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Method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase

The technology of hydroperoxide and pure amaranth is applied in the field of preparation of electrophoresis pure amaranth 13-position hydroperoxide lyase, which can solve the problems of oxidative aggregation inactivation, instability and the like, and achieves simple purification process, low cost and steps simple effect

Inactive Publication Date: 2013-02-27
JIANGNAN UNIV
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Problems solved by technology

In addition, the active center of the enzyme contains a sulfhydryl group, which is unstable and susceptible to oxidation, aggregation and inactivation by external conditions.

Method used

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  • Method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase
  • Method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase
  • Method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase

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Embodiment 1

[0044] Take amaranth leaves with a wet weight of 100 grams, cut them into small pieces, and add 300 mL of buffer A to homogenize 6 times, each time for 10 seconds, with an interval of 30 seconds. The homogenate was filtered with gauze, the filtrate was centrifuged at 4°C and 18000g for 30min, the precipitate was dissolved in 50mL of buffer B, aliquoted, and freeze-dried for 80h to obtain the freeze-dried amaranth 13-HPL microsomal enzyme. Freeze-dried amaranth 13-HPL microsomal enzyme has good resolubility, and the enzyme activity can be completely recovered. After being stored at -20°C for 50 days, the retested enzyme activity basically remained unchanged, which can provide a stable enzyme source for separation, purification and industrial production of C6 aldehyde.

[0045] Take 10 g of freeze-dried amaranth 13-HPL microsomal enzyme powder and dissolve it in 100 mL of buffer C. Centrifuge the suspension at 30,000 g for 30 min at 4°C, and dissolve the precipitate in buffer D...

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Abstract

The invention relates to a method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase (13-HPL), and belongs to the technical field of purifying membrane protein by utilizing salting out and chromatographic separation technology and membrane separation technology. The method comprises the steps of preparation, primary purification and electrophoresis purification of amaranth 13-HPL freeze-dried microsomal enzyme; the preparation of amaranth 13-HPL freeze-dried microsome comprises the following steps: taking fresh amaranth as a raw material, taking a sediment out after homogenization and centrifugalization, and performing freeze-drying on the sediment and 1 to 30 percent solution of sucrose serving as a protective agent to obtain freeze-dried microsomal lyase, wherein the freeze-dried powder has small volume and can be stored for a long period at the temperature of 20 DEG C below zero; and the lyase can be recovered at a high recovery rate, has high activity, and solves the problem of seasonal dependence of the raw material; and the purification process mainly comprises the steps of washing and dissolution of the microsomal enzyme, ammonium sulfate precipitation, dialysis, microfiltration, hydroxyapatite column chromatography, anion exchange column chromatography and ultrafiltration so as to obtain the electrophoresis pure hydroperoxide lyase. Great attention is paid on the amaranth 13-HPL in aspects of the production of food and daily commodities with high added value; and the hydroperoxide lyase is obtained from amaranth planted in China through separation and purification for the first time.

Description

technical field [0001] A method for preparing electrophoretic pure amaranth 13-hydroperoxide lyase, specifically using amaranth (Amaranthus mangostanus L.) as a raw material, and obtaining electrophoretic pure amaranth 13-hydroperoxide lyase through the method. The invention belongs to the technical field of purifying membrane protein by using salting out, chromatographic separation technology and membrane separation technology. Background technique [0002] Hydroperoxide lyase (HPL) is an enzyme downstream of lipoxygenase (LOX) in the plant lipid oxidation pathway, which can cleavage hydroperoxidized fatty acids formed by polyunsaturated fatty acids under the action of LOX to form oxoacids and volatile aldehydes kind. According to the difference in the position of the substrate peroxy group, HPL can be divided into two types: the first type can cleave 13-position hydroperoxide to generate 6C aldehydes and 12C oxyacids, called 13-HPL; the second type can cleave 9 Hydropero...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88
Inventor 华欲飞龙祯孔祥珍张彩猛
Owner JIANGNAN UNIV
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