Isolation, purification, and properties of the exocellulase from Sporotrichum ( Chrysosporium) thermophile

Abstract

The main extracellular exocellulase produced by Sporotrichum thermophile during growth on cellulose was isolated by ammonium sulfate precipitation, ion exchange chromatography, and adsorption-desorption on hydroxylapatite. The enzyme was identified on the basis of (a) its inability to liberate colored products from a dyed carboxymethyl cellulose (i.e., absence of endocellulase activity) and (b) its ability to hydrolyze the aglyconic bond of p-nitrophenyl-lactoside. The purified enzyme was homogeneous as judged by zone electrophoresis, SDS electrophoresis, electrofocalization, and titration curve. The molecular weight was 63,750 ± 4,500 and the pI was 4.52 ± 0.05. The enzyme was a glycoprotein containing 5.4% mannose and 1.7% glucose with traces of galactose and glucosamine. The enzyme was found to be very heat resistant and to have a low pH optimum (pH 3.5) on amorphous cellulose. Its activity was not affected by various cations, was only slightly inhibited by sulfhydryl reagents, but was sensitive to inhibition by ferric ions. The reactivity and mode of action on various substrates, as well as its cooperative action with other cellulolytic components from the same organism, support the evidence that the isolated exocellulase is a cellobiohydrolase (EC 3.2.1.91).