Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea

• Published in: Canadian journal of microbiology Vol. 22; no. 2; pp. 165 - 176
• Main Authors: Ong, P.S, Gaucher, G.M
• Format: Journal Article
• Language: English
• Published: Canada 01.02.1976
• Subjects: Calcium - pharmacology; Caseins - metabolism; Cell-Free System; Drug Stability; Egtazic Acid - pharmacology; Endopeptidases - biosynthesis; Endopeptidases - isolation & purification; Endopeptidases - metabolism; Ficoll - pharmacology; Fungi - enzymology; Fungi - growth & development; Fungi - metabolism; Hot Temperature; Hydrogen-Ion Concentration; Isoflurophate - pharmacology; Molecular Weight; Phenylmethylsulfonyl Fluoride - pharmacology; plant biochemistry; plant physiology
• ISSN: 0008-4166; 1480-3275
• DOI: 10.1139/m76-023

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.