Geotrichum candidum P-5 produces an intracellular serine protease resembling chymotrypsin.
A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.
Litthauer
D
D
Department of Microbiology and Biochemistry, University of Orange Free State Bloemfontein, South Africa.
Louw
C H
CH
du Toit
P J
PJ
D002918:Chymotrypsin / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D005848:Geotrichum / Q000201:enzymology*; D007700:Kinetics; D012697:Serine Endopeptidases / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*
D012697:Serine Endopeptidases; D002918:Chymotrypsin