Purification and biochemical properties of alkaline pullulanase from alkalophilic Bacillus Sp. S-1

Abstract

A thermostable DNA polymerase from Thermus caldophilus GK24 was purified to near homogeneity by chromatographic methods, including ion-exchange, gel-filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75 degrees C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose-12 gel filtration. Reaction conditions were investigated in terms of pH, metal-ion concentration and temperature. Experimental results showed that T. caldophilus (Tca) DNA polymerase had a maximum activity near pH 8.7 at 75 degrees C. The N-terminal sequence of the enzyme was highly similar to that of Thermus aquaticus (Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5'-to-3' exonuclease activity and no 3'-to-5' exonuclease activity. Gene amplification using Tca DNA polymerase resulted in longer products than amplification using Taq DNA polymerase.