公开(公告)号：US5451520A

公开(公告)日：1995-09-19

申请号：US343972

申请日：1994-11-18

申请人： Keisuke Furukawa ,  Kyoko Hashimoto ,  Masaru Suzuki

发明人： Keisuke Furukawa ,  Kyoko Hashimoto ,  Masaru Suzuki

IPC分类号： C12N9/78 ,  C12N9/80 ,  C12Q1/34 ,  C12R1/05 ,  C12N1/00 ,  C12N1/12

CPC分类号： C12Q1/34 ,  C12N9/80 ,  G01N2333/978 ,  Y10S435/829

摘要： A creatine amidinohydrolase with the following physicochemical properties is prepared:(a) action: hydrolysis of 1 mole of creatine to form 1 mole of sarcosine and 1 mole of urea;(b) substrate specificity: specific for a creatine substrate;(c) optimum pH: 7-9;(d) optimum temperature: around 35.degree.-45.degree. C.;(e) pH stability: stable in the range of pH 5.0-10.5 at 25.degree. C. for 17 hours;(f) thermal stability: stable at a temperature up to about 45.degree. C. at pH 7.5 for 30 min.;(g) inhibitors: AgNO.sub.3, HgCl.sub.2, CuSO.sub.4, etc.; and(h) molecular weight: about 80,000.+-.5000 as determined by gel filtration.The creatine amidinohydrolase is stable in high pH range and possesses a small Km value, so that it can be purified in high pH range resulting in more easy and simple production than the conventional enzyme, and the lower Km value enables reduction in the period of time and in the amount of the enzyme for each measurement. The creatine amidinohydrolase is obtained by culturing Alkaligenes sp. KS-85 FERM BP-4487.

摘要翻译： 制备具有以下物理化学性质的肌酸脒基水解酶：（a）作用：水解1摩尔肌酸以形成1摩尔肌氨酸和1摩尔尿素; （b）底物特异性：对肌酸底物有特异性; （c）最适pH：7-9; （d）最佳温度：约35°-45℃。 （e）pH稳定性：在25℃下在pH5.0-10.5的范围内稳定17小时; （f）热稳定性：在高达约45℃的温度下在pH7.5下稳定30分钟; （g）抑制剂：AgNO 3，HgCl 2，CuSO 4等; 和（h）通过凝胶过滤测定的分子量：约80,000 +/- 5000。 肌酸酰氨基水解酶在高pH范围内是稳定的，具有小的Km值，因此可以在高pH范围内进行纯化，从而比常规酶更容易和简单地生产，而较低的Km值可以减少一段时间 以及用于每次测量的酶的量。 肌酸酰胺水解酶通过培养阿尔卡氏菌属获得。 KS-85 FERM BP-4487。

公开(公告)号：US5932466A

公开(公告)日：1999-08-03

申请号：US947726

申请日：1997-10-09

申请人： Keisuke Furukawa ,  Toshio Ichikawa ,  Masaru Suzuki ,  Yasuji Koyama

发明人： Keisuke Furukawa ,  Toshio Ichikawa ,  Masaru Suzuki ,  Yasuji Koyama

IPC分类号： C12N15/09 ,  C12N1/21 ,  C12N9/78 ,  C12N9/80 ,  C12N15/55 ,  C12R1/05 ,  C12R1/19 ,  C07H21/04 ,  C12N15/63

CPC分类号： C12N9/80

摘要： The present invention relates to a creatine amidinohydrolase gene coding for the amino acid sequence of SEQ ID NO. 2, a recombinant DNA comprising the creatine amidinohydrolase gene inserted into a vector DNA, and a process for producing creatine amidinohydrolase by culturing a microorganism belonging to the genus Escherichia carrying said recombinant DNA and having the ability to produce creatine amidinohydrolase in medium and then recovering creatine amidinohydrolase from the culture.According to the present invention, creatine amidinohydrolase can be efficiently produced without adding creatine to medium.

摘要翻译： 本发明涉及编码SEQ ID NO：1的氨基酸序列的肌酸脒基水解酶基因。 2，包含插入到载体DNA中的肌酸脒基水解酶基因的重组DNA和通过培养携带所述重组DNA并具有在培养基中产生肌酸脒基水解酶的能力然后回收肌酸的属于携带所述重组DNA的埃希氏菌属的微生物来生产肌酸脒基水解酶的方法 来自培养基的脒基水解酶。 根据本发明，可以在不向培养基中添加肌酸的情况下有效地制备肌酸脒基水解酶。

公开(公告)号：US06821766B1

公开(公告)日：2004-11-23

申请号：US09869280

申请日：2001-06-27

申请人： Keisuke Furukawa ,  Yasuji Koyama ,  Masaru Suzuki

发明人： Keisuke Furukawa ,  Yasuji Koyama ,  Masaru Suzuki

IPC分类号： C12N914

CPC分类号： C12Y305/03003 ,  C12N9/78

摘要： The present invention relates to a thermostable creatine amidinohydrolase having the following physicochemical properties: (a) hydrolyzing 1 mol of creatine to give 1 mol of sarcosine and 1 mol of urea; (b) having a substrate specificity to creatine; (c) having an optimum pH of 7.0 to 8.0; (d) having a stable pH range of 4.0 to 11.0; (e) having the optimum temperature of around 45° C.; (f) being thermostable at 53° C.; (g) having a molecular weight of 92,000 Da (as determined by gel filtration), and to a process for producing the thermostable creatine aimdinohydrolase.

摘要翻译： 本发明涉及具有以下物理化学性质的热稳定性肌酸脒基水解酶：（a）水解1mol肌酸，得到1mol肌氨酸和1mol尿素; （b）对肌酸具有底物特异性; （c）具有7.0至8.0的最佳pH; （d）具有4.0至11.0的稳定pH范围; （e）具有约45℃的最佳温度; （f）在53℃热稳定; （g）分子量为92000Da（通过凝胶过滤测定），以及制备热稳定性肌酸靶标二水解酶的方法。

公开(公告)号：US07189548B2

公开(公告)日：2007-03-13

申请号：US10829427

申请日：2004-04-22

申请人： Keisuke Furukawa ,  Naoki Kajiyama

发明人： Keisuke Furukawa ,  Naoki Kajiyama

IPC分类号： C12N9/04 ,  C12N1/20 ,  C07H21/24

CPC分类号： C12N9/0034

摘要： (1) A modified sarcosine oxidase with improved stability in the acidic range compared to a wild-type sarcosine oxidase. (2) A sarcosine oxidase gene encoding a modified sarcosine oxidase of the following (a), (b), or (c): (a) protein composed of the amino acid sequence represented by SEQ ID NO: 1 (b) protein composed of an amino acid sequence wherein one or some amino acid(s) are deleted, substituted, or added from the amino acid sequence represented by SEQ ID NO: 1, and which has sarcosine oxidase activity (c) protein composed of an amino acid sequence which shows 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1, and which has sarcosine oxidase activity According to the present invention, sarcosine oxidases, in particular sarcosine oxidases which show optimal pH and high activity in the slightly acidic range and have improved stability can be prepared efficiently, thus making the invention industrially useful.

摘要翻译： （1）与野生型肌氨酸氧化酶相比，在酸性范围内具有改善的稳定性的改性肌氨酸氧化酶。 （2）编码以下（a），（b）或（c）的修饰肌氨酸氧化酶的肌氨酸氧化酶基因：（a）由SEQ ID NO：1（b）表示的氨基酸序列组成的蛋白质， 的氨基酸序列，其中一个或一些氨基酸被从SEQ ID NO：1所示的氨基酸序列中缺失，取代或添加，并具有肌氨酸氧化酶活性（c）由氨基酸序列组成的蛋白质 其显示与SEQ ID NO：1所示的氨基酸序列具有80％或更高的同源性，并且其具有肌氨酸氧化酶活性根据本发明，肌氨酸氧化酶，特别是在微酸性中显示最佳pH和高活性的肌氨酸氧化酶 可有效制备稳定性，从而使本发明在工业上有用。

公开(公告)号：US07132253B2

公开(公告)日：2006-11-07

申请号：US10990477

申请日：2004-11-18

申请人： Keisuke Furukawa ,  Naoki Kajiyama

发明人： Keisuke Furukawa ,  Naoki Kajiyama

IPC分类号： C12Q1/26 ,  C12Q1/34 ,  C12P21/06 ,  C12N9/06 ,  C07H21/04 ,  C12N15/00

CPC分类号： C12N9/0034 ,  C12Q1/26

摘要： A modified sarcosine oxidase having a lowered activity for N-ethylglycine. Such a modified oxidase may have the following physicochemical properties: (a) action: hydrolyzes 1 mol of sarcosine to produce 1 mol of glycine and 1 mol of formaldehyde; (b) substrate specificity: reactivity for N-ethylglycine is 70% or less compared with that of an unmodified protein; (c) optimal pH: around 8.0; (d) stable pH range: between 6.5 and 11.0; (e) optimal temperature: 55° C.; (f) thermostability: 55° C. or less; and (g) molecular weight: approximately 43,000 (SDS-PAGE). Genes, vectors and host cells encoding or expressing modified sarcosine oxidases. The modified sarcosine oxidases of the present invention can be used as reagents for measuring creatinine or creatine. The reagents containing the modified sarcosine oxidases used therein are hardly affected by N-ethylglycine, enabling more precise measurement than ever before.

摘要翻译： 对N-乙基甘氨酸具有降低的活性的改性肌氨酸氧化酶。 这样的改性氧化酶可以具有以下物理化学性质：（a）作用：水解1mol肌氨酸，产生1mol甘氨酸和1mol甲醛; （b）底物特异性：与未修饰蛋白质相比，N-乙基甘氨酸的反应性为70％以下; （c）最佳pH：约8.0; （d）稳定的pH范围：6.5-11.0; （e）最佳温度：55℃。 （f）热稳定性：55℃以下; 和（g）分子量：约43,000（SDS-PAGE）。 编码或表达修饰肌氨酸氧化酶的基因，载体和宿主细胞。 本发明的改性肌氨酸氧化酶可用作测量肌酸酐或肌酸的试剂。 包含其中使用的改性肌氨酸氧化酶的试剂几乎不受N-乙基甘氨酸的影响，能够比以前更精确地测量。

公开(公告)号：US20050287624A1

公开(公告)日：2005-12-29

申请号：US10990477

申请日：2004-11-18

申请人： Keisuke Furukawa ,  Naoki Kajiyama

发明人： Keisuke Furukawa ,  Naoki Kajiyama

IPC分类号： C12N15/09 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N9/06 ,  C12Q1/26 ,  C12Q1/28 ,  C12Q1/34 ,  C07H21/04 ,  C12Q1/32

CPC分类号： C12N9/0034 ,  C12Q1/26

摘要： A modified sarcosine oxidase having a lowered activity for N-ethylglycine. Such a modified oxidase may have the following physicochemical properties: (a) action: hydrolyzes 1 mol of sarcosine to produce 1 mol of glycine and 1 mol of formaldehyde; (b) substrate specificity: reactivity for N-ethylglycine is 70% or less compared with that of an unmodified protein; (c) optimal pH: around 8.0; (d) stable pH range: between 6.5 and 11.0; (e) optimal temperature: 55° C.; (f) thermostability: 55° C. or less; and (g) molecular weight: approximately 43,000 (SDS-PAGE). Genes, vectors and host cells encoding or expressing modified sarcosine oxidases. The modified sarcosine oxidases of the present invention can be used as reagents for measuring creatinine or creatine. The reagents containing the modified sarcosine oxidases used therein are hardly affected by N-ethylglycine, enabling more precise measurement than ever before.

摘要翻译： 对N-乙基甘氨酸具有降低的活性的改性肌氨酸氧化酶。 这样的改性氧化酶可以具有以下物理化学性质：（a）作用：水解1mol肌氨酸，产生1mol甘氨酸和1mol甲醛; （b）底物特异性：与未修饰蛋白质相比，N-乙基甘氨酸的反应性为70％以下; （c）最佳pH：约8.0; （d）稳定的pH范围：6.5-11.0; （e）最佳温度：55℃。 （f）热稳定性：55℃以下; 和（g）分子量：约43,000（SDS-PAGE）。 编码或表达修饰肌氨酸氧化酶的基因，载体和宿主细胞。 本发明的改性肌氨酸氧化酶可用作测量肌酸酐或肌酸的试剂。 含有其中使用的改性肌氨酸氧化酶的试剂几乎不受N-乙基甘氨酸的影响，能够比以前更精确地测量。

公开(公告)号：US07374918B2

公开(公告)日：2008-05-20

申请号：US11627484

申请日：2007-01-26

申请人： Keisuke Furukawa ,  Naoki Kajiyama

发明人： Keisuke Furukawa ,  Naoki Kajiyama

IPC分类号： C12N9/02 ,  C12N15/00 ,  C12N1/20 ,  C12P21/06 ,  C07H21/04

CPC分类号： C12N9/0034

摘要： An isolated DNA molecule encoding modified sarcosine oxidases having optimal pH, high activity in the slightly acidic range and improved stability and a method for preparing the modified sarcosine oxidases is disclosed.

摘要翻译： 公开了分离的DNA分子，其编码具有最佳pH，微酸性范围内的高活性和改善的稳定性的改性肌氨酸氧化酶和制备改性肌氨酸氧化酶的方法。

公开(公告)号：US06699700B1

公开(公告)日：2004-03-02

申请号：US09856645

申请日：2001-05-24

申请人： Keisuke Furukawa ,  Toshio Ichikawa

发明人： Keisuke Furukawa ,  Toshio Ichikawa

IPC分类号： C12N978

CPC分类号： C12N9/78 ,  C12Y305/03003

摘要： The present invention relates to a creatine amidinohydrolase having the following physicochemical properties: (a) hydrolyzing 1 mole of creatine to generate 1 mole of sarcosine and 1 mole of urea, (b) having a substrate specificity to creatine, (c) having an optimum pH ranging from 6.0 to 7.0, particularly a pH of about 6.5, (d) having a stable pH ranging from 4.0 to 11.0, (e) having an optimum temperature ranging from 50 to 55° C., and (f) having a molecular weight of approximately 92,000 daltons (as measured by gel filtration); and to a method for producing a creatine amidinohydrolase, which comprises, culturing a microorganism having an ability to produce the creatine amidinohydrolase, and recovering the creatine amidinohydrolase from the obtained culture. The creatine amidinohydrolase of the invention is characterized by being insusceptible to bilirubin when measuring creatinine, since its optimum pH is in the weakly acidic range.

摘要翻译： 本发明涉及具有以下物理化学性质的肌酸脒基水解酶：（a）水解1摩尔肌酸以产生1摩尔肌氨酸和1摩尔尿素，（b）对肌酸具有底物特异性，（c）具有最佳 pH范围为6.0-7.0，特别是约6.5的pH值，（d）pH值范围为4.0-11.0，（e）最适温度为50-55℃，（f）具有分子量 重量约92,000道尔顿（通过凝胶过滤测量）; 以及制备肌酸脒基水解酶的方法，其包括培养具有产生肌酸脒基水解酶的能力的微生物，并从所得培养物中回收肌酸脒基水解酶。 本发明的肌酸脒基水解酶的特征在于测量肌酸酐时不易胆红素，因为其最佳pH处于弱酸性范围。

公开(公告)号：US20050026265A1

公开(公告)日：2005-02-03

申请号：US10829427

申请日：2004-04-22

申请人： Keisuke Furukawa ,  Naoki Kajiyama

发明人： Keisuke Furukawa ,  Naoki Kajiyama

IPC分类号： C12N15/09 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N9/06 ,  C12N15/53 ,  C07H21/04

CPC分类号： C12N9/0034

摘要： (1) A modified sarcosine oxidase with improved stability in the acidic range compared to a wild-type sarcosine oxidase. (2) A sarcosine oxidase gene encoding a modified sarcosine oxidase of the following (a), (b), or (c): (a) protein composed of the amino acid sequence represented by SEQ ID NO: 1 (b) protein composed of an amino acid sequence wherein one or some amino acid(s) are deleted, substituted, or added from the amino acid sequence represented by SEQ ID NO: 1, and which has sarcosine oxidase activity (c) protein composed of an amino acid sequence which shows 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1, and which has sarcosine oxidase activity According to the present invention, sarcosine oxidases, in particular sarcosine oxidases which show optimal pH and high activity in the slightly acidic range and have improved stability can be prepared efficiently, thus making the invention industrially useful.

摘要翻译： （1）与野生型肌氨酸氧化酶相比，在酸性范围内具有改善的稳定性的改性肌氨酸氧化酶。 （2）编码以下（a），（b）或（c）的修饰肌氨酸氧化酶的肌氨酸氧化酶基因：（a）由SEQ ID NO：1（b）表示的氨基酸序列组成的蛋白质， 的氨基酸序列，其中一个或一些氨基酸被从SEQ ID NO：1所示的氨基酸序列中缺失，取代或添加，并具有肌氨酸氧化酶活性（c）由氨基酸序列组成的蛋白质 其显示与SEQ ID NO：1所示的氨基酸序列具有80％或更高的同源性，并且其具有肌氨酸氧化酶活性根据本发明，肌氨酸氧化酶，特别是在微酸性中显示最佳pH和高活性的肌氨酸氧化酶 可有效制备稳定性，从而使本发明在工业上有用。