聚半乳糖醛酸酶是水解D-半乳糖醛酸α-1,4-糖苷键的酶,在食品工业特别是果蔬加工中具有重要意义。该文研究棘孢木霉(14636)所产聚半乳糖醛酸酶的酶学特性,采用硫酸铵沉淀法、透析袋透析,对粗酶液进行纯化,并以聚合烯酰胺凝胶电泳(sodium dodecyl sulfate polyamide gel electrophoresis,SDS-PAGE)确定其分子质量大小。实验结果表明,该酶的活性区域在29.29~50.46 kDa,聚半乳糖醛酸酶的最适反应pH为4.0,在pH 3.0~5.0稳定性较好,最适温度为40 ℃,具有一定的热稳定性;在乙酸乙酸钠缓冲溶液中活性较高。催化性能表明,果胶是聚半乳糖醛酸酶的最佳底物,Km值为0.74 mg/mL,Vmax为3 100 μg/min; Mn2+、Mg2+、Cu2+、Ca2+和Tritonx-100对酶有激活作用,Ca2+和Tritonx-100激活作用较强,Co2+、Zn2+、Ba2+、Li+、Fe2+、SDS和Tween-80对该酶有不同的抑制作用;贮藏特性研究表明,该酶即使在30 ℃下30 d仍保留70%以上酶活性。研究结果为果蔬清洁加工领域新型酶制剂的开发提供理论依据。
Polygalacturonase is an enzyme that hydrolyzes D-galacturonic acid α-1,4- glucoside bond, which is of great significance in food industry, especially in fruit and vegetable processing. To exploit the biochemical properties of polygalacturonase produced from Trichoderma asperellum (14636), the polygalacturonase was isolated and purified by ammonium sulfate fractionation and dialysis method. Its molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). According to the experimental results, the activation range of this enzyme was from 29.29 kDa to 50.46 kDa, the optimum pH was 4.0, it had good stability within pH 3.0-5.0; the optimum temperature was 40 ℃, and it showed certain thermal stability. The enzyme activity was higher in sodium acetate buffer. The catalytic properties showed that pectin was the optimal substrate for polygalacturonase, and the Km value was 0.74 mg/ml, Vmax was 3100 μg/min. Mn2+, Mg2+, Cu2+, Ca2+ and Tritonx-100 could activate this enzyme, and the activate effect of Ca2+ and Tritonx-100 were stronger; while Co2+, Zn2+, Ba2+, Li+, Fe2+, SDS and Tween-80 exerted different inhibitory effects on its activity. Storage characteristics showed that this enzyme retained more than 70% of its activity at 30 ℃ for 30 d. This study can provide a theoretical basis for the development of new enzyme preparations in the field of fruit and vegetable clean processing.
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