阅读说明：本技术 一种热稳定性提高的玉米赤霉烯酮降解酶突变体及其应用 (Zearalenone degrading enzyme mutant with improved thermal stability and application thereof ) 是由 沐万孟 张文立 张振霞 徐炜 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种热稳定性提高的玉米赤霉烯酮降解酶突变体及其应用,属于生物工程技术领域。本发明以氨基酸序列如SEQ ID NO.2所示的玉米赤霉烯酮降解酶为亲本酶,将第134位的组氨酸His和136位的丝氨酸Ser同时替换成亮氨酸Leu,得到双突变体H134L/S136L。H134L/S136L在53℃保温2min后残余酶活提高了45％；保温5min后的残余酶活分别提高了38％；保温7min后残余酶活提高了38％。在58℃保温2min后残余酶活提高了41％,保温5min后的残余酶活提高了32％。这一发现对于制备高热稳定性的工业化玉米赤酶烯酮降解酶具有重要的应用价值。(The invention discloses a zearalenone degrading enzyme mutant with improved thermal stability and application thereof, and belongs to the technical field of biological engineering. The zearalenone degrading enzyme with an amino acid sequence shown as SEQ ID NO.2 is used as a parent enzyme, and histidine His at the 134 th site and serine Ser at the 136 th site are simultaneously replaced by leucine Leu to obtain a double mutant H134L/S136L. After the H134L/S136L is subjected to heat preservation at 53 ℃ for 2min, the residual enzyme activity is improved by 45 percent; the residual enzyme activity after 5min of heat preservation is respectively improved by 38 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 38 percent. The residual enzyme activity is improved by 41 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is improved by 32 percent after the temperature is kept for 5 min. The discovery has important application value for preparing the industrial zearalenone degrading enzyme with high thermal stability.)

1. A zearalenone degrading enzyme mutant with improved thermal stability is characterized by comprising an amino acid sequence shown as SEQ ID No. 4.

2. A gene encoding the zearalenone degrading enzyme mutant of claim 1.

3. A plasmid carrying the gene of claim 2.

4. The plasmid of claim 3, wherein the plasmid comprises pET-22b (+).

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

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

7. A method for improving the thermal stability of zearalenone degrading enzyme is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of the zearalenone degrading enzyme with an amino acid sequence shown as SEQID NO.2 are simultaneously replaced by leucine Leu.

8. A method for producing zearalenone degrading enzyme, characterized in that the cells of claim 5 or 6 are inoculated into LB medium for activation culture, and the activated seed solution is transferred into LB medium for fermentation culture.

9. The method as claimed in claim 8, wherein the cells of claim 5 or 6 are inoculated into LB medium at 35-39 ℃ and 200-220rpm for 8-14h, then the activated seed solution is inoculated into LB medium at an inoculation volume ratio of 1-10%, cultured at 35-39 ℃ for 3-4h until OD value is 0.6-0.8, cooled to 25-30 ℃, and induced by adding IPTG with final concentration of 0.5mM-1mM for 5-10 h.

10. The use of the zearalenone degrading enzyme mutant of claim 1 in the field of grain or feed.

Technical Field

The invention relates to a zearalenone degrading enzyme mutant with improved thermal stability and application thereof, belonging to the technical field of biological engineering.

Background

Zearalenone is a mycotoxin of a nonsteroidal estrogen, and has a chemical name of 6- (10-hydroxy-6-oxy-undecenyl) beta-clavulanate lactone. Zearalenone was first discovered in moldy corn in 1962 as a toxic secondary metabolite produced by fusarium, is widely present in moldy corn, wheat, barley and other grains and grain by-products, and has become a worldwide food and feed contaminant. In general, inappropriate storage conditions (temperatures of 10-30 ℃ and ambient relative humidity of 40-50%) are the primary cause of zearalenone production. Moreover, zearalenone also has a number of toxic derivatives such as: α/β -Zearalenol (α/β -Zearalenol, α/β -ZOL), α/β -Zearalanol (α/β -Zearalanol, α/β -ZAL), Zearalenone (ZAN), and the like. Zearalenone and its derivatives, an exogenous estrogen analogue, can competitively bind to estrogen receptors, causing reproductive toxicity, genotoxicity, immunotoxicity and carcinogenicity to humans and many farmed animals, causing great harm to the health of humans and livestock, and causing great economic loss to the food industry, feed industry and animal husbandry.

In recent years, the pollution degree of zearalenone in grains, foods and feeds is continuously reduced, and effective detoxification of biotoxins is listed as one of the key research and development plans of 2019 by the scientific and technological department, so that people pay more and more attention to the exploration of an effective and environment-friendly method for removing zearalenone. The prior method for removing zearalenone can be mainly divided into three types, namely physical removal, chemical decomposition and biodegradation. Wherein the physical removal is to remove the zearalenone by heating, irradiation or adsorption and other methods, but the nutrient components are usually destroyed and the removal efficiency is lower; the chemical decomposition is to detoxify the zearalenone by adopting the modes of acid/alkali hydrolysis, ammoniation or ozonization and the like, has low removal efficiency, can cause secondary pollution, and is not beneficial to environmental protection and animal and plant health. The biodegradation method utilizes the adsorption effect of microbial thalli on zearalenone or the degradation effect of enzymes generated by microorganisms on zearalenone, has the advantages of good specificity, mild action conditions, high detoxification efficiency, no damage to nutrient substances, no generation of secondary pollution and the like, and is a hotspot direction of current research.

Currently, zearalenone degrading enzymes mainly include Laccase (lacgase), Peroxidase (Peroxidase) and lactone hydrolase (Lactonase). Wherein, the laccase is mainly used for degrading aflatoxin, and an oxidation-reduction medium (methyl butyrate) is required to exist when the laccase is used for degrading zearalenone; the mechanism of degrading zearalenone by peroxidase is not clear; lactone hydrolases are currently the most interesting zearalenone degrading enzymes. In the enzymology research, the lactone hydrolase can catalyze the opening of a lactone ring of zearalenone and spontaneously decarboxylate to form a completely nontoxic degradation product.

A lactone hydrolase having the ability to degrade zearalenone was first discovered in Gliocladium roseum Clinostachys rosea IFO 7063 by Naoko in 2002, and was named ZHD 101. Followed by a number of different sources of lactonohydrolase enzymes that degrade zearalenone such as: zearalenone-jjm from Gliocladium roseum, Zhly-6 from Gliocladium roseum 31535, Cbzhd from Cladophora bataiana, Zhd518 from Rhinocladiella mackenziei CBS 650.93 and zearalenone C from Neurospora crassa were successively found. Among them, ZHD101 has been successfully expressed in various hosts such as e.coli BL21, p.pastoris GS115 and s.cerevisiae, and is the most deeply studied lactone hydrolase at present.

However, current research on zearalenone degrading enzymes has focused on the identification of enzymatic properties and the modification of substrate specificity. As a feeding enzyme, not only is it required to have high activity and broad substrate specificity, but it is also required to have high thermal stability to withstand the high temperatures of the pelleting process.

Disclosure of Invention

In order to solve the problems and obtain the zearalenone degrading enzyme more suitable for industrial application, the invention carries out molecular modification on the zearalenone degrading enzyme from Gliocladium roseum MA918 by a site-directed mutagenesis method so as to improve the thermal stability of the zearalenone degrading enzyme and obtain the zearalenone degrading enzyme more meeting the industrial requirements.

The invention aims to provide a zearalenone degrading enzyme mutant with improved thermal stability, which comprises an amino acid sequence shown in SEQ ID NO. 4.

The zearalenone degrading enzyme mutant comprises a double-mutant enzyme H134L/S136L obtained by mutating a zearalenone degrading enzyme wild enzyme derived from Gliocladium roseum MA918, the mutation mode is that histidine His at the 134 th position and serine Ser at the 136 th position of the zearalenone degrading enzyme wild enzyme are simultaneously replaced by leucine Leu, the amino acid sequence of the zearalenone degrading enzyme wild enzyme is shown as SEQ ID NO.2, the nucleotide sequence of a gene for coding the zearalenone degrading enzyme wild enzyme is shown as SEQ ID NO.1, and the number of the gene in GeneBank is KR 363960.1.

The second object of the present invention is to provide a gene encoding the zearalenone degrading enzyme mutant as described above.

In one embodiment of the invention, the nucleotide sequence of the gene is shown in SEQ ID NO. 3.

The third object of the present invention is to provide a plasmid carrying the above gene.

In one embodiment of the present invention, the plasmid comprises pET-22b (+).

The fourth purpose of the invention is to provide a cell expressing the zearalenone degrading enzyme mutant.

In one embodiment of the invention, the cell comprises escherichia coli BL21(DE 3).

The fifth purpose of the invention is to provide a method for improving the thermal stability of zearalenone degrading enzyme, which is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of the zearalenone degrading enzyme with the amino acid sequence shown as SEQ ID NO.2 are simultaneously replaced by leucine Leu.

The sixth purpose of the invention is to provide a method for producing zearalenone degrading enzyme, which comprises the steps of inoculating the cells into LB culture medium for activation culture, and transferring the activated seed liquid into LB culture medium for fermentation culture.

In one embodiment of the invention, the cells are inoculated in LB culture medium at 35-39 ℃ and 200-220rpm for 8-14h, then the activated seed liquid is inoculated into LB culture medium at an inoculation volume ratio of 1-10%, cultured at 35-39 ℃ for 3-4h until the OD value is 0.6-0.8, cooled to 25-30 ℃, and induced by adding IPTG with a final concentration of 0.5-1 mM for 5-10 h.

The seventh purpose of the invention is to provide the application of the zearalenone degrading enzyme mutant in the field of grains or feeds.

The eighth object of the present invention is the use of the cell expressing the zearalenone degrading enzyme mutant as described above in the field of grain or feed.

The ninth object of the present invention is to provide the application of the method for improving the thermal stability of zearalenone degrading enzyme in the field of grain or feed.

The invention has the beneficial effects that: the invention provides a zearalenone degrading enzyme mutant H134L/S136L, compared with wild enzyme, the optimum catalytic condition of the enzyme mutant H134L/S136L is not changed, but the residual enzyme activity of the enzyme mutant H134L/S136L is improved by 45% after the enzyme mutant H134L/S136L is subjected to heat preservation at 53 ℃ for 2 min; the residual enzyme activity after 5min of heat preservation is improved by 38 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 38 percent. The residual enzyme activity is improved by 41 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is respectively improved by 32 percent after the temperature is kept for 5 min. The discovery has important value for the industrial application of the zearalenone degrading enzyme.

Drawings

FIG. 1: comparison of the thermostability of the wild enzyme and the mutant enzyme H134L/S134L at 53 ℃.

FIG. 2: comparison of the thermostability of the wild enzyme and the mutant enzyme H134L/S134L at 58 ℃.

Detailed Description

The method for determining the activity of the zearalenone degrading enzyme comprises the following steps:

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

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 ℃. And (3) detecting the synthesis amount of the zearalenone degrading enzyme by using HPLC (high performance liquid chromatography), and calculating the enzyme activity.