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    Enzymatic reactions in the presence of keto acids
    1.
    发明授权
    Enzymatic reactions in the presence of keto acids 有权
    在酮酸存在下的酶反应

    公开(公告)号：US08163871B2

    公开(公告)日：2012-04-24

    申请号：US12284552

    申请日：2008-09-22

    申请人： Angelo P. Consalvo ,  Nozer M. Mehta ,  William Stern ,  James P. Gilligan

    发明人： Angelo P. Consalvo ,  Nozer M. Mehta ,  William Stern ,  James P. Gilligan

    IPC分类号： C07K7/00 ,  C12P13/00 ,  C12P21/06

    CPC分类号： C12Y114/17003 ,  C12P13/02 ,  C12P21/02 ,  C12Y403/02005

    摘要： Conversion in vitro of X-Gly to X-alpha-hydroxy-Gly or X-NH2 (X being a peptide or any other compound having a carbonyl group capable of forming a covalent bond with glycine) is accomplished enzymatically in the presence of keto acids, or salts or esters thereof, to provide a good yield without the necessity of catalase or similar enzymatic reaction enhancers. Peptidylglycine α-amidating monooxygenase (PAM) is a preferred enzyme for catalyzing the conversion. Alternatively, peptidylglycine α-hydroxylating monooxygenase (PHM) is utilized to convert X-Gly to X-alpha-hydroxy-Gly which may be recovered, or optionally may be simultaneously or sequentially converted to an amide by either a Lewis base or action of the enzyme peptidyl α-hydroxyglycine α-amidating lyase (PAL). Both PHM and PAL are functional domains of PAM.

    摘要翻译： 将X-Gly体外转化为X-α-羟基-Gly或X-NH2（X为肽或具有能与甘氨酸形成共价键的羰基的任何其它化合物）在酮酸存在下酶促完成 ，或其盐或酯，以提供良好的产率而不需要过氧化氢酶或类似的酶促反应增强剂。 肽基甘氨酸α-酰胺化单加氧酶（PAM）是催化转化的优选酶。 或者，使用肽基甘氨酸α-羟化化单加氧酶（PHM）将X-Gly转化成可被回收的X-α-羟基-Gly，或任选地可以通过路易斯碱同时或依次转化为酰胺，或通过 酶肽β-羟基甘氨酸α-酰胺化裂解酶（PAL）。 PHM和PAL都是PAM的功能域。

    Enzymatic reactions in the presence of keto acids
    2.
    发明申请
    Enzymatic reactions in the presence of keto acids 有权
    在酮酸存在下的酶反应

    公开(公告)号：US20090023892A1

    公开(公告)日：2009-01-22

    申请号：US12284552

    申请日：2008-09-22

    申请人： Angelo P. Consalvo ,  Nozer M. Mehta ,  William Stern ,  James P. Gilligan

    发明人： Angelo P. Consalvo ,  Nozer M. Mehta ,  William Stern ,  James P. Gilligan

    IPC分类号： C07K7/00

    CPC分类号： C12Y114/17003 ,  C12P13/02 ,  C12P21/02 ,  C12Y403/02005

    摘要： Conversion in vitro of X-Gly to X-alpha-hydroxy-Gly or X-NH2 (X being a peptide or any other compound having a carbonyl group capable of forming a covalent bond with glycine) is accomplished enzymatically in the presence of keto acids, or salts or esters thereof, to provide a good yield without the necessity of catalase or similar enzymatic reaction enhancers. Peptidylglycine α-amidating monooxygenase (PAM) is a preferred enzyme for catalyzing the conversion. Alternatively, peptidylglycine α-hydroxylating monooxygenase (PHM) is utilized to convert X-Gly to X-alpha-hydroxy-Gly which may be recovered, or optionally may be simultaneously or sequentially converted to an amide by either a Lewis base or action of the enzyme peptidyl α-hydroxyglycine α-amidating lyase (PAL). Both PHM and PAL are functional domains of PAM.

    摘要翻译： 将X-Gly体外转化为X-α-羟基-Gly或X-NH2（X为肽或具有能与甘氨酸形成共价键的羰基的任何其它化合物）在酮酸存在下酶促完成 ，或其盐或酯，以提供良好的产率而不需要过氧化氢酶或类似的酶促反应增强剂。 肽基甘氨酸α-酰胺化单加氧酶（PAM）是催化转化的优选酶。 或者，使用肽基甘氨酸α-羟化化单加氧酶（PHM）将X-Gly转化为可被回收的X-α-羟基-Gly，或任选地可以通过路易斯碱同时或顺序地转化为酰胺，或通过 酶肽肽α-羟基甘氨酸α-酰胺化裂解酶（PAL）。 PHM和PAL都是PAM的功能域。

    Purified enzymes participating in C-terminal amidation
    4.
    发明授权
    Purified enzymes participating in C-terminal amidation 失效
    纯化酶参与C-末端酰胺化

    公开(公告)号：US5871995A

    公开(公告)日：1999-02-16

    申请号：US070301

    申请日：1991-05-24

    申请人： Toshii Iida ,  Toshihiko Kaminuma ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto ,  Jiro Kishimoto ,  Ohji Ifuku ,  Ichiro Kato

    发明人： Toshii Iida ,  Toshihiko Kaminuma ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto ,  Jiro Kishimoto ,  Ohji Ifuku ,  Ichiro Kato

    IPC分类号： C07K1/107 ,  C12N9/02 ,  C12N15/53 ,  C12N15/60 ,  C12N9/48 ,  C12N9/14 ,  C12N9/78 ,  C12N9/80

    CPC分类号： C12N9/0071 ,  C07K1/107 ,  C12Y114/17003 ,  Y10S435/814 ,  Y10S435/815

    摘要： 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.

    摘要翻译： 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。

    C-terminal amidating enzyme composition, process for preparing, and use of the same
    5.
    发明授权
    C-terminal amidating enzyme composition, process for preparing, and use of the same 失效
    C-末端酰胺化酶组合物，其制备方法及其用途

    公开(公告)号：US5354675A

    公开(公告)日：1994-10-11

    申请号：US459829

    申请日：1990-01-29

    申请人： Toshii Iida ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto

    发明人： Toshii Iida ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto

    IPC分类号： C07K1/107 ,  C12N9/02 ,  C12P21/00 ,  C12N9/00

    CPC分类号： C12N9/0071 ,  C07K1/107 ,  C12Y114/17003

    摘要： A C-terminal amidating enzyme composition is disclosed having the ability to amidate the C-terminus of peptides with a C-terminal glycine. The C-terminal amidating enzyme is characterized by having a stable pH range of 5 to 9, metal ions and L-ascorbic acid as cofactors, two active fractions at molecular weights of about 50,000 and about 100,000 as determined from gel filtration, isoelectric points of about 4.5 pH for the active fraction having a molecular weight of about 50,000 and about 6.7 pH for the active fraction having a molecular weight of about 100,000 according to isoelectric point chromatography, and is activated by addition of catalase.

    摘要翻译： PCT No.PCT / JP89 / 00521 Sec。 371 1990年1月29日第 102（e）日期1990年1月29日PCT提交1989年5月25日PCT公布。 出版物WO89 / 12096 公开了C端氨酰化酶组合物，其具有用C-末端甘氨酸酰胺化肽​​的C末端的能力。 C末端酰胺化酶的特征在于具有5至9的稳定的pH范围，金属离子和L-抗坏血酸作为辅因子，分子量约50,000和约100,000的活性级分由凝胶过滤确定，等电点 根据等电点色谱法，对于分子量约为50,000的活性级分，对于分子量为约50,000的活性级分和约6.7pH的活性级分为大约4.5的pH，并且通过加入过氧化氢酶来活化。

    Method of preparing an enzyme participating in C-terminal amidation
    8.
    发明授权
    Method of preparing an enzyme participating in C-terminal amidation 失效
    制备参与C-末端酰胺化的酶的方法

    公开(公告)号：US6156555A

    公开(公告)日：2000-12-05

    申请号：US172120

    申请日：1998-10-14

    申请人： Toshii Iida ,  Toshihiko Kaminuma ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto ,  Jiro Kishimoto ,  Ohji Ifuku ,  Ichiro Kato

    发明人： Toshii Iida ,  Toshihiko Kaminuma ,  Yuka Fuse ,  Masahiro Tajima ,  Mitsuo Yanagi ,  Hiroshi Okamoto ,  Jiro Kishimoto ,  Ohji Ifuku ,  Ichiro Kato

    IPC分类号： C07K1/107 ,  C12N9/02 ,  C12N15/53 ,  C12N15/60 ,  C12N9/48 ,  C12N9/14 ,  C12N9/78 ,  C12N9/80 ,  C12N15/00

    CPC分类号： C12N9/0071 ,  C07K1/107 ,  C12Y114/17003 ,  Y10S435/814 ,  Y10S435/815

    摘要： 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.

    摘要翻译： 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。

    C-terminal .alpha.-amidating enzyme and process for production thereof
    9.
    发明授权
    C-terminal .alpha.-amidating enzyme and process for production thereof 失效
    C-末端α-酰胺化酶及其制备方法

    公开(公告)号：US5360727A

    公开(公告)日：1994-11-01

    申请号：US921600

    申请日：1992-08-03

    申请人： Hisayuki Matsuo ,  Kensaku Mizuno ,  Masayasu Kojima

    发明人： Hisayuki Matsuo ,  Kensaku Mizuno ,  Masayasu Kojima

    IPC分类号： C12N9/80 ,  C12N9/02 ,  C12N9/00 ,  C12P21/00

    CPC分类号： C12N9/0071 ,  C12Y114/17003

    摘要： The invention relates to a C-terminal .alpha.-amidating enzyme of porcine origin having the following properties: (1) the action is on a peptide or protein represented by the formula:X-R-Gly,wherein Gly represents a C-terminal glycine residue, R represents an amino acid residue to be .alpha.-amidated, and X represents a remaining portion of the peptide or protein to convert it to a peptide or protein represented by the formula:X-R-NH.sub.2,wherein R-NH.sub.2 represents a C-terminal .alpha.-amidated amino acid residue and X represents a remaining portion of the peptide or protein; (2) the optimal pH is 6.5 to 8.5; (3) the molecular weight is about 92,000 as determined by SDS-polyacrylamide gel electrophoresis; and (4) it contains the following peptide fragment:. . . Glu-Ala-Pro-Leu-Leu-Ile-Leu-Gly . . . .Further, the invention relates to a process for the production of the C-terminal .alpha.-amidating enzyme comprising the steps of extracting and purifying the enzyme from porcine atrium cordis exhibiting the enzyme activity.

    摘要翻译： 本发明涉及具有以下性质的猪来源的C-末端α-酰胺化酶：（1）作用是由下式表示的肽或蛋白：XR-Gly，其中Gly表示C-末端甘氨酸残基， R表示待酰胺化的氨基酸残基，X表示肽或蛋白质的剩余部分，以将其转化为由式XR-NH 2表示的肽或蛋白质，其中R-NH 2表示C末端α 酰胺化氨基酸残基，X表示肽或蛋白质的剩余部分; （2）最适pH为6.5〜8.5; （3）通过SDS-聚丙烯酰胺凝胶电泳测定分子量约为92,000; 和（4）它含有以下肽片段：。 。 。 Glu-Ala-Pro-Leu-Leu-Ile-Leu-Gly。 。 。 。 此外，本发明涉及一种生产C-末端α-酰胺化酶的方法，其包括从显示酶活性的猪心室提取和纯化酶的步骤。

    C-terminal alpha-amidating enzyme and process for production and use thereof
    10.
    发明授权
    C-terminal alpha-amidating enzyme and process for production and use thereof 失效
    C-末端α-酰胺化酶及其生产和使用的方法

    公开(公告)号：US4921797A

    公开(公告)日：1990-05-01

    申请号：US58919

    申请日：1987-06-05

    申请人： Hisayuki Matsuo ,  Kensaku Mizuno

    发明人： Hisayuki Matsuo ,  Kensaku Mizuno

    IPC分类号： C12N9/80 ,  C12N9/02

    CPC分类号： C12N9/0071 ,  C12Y114/17003

    摘要： C-terminal .alpha.-amidating enzyme preparations, including preparations AE-I, AE-II, AE-IIa and AE-IIb, from the skin of Xenopus laevis, wherein all components can convert a peptide having a glycine residue at its C-terminal to a C-terminal amidated peptide lacking the glycine residue, and have a common N-terminal amino acid sequence represented by Ser-Leu-Ser---, and AE-I and AE-IIa have a molecular weight of about 39,000, AE-IIb has a molecular weight of about 34,000, and AE-II comprises two components having molecular weight of about 39,000 and 34,000; a process for production of the above-mentioned enzyme preparations; and a process for .alpha.-amidation of a peptide by using the above mentioned enzyme preparations.

    摘要翻译： C-末端α-酰胺化酶制剂，包括来自非洲爪蟾皮肤的制剂AE-I，AE-II，AE-IIa和AE-IIb，其中所有组分可以在其C-末端转化具有甘氨酸残基的肽 具有缺少甘氨酸残基的C末端酰胺化肽，并且具有由Ser-Leu-Ser ---表示的常见N-末端氨基酸序列，AE-I和AE-IIa具有约39,000的分子量，AE -IIb具有约34,000的分子量，并且AE-II包含分子量为约39,000和34,000的两种组分; 制备上述酶制剂的方法; 以及通过使用上述酶制剂对肽进行α-酰胺化的方法。