摘要
经硫酸铵沉淀、疏水柱层析和分子筛层析,从黑曲霉变种RM48(Aspergillus niger v.Tiegh RM48)固体发酵后的浸提液中分离纯化了α-半乳糖苷酶.SDS-PAGE分析表明,此酶蛋白的分子量约为108kDa.酶学性质研究表明,该酶的最适反应温度是60℃,耐热温度为40℃;最适反应pH为4.5,在pH3.0～6.0之间保持90%以上的酶活性;在实验的金属离子范围内,所有金属离子对黑曲霉变种RM48α-半乳糖苷酶均有抑制作用,其中Cu2+和Fe2+抑制作用最为显著,Li2+、Zn2+、Mn2+、K+、Na+、Ca2+、Mg2+和Co2+对酶活性有轻微的抑制作用.在pH4.0和60℃反应条件下此酶的Km为7.37mmol·L-1,Vmax为5618IU·mg-1·min-1.成熟酶蛋白N端序列为DEKAYSPCYY,与已报道的α-半乳糖苷酶蛋白无同源性.
An extraeellular α-galactosidase from Aspergillus niger v. Tiegh RM48 was purified to apparent homogeneity by ammonium sulfate （80% saturation） precipitation, Phenyl Sepharose CL-4B hydrophobic column chromatography and Sephadex G-100 filtration chromatography. The specific activity of the enzyme was increased approximately 103- fold,from 26.29 IU·mg^-1 protein to 2722.3 IU· mg^-1 protein. The molecular mass of the purified enzyme as determined by SDS-PAGE was 108 kDa. The optimum pH and temperature of this a galactosidase were pH 4. 5 and 60 ℃, respectively. The enzyme was stable between pH 3. 0 and pH 6.0, however, its activity was inhibited by all tested metal ions. α-Galactosidase activity was strongly inhibited by Cu^2＋, Fe^2- and moderately inhibited by Li^2＋ , Zn^2＋ , Mn^2＋ , K^＋ , Na^＋ , Ca^2- , Mg^2- and Co^2＋ . Kinetic studies of the a-galactosidase showed that the Km and the Vmax for 4 nitrophenyl-α-D-galactopyranoside（pNPG） were 7.37 mmol·L^-1 and 5618 IU· mg^- 1 · min^-1 respectively. The N-terminal amino acids sequence of the α-galactosidase protein was determined as DEKAYSPCYY, no evidence showed the apparent sequence similarity between this enzyme and any other α-galactosidase listed in the database.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2009年第2期147-152,共6页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
浙江省重大科技攻关资助项目(2004C32049)
