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Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry

A fluorescence spectrophotometry, non-aqueous phase technology, used in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of inability to perform measurement, limit the application of fluorescence spectrophotometry, and achieve the effect of simple operation

Inactive Publication Date: 2009-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the conditions for these two reactions must be coordinated, and the conditions for the generation of fluorescent derivatives by the reaction of many fluorescent substrates are inconsistent with the conditions for enzyme-catalyzed reactions, which makes the measurement impossible, thus limiting the application of fluorescence spectrophotometry in the field of enzyme catalysis

Method used

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  • Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry
  • Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry
  • Method for screening catalysis non-aqueous phase system transesterification enzyme by fluorospectrophotometry

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Experimental program
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Embodiment 1

[0026] Embodiment 1: the ability of different reference enzymes to catalyze the transesterification reaction

[0027] The enzymes used in the experiment were all commercial enzyme preparations. Protease bacillolysin (optimum temperature 37°C, optimum pH 7.5) was purchased from Tokyo Chemical Industry Co., Ltd., Japan, and lipase LPL-3 (optimum temperature 50°C, optimum pH 7.5). 0) Purchased from Japan Amano Enzyme Co., Ltd., protease subtilisin Carlbergs (optimum temperature 37°C, optimum pH 7.5) was purchased from Sigma Company, protease Alcalase 3.0T (optimum temperature 50°C, optimum pH 8.5) was purchased from from Novozymes. NBD-H.NH 2 NH 2 Purchased from Tokyo Chemical Industry Co., Ltd., chromatographically pure. Vinyl laurate (chromatographically pure) was purchased from Tokyo Chemical Industry Co., Ltd., Japan. Vinyl propionate (chromatographic grade) was purchased from Sigma. The 1420 multilabel counter multifunctional resolution fluorometer used in the fluoresce...

Embodiment 2

[0030] Embodiment 2: Study the impact of different transesterification ester substrates on the reaction

[0031]Vinyl propionate and vinyl laurate, which have the same alcohol moiety structure and different carboxylic acid moiety carbon chain lengths, were used as substrates for the enzyme-catalyzed transesterification reaction, and the effects of ester substrates with different molecular sizes on the reaction were studied. The abilities of the four enzymes used in the experiment to catalyze the reaction of vinyl propionate and n-propanol were greater than those of vinyl laurate and n-propanol, indicating that the small molecule ester substrate is beneficial to the enzyme action.

Embodiment 3

[0032] Embodiment 3: Study the influence of different organic solvents on reaction

[0033] Using vinyl propionate and n-propanol as substrates, compare the effects of different hydrophobic organic solvents: isooctane (logP=4.5), toluene (logP=2.5), acetonitrile (logP=-0.33) on the catalytic ability of enzymes . The catalytic abilities of these four enzymes showed a consistent trend in the isooctane, toluene, and acetonitrile systems, but as the hydrophobicity of organic solvents decreased, the catalytic activities of these four enzymes decreased rapidly: protease bacillolysin, lipase LPL- 3. The catalytic ability of protease subtilisin Carlsberg and protease Alcalase 3.0T in toluene is only 11.5%, 10.1%, 10.8% and 8.8% in isooctane respectively; the catalytic ability in acetonitrile is only 1% in isooctane 5.7%, 4.1%, 5.3%, and 4.7%. Compared with toluene and acetonitrile, isooctane with stronger hydrophobicity is more suitable as a solvent for enzyme-catalyzed transesterif...

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Abstract

The invention relates to a method for filtering enzyme applied to catalyze nonaqueous phase system transesterification reaction and belongs to the technical field of enzyme reaction and fluorescence measuration. In the method, a. vinyl ester and primary alcohol substance are catalyzed by enzyme to take transesterification reaction, as one of the reaction products vinyl alcohol generates acetaldehyde by keto-enol tautomerization to ensure that the transesterification reaction is nonreversible; b. the acetaldehyde reacts with 4-diazanyl-7-nitryl-2, 1, 3-benzoxadiazole (NBD-H.NH2NH2) to generate fluorescent derivative, and the fluorescence intensity of the fluorescent derivative is mensurated to reflect the catalytic capability of the enzyme indirectly. The invention has simple operation and can intuitively and easily judge whether enzyme catalysis transesterification reaction in nonaqueous phase can occur or not, the invention can compare the catalytic activity of the enzyme catalyzing the transesterification reaction, and the method can be used for studying on the factor influencing enzymic catalytic reaction.

Description

technical field [0001] The invention relates to a screening method for an enzyme that catalyzes the transesterification reaction of a non-aqueous phase system. Specifically, it uses fluorescence spectrophotometry to determine whether the enzyme can catalyze the transesterification reaction in a non-aqueous phase, and determines the The invention relates to the strength of enzyme catalysis, and belongs to the technical field of enzyme reaction and fluorescence measurement. Background technique [0002] Some lipases, esterases and proteases catalyze decomposition reactions in aqueous phase, but can catalyze synthesis and transesterification reactions in non-aqueous systems. Enzyme-catalyzed transesterification can even modify solid polymers by introducing functional groups into the side chains of polymer compounds. Therefore, if different groups can be introduced on the surface of cotton fibers through enzyme-catalyzed transesterification, new functions can be given to cotton...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 张颖谷家栋王树根范雪荣
Owner JIANGNAN UNIV
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