Assay For Phytol-free Chlorophyll Derivatives

A technology of chlorophyll and phytol, which is applied in the fields of biomaterial analysis, microbial measurement/inspection, biochemical equipment and methods, etc., and can solve problems such as low activity of nitrophenyl esters, false negatives, and unreliability

Inactive Publication Date: 2013-01-02
DUPONT NUTRITION BIOSCIENCES APS
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Problems solved by technology

Unfortunately, this method is not very reliable as some chlorophyllases, while highly active on chlorophyll, have rather low activity on p-nitrophenyl esters and may give false negat

Method used

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  • Assay For Phytol-free Chlorophyll Derivatives
  • Assay For Phytol-free Chlorophyll Derivatives
  • Assay For Phytol-free Chlorophyll Derivatives

Examples

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example

[0086] In these examples, assays were developed to measure chlorophyllase, pheophytinase and pyropheophytinase activities. In the first step of the assay, a substrate consisting of chlorophyll, pheophytin or pyropheophytin in buffer is prepared. This substrate was added to the enzyme solution and reacted at 40°C for 10 minutes. After a reaction time of 10 min, an aliquot of the sample was transferred to the assay buffer. The measurement of enzyme activity relies on the principle that chlorophyll, pheophytin or pyropheophytin form dimers in the assay buffer (see figure 1 ). Dimer formation will quench the fluorescence signal of chlorophyll, pheophytin or pyropheophytin. The detection buffer is formulated such that the produced phytochlorophyll, pheophorbide or pyropheophorbide does not form dimers and is thus detectable by fluorescence spectroscopy.

example 1

[0087] Example 1 - Development of an Assay for Pyropheophytinase Activity

[0088] A 100 mM pH 7 phosphate buffer was prepared as a reaction buffer containing 50 mM KCl, 0.2% Triton X-100 and 5.17% acetone. Prepare the following solutions containing pyropheophytin, pyropheophytin, or mixtures thereof, and add them to the reaction buffer:

[0089] 1) Pyropheophytin solution: 500 μl reaction buffer + 50 μl water + 30 μl pyropheophytin (1mg / ml acetone solution)

[0090] 2) Pyropheophorbide solution: 500 μl reaction buffer + 50 μl water + 15 μl pyropheophorbide (2 mg / ml acetone solution) + 15 μl acetone.

[0091] 3) Pyropheophytin: 1:1 solution of pyropheophytin: 500 μl reaction buffer + 50 μl water + 15 μl pyropheophytin (1 mg / ml acetone solution) + 7.5 μl pyropheophytin ( 2 mg / ml acetone solution) + 7.5 μl acetone.

[0092] Various detection solutions (A-G) were prepared containing different concentrations of Triton X-100 (surfactant) and ethanol or isopropanol (solvent), a...

example 2

[0105] Example 2 - Development of an Assay for Pheophytinase Activity

[0106] Based on the results of Example 1, an assay for the detection of pheophytinase activity was developed. When added to detection solution E (0.015% Triton X-100; 15% IPA; 0.05M NaOH), the fluorescence emitted by pheophytin was largely quenched (in terms of pyropheophytin) at room temperature, but Pheophorbide produced a strong fluorescent signal. Therefore, a calibration curve for pheophorbide was constructed (see image 3 ).

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Abstract

The present invention provides a method for detecting a phytol-free chlorophyll derivative in a sample, comprising a step of detecting a fluorescent signal associated with the phytol-free chlorophyll derivative, wherein a fluorescent signal associated with chlorophyll or a phytol-containing chlorophyll derivative in the sample is quenched. The method may be used for quantifying activity of chlorophyllases and related enzymes in a sample without solvent fractionation of substrate and product.

Description

technical field [0001] The present invention relates to a method for detecting chlorophyll derivatives in a sample. The method can be used as an assay for determining the activity of chlorophyllase or related enzymes in a sample. Background technique [0002] Chlorophyll is a green pigment widely distributed throughout the plant kingdom. Chlorophyll is necessary for photosynthesis and is one of the most abundant organometallic compounds found on Earth. Thus many products from plants (including food and feed) contain considerable amounts of chlorophyll. [0003] In plants, chlorophyllase (chlorophyllase or chlase) is considered to be involved in the degradation of chlorophyll, and catalyzes the hydrolysis of ester bonds in chlorophyll to generate chlorophyll and phytol. Alternatively, chlorophyll can be degraded by loss of magnesium ions from the porphyrin (chlorin) ring, forming a derivative called pheophytin (see Figure 5 ). Under certain conditions, some chlorophyllas...

Claims

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Application Information

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IPC IPC(8): C12Q1/44
CPCC12Q1/44G01N2333/918
Inventor R.米克尔森T.约根森J.B.索厄
Owner DUPONT NUTRITION BIOSCIENCES APS
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