Partial characterization of a major autolysin from Mycobacterium phlei.
Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.
Li
Zusheng S
ZS
Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Beveridge
Terry J
TJ
Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Betts
Joanna
J
Immunopathology Unit, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage SG1 2NY, UK.
Clarke
Anthony J
AJ
Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001433:Bacteriolysis; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism; D009168:Mycobacterium phlei / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D017421:Sequence Analysis; D013010:Sonication; D013379:Substrate Specificity; D013997:Time Factors
D000596:Amino Acids; D010457:Peptidoglycan; D006026:Glycoside Hydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase