# US 8,785,147 B2 · L-threonine analysis method and L-threonine dehydrogenase

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- Number: US 8,785,147 B2
- Document ID: US8785147B2
- Status: Granted
- Kind: B2
- Application number: 13/602,473
- Filed: 2012-09-04
- Published: 2013-02-28
- Granted: 2014-07-22
- Earliest claimed: 2010-03-04
- Assignees: Toyama Prefecture; Ajinomoto Co., Inc.
- Applicants: Yasuhisa Asano; Techawaree Ueatrongchit
- Inventors: Yasuhisa Asano; Techawaree Ueatrongchit
- Web page: https://patentgrep.com/patent/US8785147B2

## CPC

- C12Q 1/32: involving dehydrogenase
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12Q 1/005: {involving specific analytes or enzymes (including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for analyzing L-threonine contained in an specimen, which includes the steps of mixing a sample containing the specimen with an L-threonine dehydrogenase derived from Cupriavidus necator and a coenzyme NAD+ and analyzing the amount of NADH or 2-amino-3-oxobutyric acid after a predetermined period; an L-threonine dehydrogenase derived from Cupriavidus necator, which is a novel L-threonine dehydrogenase (TDH; EC 1.1.1.103) and can be utilized in the above-mentioned analysis method; a method for preparing a gene or the like to be used in the preparation of the enzyme, or a method for preparing the enzyme; an L-threonine analysis kit which includes (A) the L-threonine dehydrogenase and (B) a coenzyme NAD+; an enzyme preparation for use in the analysis of L-threonine, which includes the L-threonine dehydrogenase contained in a buffer solution; and an enzyme sensor utilizing the L-threonine dehydrogenase.

## Claims

### Claim 1

A method for analyzing the L-threonine contained in a specimen, comprising:
A) mixing a sample comprising a specimen and L-threonine dehydrogenase, with the coenzyme NAD+; and
B) determining the quantity of a product after a time period,
wherein the product is selected from the group consisting of NADH and 2-amino-3-oxybutyric acid;
wherein said L-threonine dehydrogenase has L-threonine dehydrogenase activity that is oxidative and specific for L-threonine to 2-amino-3-oxobutyric acid, and is selected from the group consisting of:
(i) L-threonine dehydrogenase from Cupriavidus necator;
(ii) a protein comprising the amino acid sequence of SEQ ID NO: 1;
(iii) a protein comprising the amino acid sequence of SEQ ID NO: 1, but having from 1 to 30 amino acid deletions, substitutions, and/or additions; and
(iv) a protein comprising an amino acid sequence having 90% or greater homology with the amino acid sequence of SEQ ID NO: 1.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the L-threonine dehydrogenase is from Cupriavidus necator.

### Claim 3 (depends on 2)

The method of claim 2, wherein the L-threonine dehydrogenase is from Cupriavidus necator NBRC 102504 (SEQ ID NO: 1).

### Claim 4 (depends on 1)

The method according to claim 1, wherein said determining is by a method selected from the group consisting of:
A) measuring absorbance in the sample to obtain the quantity of NADH,
B) observing the pigment generated by oxidation of NADH or by using electron carriers in a NADH− tetrazolium system, and
C) measuring fluorescence of a fluorescent pigment generated by NADH to obtain the quantity of NADH.

### Claim 5 (depends on 1)

The method of claim 1, wherein said determining comprises allowing the sample to produce amino acetone from the 2-amino-3-oxobutyric acid, mixing the resulting product with monoamine oxidase to produce ammonia and hydrogen peroxide, and measuring the quantity of the ammonia or hydrogen peroxide.