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Extraction and activity determination methods of enzyme SrKA13H in stevia rebaudian

A technology of determination method and extraction method, which is applied in the field of enzyme activity detection, achieves the effect of high accuracy and increasing the content of steviol glycosides

Inactive Publication Date: 2017-11-10
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since there is no relevant method for the extraction and activity detection of SrKA13H enzyme in stevia leaves, in order to carry out the evaluation of stevia germplasm resources and accelerate the process of cultivating high-quality new varieties, the present invention discloses for the first time a preliminary separation and purification of SrKA13H enzyme from fresh stevia leaves. And analyze the HPLC detection method of its enzyme activity according to the substrate specificity of this enzyme

Method used

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  • Extraction and activity determination methods of enzyme SrKA13H in stevia rebaudian
  • Extraction and activity determination methods of enzyme SrKA13H in stevia rebaudian
  • Extraction and activity determination methods of enzyme SrKA13H in stevia rebaudian

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Embodiment 1

[0035] The present invention is a method for extracting SrKA13H enzyme in stevia leaves and measuring its enzyme activity by HPLC. Materials and reagents used: acetonitrile (chromatographically pure), methanol (chromatographically pure), ethanol, MgCl 2 , EDTA Tris, ent- KA, PVPP, NADPH, ammonium sulfate powder, mercaptoethanol, erythorbic acid. Main equipment: high performance liquid chromatography, constant temperature water bath, centrifuge, magnetic stirrer, the steps are as follows:

[0036] (1) Crude extraction of SrKA13H enzyme in stevia leaves: Collect fresh leaves in the middle of the plant at the budding stage of the stevia variety 'Zhongshan No. 8,5mM MgCl 2 , 2mM EDTA, 20mM mercaptoethanol, 4g / L isoascorbic acid, 20g / L PVPP) were shaken for 3min and mixed, centrifuged at 11000r / min at 4°C for 20min, and the supernatant was slowly added to ammonium sulfate powder to 50% saturation, at 4°C Centrifuge at 11000r / min for 15min, remove the precipitate, add ammonium s...

Embodiment 2

[0040] According to the method in Example 1, the amount of SrKA13H enzyme solution added in the reaction system was changed so that the final protein concentration of the enzyme solution was 30 μg / ml, 60 μg / ml, 90 μg / ml, 120 μg / ml, and 150 μg / ml, respectively, and the test was carried out , to calculate the SrKA13H enzyme activity. In conjunction with the test data in the example 1, obtain the substrate ent- The correlation between the consumption of KA and the amount of crude enzyme added, such as image 3 ,Depend on image 3 It can be seen that the protein concentration of the enzyme solution in the reaction system is in the range of 30-90μg / ml, and the metabolic substrate and protein concentration present a good linear relationship. The regression equation is y = 1.4167x - 10.667, and the regression coefficient is R² = 0.9836.

Embodiment 3

[0042] According to the method in Example 1, change the addition amount of the SrKA13H enzyme solution in the reaction system, so that the final protein concentrations of the enzyme solution are 50 μg / ml and 70 μg / ml respectively, and then change the enzymatic reaction time for 5 minutes, 10 minutes and 15 minutes respectively , 20min, 25min, 30min and then 100°C water bath for 10min to terminate. Test the change of SrKA13H enzyme activity at different concentrations and different reaction times, and obtain the correlation between the consumption of metabolic substrates and the reaction time. The results are shown in Figure 4 ,Depend on Figure 4 It can be seen that the longer the reaction time, the more the consumption of the metabolic substrate, but the reaction time exceeds 15min, the reduction of the metabolic substrate increases very little. Therefore, in the enzymatic reaction, the reaction time of 15 minutes is the most suitable.

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Abstract

The method discloses extraction and activity determination methods of enzyme SrKA13H in stevia rebaudian, and belongs to the technical field of enzyme activity detection. A main technology of the methods is that the enzyme SrKA13H is separated and purified from fresh leaves of the stevia rebaudian preliminarily, and activity of the enzyme is determined with high performance liquid chromatography (HPLC). The methods specifically comprise steps as follows: (1) performing crude extraction on the enzyme SrKA13H in the leaves of the stevia rebaudian; (2) preparing an enzyme SrKA13H solution of the stevia rebaudian; (3) reacting the enzyme SrKA13H of the stevia rebaudian with substrate Kaurenic acid (ent-KA), detecting changes of the content of ent-KA before and after the enzymatic reaction by a high performance liquid chromatograph, and calculating activity of the enzyme SrKA13H. The detection method has high pertinence, sensitivity and stability.

Description

technical field [0001] The invention belongs to the technical field of enzyme activity detection, in particular to a method for extracting and measuring enzyme activity of SrKA13H enzyme in stevia leaves, which can be used for detecting the activity of SrKA13H enzyme in stevia leaves. Background technique [0002] Stevia ( Stevia rebaudiana Bertoni) as an emerging natural high-sweetness and low-calorie sugar source plant, the research on its germplasm resources and the cultivation of excellent new varieties are very important to the development of stevia industry. Because the research on the biosynthetic pathway of steviol glycosides has made it clear that steviol glycosides and endogenous gibberellins share the MEP pathway until the common precursor substance endogenous root-kaurenoic acid ( ent- kaurenoic acid, ent- KA) synthesis. ent- KA endogenous-kaurenoate oxidase ( ent- kaurenoic acidoxidase, KAO) to participate in the synthesis of gibberellin, while endogenou...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12Q1/26
CPCC12N9/0073C12Q1/26G01N2333/90251
Inventor 杨永恒陆堃徐晓洋张永侠刘清泉原海燕黄苏珍
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI