A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability.

A cell-associated pullulanase (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55,000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr- Ala- Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75-80 degrees C and at 80-85 degrees C, respectively. The enzyme was stable up to 90 degrees C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae----B. acidopullulyticus----B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.