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Method for measuring activity of laminarinase in copepods

A technology of laminarin enzyme and determination method, which is applied in the field of determination of digestive enzyme activity, can solve the problems of long test cycle, difficult test, complicated operation, etc., and achieve the effects of short test time, high test efficiency and short reaction time

Inactive Publication Date: 2012-08-15
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method of analyzing the activity of digestive enzymes requires a large amount of samples, complicated operations, and a long test cycle, so the error is relatively large. This makes the test difficult for copepods that are small and difficult to grow in large quantities indoors. Therefore, it is particularly important to establish an independent method for the determination of the digestive enzyme activity of copepods.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Pick 5 individuals of the cultured Daphnia sinensis into a mortar, add 500 μl 1mM Tris-HCl buffer solution, grind in an ice bath to form a homogenate, centrifuge at 15,000 rpm, 0-4°C for 10 minutes in a refrigerated centrifuge, and take out The clear solution is ready for use. Add 2 mg of laminarin to 1 ml of acetic acid solution with a concentration of 100 mM, heat and dissolve to obtain laminarin acetic acid solution, take 40 μl of the above supernatant and mix with 460 μl of laminarin acetic acid solution, incubate in a water bath at 20°C for 1 hour, and then incubate at 95°C Water bath for 2 min, terminate the reaction to obtain the water bath product. Add 1ml of 50mM Tris-HCl buffer, 1mM MgCl to 40μl water bath product 2 solution 0.5ml, 0.5mM dithiothreitol solution (C 4 h 10 o 2 S 2 )0.5ml, 1μg ml -1 1ml of hexokinase solution, 30μM NADP + Solution 1ml, 300μM ATP solution 1ml, 0.02U ml -1 Glucose 6-phosphate dehydrogenase solution 1ml, enzymatic reaction ...

Embodiment 2

[0015] It is basically the same as in Example 1, except that Daphnia chinensis is replaced by Daphnia moxie, and its number B is 10. is 2.576, and the total concentration A is 0.2532512 (μg ml -1 ), and the activity of laminarinase in each Daphnia mexilii was 5.13(μg glucose·ind -1 h -1 ).

Embodiment 3

[0017] It is basically the same as in Example 1, except that Daphnia sinensis is replaced by Daphnia pacificus, and its number B is 15. The light absorption value of the reduced coenzyme II of the enzymatic product of Daphnia pacificis is 2.238, and the total Concentration A is 0.1607406 (μg ml -1 ), the activity of laminarin in each Daphnia pacificis was 2.17(μg glucose·ind -1 h -1 ).

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Abstract

The invention discloses a method for measuring activity of laminarinase in copepods. The method comprises the following steps of: grinding and centrifuging 5 to 15 copepods in a buffer solution, reacting with a laminarin substrate in a water bath to obtain a water bath product, mixing the water bath product, Tris-HCl, MgCl2, dithiothreitol, hexokinase, nicotinamide adenine dinucleotide phosphate (NADP)<+>, adenosine triphosphate (ATP) and glucose 6-phosphate dehydrogenase, performing enzymatic reaction, and calculating the activity of the laminarinase in each copepod according to a formula by using absorbance of reductive coenzyme II in the enzymatic reaction product and the number of the copepods as parameters. According to the method, the activity of digestive enzyme can be measured only by a few to more than ten copepods, and the absorbance of the reductive coenzyme II can be obtained by the water bath reaction and the enzymatic reaction of the laminarinase in the copepods; and the method is easy and convenient to operate, short in reaction time and less in errors, so the method has the advantages of easiness and convenience in operation, low sample consumption, short test time and high test efficiency.

Description

technical field [0001] The invention relates to the determination of digestive enzyme activity, in particular to a method for determination of laminarin enzyme activity in copepods. Background technique [0002] Copepods are a class of small lower crustaceans and are an important part of marine zooplankton. It is both a feeder of phytoplankton and a bait basis for fish, occupying a key position in marine food chains, secondary productivity and energy flow. The research on copepods often involves the analysis of the ability of copepods to ingest and digest food, the ability to adapt to the concentration of food in the surrounding environment, and the nutritional status. Indicators, due to the small size of copepods and their sensitivity to environmental factors, the activity of digestive enzymes can also be used as one of the indicators to indicate the quality of the environment; this needs to be judged by measuring the activity of digestive enzymes of copepods. The traditi...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/34C12Q1/32
Inventor 谢志浩屠燕萍俞泓伶
Owner NINGBO UNIV