Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 15, Issue 3, 2013; Page No.(459-472)

PRODUCTION AND PARTIAL CHARACTERIZATION OF β- GLUCANASE FROM ASPERGILLUS NIGER JQ1516491 UNDER SUBMERGED AND SOLID STATE FERMENTATION

AHMED A. SHINDIA, SALWA A. KHALAF AND MARWA A. YASSIN

Abstract

Aspergillus niger was the potent ß-glucanase producer, as observed from the screening profile. Based on morphological and molecular approaches (18S-28S rRNA sequence, flanking the ITS and 5.8S rRNA regions), the isolate was identified as Aspergillus niger with accession number JQ1516491. Under submerged fermentation conditions, the maximum enzyme yield (127.3 U/mg) by A. niger was obtained using 2% CMC, 0.3% yeast extract, 0.1% KH2PO4, 0.05% MgSO4.7H2O, 0.05% KCl. Among the tested ten natural agricultural byproducts, peanut shell cake was the potent substrate for induction of β-glucanase (178 U/mg) by A. niger under solid state fermentations (SSF). Under SSF conditions, the enzyme yield was increased by about 1.4 folds by supplementation of 0.1 % CMC and 0.1 % yeast extract to the same salt solution. The enzyme was purified and characterized from the solid cultures of A. niger using peanut as substrate, by salting out, gel-filtration and ion exchange chromatography. Following the chromatographic step, the enzyme activity was increased by 5.4 fold with 42.6 % yield. Using SDS-PAGE, the enzyme has molecular weight 55 and 35 kDa. The maximum enzyme activity was measured using 1% β-glucan in potassium phosphate buffer of pH 6.5-7.0, with relative pH stability at 5.4 to 6.0. Also, the highest enzyme activity was detected by incubation of the reaction mixture at 50oC, with plausible thermal stability at this degree. The enzyme thermal denaturation rate was increased subsequently with the heating temperature, as revealed from the T1/2 values that were 10, 4.7 and 2.7 hr at 50, 60 and 70oC, respectively. The enzyme has iso-electric focusing (pI) at pH 6.6-7.4. From the absorption spectra, the enzyme has a distinct peak at 230 nm for the apo- enzyme and other at 300 nm.

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