Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea.
The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 degrees C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not beta-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5-9.5 at 30 degrees C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 degrees C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/mo, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.
D002118:Calcium / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D002474:Cell-Free System; D004355:Drug Stability; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D005362:Ficoll / Q000494:pharmacology; D005658:Fungi / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology
D002364:Caseins; D007531:Isoflurophate; D005362:Ficoll; D004533:Egtazic Acid; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases; D002118:Calcium