阅读说明：本技术 咖啡酸作为漆酶降解霉菌毒素的介体的应用 (Application of caffeic acid as mediator for degrading mycotoxin by laccase ) 是由 姚斌 苏小运 王晓璐 罗会颖 黄火清 王苑 柏映国 涂涛 王亚茹 张�杰 师霞 于 2019-09-30 设计创作，主要内容包括：本发明属于农业生物领域,具体涉及参与枯草芽孢杆菌来源漆酶降解霉菌毒素的高效介体的应用。本发明提供了一种可用于霉菌毒素降解的高效漆酶介体。本发明的介体能够协助枯草芽孢杆菌来源漆酶有效地降解不同结构类型的霉菌毒素,广泛用于食品和饲料霉菌毒素脱毒领域。(The invention belongs to the field of agricultural biology, and particularly relates to application of a high-efficiency mediator participating in degradation of mycotoxin by using laccase derived from bacillus subtilis. The invention provides a high-efficiency laccase mediator for mycotoxin degradation. The mediator can assist the laccase from the bacillus subtilis to effectively degrade mycotoxins with different structural types, and is widely applied to the field of mycotoxin detoxification of foods and feeds.)

1. The application of caffeic acid as a mediator participating in the degradation of mycotoxin by laccase is disclosed, wherein the laccase is from bacillus subtilis, and the amino acid sequence of the laccase is shown as SEQ ID No. 1.

2. The use according to claim 1, wherein the mycotoxin is aflatoxin B₁And/or zearalenone.

3. Use according to claim 1, wherein the laccase and caffeic acid degrade mycotoxins in Tris-HCl solution at a concentration of 50mM and a pH of 7.0.

4. A method of increasing the degradation rate of a laccase for degrading mycotoxin, comprising the step of using caffeic acid as a mediator involved in the degradation of mycotoxin by the laccase, wherein the laccase is from bacillus subtilis and has the amino acid sequence shown in SEQ ID No. 1.

5. The method of claim 4, wherein the mycotoxin is aflatoxin B₁And/or zearalenone.

6. The method of claim 4, wherein the laccase and caffeic acid degrade mycotoxins in Tris-HCl at a concentration of 50mM and a pH of 7.0.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to application of caffeic acid as a mediator for degrading mycotoxin by laccase.

Background

Mycotoxins are secondary metabolites produced by fungi, mainly pollute stored grain and oil food and feed, and seriously harm human and livestock health. According to the structural characteristics of the mycotoxins, the mycotoxins can be divided into two main classes of aromatic rings and non-aromatic rings, wherein the aromatic rings comprise aflatoxin, zearalenone, citrinin, ochratoxin, patulin, trichothecene toxins and the like; the non-aromatic ring includes fumonisins only. Of these, aflatoxins, zearalenone and deoxynivalenol (vomitoxin) are the most common and most harmful mycotoxins. Therefore, there is a need to establish a simple, effective and environmentally friendly method for detoxification of mycotoxins.

At present, the detoxification method of the feed polluted by mycotoxin mainly comprises a physical method, a chemical method, an adsorption method, a biological method and the like. Physical and chemical detoxification methods have the defects of difficult operation, unstable effect, large loss of nutrient components, influence on the palatability of the feed and the like. Although the adsorption method is simple and easy, the method has the defects of large dosage, low economy, easy secondary pollution and the like. The microbial detoxification method has the advantages of mild action conditions, little influence on sensory properties, palatability and the like of the raw materials, increase in the nutritive value of the raw materials and the like, and is considered to be the optimal detoxification method. Biological detoxification mainly refers to the enzymatic reaction of degrading enzymes to convert toxins into low-toxicity or non-toxicity products, wherein the degrading enzymes comprise oxidases such as laccase, manganese peroxidase, hydrolases (such as esterase) and the like.

In the process of realizing large-scale application of the biological detoxification technology, bacterial strains capable of degrading mycotoxin are searched and screened, characteristic research is carried out on extracellular degrading enzymes produced by the bacterial strains, and degrading enzyme genes are cloned and expressed, so that the bacterial strains are important breakthrough points and development directions in the research field of mycotoxin biodegradation. Therefore, establishing a high-efficiency degradation system for degrading mycotoxin by an enzyme method is the key point of the biological detoxification technology.

The degradation rate of the prior laccase to the mycotoxin is generally low. It is reported in the literature that when dye decolorization is performed using a laccase-mediator system, the effect of the mediator depends on the type of dye being treated. For example, white rot fungus laccase can effectively degrade acid red 73 by taking HBT as a mediator. When the coriolus versicolor laccase is used for pesticide degradation, different pesticide substrates show selectivity for mediators, for example, when pyrimethanil and isoproturon are degraded, the best mediator is the cyanuric acid, and acetosyringone and HBT are the best mediators for degrading chlorothalonil and naprophytin. It follows that suitable mediators of laccases differ significantly by their differences in their substrate-degrading properties.

Disclosure of Invention

The invention aims to provide application of caffeic acid as a high-efficiency mediator participating in degradation of mycotoxin by using laccase derived from bacillus subtilis.

The application of caffeic acid as a mediator participating in the degradation of mycotoxin by laccase according to the embodiment of the invention is characterized in that the laccase is from bacillus subtilis, and the amino acid sequence of the laccase is shown as SEQ ID No. 1.

According to a specific embodiment of the invention, the mycotoxin is aflatoxin B₁And/or zearalenone.

According to a particular embodiment of the invention, aflatoxin B is treated in a buffered solution₁And zearalenone, wherein the buffer solution is a Tris-HCl solution with the concentration of 50mM and the pH value of 7.0.

The method for improving the degradation rate of the mycotoxin degraded by the laccase comprises the step of using caffeic acid as a mediator participating in the degradation of the mycotoxin by the laccase, wherein the laccase is from bacillus subtilis, and the amino acid sequence of the laccase is shown as SEQID No. 1.

According to a specific embodiment of the invention, the mycotoxin is aflatoxin B₁And/or zearalenone.

According to a particular embodiment of the invention, aflatoxin B is treated in a buffered solution₁And zearalenone, wherein the buffer solution is a Tris-HCl solution with the concentration of 50mM and the pH value of 7.0.

The method of the invention can efficiently degrade mycotoxin, has low cost and wide application range, and can be widely applied to the field of feed toxin degrading enzymes.

Drawings

FIG. 1 shows the Bacillus subtilis-derived laccase-caffeic acid system on aflatoxin B₁And zearalenone degradation.

FIG. 2 shows that aflatoxin B is degraded by recombinant Bacillus subtilis-derived laccase-caffeic acid system₁The result of HPLC analysis of (1);

FIG. 3 shows the HPLC analysis results of the recombinant Bacillus subtilis laccase-caffeic acid system for degrading zearalenone;

Detailed Description

Test materials and reagents

1. The strain is as follows: an engineered strain of Escherichia coli for producing laccase BsCotA derived from Bacillus subtilis.

2. Biochemical reagents: aflatoxin B₁Zearalenone, caffeic acid; chromatographic purity acetonitrile, trifluoroacetic acid and Tris.

3. Culture medium:

(1) coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, ph7.0) BsCotA amino acid sequence was as follows:

MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINASNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPVLLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK