Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity

Abstract

A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S-300. Zymogram analysis after SDS-PAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under non-denaturing conditions. The hydrolysis of xylan was optimal at temperature range of 55-75oC and pH 6.0. Xylanase activity was quite stable at 65oC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, L-tryptophan and L-cysteine and strongly inhibited by N-bromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65oC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65oC with half-life of 2.5 h and inhibited by dithiothreitol and Ca2+.