Lesk E M, Blackburn T H
J Bacteriol. 1971 May;106(2):394-402. doi: 10.1128/jb.106.2.394-402.1971.
A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.
嗜淀粉拟杆菌细胞在稳定期末期（达到最大细胞密度后约12小时）释放出一种蛋白酶。该蛋白酶通过吸附在二乙氨基乙基（DEAE）-葡聚糖上进行浓缩，并通过DEAE-葡聚糖柱层析、G-200-葡聚糖凝胶过滤和等电聚焦进行纯化，纯化倍数达532倍。纯化后的蛋白酶在pH 4.5至12.0之间具有活性，最适pH值为6.0和11.5。一些抑制剂对酯酶和蛋白酶活性的不同抑制作用表明存在不止一种蛋白酶。有证据表明只存在一种蛋白酶，因为在纯化过程中、纯化后的蛋白酶部分热失活时或用两种特异性胰蛋白酶型蛋白酶抑制剂（N-α-甲苯磺酰-L-赖氨酰氯甲基酮或苯甲磺酰氟）处理后，不同pH值下的蛋白酶活性比值没有显著变化。通过丙烯酰胺凝胶电泳发现了同一种蛋白酶的两种形式。凝胶过滤证实存在分子量为30,000和60,000的蛋白酶形式。用1 mM乙二胺四乙酸或4 M尿素处理未能将分子量为60,000的蛋白酶转化为分子量为30,000的形式。
