Journal of Pure and Applied MicrobiologyVol. 6 No. 4

Molecular Characterization of Industrially Viable Extreme Thermostable Novel a-amylase of Geobacillus sp. Iso5 Isolated from Geothermal Spring

D.M. Gurumurthy and S.E. Neelagund*

Department of PG studies and Research in Biochemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, Karnataka, India.

Received on 06 April 2012 and accepted on 12 May 2012

 

ABSTRACT

A thermotolerant alkali-resistant gram positive, rod shaped Geobacillus sp.Iso5 was isolated from the thermal water. Identification was performed by biochemical tests and 16S rRNA gene sequencing. The isolate comprises of 55%±0.2 mol % G+C content and iso-C15: 0, iso-C16: 0, iso-C17: 0 type of cellular fatty acids. Phylogenetic analysis revealed 98-99% sequence similarities was observed with thermophilic members of group Geobacillus; G.kaustophilus (99.5%), G. thermoleoverance (99%). A hyperthermostable a-amylase was purified using Sephadex G-150 gel filtration chromatography and a DEAE-cellulose column. The SDS-PAGE pattern of purified enzyme was showed an apparent molecular weight of 43 kDa. The MALDI-TOF/TOF mass finger printing analysis of purified enzyme has a 31% close homology with the surface layer protein of Geobacillus sp. The CD and FTIR spectra for secondary structure of enzyme revealed about 55% a –helix, 5% b-strand and 40% of unordered structure. The optimum activity of enzyme was determined to be 90°C and pH 8.0 and can stable up to 900C for 10 min. The DSC-TGA thermogram of purified enzyme was showed only 10% weight loss at 200°C. The effect of EDTA and Zn2+ on enzyme activity was shown maximum inhibitory activity. Whereas, Ca2+, Mg2+, Fe2+ and Cu2+ were did not affect on the activity of the purified enzyme.

Keywords : Thermal springs, Geobacillus sp. Iso5; 16S rRNA, a- amylase; MALDI-TOF/TOF, DSC-TGA, Circular Dichroism, FTIR.