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    • 5. 发明授权
    • Method of preparing an enzyme participating in C-terminal amidation
    • 制备参与C-末端酰胺化的酶的方法
    • US6156555A
    • 2000-12-05
    • US172120
    • 1998-10-14
    • Toshii IidaToshihiko KaminumaYuka FuseMasahiro TajimaMitsuo YanagiHiroshi OkamotoJiro KishimotoOhji IfukuIchiro Kato
    • Toshii IidaToshihiko KaminumaYuka FuseMasahiro TajimaMitsuo YanagiHiroshi OkamotoJiro KishimotoOhji IfukuIchiro Kato
    • C07K1/107C12N9/02C12N15/53C12N15/60C12N9/48C12N9/14C12N9/78C12N9/80C12N15/00
    • C12N9/0071C07K1/107C12Y114/17003Y10S435/814Y10S435/815
    • A purified enzyme-I is obtained that participates in C-terminal amidation by acting on a peptide C-terminal glycine adduct to form a peptide C-terminal .alpha.-hydroxyglycine adduct. The enzyme has an optimum pH of about 5 to 7, an optimum temperature of 25 to 40.degree. C. and a molecular weight of about 25 kDa or about 36 kDa, and metal ions and ascorbic acid act as a cofactor. A purified enzyme-II is obtained that participates in C-terminal amidation by acting on the peptide C-terminal .alpha.-hydroxyglycine adduct to produce a C-terminal amidated compound. The enzyme has an optimum pH of about 5 to 6, an optimum temperature of 15 to 35.degree. C. and a molecular weight of about 40 kDa or about 43 kDa. Enzyme-I does not act on the peptide C-terminal .alpha.-hydroxyglycine adduct and enzyme-II does not act on the peptide C-terminal glycine adduct. The enzymes may be purified from a biological material such as horse serum by affinity chromatography using a peptide C-terminal glycine adduct as a ligand. The enzymes may also be obtained from host cells transformed with a plasmid containing a cDNA coding for the enzymes. Assay of activity of the enzymes is carried out by measuring the C-terminal .alpha.-hydroxyglycine adduct or the C-terminal amidated compound that has been isolated such as by high performance liquid chromatography with the use of an acetonitrile-containing buffer.
    • 获得通过作用于肽C-末端甘氨酸加合物以形成肽C末端α-羟基甘氨酸加合物参与C-末端酰胺化的纯化的酶I. 酶的最佳pH为约5至7,最适温度为25至40℃,分子量为约25kDa或约36kDa,金属离子和抗坏血酸用作辅因子。 获得了通过作用于肽C-末端α-羟基甘氨酸加合物参与C-末端酰胺化产生C末端酰胺化合物的纯化的酶-II。 酶的最佳pH为约5至6,最适温度为15至35℃,分子量为约40kDa或约43kDa。 酶-I不对肽C-端α-羟基甘氨酸加合物起作用,酶-II不对肽C-末端甘氨酸加合物起作用。 可以使用肽C-末端甘氨酸加合物作为配体通过亲和层析从生物材料如马血清中纯化酶。 酶也可以从用含有编码酶的cDNA的质粒转化的宿主细胞获得。 酶的活性测定是通过使用含有乙腈的缓冲液,通过高分辨液相色谱法测定已分离的C末端α-羟基甘氨酸加成物或C末端酰胺化合物进行的。
    • 6. 发明授权
    • Purified enzymes participating in C-terminal amidation
    • 纯化酶参与C-末端酰胺化
    • US5871995A
    • 1999-02-16
    • US070301
    • 1991-05-24
    • Toshii IidaToshihiko KaminumaYuka FuseMasahiro TajimaMitsuo YanagiHiroshi OkamotoJiro KishimotoOhji IfukuIchiro Kato
    • Toshii IidaToshihiko KaminumaYuka FuseMasahiro TajimaMitsuo YanagiHiroshi OkamotoJiro KishimotoOhji IfukuIchiro Kato
    • C07K1/107C12N9/02C12N15/53C12N15/60C12N9/48C12N9/14C12N9/78C12N9/80
    • C12N9/0071C07K1/107C12Y114/17003Y10S435/814Y10S435/815
    • A purified enzyme-I is obtained that participates in C-terminal amidation by acting on a peptide C-terminal glycine adduct to form a peptide C-terminal .alpha.-hydroxyglycine adduct. The enzyme has an optimum pH of about 5 to 7, an optimum temperature of 25.degree. to 40.degree. C. and a molecular weight of about 25 kDa or about 36 kDa, and metal ions and ascorbic acid act as a cofactor. A purified enzyme-II is obtained that participates in C-terminal amidation by acting on a peptide C-terminal .alpha.-hydroxyglycine adduct to produce a C-terminal amidated compound. The enzyme has an optimum pH of about 5 to 6, an optimum temperature of 15.degree. to 35.degree. C. and a molecular weight of about 40 kDa or about 43 kDa. Enzyme-I does not act on the peptide C-terminal .alpha.-hydroxyglycine adduct and enzyme-II does not act on the peptide C-terminal glycine adduct. The enzymes may be purified from a biological material such as horse serum by affinity chromatography using a peptide C-terminal glycine adduct as a ligand. The enzymes may also be obtained from host cells transformed with a plasmid containing a cDNA coding for the enzymes. Assay of activity of the enzymes is carried out by measuring adduct (II) or the compound (III) that has been isolated such as by high performance liquid chromatography with the use of an acetonitrile-containing buffer.
    • PCT No.PCT / JP90 / 01036 Sec。 371日期1991年5月24日 102(e)日期1991年5月24日PCT提交1990年4月12日PCT公布。 公开号WO91 / 02790 日期1991年3月7日获得通过作用于肽C-末端甘氨酸加合物以形成肽C-末端α-羟基甘氨酸加合物参与C-末端酰胺化的纯化的酶-I。 酶的最佳pH为约5至7,最适温度为25至40℃,分子量为约25kDa或约36kDa,金属离子和抗坏血酸用作辅因子。 获得了通过作用于肽C-末端α-羟基甘氨酸加合物以产生C末端酰胺化合物而参与C-末端酰胺化的纯化的酶-II。 酶的最佳pH为约5至6,最适温度为15-35℃,分子量为约40kDa或约43kDa。 酶-I不对肽C-端α-羟基甘氨酸加合物起作用,酶-II不对肽C-末端甘氨酸加合物起作用。 可以使用肽C-末端甘氨酸加合物作为配体通过亲和层析从生物材料如马血清中纯化酶。 酶也可以从用含有编码酶的cDNA的质粒转化的宿主细胞获得。 酶的活性测定是通过使用含乙腈的缓冲液测定加成物(II)或分离的化合物(III),例如通过高效液相色谱法进行的。