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Modified sarcosine oxidase, process for producing the same and reagent composition using the same

A technology for sarcosine oxidase and sarcosine oxidase activity, which is applied in the field of sarcosine oxidase and can solve problems such as the unclear role of the original substrate creatine

Active Publication Date: 2005-12-21
TOYO TOYOBO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve the above problems, our group reported a sarcosine oxidase (JP-10-248572-A) with reduced effect on proline by modifying wild-type sarcosine oxidase by protein engineering, but it had no effect on the original substrate. The role of biocreatine is still unclear, and more improvements are needed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1. Construction of a plasmid expressing sarcosine oxidase

[0114] Expression plasmid pSAOEP3 for expression of sarcosine oxidase derived from Arthrobacter sp. TE1826 strain was constructed by the method described in JP-7-163341-A. This expression plasmid has an approximately 1.7 kbp DNA insert containing the gene encoding the sarcosine oxidase of TE1826 at the multiple cloning site of pUC18. Its nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence of sarcosine oxidase deduced from the nucleotide sequence is shown in SEQ ID NO:1.

Embodiment 2A

[0115] Example 2A. Preparation of modified sarcosine oxidase gene

[0116] The preparation method of the recombinant plasmid (pSAOM1A) of the modified sarcosine oxidase that encodes 89 lysines replaced by arginine in the amino acid sequence of SEQ ID NO: is to use the expression plasmid pSAOEP3 containing the sarcosine oxidase gene and Synthetic oligonucleotide of SEQ ID NO: 3 and its synthetic complementary oligonucleotide using QuickChange TM The site-directed mutation kit (provided by Stratagen) was operated according to the procedure, followed by sequencing.

[0117] The preparation method of the recombinant plasmid (pSAOM2A) of the modified sarcosine oxidase encoding the 155th cysteine ​​in the amino acid sequence of 1 replaced by isoleucine (pSAOM2A) is to use pSAOEP3 and the synthetic oligo of SEQ ID NO: 4 Nucleotides and their synthetic complementary oligonucleotides, using QuickChange TM Site-directed mutagenesis kit (provided by Stratagen), and operated in the sa...

Embodiment 3A

[0129] Example 3A. Preparation of modified sarcosine oxidase

[0130] Transform Escherichia coli JM109 competent cells with the following recombinant plasmids pSAOM1A, pSAOM2A, pSAOM3A, pSAOM4A, pSAOM5A, pSAOM6A, pSAOM7A, pSAOM8A, pSAOM9A, pSAOM10A, pSAOM11A, pSAOM12A and pSAOM13A to obtain transformants.

[0131] Put 400 mL of Terrific nutrient solution in a 2L Sakaguchi flask, sterilize at 121°C for 20 minutes, cool, and then add separately sterilized and filtered ampicillin at a final concentration of 100 μg / mL. The culture solution (5 mL) of Escherichia coli JM109 (pSAOM1A) previously cultured in LB medium containing 100 μg / mL ampicillin at 30° C. for 16 hours was inoculated into the above-mentioned medium, and cultured with shaking at 30° C. for 20 hours with aeration. After the end of the culture, the sarcosine oxidase activity in the above-mentioned activity measurement was about 9.5 U / mL of the culture solution.

[0132] The above-mentioned bacterial cells were coll...

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Abstract

The present invention discloses a protein, which still has sarcosine oxidase activity after being modified by adding, deleting, inserting or replacing at least one amino acid in the amino acid sequence constituting the protein having sarcosine ammoniaase activity, characterized by Improved stability in liquid compared to unmodified protein and / or reduced effect on L-proline compared to unmodified protein. Sarcosine oxidase has at least one of the following characteristics, namely, based on the effect of creatine on L-proline, measured at 37°C and pH 8.0, is 0.7% or less and the Km of L-proline The value is 150 mM or more; the method for producing sarcosine oxidase having excellent substrate specificity comprises culturing a microorganism capable of producing sarcosine oxidase and collecting sarcosine oxidase from the culture medium; and containing sarcosine A reagent for the detection of creatinine for amino acid oxidase.

Description

technical field [0001] The present invention relates to a sarcosine oxidase obtained by modifying a protein having sarcosine oxidase activity with protein engineering technology, characterized in that it has improved liquid stability, excellent substrate specificity and proline Reduced effect, and methods of producing sarcosine oxidase and reagent compositions using same. Background technique [0002] Sarcosine oxidase (EC 1.5.3.1) is an enzyme used together with other enzymes such as creatinase, creatinase and peroxidase to detect creatine and creatinine in body fluids, which are used in the diagnosis of muscle diseases and kidney diseases clinical indicators. Sarcosine oxidase acts on creatine as a substrate in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide. [0003] It is known that sarcosine oxidase is transmitted by Bacillus (JP-54-52789-A, JP-61-162174-A), Corynebacterium (J.Biochem.89:599, 1981), Cylindrocarpon (JP-56-92790-...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/02C12Q1/26
Inventor 岸本高英曾我部敦冈正则
Owner TOYO TOYOBO CO LTD
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