Effect of glycosylation on the catalytic and conformational stability of homologous alpha-amylases.
A thermostable alpha-amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca2+. The stability of the native enzyme was found to increase upon modification as observed from the increase in tl/2 in the absence of Ca2+ ions by about 1.5 - 13.7 times (at 85 degrees C) in the case of BLA and 5.7 - 8.4 times (at 50 degrees C) for BAA. The highest stability was observed for OSP400 modified enzyme with Delta Cm and Delta Tm values of 0.63 M and 7.92 degrees C for BLA and 0.85 M and 5.3 degrees C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.
Srimathi
Soundararajan
S
Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Chennai 600 113, India.
Jayaraman
Gurunathan
G
D001407:Bacillus / Q000201:enzymology; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D006031:Glycosylation; D006146:Guanidines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013379:Substrate Specificity; D013816:Thermodynamics; D013861:Thiocyanates / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate; D000516:alpha-Amylases