Industrial preparation of radish dual-function enzyme with chitinase and antalzyme activity

Abstract

The invention discloses an industrial preparation method of a dual-functional radish enzyme that is provided with the activity of both chitinase and muramidase and comprises the following steps: firstly raw material is washed and cut, low-temperature induction is implemented to produce the enzyme, homogenate is agitated with a homogenate liquid, a concentrated solution of the dual-functional radish enzyme is obtained through steps of centrifugation, filtering and hyperfiltration and the like, and a protecting agent and a stabilizing agent are respectively added into the concentrated solution, thus obtaining the liquid dual-functional radish enzyme or the dry dual-functional radish enzyme powder. The industrial preparation method of the dual-functional radish enzyme has the following advantages: 1. low-temperature lighting induction technique is adopted, thus raising the raw enzyme content by 2 to 3 times; 2. specific activity stabilizing agent and protecting agent of the dual-functional radish enzyme are developed, thus keeping the activity of the dual-functional radish enzyme basically unchanged in two years at room temperature and reaching the activity of over 97 percent of the raw enzyme; 3. the raw material has wide source and low price, is not affected by factors including season and weather and the like, and can be planted in all seasons; 4. the preparation method has simple technique and low price.

Description

technical field

[0001] The invention relates to a radish bifunctional enzyme with both chitinase and lysozyme activities, in particular to an industrial preparation method for the radish bifunctional enzyme with both chitinase and lysozyme activities. Background technique

[0002] Chitin is a kind of N-acetylglucosamine (N-actylglucosamine, GlcNAc) linked by β-1, 4 glycosidic bonds. important structural components. Chitin-binding proteins (CBPs) are a class of proteins that can specifically bind to chitin. They are widely found in animals and plants, and are part of the natural defense system of animals and plants against bacterial and fungal infections. The main members of the group are expressed in large quantities when animals and plants are injured, and when they are infected by diseases and insect pests.

[0003] Lysozyme can catalyze the hydrolysis of the β-1,4 glycosidic bond between N-acetylglucosamine (and N-acetylmuramic acid (NAM), which constitutes the cell wal...

Examples

Embodiment 1

[0031] Get 1000 kilograms of fresh radish leaves or tubers, wash them, and chop them; at 15°C, under 4000lux light conditions, induce for 7 days; add 4°C pre-cooled 5mM sodium bicarbonate (NaHCO 3 ) stirring homogenate; the homogenate is filtered, the filter residue is preserved, the filtrate is taken, and at room temperature, it is centrifuged at 12000r / min, and the supernatant I is collected; the filter residue is added with 4 ℃ pre-cooled 5mM sodium bicarbonate of 1 times the filter residue volume and stirred evenly slurry; the homogenate is filtered, the filter residue is discarded, and the filtrate is taken, and at room temperature, it is centrifuged at 12000r / min to collect the supernatant II; the above-mentioned supernatant I and supernatant II are combined and filtered with a 200-mesh two-layer nylon cloth, Collect the filtrate; centrifuge at 14000r / min at normal temperature to collect the supernatant III; concentrate the supernatant III by ultrafiltration to exclude pr...

Embodiment 2

[0034] Take the concentrated solution obtained in Example 1, add 4% (volume / volume) glycerin and 9% (mass / volume) glucose to obtain liquid radish bifunctional enzyme II.

[0035] The yield of radish bifunctional enzyme II is 180-220 kg / ton raw material, and the enzyme activity is 1.8-2.5 million units / g.

Embodiment 3

[0037] Take 1 obtained radish bifunctional enzyme I, add pH6.2, 0.1M Na 3 PO 4 The buffer solution was dissolved, and the supernatant was taken by centrifugation. The supernatant was prepared with chitosan as a medium for deamination and regenerated chitin gel, and then affinity chromatography was carried out to collect the eluate containing enzyme. The eluate was concentrated by ultrafiltration. Add 6% (volume / volume) glycerol and 12% (mass / volume) glucose to proteins with a molecular weight cutoff of more than 10,000 to obtain liquid radish bifunctional enzyme III.

[0038] The yield of the liquid radish bifunctional enzyme III is 45-55 liters / ton raw material, and the enzyme activity is 68,000-72,000 units / ml.

technical field

[0001] The invention relates to a radish bifunctional enzyme with both chitinase and lysozyme activities, in particular to an industrial preparation method for the radish bifunctional enzyme with both chitinase and lysozyme activities. Background technique

[0002] Chitin is a kind of N-acetylglucosamine (N-actylglucosamine, GlcNAc) linked by β-1, 4 glycosidic bonds. important structural components. Chitin-binding proteins (CBPs) are a class of proteins that can specifically bind to chitin. They are widely found in animals and plants, and are part of the natural defense system of animals and plants against bacterial and fungal infections. The main members of the group are expressed in large quantities when animals and plants are injured, and when they are infected by diseases and insect pests.

[0003] Lysozyme can catalyze the hydrolysis of the β-1,4 glycosidic bond between N-acetylglucosamine (and N-acetylmuramic acid (NAM), which constitutes the cell wal...

Examples

Embodiment 1

[0031] Get 1000 kilograms of fresh radish leaves or tubers, wash them, and chop them; at 15°C, under 4000lux light conditions, induce for 7 days; add 4°C pre-cooled 5mM sodium bicarbonate (NaHCO 3 ) stirring homogenate; the homogenate is filtered, the filter residue is preserved, the filtrate is taken, and at room temperature, it is centrifuged at 12000r / min, and the supernatant I is collected; the filter residue is added with 4 ℃ pre-cooled 5mM sodium bicarbonate of 1 times the filter residue volume and stirred evenly slurry; the homogenate is filtered, the filter residue is discarded, and the filtrate is taken, and at room temperature, it is centrifuged at 12000r / min to collect the supernatant II; the above-mentioned supernatant I and supernatant II are combined and filtered with a 200-mesh two-layer nylon cloth, Collect the filtrate; centrifuge at 14000r / min at normal temperature to collect the supernatant III; concentrate the supernatant III by ultrafiltration to exclude pr...

Embodiment 2

[0034] Take the concentrated solution obtained in Example 1, add 4% (volume / volume) glycerin and 9% (mass / volume) glucose to obtain liquid radish bifunctional enzyme II.

[0035] The yield of radish bifunctional enzyme II is 180-220 kg / ton raw material, and the enzyme activity is 1.8-2.5 million units / g.

Embodiment 3

[0037] Take 1 obtained radish bifunctional enzyme I, add pH6.2, 0.1M Na 3 PO 4 The buffer solution was dissolved, and the supernatant was taken by centrifugation. The supernatant was prepared with chitosan as a medium for deamination and regenerated chitin gel, and then affinity chromatography was carried out to collect the eluate containing enzyme. The eluate was concentrated by ultrafiltration. Add 6% (volume / volume) glycerol and 12% (mass / volume) glucose to proteins with a molecular weight cutoff of more than 10,000 to obtain liquid radish bifunctional enzyme III.

[0038] The yield of the liquid radish bifunctional enzyme III is 45-55 liters / ton raw material, and the enzyme activity is 68,000-72,000 units / ml.