Open AccessExo‐β‐ N ‐acetylmuramidase ‐ A Novel Hexosaminidase Production by Bacillus subtilis B, Purification and Characterization
Author(s) -
RÍO Luís A.,
BERKELEY Roger C. W.
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10382.x
Subject(s) - chemistry , chromatography , sephadex , diafiltration , membrane , centrifugation , size exclusion chromatography , bacillus subtilis , enzyme , lysozyme , ionic strength , molecular mass , biochemistry , aqueous solution , bacteria , biology , organic chemistry , genetics , microfiltration
Exo‐β‐N‐acetylmuramidase, or β‐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‐β‐ d ‐glucopyranosyl‐(1 → 4)‐2‐acetamido‐3‐O‐( d ‐1‐carboxyethyl)‐2‐deoxy‐ d ‐glucose and O‐[2‐acetam ido‐3‐O‐( d ‐1‐carboxyethyl)‐2‐deoxy‐β‐ d ‐glucopyranosyl]‐(1 → 4)‐2‐accetamido‐2‐deoxy‐ d ‐glucose in decreasing order of efficiency, induce the enzyme but O‐2‐acetamido‐2‐deoxy‐β‐ d ‐glucopyranosyl(1 → 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 homogeneous on polyacrylamide disc gel electrophoresis. A second heterogeneous fraction (peak B) was also collected. Exo‐β‐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 K m and V values for 4‐methylumbelliferyl‐2‐acetamido‐3‐O‐( d ‐1‐carboxyethyl)‐2‐deoxy‐β‐ d ‐glucose and O‐[2‐acetamido‐3‐O‐( d ‐1‐carboxyethyl)‐2‐deoxy‐β‐ d ‐glucopyranosyl]‐(1 → 4)‐2‐acetamido‐2‐deoxy‐D‐glucose arc respectively 0.19 and 0.65 mM and 1.50 and 16.29 μmol 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 rôles for this enzyme are discussed.