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.
Department of Agricultural Chemistry, Kyoto Prefectural University, Japan.
Y
Y
Suzuki
K
K
Hatagaki
H
H
Oda
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D000431:Ethanol / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011392:Proline / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D013439:Sulfhydryl Reagents; D012967:Sodium Dodecyl Sulfate; D000431:Ethanol; D011392:Proline; D004492:Edetic Acid; D006026:Glycoside Hydrolases; C100181:pullulanase