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[Purification and characterization of mannanase from an alkaliphilic mannanase producing bacterium HMTS15].

OBJECTIVE
To isolate and identify an alkaliphilic mannanase producing bacterium, purify and characterize mannanase thereof.


METHODS
Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia, China. The morphological, biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15. Mannanase produced by strain HMTS15 was purified by four steps including (NH4)2SO4 precipitation, cellulose DEAE-sepharose, twice Superdex 200. The enzyme properties including optimal temperature, optimal pH, thermal stability, pH stability, NaCl tolerance, metal ion tolerance, EDTA and SDS tolerance were tested.


RESULTS
Strain HMTS15 was Gram-positive rod. Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10 degrees C to 45 degrees C. The G + C content of the DNA was 40 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus. The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE. The optimal catalytic activity was showed at 75 degrees C and pH 10. The mananase was stable up to 60 degrees C and retained about 60% residual activity at 65 degrees C for 30 min. The ions Fe2+, Mn2+, Co2+, Zn2+, Ag+, Hg2+ and EDTA inhibited the acitivity of the mannanase.


CONCLUSION
Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens. The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH.