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Kit for detecting D-psicose and detection method

A technology of allulose and a kit, applied in the field of detection, can solve the problems of being unsuitable for high-throughput analysis of D-psicose, not allowing high-throughput screening, high time and cost, and achieving short detection time, The effect of fast detection and reduced detection cost

Pending Publication Date: 2022-02-08
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are accurate, they are not suitable for high-throughput analysis of D-psicose, and because the determination process requires a lot of time and high cost, it does not allow large-scale variation produced in enzyme directed evolution screening campaigns. high-throughput screening

Method used

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  • Kit for detecting D-psicose and detection method
  • Kit for detecting D-psicose and detection method
  • Kit for detecting D-psicose and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Determination of the concentration of D-psicose in the sample:

[0027] In this example, reagent I is a phosphate buffer, and its composition is: NaCl 3.0-10.0 mg / mL, KCl 0.05-1.0 mg / mL, NaCl 2 HPO 4 0.3~2.0mg / mL, KH 2 PO 4 0.01~0.5mg / mL; Reagent II is NADH dissolved in Reagent I, where the concentration of NADH is 50mg / mL; Reagent III is ribitol dehydrogenase dissolved in Reagent I, where the concentration of ribitol dehydrogenase Reagent IV is D-psicose dissolved in reagent I, wherein the concentration of D-psicose is 5 mg / mL.

[0028] 1) The pH of reagent I was adjusted to 8.0, and reagent II and reagent I were mixed to obtain a mixed solution. The concentration of NADH in the mixed solution was 2.0 μM. In this example, the Infinite M200 Pro multi-functional microplate reader was used to detect the relative fluorescence value.

[0029] 2) Dilute reagent V with reagent I to obtain a set of diluted solutions with different concentrations of D-psicose, in which th...

Embodiment 2

[0040] Determination of D-psicose concentration in KEase enzymatic reaction:

[0041] The kit used in this example was the same as that in Example 1. Before use, the pH of reagent I was adjusted to 7.4, and reagent II was diluted with reagent I until the concentration of NADH was 2.0 mg / mL.

[0042] 1) According to the gene sequence of D-psicose-3-epimerase (AgDAEase, which can catalyze the conversion of D-fructose into D-psicose) of Arthrobacter globiformis M30, the The AgDAE-pET-28a recombinant plasmid synthesized by the whole gene was transformed into Escherichia coli BL21(DE3), and the transformants were screened by kanamycin sulfate resistance plate to construct the Escherichia coli AgDAEase recombinant expression bacteria.

[0043] 2) Pick a single colony of recombinant AgDAEase recombinant expression bacteria and inoculate it in 5 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate, and culture it with shaking at 37 °C and 220 r / min until the OD of the bacteria...

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Abstract

The invention provides a kit for detecting D-psicose and a detection method, and relates to the technical field of detection. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V. The reagent I is a phosphate buffer solution, the main component of the reagent II is reduced nicotinamide adenine dinucleotide (NADH), the main component of the reagent III is ribitol dehydrogenase, and the main component of the reagent IV is D-psicose. By utilizing the kit, 96 samples can be detected within 30 minutes, and the concentration or content of D-psicose in the samples can be quickly and accurately reflected. The kit is not influenced by interferents such as D-fructose and other monosaccharides in the detection process, and can be applied to detection of the conversion amount of D-fructose to D-psicose in the KEase enzymatic reaction process and screening of KEase enzyme variants. In the detection process by using the kit, large-scale instruments and equipment are not needed, so that the detection cost can be remarkably reduced.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a kit and a detection method for detecting D-psicose. Background technique [0002] Allulose is an important member of the rare sugar family, which only exists in a few plants and some bacteria at extremely low levels. With the incidence of metabolic syndromes such as obesity and diabetes increasing rapidly worldwide, nutrition (functional foods) and health issues are gaining more and more attention. D-psicose has gradually become a research hotspot in the pharmaceutical, health care and food industries due to its many functions such as blood sugar control, protection of islets, and anti-oxidation. [0003] However, the application of D-allose is limited by its scarcity. It is unrealistic to extract D-psicose from natural resources, which will consume a lot of resources, increase production costs and damage the environment. On the other hand, the chemical synthesis of D-psic...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/31
CPCG01N21/6428G01N21/6486G01N21/31
Inventor 秦慧民路福平吴冕
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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