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A procedure with four chromatography steps was developed for the purification of DNA polymerase from Bacillus caldotenax by using fast protein liquid chromatography. The procedure was suitable for use with process-scale media. Elution profiles obtained from ion-exchange chromatography and triazine-dye affinity chromatography with fast protein liquid chromatography and process-scale media were similar. The enzyme showed stronger interaction, however, with phenyl-Sepharose FF in the scaled-up process than with the phenyl-Superose used in fast protein liquid chromatography. The surprising binding of the DNA polymerase to sulphonated ion-exchange media at pH 7.5 may be explained by the structure of the enzyme.

A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure. The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3'-5' or 5'-3'-exonuclease activities or endonuclease activity. The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5. The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2. Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively. Enzyme activity was inhibited in the presence of 2',3'-dideoxy-TTP, arabinosyl-CTP and aphidicolin. Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity. The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10. The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer. The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP). The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM. The kcat. value was about 1.05 s-1. The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs. Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.