| Beta-1, 3-glucanases belong to enzymes hydrolyzing beta-1, 3-D-glucan, which can hydrolyze the O-glycosidic linkages of 1,3- beta–linked glucan. Beta-1, 3-glucanases are widely distributed in nature. The enzymes have been isolated from many kinds of organisms, such as higher plants, yeast, fungi, bacteria and virus. Beta-1, 3-glucanases play an important physiological role in cell growth and defense against fungal pathogens in plant and fungi. Beta-glucan, the production of beta-1, 3-glucanases, is known to be able to stimulate plant and animal defense mechanisms. In addition these enzymes have been found many applications in pharmaceutical industry, biological control, fungal and yeast cell wall degradation and medical fields.In recent years, enzymes from thermophilic fungi, have aroused increasing attention among researchers. Although there are a number of reports describing beta-1, 3-glucanases from fungi, but no thermostable exo-beta-1,3-glucanases from thermophilic fungi have been described. Chaetomium thermophilum is a widely distributed soil-inhibiting thermophilic fungus. In this report, we described the purification, partial characterization and cDNA cloning of an extracellular exo-beta-1, 3-glucanase from Chaetomium thermophilum.A thermostable extracellular beta-1, 3-glucanase from culture supernatant of the thermophilic fungus Chaetomium thermophilum was purified. The exo-beta-1, 3-glucanase was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by fractional ammonium sulphate precipitation, Pheny1-Sepharose Fast Flow hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose Fast Flow chromatography, and gel filtration on Sephacryl S-100. At the end of the steps, a purified enzyme preparation was obtained showing marked exo-beta-1, 3-glucanase activity, capable of hydrolyzing laminarin. SDS-PAGE of the purified enzyme showed a single protein band.at 76.3 kDa. The molecular mass of the purified enzyme was also estimated to be 77.6 kDa by gel filtration on Sephacryl S-100, with standard protein markers from Pharmacia, suggesting that the enzyme may be a monomeric protein. The exo-beta-1,3-glucanase was purified 26.24 fold with a yield of 17.2% to a specific activity of 45.4 U·mg-1 of protein. The optimum pH and temperature obtained for the beta-1,3-glucanase was 6.0 and 60 oC. It was thermostable at 50 oC and 60 oC. The enzyme was relatively stable at 50 oC. The half-life was 20 min at 70 oC, and retained 90% activity after 60 min at 60 oC. It can be deduced that the enzyme is somewhat thermally stable. Ca2+ and Na+ enhanced the enzyme activity, whereas Fe2+, Ag+, Al3+ and Cu2+ caused obvious inhibition, and the enzyme was not significantly influenced by Ba2+, K+, and Mn2+.The HPLC analysis of the enzymic products formed from the laminarin by the purified exo-beta-1, 3-glucanase showed that the enzyme can successive hydrolysisβ-D-glucose units from the non-reducing ends of laminarin, releasingα-glucose.The N-terminal amino acid analysis showed that the sequence of first 8 residues from N-terminal of the exo-beta-1, 3-glucanase from C. thermophilum were HWLGDIPH. Using database search (BLAST), the segment revealed no homology with the N-terminal sequences of others.Degenerate primers designed based on the N-terminal sequences of purified beta-1, 3-glucanase and the conserved amino acid sequence of other fungi amino acid. Two cDNA fragments encoding the beta-1, 3-glucanase gene were amplified and registered in Genbank with accession number EU744253and EU746500 respectively. The sequencing analysis showed that the two cDNA fragments were composed of 1273bp and 1267bp which coding 423 and 421 amino acids. The amino acids fragment was subsequently analyzed in GenBank and Swiss-Port date-bases, the alignment results of putative amino acids sequence showed the catalytic domain were homology with the catalytic domains of the beta-1, 3-glucanases.
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