Purification coupled fixation method of myrosinase

A technology of myrosinase and seeds is applied in the field of myrosinase purification and coupling immobilization, which can solve the problems of complicated experiments, inability to completely solve the ESP protein, and high cost, so as to reduce the difficulty of purification, improve the immobilization efficiency, and increase the conversion rate. Effect

Active Publication Date: 2018-10-09
BEIJING UNIV OF CHEM TECH
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Chinese invention patent application with the application number 200910037363.1 discloses a method for extracting multifunctional sulforaphane from broccoli sprouts. This method needs to consider the growth time of the sprouts and pretreat the sprouts. The experiment is complicated , and also cannot completely solve the problem of ESP protein, so the yield is low and the cost is high

Method used

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  • Purification coupled fixation method of myrosinase
  • Purification coupled fixation method of myrosinase
  • Purification coupled fixation method of myrosinase

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preparation example Construction

[0042] (2) Preparation of ferric oxide nanospheres

[0043] Use FeCl 3 ·6H 2 O and FeCl 2 4H 2 O, the molar ratio is 2:1 (also can be 0.1:1-10:1), in N 2 Dissolve it in 50ml (or 10-1000ml) of 1.5M (or 0.1-5) ammonia water under protection, and keep it warm at 40°C (or 5-50°C) for 30 minutes to prepare ferric oxide nanoparticles. The supernatant was removed by centrifugation, and the precipitate was washed 3 times with water for later use.

[0044] (3) Preparation of silica nanospheres wrapped with ferric oxide

[0045] Take 100 mg of ferric oxide nanospheres prepared by ultrasonication for 20 minutes, and add 200 ml (or 10-1000 ml) of ethanol, 6.8 ml (or 1-100 ml) of ammonia water, and 1.5 ml ( It can also be 0.1-10ml) TEOS and 10ml (also can be 1-100ml) water, after reacting for 20 minutes (also can be 1-100 minutes), add ferric oxide ball after ultrasonic, mechanically stir for 15 hours (also can be After 1-50 hours), add 140ul (or 10-1000ul) of APTES, react for 8 hou...

Embodiment 1

[0059] Broccoli seeds without high temperature treatment were crushed, ultrasonically crushed, filtered with gauze, and then centrifuged to obtain a crude enzyme solution containing myrosinase activity; use FeCl 3 ·6H 2 O and FeCl 2 4H 2 O, the molar ratio is 2:1, in N 2 It was dissolved in 50ml of 1.5M ammonia water under protection, and kept at 40°C for 30 minutes to prepare ferric oxide nanoparticles. After that, the supernatant was removed by centrifugation, and the precipitate was washed 3 times with water before use.

[0060] Take 100mg of ferric oxide nanospheres prepared by ultrasonication for 20 minutes for use, add 200ml of ethanol, 6.8ml of ammonia water, 1.5ml of TEOS and 10ml of water at 40°C, and react for 20 minutes, then add the ultrasonicated trioxide Iron balls, after mechanical stirring for 15 hours, add 140ul APTES, react for 8 hours, centrifuge to remove the supernatant, wash the precipitate with water for 3 times and set aside.

[0061] Add 80 mg EDC ...

Embodiment 2

[0066] The radish seeds without high temperature treatment were crushed, ultrasonically crushed, filtered with gauze, and then centrifuged to obtain a crude enzyme solution containing myrosinase activity; using FeCl3 6H2O and FeCl2 4H2O with a molar ratio of 2:1, protected under N2 Dissolve it in 50ml of 1.5M ammonia water, incubate at 40°C for 30 minutes to prepare ferric oxide nanoparticles, then centrifuge to remove the supernatant, wash the precipitate with water three times and set aside.

[0067] Take 100mg of ferric oxide nanospheres prepared by ultrasonication for 20 minutes for use, add 200ml of ethanol, 6.8ml of ammonia water, 1.5ml of TEOS and 10ml of water at 40°C, and react for 20 minutes, then add the ultrasonicated trioxide Iron balls, after mechanical stirring for 15 hours, add 140ul APTES, react for 8 hours, centrifuge to remove the supernatant, wash the precipitate with water for 3 times and set aside.

[0068] Add 80 mg EDC and 10 mg concanavalin according t...

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Abstract

The invention provides a purification coupled fixation method of myrosinase. By means of concanavalin combined with myrosinase, concanavalin is loaded on a base, myrosinase is linked to the concanavalin, and therefore, the myrosinase is specifically fixed to the base; purification and fixation of the myrosinase are combined together such that difficulty in purifying the myrosinase is greatly reduced, and fixing efficiency is improved; the concanavalin adsorbs glycosyl on the surface of the myrosinase so as to adsorb the myrosinase but not adsorb ESP protein, fewer nitriles are generated, the yield of isothiocyanates is increased, and conversion rate of glucosinolates into isothiocyanates is greatly increased.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for purifying, coupling and immobilizing myrosinase. Background technique [0002] Cruciferous plants are a class of plants that are widely believed to have anti-cancer functions. This is mainly because cruciferous plants contain a large amount of glucosinolates (glucosinolates, referred to as GLS), glucosinolates are a sulfur-containing secondary metabolite of plants, and more than 120 kinds of sulfur have been found in plants. Glucoside, its basic structure is shown in the following formula. Glucosinolates are water-soluble, non-volatile, heat-stable ionic compounds. If plant cells are damaged, glucosinolate hydrolase, myrosinase (thioglucoside glucohydrolase; EC3.2.3.1) is released, and hydrolyzes glucosinolates to generate bisulfate, glucose and a series of Glucosides, these ligands undergo intramolecular rearrangement to form isothiocyana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N11/14C12P11/00
CPCC12N9/2402C12N11/14C12P11/00C12Y302/01147
Inventor 袁其朋程立梁浩
Owner BEIJING UNIV OF CHEM TECH
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