Creatine amidinohydrolase, production thereof and use thereof

a technology of amidinohydrolase and creatine, which is applied in the field of creatine amidinohydrolase, can solve the problems of reducing the heat stability and the km value of creatine, affecting the stability of creatine amidinohydrolase, and affecting the use of enzymes as reagents

Inactive Publication Date: 2006-10-17
TOYOBO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]The present invention is based on the successful provision of a creatine amidinohydrolase gene which expresses a novel creatine amidinohydrolase having a small Km value for creatine, by introducing a mutation, by genetic engineering and protein engineering, into a creatine amidinohydrolase gene derived from conventionally known bacteria belonging to the genus Alcaligenes, which is a known creatine amidinohydrolase having a rather small Km value. The creatine amidinohydrolase of the present invention can be produced in large amounts by culturing a microorganism capable of expressing said gene in a nutrient medium.
[0012]The novel creatine amidinohydrolase of the present invention has a very small Km value for creatine as compared to conventionally known enzymes, and shows superior reactivity to creatine contained in a trace amount in a sample. Thus, it is useful as a reagent for determining creatine or creatinine with high sensitivity and high precision.

Problems solved by technology

Yet, the creatine amidinohydrolase produced from various known cell lines show lower heat stability and greater Km value for creatine.
The enzymes derived from the bacteria belonging to the genus Corynebacterium, Micrococcus, Actinobacillus or Bacillus (Japanese Patent Examined Publication No. 76915 / 1991) is thermally stable at a temperature not more than 50° C., whereas Km value for creatine is as great as about 20 mM, and these enzymes are not suitable for use as reagents for clinical tests.

Method used

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  • Creatine amidinohydrolase, production thereof and use thereof
  • Creatine amidinohydrolase, production thereof and use thereof
  • Creatine amidinohydrolase, production thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

reference example 1

Isolation of chromosomal DNA

[0107]The chromosomal DNA of Alcaligenes•faecalis TE3581 was isolated by the following method.

[0108]The cells (FERM P-14237) were shake-cultured overnight at 30° C. in a nutrient broth (150 ml) and the cells were collected by centrifugation (8000 rpm, 10 min). The cells were suspended in a solution (5 ml) containing 10% sucrose, 50 mM Tris-HCl (pH 8.0) and 50 mM EDTA, and a lysozyme solution (1 ml, 10 mg / ml) was added. The mixture was incubated at 37° C. for 15 min. Then, 10% SDS solution (1 ml) was added. An equivalent amount (1 ml) of a chloroform•phenol solution (1:1) was added to this mixture. The mixture was stirred and separated into an aqueous layer and a solvent layer by centrifugation at 10,000 rpm for 3 min. The aqueous layer was separated, and onto this aqueous layer was gently layered a 2-fold amount of ethanol. The content was slowly stirred with a glass rod to allow the DNA to wind around the rod.

[0109]This DNA was dissolved in 10 mM Tris-HC...

reference example 2

Preparation of DNA fragment containing a gene encoding creatinine amidinohydrolase and recombinant vector containing said DNA fragment

[0110]The DNA (20 μg) obtained in Reference Example 1 was partially cleaved with restriction enzyme Sau3AI (Toyo Boseki Kabushiki Kaisha) and 2-10 kbp fragments were recovered by sucrose density gradient centrifugation. Meanwhile, pBluescript KS(+) cleaved with restriction enzyme BamHI (Toyo Boseki Kabushiki Kaisha) was dephosphorylated with bacterial alkaline phosphatase (Toyo Boseki Kabushiki Kaisha). Then, the both DNAs were treated with T4DNA ligase (1 unit, Toyo Boseki Kabushiki Kaisha) at 16° C. for 12 hr to ligate the DNA. Escherichia coli JM109 competent cell (Toyo Boseki Kabushiki Kaisha) was transformed with the ligated DNA and plated onto a creatine amidinohydrolase activity detection agar medium [0.5% yeast extract, 0.2% meat extract, 0.5% polypeptone, 0.1% NaCl, 0.1% KH2PO4, 0.05% MgSO4 / 7H2O, 1.15% creatine, 10 U / ml sarcosine oxidase (Toy...

example 1

Preparation of recombinant plasmid pCRH273 by mutating creatine amidinohydrolase gene

[0113]The region of from β-galactosidase structural gene derived from the vector to the upstream region of the creatine amidinohydrolase structural gene of the insert DNA was deleted from the recombinant plasmid pCRH173 of Reference Example 2, using the synthetic DNA depicted in SEQ ID No:3 and a commercially available mutation introduction kit (Transformer™; Clonetech) to prepare recombinant plasmid pCRH173M. The detailed method for introducing the mutation was given in the protocol attached to the kit.

[0114]The pCRH173M was cleaved with restriction enzyme EcoRI (Toyo Boseki Kabushiki Kaisha) and self-ligated to prepare pCRH273 (FIG. 1).

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Abstract

A creatine amidinohydrolase having the following physicochemical properties:Action: catalyzing the following reaction;creatine+H2O→sarcosine+ureaOptimum temperature: about 40-50° C.Optimum pH: pH about 8.0-9.0Heat stability: not more than 50° C. (pH 7.5, 30 min)Km value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: about 3.5-10.00 mMMolecular weight: about 43,000 (SDS-PAGE)Isoelectric point: 3.54.5,a method for producing said enzyme, comprising culture of microorganism producing said enzyme, a method for the determination of creatine or creatinine in a sample using said enzyme, and a reagent therefor.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001]More than one reissue application has been filed for the reissue of U.S. Pat. No. 6,080,553. The reissue applications are the present application and application Ser. No. 09 / 940,941, each of which is a divisional reissue of U.S. Pat. No. 6,080,553. <?insert-end id="INS-S-00002" ?>FIELD OF THE INVENTION[0002]The present invention relates to a novel creatine amidinohydrolase, specifically, a novel creatine amidinohydrolase having a very low Km value for creatine, and a method for producing said enzyme. The present invention also relates to a method for the determination of creatine or creatinine in a sample by the use of said enzyme, and a reagent therefor.BACKGROUND OF THE INVENTION[0003]A creatine and a creatinine are found in blood and urine. A quick and accurate determination of their amounts is very important in making diagnosis of the diseases such as uremia, chronic nephritis, acute nephritis, giantism, tonic muscular dystroph...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N9/78C07H21/04C12N1/21C12N15/01C12Q1/34C12N15/09C12N15/00C12N15/52C12R1/05C12R1/19G01N33/52
CPCC12Q1/34G01N33/52G01N2333/978Y10S435/829
Inventor SOGABE, ATSUSHIHATTORI, TAKASHINISHIYA, YOSHIAKIKAWAMURA, YOSHIHISA
Owner TOYOBO CO LTD
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