Composition for measuring glucose having improved substrate specificity

A technology for glucose and glucose dehydrogenase, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of the action of maltose and other problems, and achieve the effect of high-precision analysis

Inactive Publication Date: 2007-02-14
TOYO TOYOBO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with glucose oxidase, the wild-type PQQGDH has a problem in its activity on sugar substrates other than glucose (so-called substrate specificity), especially in its activity on maltose.

Method used

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  • Composition for measuring glucose having improved substrate specificity
  • Composition for measuring glucose having improved substrate specificity
  • Composition for measuring glucose having improved substrate specificity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0189] Embodiment 1: The gene recombination production bacterium of modified PQQ-dependent glucose dehydrogenase makes the expression plasmid pNPG5 of wild-type PQQ-dependent glucose dehydrogenase, which is a multiple cloning site in vector (vector) pBluescript SK (-), A substance marking a PQQ-dependent glucose dehydrogenase structural gene derived from Acinetobacter baumannii NCIMBI1517 strain was inserted. The triplet of the recombinant plasmid pNPG5 and the amino acid labeled with the mutation introduction site was performed based on the synthetic oligonucleotide of about 40mer contained in the center, using the QuickChangTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) according to its protocol. The mutation processing operation obtains the markers that replace 168-position Gln with Ala, 169-position Leu with Pro, 170-position Ala with Met, 245-position Glu with Asp, 342-position Met with Ile, and 351-position Ala with Thr Recombinant plasmid of modified PQQ-...

Embodiment 2

[0231] Example 2: Preparation of whole enzyme expression purified enzyme

[0232] Put 500ml of Terrific broth into a 2L Sakaguchi shake flask, autoclave at 121°C for 20 minutes, and add streptomycin after cooling to make the concentration reach 100μg / ml. Use 5 ml of the culture solution of Pseudomonas putida (Pseudomonas putida) TE3493 (pNPG6-Q168A+L169P+A170M+E245D+M342I+A351T) cultured in PY medium containing 100 μg / ml streptomycin in advance for 24 hours at 30°C Inoculate and culture at 30°C with aeration and stirring for 40 hours. At the end of the culture, the activity of PQQ-dependent glucose dehydrogenase was determined according to the above-mentioned activity, and it was about 5 U / ml per 1 ml of culture solution.

[0233] The bacteria were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.0), and disrupted by ultrasonic waves. Centrifuge again, and the obtained supernatant is the crude enzyme liquid. The obtained crude enzyme solution was chrom...

Embodiment 3

[0234] Example 3: Confirmation of substrate specificity of modified PQQGDH

[0235] The activity of the purified modified PQQGDH produced in Example 2 on maltose was compared with that of wild-type PQQGDH (GLD-321 manufactured by Toyobo). Using the method of Test Example 1 (wherein, the substrate concentration in the assay mixture is 4.8 mM), the activities of the case where glucose and maltose were used as the substrate were respectively calculated, and the activity value when maltose was used was calculated. Relative to the ratio when glucose is used, it is used as maltose activity. As a result, the maltose activity of the wild type was 110%, whereas that of the modified PQQGDH was 13%, and it was confirmed that substrate specificity was improved.

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Abstract

The present invention relates to a method for lowering activity with respect to maltose in glucose measurement comprising a step of reacting modified pyrroloquinoline quinone dependent glucose dehydrogenase subjected to amino acid sequence modification, wherein pyrroloquinoline quinone dependent glucose dehydrogenase is reacted in the presence of at least one type of substance selected from the group comprising succinic acid, malonic acid, glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid, maleic acid, potassium chloride, ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium alpha-ketoglutarate, fumaric acid, glycine, glutamic acid, serine and citric acid.

Description

technical field [0001] The invention relates to a modified pyrroloquinoline quinone-dependent glucose dehydrogenase comprising a modified amino acid sequence (hereinafter, pyrroloquinoline quinone is described as PQQ, glucose dehydrogenase is described as GDH, pyrrole Method for reducing reactivity to maltose in glucose measurement described as PQQGDH) reaction process of quinoline quinone-dependent glucose dehydrogenase, composition for glucose measurement with reduced reactivity to maltose, glucose sensor, and method for producing the same . Background technique [0002] Pyrroloquinoline quinone-dependent glucose dehydrogenase (also referred to as PQQGDH in this application) is glucose dehydrogenase (GDH) with pyrroloquinoline quinone (PQQ) as a coenzyme. Because it catalyzes the oxidation of glucose to gluconolactone, it can be used for the determination of glucose. The concentration of glucose in the blood is an important sign of diabetes, and it is a very important in...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12Q1/54
Inventor 北林雅夫相场洋志
Owner TOYO TOYOBO CO LTD
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