Characterization of a neutral and thermostable glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae: activity, stability, and structural correlation.
Glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae was purified by anion exchange and gel filtration chromatographic techniques using a fast protein liquid chromatographic system. The structure and thermal stability of this unique 'thermostable and neutral glucoamylase' were analyzed by circular dichroism (CD). T. indicae-seudaticae glucoamylase (TGA) contained typical aromatic amino acid (tryptophan/tyrosine) fingerprints in its tertiary structure. Analysis of the far-UV CD spectrum at pH 7.0 and 25 degrees Celsius revealed the presence of 45% alpha-helix, 43% beta-sheet, and 12% remaining structures. The alpha-helix content was highest at pH 7.0, where glucoamylase is optimally active. This observation points towards the possible (alpha/alpha)(6) barrel catalytic domain in TGA, as reported in microbial glucoamylases. Thermal denaturation curves of the pure protein at different pH values revealed maximum stability at pH 7.0, where no change in the secondary structure was observed upon heating in the temperature range between 20 degrees Celsius and 60 degrees Celsius. The observed midpoint of thermal denaturation (T (m)) of glucoamylase at pH 7.0 was 67.1 degrees Celsius, which decreased on either sides of this pH. Thermostability of TGA enhanced in the presence of starch (0.1%) as no transition curve was obtained in the temperature range between 20 degrees Celsius and 85 degrees Celsius. The only product of TGA action on starch was glucose, and it did not exhibit transglycosylation activity even at 40% glucose that can also be considered as an advantage during starch saccharification.
Kumar
Pardeep
P
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India.
Islam
Asimul
A
Ahmad
Faizan
F
Satyanarayana
T
T
D000595:Amino Acid Sequence; D055162:Biocatalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D009090:Mucorales / Q000201:enzymology*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013696:Temperature
D005087:Glucan 1,4-alpha-Glucosidase