ハイブリド酵素(ナイロンオリゴマー分解酵素EIIと,その進化起源酵素間の)作成と性質

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The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase, Ell, (nylB gene) and its probable evolutionary antecedent, Ell', (nylB' gene) have a single open reading frame encoding a peptide of 392 amino acids of which 47 are different, and conserved restriction sites. The specific activity of Ell toward 6-aminohexanoate-dimer is about 100 folds of that of Ell'. Alteration from Gly181 to Asp181 is essential for the high activity of the Ell, and its effect is enhanced by one or more of 15 amino acid alterations encoded in 370 bp Sall-BamHl fragment. To determine the essential amino acid alteration encoded in this fragment, we constructed hybrid genes by exchanging fragments flanked by conserved restriction sites of Sall, Sau3Al, Nael and BamHl (at 771,825,915 and 1141 bp downstream from the initiation codon, respectively) from nylB gene and nylB' gene. Constructed Hyb-19 enzyme, which carried 6 amino acid alterations encoded in 144 bp Sall-Nael fragment, shows a high enzyme activity as determined by paper chromatography. It lead us to construct Hyb-20 enzyme which carried 2 amino acid alterations encoded in 54 bp Sall-Sau3Al fragment. Hyb-20 and Ell enzyme purified by three passages through DEAE-Sephadex A-50 colume had almost the same specific activity (1.8 units/mg protein). From these results, we concluded that either alteration from Thr259 to Gln259 or alteration from His266 to Asn266 is essential for increasing the enzyme activity. To determine one essential amino acid alteration from these two, we constructed hybrid plasmids carrying mutation encoding for each of the two alterations. Hybrid enzvme carrying mutation from His266 to Asn266 shows a hmgh ectivity as determined by paper chromatography. It suggests that this alteration is essential for enhancing the effect of alteration from from Gly181 to Asp181.