Partial purification and characterization of alpha-amylase from Bacillus Amyloliquefaciens UMAS1002

Marilyn, Jaoi @ Edward. (2007) Partial purification and characterization of alpha-amylase from Bacillus Amyloliquefaciens UMAS1002. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Confirmation test using several biochemical, physiological and characteristics methods has confirmed that the bacterium used in this study is the same isolate used in previous study, which was designated as Bacillus amyloliquefaciens UMASI002. The nucleotide sequence of the 16S rRNA gene of this bacterium was determined. Construction of phylogenetic tree revealed very close relationship of this isolate with B. subtilis strain CICCI0163 and another Bacillus sp. strain Q-12~ This findings correlate with other findings by other investigators. The only difference they could detect is that there was very low sequence homology between liquefYing amylase of B. amyloliquefaciens and saccharifying amylase of B. subtilis. Optimization production of alpha-amylase enzyme from B. amyloliquefaciens UMASI002 was also determined by a series of optimization schemes. The outcome revealed that maximum activity of alpha-amylase was obtained using Bacillus defined medium at pH6.0, with soluble starch concentration of 2% (w/v) and incubated at 45°C for 12 hours. Highest specific activity obtained using these defined conditions are 34771.2 U/mg. An extra cellular alpha-amylase from B. amyloliquefaciens UMASI002 was partially purified through a series of four steps and the purity of enzymes was analyzed using SDS-PAGE. SDS-PAGE showed multiple bands for the partially purified enzyme, with an apparent molecular weight of 75 kDA, 55 kDA and 20 .... kDA. The optimum pH and temperature for the enzyme action on starch was 5.5 - 6.5 and 55°C - 65°C, respectively. This enzyme is stable in the pH range of 5.5 to 6.5 and the temperature range of 30°C to 45°C (after incubation at defined temperature for 2 hours). A molecular manipulation on the enzyme could lead to an enhanced thermo stability of this enzyme. The gene encoding putative alpha-amylase from B. amyloliquefaciens UMASI002 was cloned in pGEMT vector and sequenced. The fragment was obtained by peR method and expression study was done by cloning the fragment into pET41a (+) expression vector and transformed in Escherichia coli BL21 (DE3). The nucleotide sequence of the gene predicts a 179-amino acid protein with an estimated molecular mass of 19481 Da, which corresponds well with the value obtained from the third band of the partially purified enzyme using SDS-PAGE. The predicted amino acid sequence showed low homology with Arabidopsis thaliana, Corynebacterium diptheriae and Arthrobacter sp. FB24 alpha-amylases. The positive clones obtained failed to express alpha-amylase and this might be due to lack of conserved region present in the sequence which responsible for the catalytic activity in alpha-amylase.

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