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）及其可能的进化前基因Ell‘, （nylB’基因）的结构基因具有一个开放的阅读框，编码392个氨基酸的肽，其中47个是不同的，并且具有保守的限制性位点。Ell对6-氨基己酸二聚体的比活性约为Ell的100倍。从Gly181到Asp181的改变对于Ell的高活性是必不可少的，并且在370 bp的small - bamhl片段中编码的15个氨基酸中的一个或多个氨基酸改变增强了其作用。为了确定该片段编码的必需氨基酸改变，我们通过交换nylB基因和nylB'基因中Sall、Sau3Al、Nael和BamHl的保守限制性内切位点（分别位于起始密码子下游771,825,915和1141 bp处）的片段，构建了杂交基因。构建的Hyb-19酶在144 bp的small - nael片段中编码了6个氨基酸的改变，经纸层析分析显示其具有较高的酶活性。由此构建了携带2个氨基酸突变的Hyb-20酶，该酶编码在54 bp的small - sau3al片段中。通过DEAE-Sephadex A-50柱纯化的Hyb-20和Ell酶具有几乎相同的比活性（1.8单位/mg蛋白）。从这些结果中，我们得出结论，从Thr259到Gln259的改变或从His266到Asn266的改变对于提高酶的活性是必不可少的。为了从这两种改变中确定一种必需氨基酸的改变，我们构建了携带两种改变中每一种的突变编码的杂交质粒。用纸层析法测定了携带His266到Asn266突变的杂合酶的高通透性。这表明该突变是增强Gly181到Asp181突变效应的必要条件。

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.