黄豆脲酶的提取与性质研究

PURIFICATION AND PROPERTIES OF UREASE FROM GLYCINE MAX

为解决脲酶的国产化问题 ,建立了从黄豆中提取脲酶的全套流程 ,主要包括醋酸盐缓冲液提取、亚硫酸钠盐析和离子交换色谱等步骤 ,操作简便 ,成本低廉。收率达 44 .0 9%。产品比活性达 6 7.15 U/ mg蛋白 ,可满足临床测定尿素的需要。再经分子筛色谱 ,可获得更纯的黄豆脲酶 (比活达 115 9.6 7U/ mg蛋白 )。酶性质研究结果显示 ,黄豆脲酶的分子量为 2 935 0 0 ,p I为 3.9,在 EDTA和 Tris-琥珀酸缓冲系统中的Km 值分别为 0 .0 1176 mol.L-1和 0 .0 1818mol.L-1 ,Vm ax分别为 3.42 μmol.min-1和4.35 μmol.min-1 ,在 EDTA、磷酸盐和 Tris-琥珀酸缓冲系统中的最适 p H分别为6 .0 0、7.0 0和 6 .5 0 ,最适温度为 6 5℃。这些性质与巨豆脲酶有显著差异。

A procedure for purifying the urease from Glycine max was developed. It was simple and economical and mainly included two steps, i.e salting\|out and column chromatograghy on DEAE\|cellulose. The specific activity of the product attained to 67.15 U/mg protein, which was fit for determining urea in clinical situation. By the column chromatograghy on Sephacryl 200, a specific activity of 1159.67 U/mg protein was obtained. The relative molecular weight of the enzyme was estimated to be 293 500 by SDS\|PAGE and column chromatograghy on Sephadex G\|200. The isoelectric point (pI) was 3.9 as judged by isoelectric focusing experiments. In EDTA buffer and Tris\|succinic acid buffer, the K \-m was 0.011 76 mol.L\+\{-1\} and 0.018 18 mol.L\+\{-1\}, the V \-\{max\} was 3.42 μmol.min\+\{-1\} and 4.35 μmol.min\+\{-1\},respectively.In EDTA buffer and phosphate buffer and Tris\|succinic acid bufer, the optimum pH was 6.0,7.0 and 6.5 respectively. The optimum temperature was 65℃。These properties markedly differ from those of urease from jack bean.