Fructosylamine oxidase

A technology based on amine oxidase and fructosyl, which is applied in the fields of enzymes, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2005-11-23
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no method is provided to enable the glycated amino acid of interest to a...

Method used

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  • Fructosylamine oxidase
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 : Screening and identification of microorganisms producing FAO

[0082] (1) Screening of microorganisms producing FAO

[0083] Glycation of VHL produces fructosyl valine-histidine-leucine (FVHL) identical to the N-terminal sequence of the glycohemoglobin beta chain. Methods for this are known to those skilled in the art.

[0084] FVHL medium

FVHL

Dipotassium phosphate

Sodium dihydrogen phosphate

Magnesium Sulfate Heptahydrate

calcium chloride dihydrate

vitamin mix *

metal solution **

distilled water

5g

1g

1g

0.5g

0.1g

0.1% (v / v)

1.0% (v / v)

q.s

total capacity

1000ml

[0085] Thiamine Hydrochloride

riboflavin

1mg

2

thbrthdrexvbdr

Vitamin B 6 HCl

Biotin

p-aminobenzoic acid

Niacin

folic acid

distilled water

2

2

0.1

1

2

0.1

q.s.

t...

Embodiment 2

[0110] Example 2 : Preparation and identification of FAO using GL2-1

[0111] (1) Partial purification of FAO

[0112] 1) Culture and prepare cell-free extract

[0113] Under the same medium composition and culture conditions, the GL-2 bacterial strain identified in Example 1 was cultured in the 100 ml GV browning medium described in Example 1 (2).

[0114] After culturing, the medium was filtered through a filter to collect mycelia. The obtained mycelium (0.6g) was suspended in 0.1M Tris-HCl buffer solution (pH8.0) containing 1mM DTT, and the Mini-BeadBeater TM (0.5 mm glass beads) and centrifuge (4° C., 10,000×g, 10 minutes) to obtain the supernatant, which is the cell-free extract.

[0115] 2) Ammonium sulfate classification

[0116] The cell-free extract obtained from 1) was dissolved in 50 mM Tris-HCl buffer containing 1 mM DTT and dialyzed against the same buffer for ammonium sulfate (30-80% saturation) fractionation.

[0117] 3) Resource Q column chromatography ...

Embodiment 3

[0146] Example 3 : Cloning of FAO cDNA

[0147] Genomic DNA of GL2-1 was prepared. Using genomic DNA as a template, the cDNA of FAO can be obtained by PCR.

[0148] (1) Genomic DNA preparation of GL2-1 strain

[0149] Genomic DNA was prepared from the GL2-1 strain according to a method comprising the following steps.

[0150] 1. GL2-1 strain was cultured in 15 ml of DP medium (1% Dextone, 1% peptone, 0.5% sodium chloride, pH7.4) at 30°C for 2-3 days.

[0151] 2. Collect fungal cells (wet weight, 0.3 g) by filtering through a glass filter (3GL).

[0152] 3. The resulting fungal cells are homogenized in a mortar containing liquid nitrogen with a pestle, further crushed under the action of a motor or the like, and collected into Corning tubes.

[0153] 4. After adding 2 ml of ice-cold TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), the mixture was vortexed slightly.

[0154] 5. After adding 2 ml of 50 mM EDTA and 0.5% SDS solution, the mixture was stirred several times by ...

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Abstract

The present invention provides a fructosylamine oxidase which is obtainable by culturing Fusarium proliferatum, and purifying two types of fructosylamine oxidase (FAO) with different substrate specificities from the culture, and which is useful in the measurement of amadori compounds.

Description

technical field [0001] The present invention relates to novel fructosylamine oxidases, and more particularly to fructosylamine oxidases derived from Fusarium proliferatum, methods for their preparation, and their use in the determination of amador (amador) ) applications in compounds. Background technique [0002] Amador compounds are formed when reactive substances with amino groups such as proteins, peptides or amino acids coexist with reducing sugars such as glucose sugar in blood or food. In this way, they combine non-enzymatically and irreversibly via amino and aldehyde groups, followed by Amador rearrangement to form Amador compounds. Since the rate of formation of the Amador compound is a function of the concentration of the reactive species, contact time, temperature, etc., various information about the sample containing this reactive species can be obtained from the amount of the Amador compound. Therefore, the analysis of Amador compounds is used in fields relate...

Claims

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Application Information

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IPC IPC(8): C12N15/53
Inventor 吉田信行谷吉树米原聪
Owner ARKRAY INC
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