WO2024125165A1 - 褐色绿僵菌ifst-ot3及其产生的脱毒酶与应用 - Google Patents

褐色绿僵菌ifst-ot3及其产生的脱毒酶与应用 Download PDF

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WO2024125165A1
WO2024125165A1 PCT/CN2023/130411 CN2023130411W WO2024125165A1 WO 2024125165 A1 WO2024125165 A1 WO 2024125165A1 CN 2023130411 W CN2023130411 W CN 2023130411W WO 2024125165 A1 WO2024125165 A1 WO 2024125165A1
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ifst
enzyme
amh1
ochratoxin
detoxification
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邢福国
王刚
吴文庆
郭旭
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中国农业科学院农产品加工研究所
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  • the invention relates to the field of microbiology and biological detoxification technology, and in particular to a brown green anisopliae IFST-OT3 and a detoxification enzyme produced by the same and its application.
  • Ochratoxin A is a secondary metabolite produced by Aspergillus and Penicillium fungi. In 1993, the International Agency for Research on Cancer classified OTA as a Class 2B carcinogen. In addition, studies have shown that OTA has nephrotoxicity, neurotoxicity, immunotoxicity, etc. (Malir, et al., 2016, Toxins 8:191). Ochratoxin B (OTB) is a precursor of OTA, and its molecular structure has one less chlorine atom than OTA.
  • OTA and OTB can contaminate a variety of agricultural products such as corn, wheat, sorghum, fruits, dairy products, and meat products (Wang, et al., 2022, Frontiers in Microbiology, 13:857726), causing serious food safety problems.
  • OTA in food and feed can cause import and export trade obstructions, human and animal medical expenses, and cause huge economic losses.
  • Biological detoxification methods can be divided into two categories. One is based on the adsorption of microorganisms themselves, and the other is the degradation of OTA by metabolites produced by microorganisms, such as enzymes. There are widespread microbial resources that can degrade OTA in nature, and some bacteria have a degradation rate of OTA as high as 90%.
  • Bacillus amyloliquefaciens has a degradation rate of OTA of 98.5% within 24 hours (Chang et al., 2015, Food Additives & Contaminants: Part A, 32, 564-571).
  • Bacillus subtilis (B.subtilis) isolated from elk feces has a degradation rate of OTA of 97.6% within 24 hours (Shi, et al., 2014, Journal of the Science of Food and Agriculture, 94: 1879-1885).
  • Alcaligenes faecalis can degrade 97% of OTA within 48 hours (Zhang, et al., 2017, Journal of Applied Microbiology, 123: 661-668).
  • the purpose of the present invention is to provide a brown green muscardine Metarhizium brunneum IFST-OT3 and its application in biological detoxification.
  • Another object of the present invention is to provide a detoxification enzyme produced by Metarhizium anisopliae IFST-OT3 and its application.
  • the present invention provides a strain of Metarhizium brunneum IFST-OT3 isolated and purified from a soil sample, which has been deposited in the General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with a deposit number of CGMCC No. 40349 and a deposit date of October 28, 2022.
  • the present invention provides a bacterial agent containing the brown Metarhizium IFST-OT3.
  • the present invention provides the use of the brown Metarhizium IFST-OT3 or its bacterial agent in the detoxification of ochratoxins A and B.
  • the present invention provides a detoxification enzyme Amh1 produced by the brown Metarhizium IFST-OT3, wherein the detoxification enzyme Amh1 comprises or consists of the following amino acid sequence:
  • ii an amino acid sequence obtained by connecting a tag to the N-terminus and/or C-terminus of i);
  • the present invention provides a nucleic acid molecule encoding the detoxification enzyme Amh1, and its nucleotide sequence is shown in SEQ ID NO:2.
  • the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.
  • the present invention provides any of the following applications of the detoxification enzyme Amh1:
  • the present invention provides a method for degrading ochratoxin A, wherein the detoxifying enzyme Amh1 is contacted with ochratoxin A in a buffer system at 30-80° C. and pH 4.0-8.0.
  • the concentration of ochratoxin A in the system is 1-5 ⁇ g/ml.
  • the molar ratio of detoxifying enzyme Amh1 to ochratoxin A is 1:130.
  • the invention realizes heterologous high expression of the detoxification enzyme in a prokaryotic expression system (Escherichia coli), and has extremely high degradation efficiency for ochratoxin.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention provides a brown green anisopliae fungus IFST-OT3 with strong degradation ability for ochratoxin A and B, as well as a detoxification enzyme (degradation enzyme) produced by the fungus and a nucleotide sequence encoding the amidase, and also provides the optimal physicochemical properties of the enzyme, including temperature and pH.
  • the amidase treats a substrate concentration of 1 ⁇ g/ml OTA toxin in a buffer system of 60°C and pH 6.0, and the degradation efficiency is as high as 100% within 3 minutes.
  • the strain IFST-OT3 and the detoxification enzyme produced by the strain can be used for the degradation of ochratoxin in agricultural products, and has broad application prospects.
  • Figure 1 is a HPLC test chart of the IFST-OT3 crude enzyme solution in a preferred embodiment of the present invention when incubated with OTA and OTB standard samples with a substrate concentration of 1 ⁇ g/ml for 24 hours and the molecular mass identified by HPLC-MS.
  • a and B are the HPLC and HPLC-MS test results of the OTA standard
  • C and D are the HPLC and HPLC-MS test results of OTA and IFST-OT3 crude enzyme solution after incubation for 24 hours
  • E and F are the HPLC and HPLC-MS test results of OTB standard
  • G and H are the HPLC and HPLC-MS test results of OTB and IFST-OT3 crude enzyme solution after incubation for 24 hours.
  • FIG. 2 shows the cloning, prokaryotic expression and purification of the detoxification enzyme Amh1 gene in a preferred embodiment of the present invention.
  • A is the agarose gel electrophoresis of the Amh gene cloned from the cDNA of the strain IFST-OT3;
  • B is the SDS-PAGE electrophoresis after Escherichia coli expressed Amh1, and the empty expression vector (vector) was used as a control;
  • C is the SDS-PAGE electrophoresis after Amh1 purification.
  • Figure 3 shows the degradation efficiency of amidase Amh1 in a preferred embodiment of the present invention.
  • A is the effect of different pH on the degradation efficiency;
  • B is the effect of different temperatures on the degradation efficiency;
  • C is the effect of different metal ions on the degradation efficiency;
  • D is the degradation time of 1.0 ⁇ g/ml, 2.5 ⁇ g/ml and 5.0 ⁇ g/ml OTA under the optimum pH 6.0 and optimum temperature (60°C).
  • Example 1 Isolation and identification of Metarhizium anisopliae IFST-OT3
  • IFST-OT3 was isolated from Beijing farmland soil samples by the following method: 5 g of soil was added with 5 mL of sterile water, shaken on a shaker for 30 min (180 rpm), gradiently diluted 10, 100, and 1000 times, respectively, and spread on PDA culture medium, inverted and cultured at 28°C for 72 h, and the grown colonies were purified by the plate streak method, and the obtained single colonies were inoculated on PDA plates for culture.
  • strain IFST-OT3 On PDA medium, the colonies of strain IFST-OT3 were villi or flocculent, initially white, dark green after spore production, and the edges of the colonies were regular and flat. The hyphae were septate, smooth, and transparent, with 2-3 phialides on the conidiophores, and the conidia were unicellular.
  • the strain IFST-OT3 was cultured in PDB medium, and DNA was extracted after 3 days.
  • the EF-1a (primer sequences: 5'-GTACCTCCCAGGCTGACTGC-3';5'-GTTCTTGGAGTCACCAGCAACG-3'), RPB2 (5'-GAGGAGCTGAAGAGGACCGG-3';5'-AATCGGTGTGTTGGTTCGTCG-3'), beta-tub (5'-CCGTCCATCAGCTCGTTGAG-3';5'-GGAACATGGCAGTGAACTGCTC-3') genes were amplified and sequenced.
  • the accession numbers of the three genes in NCBI are OP947525, OP947524, and OP947526. All three genes had the highest sequence identity with the homologous genes in Metarhizium brunneum ARSEF 3297.
  • strain IFST-OT3 was identified as Metarhizium brunneum, with the deposit number CGMCC No.40349.
  • Example 2 Degradation ability of OTA and OTB by Metarhizium anisopliae IFST-OT3
  • brown green anisopliae IFST-OT3 was cultured in PDB liquid medium at 180 rpm for 7 days.
  • the collected mycelium was ground into powder in a mortar with continuous addition of liquid nitrogen.
  • 1 g of powder was added to 20 ml Lysis Buffer (1 mM PMSF, 1% anisole hydrochloride, 10 mM Tris-Hcl (pH7), 150 mM NaCl, 0.5 mM EDTA, 0.01% Triton X-100, 1 mM DTT), and vortexed to mix. Centrifuge at 8000 rpm at 4°C for 15 min, and the supernatant obtained was the crude enzyme solution.
  • the present invention extracts fungal crude protein to determine the degradation activity and finds that brown green anisopliae IFST-OT3 has the ability to degrade OTA into OT ⁇ .
  • an amidase (detoxification enzyme) with degradation activity is finally obtained.
  • Gene Amh1 (GenBank: OP947527) was amplified from reverse transcribed cDNA of Metarhizium brunneum strain ( Figure 2, A). When designing primers, a BamHI restriction site was introduced in the upstream primer and a NotI restriction site was introduced in the downstream primer.
  • the upstream primer was 5'- GGATCC ATGACTAGACGGGTGATTCCCC-3'; the downstream primer was 5'- GCGGCCGC TCACAACTCCTCTCCCCAAGG-3'.
  • the restriction endonuclease sequence was underlined. A high-fidelity DNA polymerase with blunt ends was used for the amplified product.
  • the target fragment was recovered by gel recovery kit and mixed with 1 ⁇ l of vector at a ratio of 40 ng. -Blun Cloning Vector connection, blue-white screening sequencing to obtain the correct sequence of the gene fragment.
  • the pET28a (+) vector was linearized by BamHI and NotI restriction endonucleases, and the vector and fragment after restriction digestion were connected at 16 ° C for 3 h using DNA Ligation Kit (Mighty Mix, Takara, Japan) to construct a recombinant plasmid.
  • the recombinant plasmid was transformed into Escherichia coli E.coli DH5 ⁇ , and the correct expression recombinant plasmid was obtained by restriction digestion sequencing verification, and finally the plasmid was transferred into the expression strain E.coli BL21 (DE3).
  • the transformed strain E. coli BL21 (DE3) monoclone in Example 2 was selected and shaken overnight in 10 mL LB liquid medium with a final kanamycin concentration of 80 ⁇ g/ml, and inoculated into 1 L LB liquid medium (containing kanamycin) at a 1% inoculum, and shaken at 180 rpm at 37° C.
  • the bacterial solution concentration OD 600 was measured, and when the OD value was 0.6-0.8, 1 mg/ml IPTG solution (IPTG final concentration was 1 mM) was added at a ratio of 1/2000, and the shaker temperature was adjusted to 16° C. After the addition, the bacterial solution was cooled to 16° C. and induced at 100 rpm for about 16 hours.
  • the SDS-PAGE electrophoresis of BL21 expressing Amh1 shows an additional band between 55kDa and 70kDa compared with BL21 expressing an empty vector, which is presumed to be the protein expressed by the Amh1 gene.
  • Ni-Elute buffer 50 mM Tris-HCl pH 8.0, 300 mM imidazole
  • ddH 2 O two column volumes of ddH 2 O were used to wash.
  • W1 buffer in Example 3 was used to wash, and the supernatant retained in Example 3 was loaded into the chromatography column.
  • one column volume of W1 buffer was added to the chromatography column after the sample was drained, and one column volume of W2 buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole) was added after the W1 buffer was drained.
  • W3 buffer 50 mM Tris-HCl pH 8.0
  • Ni-Elute buffer was added to the chromatography column to elute the target protein and the effluent was collected. The eluate was detected by Bradford until the blue color was lighter.
  • the purified protein collected in the previous step was concentrated by an anion exchange chromatography column, and then a suitable ultrafiltration tube was selected for ultrafiltration according to the molecular weight of the protein (using a 30KDa ultrafiltration tube). After ultrafiltration and concentration, the purified protein was re-concentrated by gel filtration chromatography protein purification method (molecular sieve) and the concentration was determined. The purified protein results were detected by SDS-PAGE electrophoresis. As shown in Figure 2C, the apparent molecular weight of the Amh1 protein was consistent with the theoretical molecular weight (55.4KDa). The purified protein was quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for later use.
  • the purified protein Amh1 in Example 5 was diluted to 2.5 ⁇ g/mL for enzyme activity determination (the dilution buffer was Tris-HCl pH6.0).
  • the reaction system was 1 mL, and the purified protein Amh1 at a concentration of 2.5 ⁇ g/mL was mixed with 1 ⁇ L of 1000 ⁇ g/ml OTA toxin standard sample, and the pH gradient was set to 3, 4, 5, 6, 7, 8, 9, 10, and the degradation experiment was carried out at 28°C.
  • the results showed that the enzyme had a high degradation efficiency for OTA in a slightly acidic environment of pH4-7. After incubation for 40 minutes, OTA could be completely degraded under the condition of pH5-7 ( Figure 3, A).
  • the temperature gradient was set to 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C, respectively, and the degradation experiment was carried out under pH6.
  • OTA could be completely degraded under the condition of 30-70°C; when incubated for 20 minutes, OTA could be completely degraded under the condition of 40-70°C; when incubated for 10 minutes, OTA could be completely degraded under the condition of 60°C ( Figure 3, B).
  • the above results show that the enzyme can efficiently degrade OTA under the conditions of pH 4-7 and temperature 30-70°C. Good acid and heat resistance.
  • the degradation rate of Amh1 on 1 ⁇ g/ml OTA was tested at the optimal temperature (60°C) and pH 6.0. The results showed that when the concentration of Amh1 was 1 ⁇ g/ml, OTA was completely degraded within 11 minutes; when the concentration was 2.5 ⁇ g/ml, OTA was completely degraded within 5 minutes; when the concentration was 5 ⁇ g/ml, OTA was completely degraded within 3 minutes ( Figure 3, D). Compared with other degradation enzymes, Amh1 has a very high degradation efficiency.
  • the present invention provides a brown Metarhizium IFST-OT3 and a detoxification enzyme produced by it and its application.
  • the present invention provides a brown Metarhizium IFST-OT3 (preservation number CGMCC No. 40349) with strong degradation ability for ochratoxins A and B, as well as a detoxification enzyme (degradation enzyme) Amh1 produced by it and a nucleotide sequence encoding the amidase, and also provides the optimal physicochemical properties of the enzyme, including the influence of temperature, pH and metal ions.
  • the amidase treats a substrate concentration of 1 ⁇ g/ml OTA toxin in a buffer system of 60°C and pH 6.0, and the degradation efficiency is as high as 100% within 3 minutes, and Amh1 has strong adaptability to temperature and acidic pH.
  • the strain IFST-OT3 and the detoxification enzyme produced by it can be used for the degradation of ochratoxins in agricultural products, and have good economic value and application prospects.

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Abstract

提供一株褐色绿僵菌IFST-OT3及其产生的脱毒酶与应用。提供一株对赭曲霉毒素A和B具有较强降解能力的褐色绿僵菌IFST-OT3（保藏编号CGMCC No.40349），以及由其产生的脱毒酶（降解酶）Amh1以及编码该酰胺酶的核苷酸序列，还提供了该酶的最适理化特性，包括温度、pH和金属离子的影响。该酰胺酶在60℃，pH6.0的缓冲体系中处理底物浓度为1μg/ml OTA毒素，3min内降解效率高达100％，Amh1对温度和酸性pH具有较强的适应性。菌株IFST-OT3及由其产生的脱毒酶可用于农产品中赭曲霉毒素的降解。

Description

褐色绿僵菌IFST-OT3及其产生的脱毒酶与应用
交叉引用
本申请要求2022年12月15日提交的专利名称为“褐色绿僵菌IFST-OT3及其产生的脱毒酶与应用”的第2022116177171号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及微生物学及生物脱毒技术领域，具体地说，涉及一株褐色绿僵菌IFST-OT3及其产生的脱毒酶与应用。
背景技术
赭曲霉毒素A(OTA)是曲霉和青霉属真菌产生的次级代谢产物。1993年，国际癌症研究机构将OTA列为2B类致癌物质。此外，研究证明OTA具有肾毒性、神经毒性、免疫毒性等(Malir,et al.,2016,Toxins 8:191)。赭曲霉毒素B(OTB)是OTA的前体，其分子结构比OTA少一个氯原子。OTA和OTB可污染玉米、小麦、高粱、水果、奶制品、肉制品等多种农产品(Wang,et al.,2022,Frontiers in Microbiology,13:857726)，引起严重的食品安全问题。食品及饲料中OTA会造成进出口贸易受阻、人畜医疗费用，造成巨大的经济损失。
采取多种方式脱除农产品中污染的OTA是避免经济损失不可或缺的方式。目前，食品和饲料工业中主要通过物理、化学和生物的方法脱毒，其中生物脱毒具有安全、高效、无污染的特征，最具应用前景。生物脱毒方法可分为两类，一是基于微生物本身的吸附作用，二是微生物产生的代谢物，例如酶对OTA的降解作用。自然界中广泛存在可降解OTA的微生物资源，部分细菌对OTA的降解率高达90％。解淀粉芽孢杆菌(Bacillus amyloliquefaciens)在24h内对OTA降解率达到98.5％(Chang et al.,2015,Food Additives & Contaminants:Part A,32,564-571)。分离自麋鹿粪便的枯草芽孢杆菌(B.subtilis)，在24h内对OTA的降解率达到97.6％(Shi, et al.,2014,Journal of the Science of Food and Agriculture,94:1879-1885)。粪产碱菌(Alcaligenes faecalis)48h内可降解97％的OTA(Zhang,et al.,2017,Journal of Applied Microbiology,123:661-668)。
目前，挖掘生物脱毒酶及其编码基因，可实现OTA的精准高效脱除，提高脱毒效果的稳定性，为生物解毒方法积累关键性的核心技术。目前，对于脱毒机制和毒素降解基因报道较少，亟需挖掘新的OTA生物降解酶。
发明内容
本发明的目的是提供一株褐色绿僵菌Metarhizium brunneum IFST-OT3及其在生物脱毒中的应用。
本发明的另一目的是提供由褐色绿僵菌IFST-OT3产生的脱毒酶及其应用。
为了实现本发明目的，第一方面，本发明提供从土壤样品分离纯化获得的一株褐色绿僵菌Metarhizium brunneum IFST-OT3，该菌现已保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101，保藏编号CGMCC No.40349，保藏日期2022年10月28日。
第二方面，本发明提供含有所述褐色绿僵菌IFST-OT3的菌剂。
第三方面，本发明提供所述褐色绿僵菌IFST-OT3或其菌剂在赭曲霉毒素A和B脱毒中的应用。
第四方面，本发明提供由所述褐色绿僵菌IFST-OT3产生的脱毒酶Amh1，所述脱毒酶Amh1包含如下的氨基酸序列或由其组成：
i)SEQ ID NO:1所示的氨基酸序列；或
ii)在i)的N端和/或C端连接标签得到的氨基酸序列；或
iii)i)或ii)的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的酶。
第五方面，本发明提供编码所述脱毒酶Amh1的核酸分子，其核苷酸序列如SEQ ID NO:2所示。
第六方面，本发明提供含有所述核酸分子的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第七方面，本发明提供所述脱毒酶Amh1的以下任一应用：
1)用于降解赭曲霉毒素A和B；
2)用于制备赭曲霉毒素A和B的脱毒剂。
第八方面，本发明提供赭曲霉毒素A的降解方法，在30-80℃，pH4.0-8.0的缓冲体系中，使所述脱毒酶Amh1与赭曲霉毒素A接触。
优选地，体系中赭曲霉毒素A的浓度为1-5μg/ml。
优选地，脱毒酶Amh1与赭曲霉毒素A的摩尔比为1:130。
本发明在原核表达系统(大肠杆菌)中实现了该脱毒酶的异源高表达，且对于赭曲霉毒素的降解效率极高。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供一株对赭曲霉毒素A和B具有较强降解能力的褐色绿僵菌IFST-OT3，以及由其产生的脱毒酶(降解酶)以及编码该酰胺酶的核苷酸序列，还提供了该酶的最适理化特性，包括温度和pH。该酰胺酶在60℃，pH6.0的缓冲体系中处理底物浓度1μg/ml OTA毒素，3min内降解效率高达100％。菌株IFST-OT3及由其产生的脱毒酶可用于农产品中赭曲霉毒素的降解，应用前景广阔。
附图说明
图1为本发明较佳实施例中IFST-OT3粗酶液与底物浓度为1μg/ml的OTA和OTB标准样品孵育24h时的HPLC检测图以及HPLC-MS鉴定分子质量。其中，A和B为OTA标准品HPLC和HPLC-MS检测结果；C和D为OTA和IFST-OT3粗酶液孵育24h后，HPLC和HPLC-MS检测结果；E和F为OTB标准品HPLC和HPLC-MS检测结果；G和H为OTB和IFST-OT3粗酶液孵育24h后，HPLC和HPLC-MS检测结果。
图2为本发明较佳实施例中脱毒酶Amh1基因克隆及原核表达纯化。 其中，A为从菌株IFST-OT3的cDNA中克隆的Amh基因琼脂糖凝胶电泳图；B为大肠杆菌表达Amh1后的SDS-PAGE电泳图，表达空载体(vector)作为对照；C为Amh1纯化后的SDS-PAGE电泳图。
图3为本发明较佳实施例中酰胺酶Amh1的降解效率。其中，A为不同pH对降解效率的影响；B为不同温度对降解效率的影响；C为不同金属离子对降解效率的影响；D为在最适pH6.0和最适温度(60℃)条件下，1.0μg/ml、2.5μg/ml和5.0μg/ml的OTA的降解时间。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J & Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1：褐色绿僵菌IFST-OT3的分离鉴定
褐色绿僵菌IFST-OT3的分离纯化：IFST-OT3从北京农田土壤样品分离，具体方法为：取土壤5g加入5mL无菌水，摇床震荡30min(180rpm)，梯度稀释10、100、1000倍，分别涂布至PDA培养基，28℃倒置培养72h，对长出的菌落通过平板划线法纯化，获得的单菌落接种到PDA平板上培养。
在PDA培养基上，菌株IFST-OT3菌落呈绒毛状或絮状，最初呈白色，产孢后呈暗绿色，菌落边缘规则平整。菌丝有隔、光滑、透明，分生孢子梗上多2-3个瓶梗，分生孢子单细胞。用PDB培养基摇培菌株IFST-OT3，3d后提取DNA，扩增EF-1a(引物序列：5’-GTACCTCCCAGGCTGACTGC-3’；5’-GTTCTTGGAGTCACCAGCAACG-3’)、RPB2(5’-GAGGAGCTGAAGAGGACCGG-3’；5’-AATCGGTGTGTTGGTTCGTCG-3’)、beta-tub(5’-CCGTCCATCAGCTCGTTGAG-3’；5’-GGAACATGGCAGTGAACTGCTC-3’)基因，并测序。三个基因在NCBI中登录号分别为OP947525、OP947524、OP947526。经BLAST比对分析， 三个基因均与Metarhizium brunneum ARSEF 3297中同源基因的序列一致性最高。
根据测序结果以及上述微生物学特征，将菌株IFST-OT3鉴定为褐色绿僵菌(Metarhizium brunneum)，保藏编号CGMCC No.40349。
实施例2：褐色绿僵菌IFST-OT3对OTA和OTB的降解能力
28℃条件下，褐色绿僵菌IFST-OT3在PDB液体培养基中180rpm摇培7d。将收集到的菌丝用研钵在不断添加液氮条件下研磨成粉，取1g菌粉加入到20ml Lysis Buffer(1mM PMSF，1％盐酸苯甲醚，10mM Tris-Hcl(pH7)，150mM NaCl，0.5mM EDTA，0.01％曲拉通X-100，1mM DTT)中，涡旋震荡混匀。4℃8000rpm离心15min，获得的上清液为粗酶液。吸取1ml粗酶液，加入OTA或OTB标品(终浓度为1μg/ml)，28℃恒温孵化24h，加入1ml甲醇终止反应，高效液相色谱(HPLC)和高效液相色谱串联质谱(HPLC-MS)检测产物。
结果如图1(A～H)所示，OTA标准品出峰时间为15.1min(图1A)，经质谱鉴定其分子量为403([M+H]+＝404，图1B)；OTA与IFST-OT3孵化24h后，OTA的峰消失，在6.2min出现一个新的峰(图1C)，其分子量为256([M-H]+＝255，图1D)，出峰时间和分子量均与OTA的酰胺键断裂后降解产物OTα一致；OTB标准品出峰时间为8.9min(图1E)，经质谱鉴定其分子量为369([M+H]+＝370，图1F)；OTB与IFST-OT3孵化24h后，OTB峰消失，在4.6min出现一个新的峰(图1G)，其分子量为222([M-H]+＝221，图1H)，出峰时间和分子量均与OTB的酰胺键断裂后降解产物OTβ一致。以上结果表明，褐色绿僵菌IFST-OT3可将OTA和OTB降解为OTα和OTβ。
实施例3：表达载体的构建
本发明通过提取真菌粗蛋白测定降解活性发现褐色绿僵菌IFST-OT3具有将OTA降解为OTα的能力，经蛋白分离活性测定、质谱分析、基因本体论分析和蛋白重组表达，最终获得具有降解活性的酰胺酶(脱毒酶)。 将基因Amh1(GenBank：OP947527)从Metarhizium brunneum菌株反转录cDNA中扩增出来(图2，A)，引物设计时在上游引物中引入BamHI酶切位点，下游引物引入NotI酶切位点。上游引物5’-GGATCCATGACTAGACGGGTGATTCCCC-3’；下游引物5’-GCGGCCGCTCACAACTCCTCTCCCCAAGG-3’，已用下划线标出限制性内切酶序列，使用扩增产物为平端的高保真DNA聚合酶。通过胶回收试剂盒回收目的片段，按照40ng的比例与1μl载体-Blun Cloning Vector连接，经过蓝白斑筛选测序获得序列正确的基因片段。通过BamHI和NotI限制性内切酶线性化pET28a(+)载体，使用DNA Ligation Kit(Mighty Mix，Takara，日本)于16℃，3h下连接酶切处理后的载体与片段，构建重组质粒。将重组质粒转化至大肠杆菌E.coli DH5α中，经过酶切测序验证获得正确的表达重组质粒，最终将质粒转入表达菌株E.coli BL21(DE3)中。
实施例4：酰胺酶(脱毒酶)基因的异源表达
挑取实施例2中的转化菌株E.coli BL21(DE3)单克隆在卡那霉素终浓度为80μg/ml的10mL LB液体培养基摇培过夜，按照1％接种量接种到1L LB液体培养基中(含卡那霉素)，37℃条件下以180rpm摇培。测定菌液浓度OD600，在OD值为0.6-0.8时按照二千分之一的比例添加1mg/ml的IPTG溶液(IPTG终浓度为1mM)，加入后调整摇床温度至16℃，待菌液冷却至16℃时以100rpm摇培诱导16h左右。将1L菌液在4℃，4000rpm条件下离心10min，弃上清，将菌体溶于溶菌液中搅拌15-20min(30mL W1 buffer：50mM Tris-HCl pH 8.0，300mM NaCl；1mM PMSF；10mM MgCl2；20μg/mL Lysozyme；1μg/mL DNase I)。4℃高压(加压1000MPa)均质10-15min(视菌量而定)，至菌液澄清。将澄清的菌液在4℃，12000rpm条件下离心45min，保留上清为粗酶液。如图2中B所示，BL21表达Amh1后的SDS-PAGE电泳图，与表达空载体的BL21相比，在55kDa到70kDa之间多了一个条带，推测为Amh1基因表达的蛋白。
实施例5：蛋白纯化
将带有1.5mL左右Ni填料的层析柱进行前处理，使用Ni-Elute buffer(50mM Tris-HCl pH 8.0,300mM咪唑)冲洗2个柱体积，ddH2O冲洗2个柱体积。使用实施例3中的W1 buffer冲洗1个柱体积，将实施例3中所保留的上清液上样至层析柱中。为了洗去非特异性结合的杂蛋白，待样品流干后加入1个柱体积的W1 buffer至层析柱中，待W1 buff'er流干后加入1个柱体积的W2 buffer(50mM Tris-HCl pH 8.0,500mM NaCl,20mM咪唑)。为了洗去纯化蛋白中的高盐，待W2 buffer流干后加入1个柱体积W3 buffer(50mM Tris-HCl pH 8.0)。最后向层析柱中加入Ni-Elute buffer洗脱目的蛋白并收集流出液，使用Bradford检测洗脱液至蓝色较浅为止。
将上一步收集的纯化蛋白通过阴离子交换层析柱进行浓缩，再根据蛋白分子量大小选择合适的超滤管进行超滤(使用30KDa的超滤管)。超滤浓缩后将纯化蛋白通过凝胶过滤色谱蛋白纯化法(分子筛)进行再次浓缩并测定浓度。通过SDS-PAGE电泳检测纯化蛋白结果，如图2中C所示，Amh1蛋白表观分子量与理论分子量相符(55.4KDa)。用液氮将纯化蛋白速冻后存于-80℃冰箱保存备用。
实施例6：酶活测定
将实施例5中的纯化蛋白Amh1稀释至2.5μg/mL进行酶活测定(稀释buffer为Tris-HCl pH6.0)。反应体系为1mL，将浓度为2.5μg/mL的Amh1纯化蛋白与1μL 1000μg/ml的OTA毒素标准样品混合，设置pH梯度为3、4、5、6、7、8、9、10，28℃条件下进行降解实验。结果表明，该酶在pH4-7的偏酸性环境时对OTA的降解效率很高，孵化40min后，pH5-7的条件下可完全降解OTA(图3，A)。分别设置温度梯度20℃、30℃、40℃、50℃、60℃、70℃、80℃的范围，pH6条件下进行降解实验，孵化40min后，30-70℃条件下OTA能全部降解；孵化20min时，40-70℃条件下OTA能全部降解；孵化10min时，60℃条件下OTA能全部降解(图3，B)。以上结果表明，该酶可在pH4-7、温度30-70℃条件下高效降解OTA，表现 良好的耐酸耐热活性。
进一步研究9种金属离子对Amh1活性的影响，向反应体系中分别加入ZnSO4、MnSO4、FeSO4、Fe2(SO4)3、NiSO4、CuSO4、MgSO4、LiCl2、CaCl2，使金属离子终浓度为50mM，在28℃和pH 6条件下反应20分钟。结果表明，Li2+、Zn2+、Mn2+、Ca2+显著提高没得降解活性，Ni2+使酶活性降低，而在Fe2+、Fe3+、Cu2+存在的条件下，Amh1不能降解OTA(图3，C)。测试最适温度(60℃)和pH6.0条件下，Amh1对1μg/ml OTA的降解速率。结果表明，Amh1浓度为1μg/ml时，OTA在11min内全部降解；浓度为2.5μg/ml时，OTA在5min内全部降解；浓度为5μg/ml时，OTA在3min内全部降解(图3，D)。同其他降解酶相比，Amh1具有极高的降解效率。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种褐色绿僵菌IFST-OT3及其产生的脱毒酶与应用。本发明提供一株对赭曲霉毒素A和B具有较强降解能力的褐色绿僵菌IFST-OT3(保藏编号CGMCC No.40349)，以及由其产生的脱毒酶(降解酶)Amh1以及编码该酰胺酶的核苷酸序列，还提供了该酶的最适理化特性，包括温度、pH和金属离子的影响。该酰胺酶在60℃，pH6.0的缓冲体系中处理底物浓度为1μg/ml OTA毒素，3min内降解效率高达100％，Amh1对温度和酸性pH具有较强的适应性。菌株IFST-OT3及由其产生的脱毒酶可用于农产品中赭曲霉毒素的降解，具有较好的经济价值和应用前景。

Claims (10)

  1. 褐色绿僵菌Metarhizium brunneum IFST-OT3，其特征在于，保藏编号为CGMCC No.40349。
  2. 含有权利要求1所述褐色绿僵菌IFST-OT3的菌剂。
  3. 权利要求1所述褐色绿僵菌IFST-OT3或其菌剂在赭曲霉毒素A和B脱毒中的应用。
  4. 由权利要求1所述褐色绿僵菌IFST-OT3产生的脱毒酶Amh1，其特征在于，所述脱毒酶Amh1包含如下的氨基酸序列或由其组成：
    i)SEQ ID NO:1所示的氨基酸序列；或
    ii)在i)的N端和/或C端连接标签得到的氨基酸序列；或
    iii)i)或ii)的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的酶。
  5. 编码权利要求4所述脱毒酶Amh1的核酸分子。
  6. 含有权利要求5所述核酸分子的生物材料，其特征在于，所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
  7. 权利要求4所述脱毒酶的以下任一应用：
    1)用于降解赭曲霉毒素A和B；
    2)用于制备赭曲霉毒素A和B的脱毒剂。
  8. 赭曲霉毒素A的降解方法，其特征在于，在30-80℃，pH4.0-8.0的缓冲体系中，使权利要求4所述脱毒酶Amh1与赭曲霉毒素A接触。
  9. 根据权利要求8所述的方法，其特征在于，体系中赭曲霉毒素A的浓度为1-5μg/ml。
  10. 根据权利要求8所述的方法，其特征在于，所述脱毒酶Amh1与赭曲霉毒素A的摩尔比为1:130。
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