Rapid determination method of lipase synthetase vitality in non-aqueous phase

A rapid determination and lipase technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of expensive fluorescent substrates and high requirements for reaction solvents, and achieve the effect of simple and purposeful determination methods.

Inactive Publication Date: 2007-03-14
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, not all laboratories have fluorescence spectrophotometers, and fluorescent substrates are usually expensive and require high reaction solvents

Method used

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  • Rapid determination method of lipase synthetase vitality in non-aqueous phase
  • Rapid determination method of lipase synthetase vitality in non-aqueous phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 uses the inventive method to verify 6 kinds of lipases

[0039] Select above-mentioned 6 kinds of lipases, verify with assay method described in the present invention, and provide convincing proof for the feasibility that this assay method is used for lipase non-aqueous phase synthetase activity assay, the results are shown in Table 1. Since the above reactions are not the results under the optimal enzyme activity assay conditions, the conversion rate is used to represent the catalytic synthesis activity of the enzyme. The result (method II in table 1) of gas chromatography detection ethyl palmitate and the result (method I in table 1) that adopt colorimetry to measure are 90% correlation degree, illustrate that assay method of the present invention is feasible and the detection method adopted in the present invention can effectively measure the lipase-catalyzed transesterification reaction between p-nitrophenol palmitate (pNPP) and ethanol. The result of ...

Embodiment 2

[0044] Embodiment 2 carries out L-PS-C lipase enzyme activity assay with the inventive method

[0045] Weigh 2.5mg of L-PS-C lipase and place it in a 2mL stoppered plastic tube, add 0.5mL of 10mM pNPP n-heptane solution and 30μL of absolute ethanol that has been dehydrated by 4 Å molecular sieves, and react at 200rmp at 50°C After 5 minutes, 50 μL of the reaction solution was extracted with 1 mL of 0.1M NaOH solution, and the absorbance was measured at 410 nm, and the measured enzyme activity was 140.1 U / g.

Embodiment 3

[0046] Embodiment 3 uses the inventive method to carry out L-Rh whole cell lipase enzyme activity assay

[0047] Weigh 10 mg of L-Rh whole cell lipase and place it in a 2 mL stoppered plastic tube, add 0.5 mL of 10 mM pNPP n-heptane solution and 30 μL of absolute ethanol that has been dehydrated by 4 Å molecular sieves, and react at 200 rpm for 30 min at 40 °C , Take 50μL of the reaction solution and extract it with 1mL of 0.1M NaOH solution, measure the absorbance at 410nm, and the measured enzyme activity is 5.6U / g.

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PUM

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Abstract

The fast detection process of lipase synthesis activity in nonaqueous phase belongs to the field of nonaqueous enzyme catalysis technology. Lipase is used in catalyzing the esterification between p-nitro phenol ester and alcohol in nonaqueous phase, and after reaction for certain time, reacted liquid in certain amount is extracted with 0.1M concentration NaOH solution and the absorbance at 410 nm is measured in a colorimetric process so as to determine the amount of produced p-nitro phenol. The lipase activity unit is defined as the amount of lipase required for producing 1 micromol of p-nitro phenol. The present invention is used for the direct screening of lipase producing bacteria with high ester synthesizing activity and has relatively low cost.

Description

technical field [0001] The invention relates to a rapid assay method for lipase synthetase activity in a non-aqueous phase, which can be used in the screening of lipase-producing bacteria with high ester synthesis activity in the non-aqueous phase and rapid assay of the enzyme activity, belonging to the non-aqueous phase lipase catalysis technology field. Background technique [0002] As a triglyceride hydrolase, lipase has become an efficient catalyst for organic synthesis in the process of non-aqueous biocatalysis with the continuous deepening and development of non-aqueous phase enzymology. It can effectively catalyze esterification and transesterification The reaction has been successfully used in the production of many important compounds, such as aromatic esters, monoglycerides, chiral compounds, and biodiesel. There are many kinds of lipases with different catalytic properties, so the key to the catalytic process is to select the desired lipases purposefully. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/44
Inventor 徐岩滕云
Owner JIANGNAN UNIV
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