Improvement of Thermostable Enzyme with Sugar Metabolic Activity by Targeted Mutagenesis

Abstract

It was well known that improvement of enzymatic activity and stability is very difficult. For most enzymes, introduction of mutation into the amino acid residues located within the reaction center usually disappears their activity. Conversely, it should be useful for application of enzymes if enzymatic activity and stability are artificially enhanced. The enzyme isolated from thermophilic archaea generally possesses absolute stability. The nucleotide-sugar molecule is a powerful material for artificial construction of polymer structure of sugar. The ST0452 protein, an enzyme with sugar-1-phosphate nucleotidylyltransferase activity from Sulfolobus tokodaii, was chosen as target for introduction of targeted mutagenesis into the reaction center. All mutant ST0452 enzymes exhibited the same thermostability as shown by the parental ST0452 enzyme. Among 11 mutant ST0452 proteins with substitution of the amino acid residues located at the reaction center by alanine and other amino acids, five mutant ST0452 proteins showed kcat values larger than the original value, revealing that in these mutant ST0452 proteins, reactions progress faster than the original enzyme. Even though these mutant ST0452 proteins showed higher Km values than that of the original enzyme, these improved mutant ST0452 proteins were capable of exhibiting a higher activity than that of the wild-type ST0452 protein under the presence of high concentration of substrate. These results indicate that thermostable enzymes with higher activity were constructed from S. tokodaii ST0452 enzyme by substitution of amino acid residues at the reaction center. These improved enzymes are expected to be useful for application.