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

6-氨基己酸二聚体水解酶Ell(nylB基因)及其可能的进化前驱Ell‘(nylB’基因)的结构基因都有一个单一的开放阅读框，编码392个氨基酸的多肽，其中47个氨基酸是不同的，并且具有保守的限制性内切酶切点。Ell对6-氨基己酸二聚体的比活性约为Ell‘的100倍。从Gly181到Asp181的改变是Ell高活性所必需的，其作用被370bpSall-BamH1片段中15个氨基酸改变中的一个或多个所增强。为了确定该片段中编码的必需氨基酸的改变，我们通过交换nylB基因和nylB‘基因中Sall、Sau3Al、Nael和BamH1(分别位于起始密码子下游的771、825、915和1141bp的保守限制位点)两侧的片段构建了杂交基因。构建的Hyb-19酶带有6个氨基酸残基，编码144个碱基的Sall-Nael片段，纸层析检测显示该酶具有较高的酶活性。构建了含2个氨基酸残基的Hyb-20酶，编码54个碱基的Sall-Sau3Al片段。经DEAE-Sephadex A-50柱三代纯化的Hyb-20和Ell酶的比活力几乎相同(1.8U/mg蛋白)。根据这些结果，我们得出结论，Thr259到Gln259的改变或His266到Asn266的改变对于提高酶的活性是必不可少的。为了确定这两个改变中的一个必需氨基酸改变，我们构建了携带编码这两个改变中每一个的突变的杂交质粒。从His266突变到Asn266的杂交酶经纸层析检测显示有很高的活性。这表明，这种改变对于提高从Gly181到Asp181的改变的效果是必不可少的。

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.