Abstract:
Amylases play a major role as hydrolytic enzymes in starch-based industries. It is
desirable for enzymes used in industry to have thermo-tolerant properties to withstand
temperatures encountered in industrial processes. Thermophiles are naturally
endowed with thermo-stable enzymes which are suited for specialized industrial
applications. In the present study we attempted to screen and isolate a thermophilic
bacterium that produced a novel thermostable alpha-amylase and to perform
purification and characterization processes of the enzyme. Nelum-wewa hot water
springs in Sewanapitiya, Polonnaruwa has one of the highest recorded temperatures of
a water body in Sri Lanka. Water and soil samples were collected, under sterile
conditions, from four distinct sampling sites and were transported to the laboratory in
a cold box (0°C). Water temperature and pH were recorded to be 52°C and pH 7
respectively. Samples collected were inoculated on to culture agar plates and broth
containing 0.5% (w/v) peptone and 0.2% (w/v) yeast extract, supplemented with a salt
solution. Soluble starch (1% w/v) was added to induce the production of alpha amylase.
Cultivation of bacteria was done at 50°C under high agitation in shaker water bath and
the pH was maintained at 6.9 during the culture. Isolation of bacteria was done by streak
plate and dilution plate methods. Amylase producing bacteria were identified by the
clearance zones produced on starch agar plates visualized with iodine solution. The
bacteria with the highest amylase activity was identified by morphological and
biochemical tests and 16s rRNA analysis as Caldimonas manganoxidans NMS1. Alpha
amylase activity was assayed by the method described by Bernfeld (1955) and the
maximum supernatant alpha amylase activity of 56 U/ml was obtained on incubation at
50°C for 20 hours. The extracellular amylase enzyme was purified by ammonium
sulphate fractionation and DEAE ion exchange chromatography. The specific activity of
the purified enzyme was observed to be 2143U/mg with 21-fold purification and 57%
of amylase activity retention. Polyacrylamide gel electrophoresis showed a single band
of protein indicating that the enzyme was purified to homogeneity. The purified enzyme
had the highest activity at 50°C and was stable up to 60°C. The enzyme showed a pH
range of 6 to 9 with maximum amylase activity observed at pH 6.9 and was stable within
the pH values of 6 to 9. The Km and Vmax values calculated from Lineweaver-Burk plot
were 1.7mg/ml and 217µmol/min/mg of protein respectively. The optimum
production of extracellular α-amylase was shown in a media comprising of 10% soya
powder and 1% soluble starch solution. The enzyme showed a relative enhancement of
activity with calcium ions (Ca2+) and manganese ions (Mn2+).
