del Rio L A, Berkeley R C
Eur J Biochem. 1976 May 17;65(1):3-12. doi: 10.1111/j.1432-1033.1976.tb10382.x.
Exo-beta-N-acetylmuramidase, or beta-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, is produced by Bacillus subtilis B, growing in a succinate/peptone/salts medium, at the end of exponential growth and occurs partly in the medium and partly bound to the cells. A lysozyme digest of Micrococcus lysodeikticus cell walls, O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucose and O-[2-acetamide-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose in decreasing order of efficiency, induce the enzyme but O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose does not do so. The enzyme was purified from the growth medium, after removal of the cells by continuous centrifugation, by ammonium sulphate precipitation, continuous filtration through XM-300 membranes (to remove the high-molecular-weight material which renders the enzyme sedimentable in low-ionic-strength solutions), diafiltration through PM-30 membranes and ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex. Two peaks of activity were obtained. Peak A was purified 1800-fold and was homogenous on polyacrylamide disc gel electrophoresis. A second heterogeneous fraction (peak B) was also collected. Exo-beta-N-acetylmuramidase is most stable at pH 8.0 and has a molecular weight of about 90000. The results of studies on its ability to attack several synthetic and natural substrates are given. The Km and V values for 4-methylumbelliferyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucose and O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose are respectively 0.19 and 0.65 mM and 1.50 and 16.29 mumol min(-1) mg(-1). From these results and those of inhibition studies it is concluded that the enzyme is specific for substrates with non-reducing N-acetylmuramic acid end groups. Possible roles for this enzyme are discussed.
外-β-N-乙酰胞壁酸酶，即β-2-乙酰氨基-3-O-（D-1-羧乙基）-2-脱氧-D-葡萄糖苷乙酰氨基脱氧葡糖苷水解酶，由枯草芽孢杆菌B在琥珀酸盐/蛋白胨/盐培养基中指数生长末期产生，部分存在于培养基中，部分与细胞结合。溶壁微球菌细胞壁的溶菌酶消化产物，O-2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖基-（1→4）-2-乙酰氨基-3-O-（D-1-羧乙基）-2-脱氧-D-葡萄糖和O-[2-乙酰酰胺-3-O-（D-1-羧乙基）-2-脱氧-β-D-吡喃葡萄糖基]-（1→4）-2-乙酰氨基-2-脱氧-D-葡萄糖，按效率递减顺序可诱导该酶产生，但O-2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖基-（1→4）-2-乙酰氨基-2-脱氧-D-葡萄糖则不能。通过连续离心去除细胞后，从生长培养基中纯化该酶，依次经过硫酸铵沉淀、通过XM - 300膜连续过滤（以去除使酶在低离子强度溶液中可沉淀的高分子量物质）、通过PM - 30膜进行渗滤以及在DEAE - 葡聚糖凝胶和CM - 葡聚糖凝胶上进行离子交换色谱。获得了两个活性峰。峰A纯化了1800倍，在聚丙烯酰胺圆盘凝胶电泳上呈均一性。还收集了第二个不均一的组分（峰B）。外-β-N-乙酰胞壁酸酶在pH 8.0时最稳定，分子量约为90000。给出了关于其作用于几种合成和天然底物能力的研究结果。4-甲基伞形酮基-2-乙酰氨基-3-O-（D-1-羧乙基）-2-脱氧-β-D-葡萄糖和O-[2-乙酰氨基-3-O-（D-1-羧乙基）-2-脱氧-β-D-吡喃葡萄糖基]-（1→4）-2-乙酰氨基-2-脱氧-D-葡萄糖的Km值和V值分别为0.19和0.65 mM以及每分钟每毫克1.50和16.29 μmol。从这些结果以及抑制研究结果可以得出结论，该酶对具有非还原性N-乙酰胞壁酸端基的底物具有特异性。讨论了这种酶可能的作用。
