灰色链霉菌RX-17溶菌酶R2的纯化及其酶学鉴定

Purification and Enzymatic Characterization of Bacteriolytic Enzyme R2 from Streptomyces griseus RX-17

从灰色链霉菌 (Streptomycesgriseus)RX 1 7的发酵液中 ,通过硫酸铵分级沉淀 ,CM SephadexC 5 0和CM SepharoseFastFlow离子交换层析 ,纯化得到了溶菌酶R2 .该酶分子量约为 2 4 8kD ,等电点约为 9 7,N端 1 5个氨基酸的顺序为DTSGVQGIDVSHWQG .R2酶溶解变链球菌Ingbritt(StreptococcusmutansIngbritt)的最适作用温度为 5 5℃ ,最适pH为 7 0 .5 0℃处理 1h ,R2酶残存酶活74 % ,碱性条件 (pH >9)下该酶保持稳定 .Zn2 + 、Cu2 + 、Fe2 + 、Cd2 + 、Pb2 + 可使酶完全失活 ,螯合剂、盐酸羟胺、溴替丁二酰亚胺及离子型去垢剂SDS抑制R2酶的溶菌作用 ,而非离子型去垢剂TritonX 1 0 0等则能促进溶菌 .R2酶溶菌谱广泛 ,能够溶解多种鸡卵清溶菌酶不能作用的革兰氏阳性菌和革兰氏阴性菌 .从对金黄色葡萄球菌 (Staphylococcusaureus)的高活性来看 ,该酶应分类为 β 1 ,4 N ,6 O 二乙酰胞壁质酶 (β 1 ,4 N ,6 O diacetylmuramidase) .

A novel bacteriolytic enzyme R2 was purified from the crude culture of Streptomyces griseus RX 17 by ammonium sulfate precipitation, CM Sephadex C 50 and CM Sepharose Fast Flow ion exchange chromatography. The molecular weight was 24 8 kD and pI 9 7. The 15 amino acid residues of N terminal was DTSGVQGIDVSHWQG. The optimal temperature for the lytic activity against S.mutans Ingbritt was 55℃ and the optimal pH 7\^0. About 74% of the original activity remained unchanged after 1 hour at 50℃, and the enzyme activity was stable in alkaline solution. Zn 2+ , Cu 2+ , Fe 2+ , Cd 2+ , Pb 2+ could inactivate the enzyme. Chelating agents, hydroxylamine hydrochloride, N bromosuccinimide and SDS inhibited the lytic activity, whereas non ionic surfactant such as Triton X 100 could stimulate the activity. The enzyme had a broad bacteriolytic spectrum against many G\++ and G\+- bacteria which were resistant to hen egg white lysozyme. Since the high activity on Staphylococcus aureus, bacteriolytic enzyme R2 should be classified to β 1,4 N ,6 O diacetylmuramidase.