Purification and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103

Appl Microbiol Biotechnol. 2006 Dec;73(3):591-7. doi: 10.1007/s00253-006-0513-4. Epub 2006 Jul 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Bread / microbiology
  • Chromatography, Ion Exchange
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Oligosaccharides / metabolism*
  • Soil Microbiology*
  • Temperature
  • alpha-Amylases / chemistry*
  • alpha-Amylases / isolation & purification*
  • alpha-Amylases / metabolism


  • Oligosaccharides
  • maltooligosaccharides
  • alpha-Amylases