阅读说明：本技术 一种真菌毒素zen降解酶突变体及其应用 (Mycotoxin ZEN degrading enzyme mutant and application thereof ) 是由 沐万孟 张文立 徐炜 张振霞 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种真菌毒素ZEN降解酶突变体及其应用,属于生物工程技术领域。本发明公开了来源于Gliocladium roseum MA918的玉米赤霉烯酮降解酶(简称ZENG酶)作为亲本,利用基因突变技术,第134位的组氨酸His和136位的丝氨酸Ser同时替换成苯丙氨酸Phe,得到双突变体H134F/S136F。最适催化条件下,酶在53℃保温2min后残余酶活提高了36％；保温5min后的残余酶活提高了33％；保温7min后残余酶活提高了12％。在58℃保温2min后残余酶活提高了34％,保温5min后的残余酶活提高了13％。(The invention discloses a mycotoxin ZEN degrading enzyme mutant and application thereof, and belongs to the technical field of biological engineering. The invention discloses zearalenone degrading enzyme (ZENG enzyme for short) derived from Gliocladium roseum MA918 as a parent, and a double mutant H134F/S136F is obtained by simultaneously replacing histidine His at a 134 th position and serine Ser at a 136 th position with phenylalanine Phe by using a gene mutation technology. Under the most suitable catalysis condition, the residual enzyme activity of the enzyme is improved by 36 percent after the enzyme is kept at 53 ℃ for 2 min; the residual enzyme activity after 5min of heat preservation is improved by 33 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 12 percent. The residual enzyme activity is improved by 34 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is improved by 13 percent after the temperature is kept for 5 min.)

1. A fungal toxin ZEN degrading enzyme mutant with improved thermostability, characterized in that its amino acid sequence is:

(a) as shown in SEQ ID NO. 4;

(b) or (b) the protein which is derived from the (a) and has ZEN degrading enzyme activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

2. A gene encoding the mycotoxin ZEN degradative enzyme mutant of claim 1.

3. A plasmid containing the gene of claim 2.

4. The plasmid of claim 3, wherein the plasmid comprises, but is not limited to, pET series plasmids.

5. A cell expressing the mycotoxin ZEN degrading enzyme mutant of claim 1.

6. The cell of claim 5, wherein the cell is E.coli BL21(DE 3).

7. A method for improving the heat stability of ZEN degrading enzyme is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of zearalenone degrading enzyme with an amino acid sequence shown as SEQ ID NO.2 are simultaneously replaced by phenylalanine Phe.

8. A method for degrading ZEN is characterized in that ZEN is used as a substrate, and crude enzyme solution or whole cells containing the mycotoxin ZEN degrading enzyme mutant are added.

9. Use of the ZEN degrading enzyme mutant of claim 1 in the field of agricultural products.

10. The use of the ZEN degrading enzyme mutant of claim 1 in the field of feed.

Technical Field

The invention relates to a mycotoxin ZEN degrading enzyme mutant and application thereof, belonging to the technical field of biological engineering.

Background

Zearalenone (ZEN), also known as F-2 toxin, is a fungal toxin produced by many fusarium species such as fusarium graminearum, fusarium flavum and fusarium graminearum and released into the soil environment. The chemical structure of ZEN was determined by Urry in 1966 using techniques such as nuclear magnetic resonance, classical chemistry and mass spectrometry and was named: 6- (10-hydroxy-6-oxy-undecenyl) beta-resorcinolic acid lactone. ZEN has wide pollution in grains and byproducts thereof all over the world, brings huge loss to the planting industry and the breeding industry, and also poses serious threat to food safety.

Currently, the degradation method of ZEN can be divided into three categories, namely physical, chemical and biological.

The physical method mainly comprises manual removal, water washing, shelling, high temperature, pressure boiling, adsorbent adsorption and the like. Removing ZEN from slightly ZEN-polluted grains by means of removing, washing and the like; because the ZEN has good thermal stability and does not decompose after being heated for 4 hours at 120 ℃, the modes of heat treatment, pressure cooking and the like mainly have a killing effect on fungi generating the ZEN, the damage effect on the ZEN is small, and the high temperature damages the nutritional value of food and feed and influences the taste of the food; the adsorption method mainly utilizes the hydrophobic effect to achieve the purpose of removing mycotoxin in food, and common adsorbents comprise yeast cell walls, activated carbon, montmorillonite, particularly modified montmorillonite, kaolin, cholestyramine and the like, but the adsorbents can combine some important nutrient substances such as amino acid, vitamin and the like in feed while adsorbing the toxin, influence nutrient substances in food, reduce the product quality, and can not be completely degraded, so that the environment can be seriously influenced.

The degradation principle of the chemical method is oxidative degradation, and the chemical structure of ZEN is O₃，H₂O₂And the like, and finally becomes a nontoxic ZEN byproduct. However, the chemical method has large workload and long operation time, and the ZEN is oxidized and degraded and can damage the nutrient components in the feed, so the improvement is still needed.

The principle of the biological method is to degrade ZEN into a nontoxic product by utilizing the adsorption of microbial cells and the degradation of the ZEN by the microbes or enzymes generated by the microbes. Particularly, the latter, the key enzyme gene with the ability to degrade ZEN is the focus of the current biological method research. At present, the research on the zearalenone biodegradation technology is still incomplete, for example, the principle of enzyme degradation and conversion of ZEN is not deeply researched, and meanwhile, the problems that the function of ZEN degrading bacteria is easy to decline and is unstable are endless and need to be improved.

Therefore, an effective method for biodegrading ZEN is provided, and the method has a wide application prospect.

Disclosure of Invention

One of the purposes of the invention is to provide a fungal toxin ZEN degrading enzyme mutant with improved thermal stability, and the amino acid sequence of the mutant is as follows:

(a) as shown in SEQ ID NO. 4;

(b) or (b) the protein which is derived from the (a) and has ZEN degrading enzyme activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

One of the objects of the present invention is to provide a gene encoding the above-mentioned mycotoxin ZEN degrading enzyme mutant.

Furthermore, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

It is an object of the present invention to provide plasmids containing the above genes, including but not limited to pET series plasmids.

Further, the plasmid includes pET-22b (+).

One of the purposes of the invention is to provide Escherichia coli expressing the fungal toxin ZEN degrading enzyme mutant.

Further, the Escherichia coli is Escherichia coli BL21(DE 3).

One of the purposes of the invention is to provide a method for improving the thermal stability of ZEN degrading enzyme, which is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of zearalenone degrading enzyme parent enzyme with amino acid sequences shown as SEQ ID NO.2 are simultaneously replaced by phenylalanine Phe. The nucleotide sequence of the gene for coding the parent enzyme of the zearalenone degrading enzyme is shown as SEQID NO.1, and the number of the gene is KR363960.1 in GeneBank

One of the purposes of the invention is to provide a method for degrading ZEN, which takes ZEN as a substrate and adds crude enzyme solution or whole cells containing the mycotoxin ZEN degrading enzyme mutant.

Further, the addition amount of the mycotoxin ZEN degrading enzyme mutant is 1-10000U/g substrate.

One of the purposes of the invention is to provide the application of the ZEN degrading enzyme mutant in the field of agricultural products.

One of the purposes of the invention is to provide the application of the ZEN degrading enzyme mutant in the field of feed.

The invention has the beneficial effects that: the invention provides a mutant enzyme H134F/S136F of ZENG, the optimum catalytic condition of which is not changed, compared with the parent enzyme ZENG, the optimum catalytic condition of the mutant enzyme H134F/S136F of ZENG is not changed, but the residual enzyme activity is improved by 36 percent after the mutant enzyme is insulated for 2min at 53 ℃; the residual enzyme activity after 5min of heat preservation is improved by 33 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 12 percent. The residual enzyme activity is improved by 34 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is respectively improved by 13 percent after the temperature is kept for 5 min. The finding has important value for the industrial application of the ZEN in degrading the lactonohydrolase.

Drawings

FIG. 1: comparison of the thermostability of the parent enzyme and the mutant enzyme H134F/S136F at 53 ℃.

FIG. 2: comparison of the thermostability of the parent enzyme and the mutant enzyme H134F/S136F at 58 ℃.

Detailed Description

Enzyme activity determination method of (I) ZEN degrading enzyme

The reaction system was 250. mu.L, and consisted of 5. mu.L of ZEN in methanol (4mg/mL), 5. mu.L of the enzyme solution (0.5mg/mL) and 240. mu.L of phosphate buffer (50mM, pH 7.0) and stopped by adding 50. mu.L of hydrochloric acid (1mol/L) and 300. mu.L of methanol at 38 ℃ for 10 min.

1U total enzyme activity is defined as the amount of enzyme required to consume 1. mu.g of substrate per minute for the reaction at pH 7.0, 38 ℃.

(II) protein purification method

A nickel ion affinity chromatography column was prepared by first pumping deionized water into the column (about 6-12 column volumes) using a constant flow pump at room temperature, and then equilibrating the column environment with buffer A (500mmol/L NaCl, 50mM Tris-HCl, pH 8.0). When the effluent at the lower end of the column and buffer A pumped into the column have the same pH value (about 5 column volumes of buffer), the resulting membrane-passed crude enzyme solution is added to the column. The heteroproteins are first washed with buffer B (500mmol/L NaCl, 50mmol/L imidazole, 50mM Tris-HCl, pH 8.0) to baseline equilibrium and then eluted with an eluent containing high concentrations of imidazole (500mmol/L NaCl, 500mmol/L imidazole, 50mM Tris-HCl, pH 8.0). Collecting the eluate of the absorption peak, and determining the enzyme activity to obtain the target protein.