanase type I.The activity of PulXF could be promoted by Ca2+,Zn2+and Mg2+. Among them,Ca2+had the most significant effect. The enzyme had a tolerance for surfactants,which was relatively stable in the presence of 5% SDS,5% CTAB,5% Tween 80 and 30% ethanol.The excellent physical and chemical properties make PulXF suitable in the fields of starch processing,food and beverage and biotechnological industries that require high activity at high temperature and acid conditions.