Method for detecting lipase activity

A technology of lipase and activity, which is applied in the field of analytical chemistry, can solve the problems of complex preparation of solutions, dependence, and inconspicuous color changes, and achieve the effects of large-scale use, reduced experimental errors, and good water solubility of substrates

Active Publication Date: 2013-04-24
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most classic method at present is to use pNPP (p-nitrophenyl ester), which is simple to operate and has good optical properties. After hydrolysis by lipase, yellow p-nitrophenol is produced, but the yellow color is colorless and transparent compared to the previous one. The color change of the substrate is not very obvious, which is not conducive to direct observation with the naked eye, and it is also very complicated to prepare the solution
The above methods have their own advantages under different detection conditions, but they also have their limitations, such as insufficient sensitivity or too much dependence on instruments, which cannot realize real-time detection, etc., so a simple, sensitive, and visual detection of fat Enzyme activity methods urgently need to be established
[0004] In the prior art, the exploration of the detection of lipase activity described in the present invention has attracted more and more attention in recent years, but the method of visually detecting lipase activity using gold nanoparticles has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Preparation and characterization of nano-gold solution

[0036] First, accurately weigh the HAuCl 4 .4H 2 O 0.0123 g was dissolved in 100 mL deionized water, and then added to a 250 mL fixed three-neck flask. Stir vigorously and heat to reflux. Then accurately weigh 0.2849 g of sodium citrate and dilute to volume in a 25 mL volumetric flask. After the water bath was heated to 50 °C, a certain volume of sodium citrate solution was accurately pipetted into the flask with a pipette. The solution changed from colorless to light blue to purple and finally to wine red. Continue heating for 10 minutes, then stop heating, continue stirring for 10 minutes and then cool to room temperature to obtain the required 13 ± 2.5 nm gold colloid. The diameter of gold nanoparticles was finally determined using a transmission electron microscope (JEOL JEM-200CX, Japan).

[0037] (2) modifying the nano-gold solution obtained in step (1) with methyl thioglycolate to obtain a function...

Embodiment 2

[0040] The method for visually detecting lipase activity based on gold nanoparticles described in this embodiment comprises the following steps:

[0041] (1) adopting sodium citrate reduction chloroauric acid to make concentration is the wine red nano-gold solution of 2.5 nmol / L,

[0042] (2) Modify the nano-gold solution obtained in step (1) with methyl thioglycolate to obtain a functional nano-gold solution; wherein, the molar ratio of nano-gold and methyl thioglycolate modification is 1:60; the modification time is 24h;

[0043] (3) Add a buffer solution to the nano-gold solution obtained in step (3) to adjust the pH, adjust the pH to 4.5; then add the lipase solution to be tested, mix well, adjust the reaction temperature to 35 ° C, and carry out the solution Detect, get the UV-Vis spectrum corresponding to different times, and then use the absorbance ratio E 650 / E 520 As the ordinate and time as the abscissa, draw the kinetic curve of lipase activity 。

Embodiment 3

[0045] The method for visually detecting lipase activity based on gold nanoparticles described in this embodiment comprises the following steps:

[0046] (1) adopting sodium citrate reduction chloroauric acid to make concentration is the wine red nano-gold solution of 1.8 nmol / L,

[0047] (2) modifying the nano-gold solution obtained in step (1) with methyl thioglycolate to obtain a functional nano-gold solution; wherein, the molar ratio of nano-gold and methyl thioglycolate modification is 1:40; the modification time is 1h;

[0048] (3) Add a buffer solution to the nano-gold solution obtained in step (3) to adjust the pH, adjust the pH to 4.0; then add the lipase solution to be tested, mix well, adjust the reaction temperature to 45 ° C, and carry out the solution Detect, get the UV-Vis spectrum corresponding to different times, and then use the absorbance ratio E 650 / E 520 As the ordinate and time as the abscissa, draw the kinetic curve of lipase activity 。

[0049]

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Abstract

The invention provides a method for detecting lipase activity. The method comprises the following steps: mercaptoacetic acid methyl ester is utilized for modifying nanogold; in the presence of lipase, mercaptoacetic acid is generated by hydrolysis, and thus carboxylate radicals are exposed; intense hydrogen-bond interaction exists among carboxylate radicals, and thus the distance between nanogold is shortened to gather and blue the nanogold. On one hand, the strength of the lipase activity can be directly identified by naked eyes from the color and on the other hand, the lipase activity can be sensitively detected by an ultraviolet and visible spectrophotometer. The method can be used for detecting the lipase activity from the aspects of time, temperature, quantity and the like and the experimental phenomenon is obvious. The method has the advantages of being simple to operate without substrate emulsification, small in dosage, being beneficial to being obviously observed by naked eyes and low in cost. The method has not only good research value academically, but also wide application in production application.

Description

technical field [0001] The invention belongs to the field of analytical chemistry and relates to a method for detecting lipase activity. Background technique [0002] Lipase is a large class of biocatalysts that can catalyze various chemical reactions such as hydrolysis, esterification, and transesterification. It has chemical, stereo, and site selectivity, high activity, few side reactions, and no cofactors. Widely used in many industrial fields such as food processing, biomedicine, biodiesel, fine chemicals, etc. Compared with ordinary chemical catalysts, it has the characteristics of high efficiency, stability, mildness and cleanliness, so it is a kind of "green" with great potential Therefore, it is very important to establish a convenient and fast method for detecting lipase activity. [0003] At present, there are many methods for detecting lipase activity, such as physical and chemical methods (detecting the amount of reacted substrate and the amount of products prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33G01N21/31
Inventor 田丹碧梅亚军黄和江凌左焕桢
Owner NANJING UNIV OF TECH
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