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A Colorimetric Method for Rapid Quantitative Detection of Amino Acid Decarboxylase
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A technology for quantitative detection of amino acid decarboxylase, applied in the field of enzyme activity detection, can solve the problems of difficult operation, insufficient details of operation methods, high cost, etc., and achieve the effect of good repeatability, good linear relationship and low cost
Active Publication Date: 2017-05-17
SOUTHWEST UNIV
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However, there is no detailed method and commercial reagent test kit for rapid quantitative detection of amino acid decarboxylase activity.
In order to replace the traditional detection methods such as high cost and radioactive substance contamination (requires a liquid scintillation instrument), there are currently patent reports using carboxylase enzyme coupling to fix CO 2 The technology detects the activity of amino acid decarboxylase, but the operation method is not detailed enough, and it is very difficult to operate
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Embodiment 1
[0045] A colorimetric method for rapid quantitative detection of amino acid decarboxylase, the specific steps are as follows:
[0046] Taking pig colon microbial cells as an example, the colonic microbial cells lysine decarboxylase, glutamic acid decarboxylase, tyrosine decarboxylase, glycine decarboxylase, tryptophan decarboxylase, histidine decarboxylase, formazan Thionine decarboxylase and arginine decarboxylase.
[0047] 1. Reagents
[0048] (1) Reagent composition and preparation of reaction solution and chromogenic solution
[0049] The reagent formula of the colorimetric method for rapid quantitative detection of decarboxylase is shown in Table 1.
[0050] Reaction solution: ready to use, just dissolve reagent 2 in reagent 1.
[0051] Reagent 1 (100ml): 50mmol / L Tris (pH7.5), 10mmol / L MgCl 2, 2mmol / LPEP monocyclohexylamine salt, 5mmol / L amino acid, using CO-free 2 Distilled water preparation;
[0052] The amino acid is the amino acid corresponding to the amino aci...
Embodiment 2
[0114] Determination of lysine decarboxylase, glutamic acid decarboxylase, tyrosine decarboxylase, glycine decarboxylase, tryptophan decarboxylase, histidine decarboxylase and arginine decarboxylase in pig brain tissue samples enzyme.
[0115] 1. Reagents
[0116] (1) Reagent composition and preparation of reaction solution and chromogenic solution
[0120] (1) Preparation of microbial samples before determination
[0121] Weigh 1 g of the brain tissue pieces of 30 kg pigs and 90 kg pigs, and rinse them with ice saline or PBS buffer solution for 2 to 3 times after stabilizing at 4 °C, each time not less than 5 mL, remove all impurities such as blood, and blot dry with filter paper. Weigh again, place in a 10mL centrifuge tube and insert into ice for later use. Put the tissue sample into a 10mL centrifuge tube, take the pre-cooled homogenization medium (PH7.4, 0....
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Abstract
The invention discloses a colorimetric method for rapid quantitative detection of amino acid decarboxylases, through the configuration of a reaction liquid and a developing liquid, a standard curve is prepared, and enzyme activity of the amino acid decarboxylases of a to-be-detected sample can be obtained. The method has the advantages of being low in cost, good in repeatability, good in linear relationship in the enzyme concentration of 0.001U-0.006U / muL, simple in operation, low in instrument requirements, reliable, wide in applicable range, and capable of detecting a variety of amino acid decarboxylases. The method is very important for change of the enzyme activity of the amino acid decarboxylases in animal tissues, microbial culture fluids, cell culture and blood samples.
Description
technical field [0001] The invention relates to the field of enzyme activity detection, in particular to a colorimetric method for rapid quantitative detection of amino acid decarboxylase. Background technique [0002] Amino acid decarboxylase (amino acid decarboxylase) is a general term for lyases that catalyze the removal of the carboxyl group of an amino acid to generate the corresponding amine (as shown in the following brackets): NH 2 CHRCOOH→NH 2 CH 2 R+CO 2 . It is mainly found in bacteria growing on acidic medium, although it is rare in animals and plants, it can also be found. Lysine (cadaverine), tyrosine (tyramine), arginine (herringspermine), ornithine (putrescine), glutamic acid (γ-aminobutyric acid), etc. Corresponding specificity decarboxylase, these enzymes all use pyridoxalphosphate as coenzyme. In animal tissues, glutamic acid (γ-aminobutyric acid), tyrosine (tyramine), histidine (histamine), cysteine (taurine), 5-hydroxytryptophan (5 -HT) corres...
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