Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313.
A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48 degrees C and 4.9, respectively. The enzyme was stable up to 60 degrees C and its pH stability was in the range of 5.0-8.0. The Km and Vmax of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, Ea, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a Ki of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a Ki of 9.5 mg/ml. The enzyme was inhibited by Hg2+, Mn2+, Fe3+ and Cu2+ and enhanced by Co2+ and Mg2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate.
Dey
G
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Microbial Biotechnology and Downstream Processing Laboratory, Agricultural and Food Engineering Department, IIT-Kharagpur 721302, India.
Palit
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S
Banerjee
R
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Maiti
B R
BR
D000681:Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D002850:Chromatography, Gel; D003912:Dextrins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015233:Models, Statistical; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis*; D013816:Thermodynamics
D003912:Dextrins; D009844:Oligosaccharides; C021705:maltooligosaccharides; D000681:Amylases; D005947:Glucose