Production, purification and characterisation of alkali stable xylanase from Cellulosimicrobium sp. MTCC 10645

Abstract

ObjectiveThe aim of this experimental study was production, purification and characterization of alkali stable xylanase from locally isolated Cellulosimicrobium sp. MTCC 10645, which is an important industrial enzyme used in the pulp and paper industry. MethodsThe enzyme was produced in Erlenmeyer flasks containing fresh basal salt medium supplemented with 1% oat spelt xylan. The enzyme was extracted and isolated using ammonium sulphate precipitation and dialysis. It was further purified using DEAE cellulose chromatography and purity was checked by SDS-PAGE. Effect of temperature and pH on activity and stability of enzyme was studied. The enzyme was laso studied for its substrate specificity and kinetic parameters. ResultsThe isolate was identified on the basis of cultural, morphological, physiological and biochemical properties as well as 16S rRNA sequencing. Among the carbon sources tested, birchwood xylan found prominent for increased level of xylanase i. e. 96.33 U/ml. The enzyme was purified by DEAE cellulose chromatography at NaCl concentration of 0.25 M and had a molecular mass of 78.0 kDa. Xylanase was purified sixteen fold with a specific activity of 246.6 U/mg. Xylanase activity was maximum at 50°C. The enzyme was thermostable retaining 8% of the original activity after incubation at 60°C of 4 h. The enzyme was active over a pH range of 6.0-11.0, although its activity was optimal at pH 7.0. About 48.52% of the enzyme activity was retained after 4 h at pH 11.0. The enzyme was active on oat spelt and birchwood xylans but not on avicel, CMC, cellobiose, starch or p-nitrophenyl xylopyranoside. The xylanase had Km and Vmax values of 4.76 mg/ml and 232.5 μmol/min/mg, respectively when birchwood xylan used as substrate. ConclusionsThe xylanase showed a unique pattern of xylan hydrolysis releasing a large amount of intermediate products (xylotriose and xylobiose) with small quantity of xylose. Some of these characteristics make this enzyme potentially effective in xylan biodegradation and pulp bleaching.