阅读说明：本技术 腈水解酶BnNIT2的应用及消除饲料中腈化物毒性的方法 (Application of nitrilase BnNIT2 and method for eliminating toxicity of nitrile compounds in feed ) 是由 罗会颖 张亨 姚斌 王亚茹 柏映国 黄火清 苏小运 王苑 涂涛 张�杰 于 2020-08-13 设计创作，主要内容包括：本发明涉及农业生物技术领域,具体涉及腈水解酶RmNIT的应用及消除饲料中腈化物毒性的方法。本发明确定Brassica napu来源的氨基酸序列如SEQ ID NO：1所示的蛋白为腈水解酶,其最适pH为7.0,最适温度为45℃。50℃处理10 min约剩余50%酶活力,50℃处理1 h约剩余10%酶活力,在pH 6-8范围内37℃处理1 h,剩余80%酶活力,适合于在食品、饲料、医药等行业中应用,消除菜籽粕中的抗营养物质。(The invention relates to the technical field of agricultural biology, in particular to application of nitrilase RmNIT and a method for eliminating toxicity of nitrile compounds in feed. The amino acid sequence of Brassica napu source is determined to be shown in SEQ ID NO: the protein shown in 1 is nitrilase, the optimum pH value is 7.0, and the optimum temperature is 45 ℃. 50 percent of enzyme activity is remained after 10 min of 50 ℃, 10 percent of enzyme activity is remained after 1 h of 50 ℃, 80 percent of enzyme activity is remained after 1 h of 37 ℃ of pH 6-8, and the method is suitable for application in food, feed, medicine and other industries and can eliminate anti-nutritional substances in rapeseed dregs.)

1. The amino acid sequence is shown as SEQ ID NO: 1 as a nitrilase.

2. Use according to claim 1, wherein the protein exerts nitrilase activity at a temperature of 30-50 ℃.

3. Use according to claim 2, wherein the protein exerts nitrilase activity at a temperature of 45 ℃.

4. The use according to claim 1, wherein the protein exerts nitrilase activity at ph6.0 to ph 8.0.

5. The use according to claim 4, wherein the protein exerts nitrilase activity at pH 7.0.

6. A method for eliminating the toxicity of nitriles in feed, which comprises using a peptide having the amino acid sequence shown in SEQ ID NO: 1, hydrolyzing nitrile compounds in the feed by using the enzyme.

7. The method for eliminating toxicity of nitrile compounds in feedstuff as claimed in claim 6, wherein said enzyme hydrolyzes nitrile compounds in feedstuff at 30-50 ℃ and pH 6.0-8.0.

8. The method for eliminating toxicity of nitrile compounds in feed according to claim 6, wherein the enzyme hydrolyzes nitrile compounds in feed at 45 ℃ and pH 7.0.

9. The method for eliminating toxicity of nitrile compounds in feed according to claim 6, wherein the feed is rapeseed meal.

Technical Field

The invention relates to the technical field of agricultural biology, in particular to application of nitrilase RmNIT and a method for eliminating toxicity of nitrile compounds in feed.

Background

China has abundant miscellaneous meal protein resources such as: cotton seed meal, rapeseed meal, palm meal, peanut meal, and the like. Under the condition of severe shortage of high-quality protein feed resources in the current market, the miscellaneous meal protein resources have good application prospects. But the addition of the miscellaneous meal in the feed is seriously influenced by the existence of anti-nutrient substances in the miscellaneous meal. Polyphenol compounds such as gossypol and coagulating agent can reduce protein digestibility in feed; phytic acid, thioglucoside and the like influence the absorption of trace elements by animals and the like. At the present stage, the methods for eliminating the anti-nutritional substances include physical methods, chemical methods and biological methods, wherein the application of the feed enzyme preparation is an effective way for eliminating the anti-nutritional substances.

The rapeseed dregs are a large category of miscellaneous dregs resources, and the elimination of anti-nutritional substances in the rapeseed dregs has a great promotion effect on the application of the rapeseed dregs in the feed. One type of anti-nutritional substance in the rapeseed dregs is thioglucoside, which is called thioglycoside for short and is a sulfur-containing secondary metabolite widely existing in cruciferae and related species, and more than 120 thioglycosides are found in hundreds of plants at present. The glucosinolate can cause the thyroid enlargement of the feeding animals, thereby causing the growth and development retardation of the feeding animals, and the rapeseed dregs are difficult to be fully applied as high-quality feed protein resources. Thioglucoside itself is non-toxic and its degradation products, such as nitrile compounds, are harmful to animal growth. There are many kinds of nitriles in rapeseed dregs, mainly: 3-hydroxy-4-pentenenitrile, 3-butenenitrile, 3-indoleacetonitrile, and the like.

Nitrilase (Nitrilase; EC 3.5.5.1) is an important catalyst in Nitrilase super family, and as an important industrial enzyme, the Nitrilase not only can convert nitrile compounds into intermediates of products with high added values, such as industry, food, medicine and the like, but also can directly convert toxic nitrile compounds into corresponding non-toxic acid and ammonia to treat nitrile pollutants in the environment.

Disclosure of Invention

The object of the present invention is to provide the use of a Brassica napu-derived enzyme as a nitrilase.

It is still another object of the present invention to provide a method for eliminating the toxicity of nitriles in feed.

The amino acid sequence of Brassica napu is shown as SEQ ID NO: 1 is a nitrilase. Wherein the enzyme has a full length of 343 amino acids and a theoretical molecular weight of 37 kDa.

The nitrilase BnNIT2 had an optimum pH of 7.0 and an optimum temperature of 45 ℃. 50% of enzyme activity is remained after 10 min of 50 ℃ treatment, and 10% of enzyme activity is remained after 1 h of 50 ℃ treatment. Treating at 37 deg.C within pH 6-8 for 1 h to obtain 80% enzyme activity.

The DNA complete sequence analysis result of the nitrilase gene shows that the overall length of the nitrilase BnNIT2 structural gene is 1029bp, and the DNA complete sequence analysis result of the nitrilase gene is obtained by a complete synthesis method through the conventional operation of genetic engineering, wherein the DNA complete sequence analysis result corresponds to the sequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, respectively.

The method for eliminating toxicity of nitrile compounds in feed according to the present invention comprises using a peptide having an amino acid sequence as set forth in SEQ ID NO: 1, hydrolyzing nitrile compounds in the feed by using the enzyme.

According to the method for eliminating the toxicity of the nitrile compounds in the feed, the enzyme hydrolyzes the nitrile compounds in the feed at the temperature of 30-50 ℃ and the pH value of 6.0-8.0.

According to the method for eliminating the toxicity of the nitrile compounds in the feed, the enzyme hydrolyzes the nitrile compounds in the feed at 45 ℃ and pH 7.0.

According to the method for eliminating the toxicity of the nitrile compounds in the feed, the feed is rapeseed dregs.

The invention provides the application of the nitrilase. The nitrilase is industrially produced by using a genetic engineering means. The nitrilase can effectively catalyze 3-hydroxy-4-pentenenitrile, 3-butenenitrile, 3-indoleacetonitrile and the like in rapeseed dregs, so that the nitrilase can be applied to industries such as feed, food, medicine and the like. According to the technical scheme of the invention, nitrilase with excellent property and suitable for industrial application can be produced by utilizing a genetic engineering means.

Drawings

FIG. 1 shows the optimum pH for nitrilase BnNIT 2;

FIG. 2 shows the pH stability of nitrilase BnNIT 2;

FIG. 3 shows the optimum reaction temperature for nitrilase BnNIT 2;

FIG. 4 shows the thermostability of nitrilase BnNIT 2.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: pET28a vector, E.coli expression strain BL21 (DE 3).

2. Enzymes and other biochemical reagents: restriction enzyme, FastpfuDNA polymerase, DNA recovery kit and plasmid miniextraction and miniextraction kit.

3. Culture medium:

(1) LB liquid medium: 5 g/L yeast powder, 10 g/L peptone and 10 g/L NaCl.

(2) LB solid medium: 15 g/L agar powder is added on the basis of the solid culture medium.

(3) Kanamycin: 100 mg/mL, 0.22. mu.M filter membrane was filtered out.

(4) 50 × TAE: 57.1 mL/L glacial acetic acid, 242 g/L Tris base, 50M EDTA, adjusted to pH8.0

(5) Solution A: 2.5 g of sodium hydroxide, 18.7 g of disodium hydrogen phosphate and 15.9 g of sodium phosphate, sodium hypochlorite solution containing 250 mg of available chlorine, wherein the pH value is 11.7, and ddH2O is added to a constant volume of 500 mL to obtain a sodium hydroxide and sodium hypochlorite compound solution.

And B, liquid B: 5.0 g of phenol, 0.025 g of sodium nitroferricyanide and ddH2O were added to a constant volume of 500 mL to prepare a complex solution of phenol and sodium nitroferricyanide.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.