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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

杂化酶（尼龙低聚物降解酶EII与其进化起源酶之间）的创建及性质

基本信息

批准号：
60440007
负责人：
OKADA Hirosuke
金额：
$ 11.14万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1988
项目状态：
已结题

项目摘要

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.

两种同源酶的结构基因，6-氨基己酸二聚体水解酶，Ell，（nylB基因）和其可能的进化前体，Ell '，（nylB'基因）具有编码392个氨基酸的肽的单个开放阅读框，其中47个是不同的，和保守的限制性位点。E11对6-氨基己酸二聚体的比活性约为E11 ′的100倍。从Gly 181到Asp 181的改变对于Ell的高活性是必需的，并且其效果通过370 bp Sall-BamHI片段中编码的15个氨基酸中的一个或多个改变而增强。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'基因两侧的萨尔、Sau 3Al、Nael和BamH 1保守限制性位点（分别在起始密码子下游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Sall-Nael片段中编码6个氨基酸的改变，通过纸层析测定显示出高的酶活性。因此，我们构建了Hyb-20酶，该酶在54 bp的Sall-Sau 3Al片段中编码2个氨基酸改变。Hyb-20和Ell酶经DEAE-SephadexA-50柱层析三次纯化后，比活力基本相同（1.8U/mg蛋白）。根据这些结果，我们得出结论，从Thr 259到Gln 259的改变或从His 266到Asn 266的改变对于增加酶活性是必需的。为了从这两个中确定一个必需的氨基酸改变，我们构建了携带编码两个改变中的每一个的突变的杂交质粒。经纸层析法测定，His 266突变为Asn 266的杂合酶具有较高的活性。这表明这种改变对于增强从Gly 181到Asp 181的改变的效果至关重要。