Purification and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103.
A maltooligosaccharide-forming alpha-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of alpha-amylase in KCC103 was not catabolite-repressed. The alpha-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified alpha-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65-70 degrees C, it was rapidly deactivated at 70 degrees C with a half-life of 7 min and at 50 degrees C, the half-life was 94 min. The K (m) and V (max) for starch hydrolysis were 2.6 mg ml(-1) and 909 U mg(-1), respectively. Ca(2+) did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg(2+), Ag(2+), and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The alpha-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2-D4) were formed more predominantly than larger maltooligosaccharides (D5-D7). This maltooligosaccharide forming endo-alpha-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.
Nagarajan
Dilli Rani
DR
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India.
Rajagopalan
Gobinath
G
Krishnan
Chandraraj
C
D001412:Bacillus subtilis / Q000201:enzymology*; D001939:Bread / Q000382:microbiology; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D012988:Soil Microbiology; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D009844:Oligosaccharides; C021705:maltooligosaccharides; D000516:alpha-Amylases