构巢曲霉产植酸酶的酶学特性分析

从构巢曲霉AnP-16菌株发酵液中纯化单一组分的耐热耐酸植酸酶,并对酶学特性进行分析,为其在食品工业中的应用提供理论依据。通过硫酸铵盐析、离子交换层析和疏水层析纯化植酸酶,SDS-PAGE电泳测定其分子量。结果表明,从该菌株发酵液中纯化到了单一组分的植酸酶,纯化倍数60.8倍、回收率41.6%,酶分子量约52 kDa。植酸酶最适作用温度和pH分别为55 ℃和pH4.0,在pH3.0~6.0范围酶活性较高,pH2.0~7.0下孵育3 h,仍能保持80%以上活性。植酸酶耐热性好,70 ℃孵育1 h仍能保持81%活性。Hg2+、Mn2+、Fe2+、Cu2+和Zn2+ 5 mmol/L浓度下对酶活性有明显抑制作用,但Ca2+和Mg2+在1和5 mmol/L浓度时均增强酶活性;有机溶剂甲醇和乙醇在2%浓度有激活作用;SDS抑制酶活性,但其它表面活性剂(Triton X-100和Tween 80)和有机溶剂(丙酮和异戊醇)对酶活性无明显影响。植酸酶有宽泛的底物特异性,但对植酸钠的催化活性最强,其K_m值为0.576 mmol/L。基于植酸酶耐酸耐热及宽广的底物催化活性,其有望应用于粮油食品加工领域,提高食品的营养价值。

Enzymatic Characterization of a Novel Acid and Thermostable Phytase from Aspergillus nidulans

To purify a single heat and acid-resistant phytase from Aspergillus nidulans AnP-16 and to characterize the enzyme in order to provide theoretical basis for its application in food industry. Phytase was purified by ammonium sulfate fractional precipitation,ion exchange chromatography and hydrophobic chromatography,and its molecular weight was determined by sds-page electrophoresis. Results showed that,this phytase was purified to homogeneity with 60.8 fold purification and 41.6% recovery yield. The molecular mass of the enzyme was estimated as about 52 kDa. The purified phytase had an optimal activity at 55 ℃ and pH4.0,and it exhibited high enzymatic activity between pH3.0 and 6.0. The enzyme retained more than 80% of its initial activity after being incubated under varying pH conditions(pH2.0~7.0)for 3 h. The purified phytase was resistant to heat inactivation,as it retained 81% of its initial activity after incubation at 70 ℃ for 1 h. The inhibition of phytase activity by metal ions was observed to be drastically inhibited by Hg2+,Mn2+,Fe2+,Cu2+ and Zn2+ at 5 mmol/L concentrations. A positive effect was obtained with Ca2+ and Mg2+ at 1 and 5 mmol/L,methanol and ethanol at 2% concentration. The acidic phytase activity was also inhibited by SDS while the other detergents(Triton X-100 and Tween 80)and solvents(acetone and isoamylol)had no obvious effect on its activity.The phytase displayed broad substrate specificity while the enzyme was more specific for sodium phytate and the K_m value for phytate was 0.576 mmol/L. Based on phytase’s resistance to acid and heat and broad substrate catalytic activity,it is expected to be applied in the field of grain,oil and food processing to improve the nutritional value of food.