Extracellular cellulolytic enzyme system of Aspergillus japonicus: 3. Isolation, purification and characterization of multiple forms of endoglucanase

Abstract

The crude culture filtrate of Aspergillus japonicus exhibits marked cellulose and xylan degrading activities. Affinity chromatography of the crude enzyme preparation on concanavalin A (Con A)-Sepharose 4B resolves it into three fractions. The eluted fraction A contains CMCase (CMCase I) and xylanase activities; fraction B shows only CMCase activity (CMCase II); and fraction C exhibits CMCase (CMCase III) and xylanase activities. On further purification by gel filtration, only fraction B imparts a homogeneous preparation that gives a single band on polyacrylamide gel electrophoresis at pH 8.3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of this homogeneous form shows the molecular weight about 57 000 daltons. Sephadex G-100 column chromatography also supports the molecular weight. A low molecular weight endoglucanase, of about 12 700 daltons, was obtained from fraction A by Sephadex G-75 column chromatography whereas the molecular weight of the endoglucanase from fraction C was higher (77 000 daltons). The silver ion (Ag 1+) strongly inhibits the endoglucanase I activity but has no effect on II and III. Hg 2+ inhibits all the three forms. The pH optimum for endoglucanase I, II and III is 4.5. The endoglucanase II shows the highest temperature optimum of 65°C whereas those of endoglucanase I and III are 50°C and 55°C, respectively. The first two forms have the least activity toward tamarind kernel polysaccharide (TKP) but the last form, endoglucanase III, is highly reactive to it.