Fructosyl amino acid oxidase

An amino acid, fructosyl technology, applied in the direction of enzymes, biochemical equipment and methods, instruments, etc., can solve problems such as affecting the measurement results

Active Publication Date: 2012-05-23
F HOFFMANN LA ROCHE & CO AG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when fructosyl amino acids are determined by fructosyl amino acid oxidase containing a medium, the dissolved oxygen level in the reaction system will greatly affect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Preparation and Characterization of N1-1 FAOD Mutant

[0076] Structure-function studies of monomeric sarcosine oxidase (MSOX) revealed that the proton transfer system (PRS) involved in transferring electrons from FAD to oxygen contains Thr48 and Lys 265 as well as four H 2 O molecules (Trickey et al., Structure, 7, 331-345, 1999). The active site of fructosyl amino acid oxidase from Pichia pastoris N1-1 strain (N1-1 FAOD, SEQ ID NO: 2) has high homology to MSOX, especially in the conserved residues expected to be responsible for the proton transport system .

[0077] Using the MSOX structure, a predicted structural model of N1-1 FAOD was constructed, predicting that amino acid residues Asn44, Ser46, Asn47, Lys48 and Lys269 are involved in proton and electron transport from FAD. Single or double mutations were introduced into those amino acid residues with the aim of modifying the electron acceptor availability of N1-1 FAOD.

[0078] Use QuickChange ? Methods (Strat...

Embodiment 2

[0084] Purification of N1-1 wild type and N47 mutant FAOD

[0085] Recombinant FAOD was purified as follows. First, an enzyme-containing water-soluble fraction was prepared from recombinant Escherichia coli. Escherichia coli containing the expression vector was cultured in 7L LB medium (37°C, 50 μg / ml ampicillin in a 10L fermenter), and then at about OD 660 = 0.7, the expression was induced with IPTG (final concentration: 0.3 mM), and the culture temperature was lowered to 30°C. Cells were suspended in 100 mM PPb (pH 7.0) and disrupted 4 times with a French cell press. The supernatant was subjected to ultracentrifugation (40,000 g, 90 minutes) and dialyzed against 10 mM PPb (pH 7.0) overnight at 4°C to prepare a water-soluble fraction.

[0086] The water-soluble fraction was further subjected to liquid chromatography to prepare purified enzyme. The enzyme was further purified by anion-exchange chromatography (DEAE-5PW). The water-soluble fraction was adsorbed onto an ani...

Embodiment 3

[0093] Preparation of PnFPOX mutants

[0094] prepared and characterized from Phaeosphaeria nolorum A mutant of fructosyl amino acid oxidase (PnFPOX , SEQ ID NO: 1).

[0095] Alignment based on N1-1 FAOD and PnFPOX ( figure 1 ) and results from Examples 1 and 2 predict that Asn56 is involved in proton and electron transfer from FAD. Different mutations were introduced into Asn56 with the aim of modifying the electron acceptor availability of PnFAOD.

[0096] Mutations were introduced using site-directed mutagenesis as described in Example 1, and BL21 (DE3) cells were transformed with expression vectors containing wild-type or mutant PnFPOX. Cultured cells were resuspended in 10 mM PPB, pH 7.0, and lysed by sonication. Centrifuge the lysate at 10,000 g for 20 min at 4°C and the supernatant at 50,000 rpm for 60 min at 4°C. Oxidase and dehydrogenase activities were measured as described in Example 1.

[0097] The activities of crude PnFPOX wild-type and Asn56 variants are...

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Abstract

Disclosed is a mutant fructosyl amino acid oxidase modified at an amino acid residue involved in proton relay system. The mutant fructosyl amino acid oxidase has reduced oxidase activity while substantially maintaining its dehydrogenase activity. The invention also provides an assay device and assay method for measuring glycated protein.

Description

technical field [0001] The present invention relates to fructosyl amino acid oxidase (also known as fructosylamine oxidase) for use in kits and sensors for measuring glycated proteins such as glycated albumin, fructosyl peptides, HbA1c and fructosyl valine (FV). More specifically, the present invention relates to mutant fructosyl amino acid oxidases with reduced oxidase activity. Background technique [0002] Glycated proteins are produced non-enzymatically via a covalent bond between the amino group on the protein and the reducing end of the sugar, also known as Amadori compounds. In blood, glucose binds to valine at the N-terminal of the β-chain of hemoglobin to generate glycated hemoglobin (glycohemoglobin; HbA1c). The abundance ratio of HbA1c to hemoglobin is higher in patients suffering from diabetes compared to normal healthy individuals, and the concentration of HbA1c in the blood is known to reflect blood glucose levels over the past few weeks. Thus, the concentra...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12Q1/26
CPCC12Q1/005G01N33/6803C12N9/0022G01N33/723G01N33/6806
Inventor K.索德K.伊克布库罗
Owner F HOFFMANN LA ROCHE & CO AG
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