Purification and medium optimization of α-amylase from Bacillus subtilis 168

  • A Sumrin
  • W Ahmad
  • B Ijaz
  • MT Sarwar
  • S Gull
  • H Kausar
  • I Shahid
  • S Jahan
  • S Asad
  • M Hussain
  • S Riazuddin
Keywords: Bacillus, α-amylase, optimization, Plackett-Burman design, response surface methodology


α-Amylase was first time isolated and purified from Bacillus subtilis 168 (1A1). Purified α-amylase fraction showed a single protein band with a molecular weight of 55 kD. Chemical characterization of the purified α-amylase revealed optimum amylolytic activity at 37°C and pH 7.0 using starch as substrate. It was stable at pH 5.0 to 9.0 and at temperatures 25–70°C. Culture conditions were optimized by using statistics-based experimental designs to enhanced α-amylase (EC. production. A two level fractional factorial Plackett-Burman design was used for the preliminary screening significant media components and conditions. Response surface methodology (RSM) involving a 24 full-factorial central composite design (CCD) and a second-order polynomial equation was then employed to identify the relationship between the α-amylase yield and the four significant variables. Optimal levels of the significant variables for the maximum α-amylase yield were starch 2.55 g/l, yeast extract 8.4 g/l, sodium chloride 8.1% and 48 h of incubation. Mean value of α-amylase yield was 639.7 IU/ml, which was in excellent agreement with the predicted value (633.5 IU/ml).

Key words: Bacillus, α-amylase, optimization, Plackett-Burman design, response surface methodology.


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eISSN: 1684-5315