米根霉葡萄糖淀粉酶的分离纯化及特性研究

Purification and Characterization of Glucoamylase from Rhizopus oryzae

葡萄糖淀粉酶是米根霉在淀粉质培养基中诱导分泌的一类胞外酶,其表达对米根霉转化淀粉生成L-乳酸的效率,以及L-乳酸分批发酵周期的长短有很大影响。本文对米根霉葡萄糖淀粉酶进行分离纯化,并研究其酶学性质,结论如下:经硫酸铵盐析、透析脱盐、SephadexG-100柱层析等纯化步骤,葡萄糖淀粉酶比活力提高23倍。SDS-PAGE显示纯化后的酶为一条带,相对分子量约75.5kD。该酶以可溶性淀粉为底物时最适催化反应pH值为4.0～6.0,最适催化反应温度为40～50℃,在50℃下保持稳定。钙离子和锰离子对该酶活力有一定的增强作用,而铅离子和亚铁离子对其活力有明显的抑制作用。

Glucoamylase is a kind of extracellular enzyme of Rhizopus oryzae induced in amylum culture medium. Its expression has a great impact on the production efficiency of L-lactic acid and the length of L-lactic acid batch fermentation cycle by Rhizopus oryzae. Purification and characterization of glucoamylase from Rhizopus oryzae were carried out in this dissertation. The results indicated that ammonium sulphate fractional precipitation, dialysis and Sephadex G-100 gel filtration could purify the glucoamylase from the extract of Rhizopus oryzae and increase its specific activity by 23 times. The SDS-PAGE showed that the purified enzyme was a homogeneous protein with molecular weight around 75.5kD. The optimum reaction pH value and temperature was about 4.0～6.0 and 40～50℃, respectively. It was stable under 50℃. The glucoamylase was activated by Ca 2+ and Mn2+,but inhibited by Pb2+ and Fe2+.