Method for detecting D-2-hydroxyglutarate by using FAD dependent D-2-hydroxyglutarate dehydrogenase and resazurin

A technology of hydroxyglutarate dehydrogenase and hydroxyglutarate, which is applied in botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problem of poor substrate specificity, high detection cost, and sensitivity low cost, low cost, high sensitivity and low cost

Active Publication Date: 2021-04-02
THE SECOND HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since this method is a two-step reaction (the first step is N-D2HGDH catalyzing the oxidation of D-2-HG, and simultaneously reducing NAD to generate NADH; the second step is diaphorase oxidizing NADH, and simultaneously reducing resazurin), the detection There are many factors that affect the results. The two catalyzed two-step reaction coupling can realize the final electron transfer to resazurin to generate fluorescence, which introduces a lot of interference and low sensitivity; the detection system needs to use two kinds of enzymes, and needs to be added. High cofactor NAD, the detection cost is high; in addition, the NAD-dependent dehydrogenase is not only active on D-2-HG, but also active on D-2-hydroxyadipate, and the substrate of the enzyme used for detection is specific The oneness is not good, and there is a risk of interference in the detection

Method used

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  • Method for detecting D-2-hydroxyglutarate by using FAD dependent D-2-hydroxyglutarate dehydrogenase and resazurin
  • Method for detecting D-2-hydroxyglutarate by using FAD dependent D-2-hydroxyglutarate dehydrogenase and resazurin
  • Method for detecting D-2-hydroxyglutarate by using FAD dependent D-2-hydroxyglutarate dehydrogenase and resazurin

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preparation example Construction

[0071] 1. Preparation of AX-F-D2HGDH protein

[0072] 1.1 Acquisition of gene sequence

[0073] Achromobacter xylosoxidansATCC27061 is a common model strain of Achromobacter, and the A-F-D2HGDH derived from this Achromobacter is named AX-F-D2HGDH.

[0074] Enter Gene ID: 29509945 into the NCBI database to obtain the gene sequence (SEQ ID NO: 1, 1413bp) and amino acid sequence (SEQ ID NO: 2, 470aa) of AX-F-D2HGDH in Achromobacter xylosoxidansATCC27061.

[0075] Using sequence alignment tools well-known to researchers in the field (such as Nucleotide Blast in NCBI, etc.), the gene sequence of AX-F-D2HGDH was compared to obtain a sequence with an identity greater than 70%, and the encoded protein had D-2- Hydroxyglutarate dehydrogenase activity. Using sequence alignment tools well known to researchers in the field (such as Protein Blast in NCBI, etc.), compare the amino acid sequence of AX-F-D2HGDH to obtain a sequence with more than 50% identity, and the protein has D-2-hydrox...

Embodiment

[0128] 1. Preparation of AX-F-D2HGDH protein

[0129] 1.1 Acquisition of gene sequence

[0130] Achromobacter xylosoxidansATCC27061 is a common model strain of Achromobacter, and the A-F-D2HGDH derived from this Achromobacter is named AX-F-D2HGDH.

[0131]Enter Gene ID: 29509945 into the NCBI database to obtain the gene sequence (SEQ ID NO: 1, 1413bp) and amino acid sequence (SEQ ID NO: 2, 470aa) of AX-F-D2HGDH in Achromobacter xylosoxidansATCC27061.

[0132] Using sequence alignment tools well-known to researchers in the field (such as Nucleotide Blast in NCBI, etc.), the gene sequence of AX-F-D2HGDH was compared to obtain a sequence with an identity greater than 70%, and the encoded protein had D-2- Hydroxyglutarate dehydrogenase activity, and has high substrate specificity, only active on D-2-HG. Utilize the sequence alignment tools well-known to researchers in the field (such as ProteinBlast in NCBI, etc.), compare the amino acid sequence of AX-F-D2HGDH, and obtain a seq...

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Abstract

The invention discloses a method for detecting D-2-hydroxyglutarate by using FAD dependent D-2-hydroxyglutarate dehydrogenase and resazurin, and relates to the technical field of enzymatic detection.The method uses FAD dependent D-2-hydroxyglutarate dehydrogenase, resazurin and a PBS buffer solution to realize detection of D-2-hydroxyglutarate; and the FAD dependent D-2-hydroxyglutarate dehydrogenase is an AX-F-D2HGDH protein or a P-D2HGDH protein. The method is simpler in composition, lower in cost and simpler and more convenient to operate. Since no cofactor, coenzyme or diaphorase needs tobe added, introduced interference is less, sensitivity is higher, only one enzyme needs to be prepared, no exogenous cofactor NAD and the like need to be added, and meanwhile, the buffer solution canbe a PBS buffer solution which is lower in cost and easy to obtain, so that cost is lower.

Description

technical field [0001] The invention relates to the technical field of enzymatic detection, in particular to a method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin. Background technique [0002] 2-Hydroxyglutarate (2-HG) is generally the reduction product of 2-ketoglutarate. The 2-HG molecule contains an asymmetric carbon atom, which is divided into D-2- Hydroxyglutarate (D-2-HG or R-2-HG) and L-2-hydroxyglutarate (L-2-HG or S-2-HG), responsible for catalyzing the oxidation of D-2-HG in vivo The enzyme into 2-oxoglutarate is D-2-hydroxyglutarate dehydrogenase (D-2-hydroxyglutarate dehydrogenase; D2HGDH). [0003] It has been reported in the literature that the D2HGDH of a 70kg adult can decompose 40 mmol of D-2-HG per day. Mutations in the D2HGDH coding gene can cause an increase in the concentration of D-2-HG, resulting in D-2-hydroxyglutaric acidemia (D-2-HGA). D-2-HGA is an autosomal recessive genetic disease....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C12N9/04C12N15/53C12N15/70G01N21/64
CPCC12Q1/32C12N9/0006C12N15/70G01N21/6428C12Y101/99002G01N2333/904
Inventor 张文
Owner THE SECOND HOSPITAL OF SHANDONG UNIV
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