CN114763540B - 一种酶及其制备方法和应用 - Google Patents
一种酶及其制备方法和应用 Download PDFInfo
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- CN114763540B CN114763540B CN202110033327.9A CN202110033327A CN114763540B CN 114763540 B CN114763540 B CN 114763540B CN 202110033327 A CN202110033327 A CN 202110033327A CN 114763540 B CN114763540 B CN 114763540B
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- aflatoxin
- leu
- mycotoxin
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Abstract
本发明属于农业畜牧生产领域，具体涉及一种酶及其制备方法和应用；本发明还公开了含有上述酶的复合酶的制备方法及其在真菌毒素脱毒上的应用；本发明还公开了含有上述酶的真菌毒素生物降解剂的制备方法及其在真菌毒素脱毒上的应用；本发明所涉及的酶，其氨基酸序列如SEQ ID No.1或SEQ ID No.2所示；本发明还公开了编码上述酶的基因，其DNA序列如SEQ ID No.3或SEQ ID No.4；本发明提供的酶在真菌毒素脱毒领域具有广泛的应用前景。
Description
技术领域
本发明涉及农业畜牧生产领域，特别涉及具有降解真菌毒素功能的酶及其制备方法和应用。
背景技术
真菌毒素(也称为霉菌毒素)是真菌(霉菌)在生长的过程中产生的有毒的次级代谢产物，对人和动物具有致癌、遗传、生殖、神经和免疫毒性。目前已经分离和鉴定的真菌毒素有近400种，其中污染最广泛、毒性最大，关注最多的是玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素、呕吐毒素、T-2毒素、赭曲霉毒素、烟曲霉毒素、展青霉素、交链孢毒素和杂色曲霉素等。鉴于此，科研工作者越来越多的关注食品和饲料中真菌毒素的生物降解，主要指通过生物酶的特异性催化作用，对毒素分子结构进行化学修饰和改造使其生成无毒或低毒的产物。
研究报道植物、动物和微生物体内含有很多酶类，其中一些酶类被报道具有降解真菌毒素的功效，比如漆酶、锰过氧化物酶、内酯水解酶、脱氢酶等都对某类特定的真菌毒素有降解作用，生成的降解产物毒性降低或者消除，对饲料和食品中真菌毒素的生物降解具有很好的应用前景。目前报道的降解真菌毒素的酶主要来源于微生物，植物和动物体来源的酶的催化降解真菌毒素的研究非常少，还没有大豆来源的酶降解真菌毒素的研究报道。我国大豆产量巨大，如果能充分开发和利用大豆资源，从大豆或其副产物中提取具有某些特定功效的生物酶具有非常重要的意义，可以推进大豆的高值化利用，将其应用在食品和饲料领域进行有毒有害物的生物脱毒，对我国整个农业和畜牧业的健康发展都有非常重要的作用。
发明内容
有鉴于此，本发明提供了一种酶及其制备方法和应用。该酶可以降解真菌毒素(霉菌毒素)。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种酶，具有降解真菌毒素功能；
所述酶具有：
(Ⅰ)、如SEQ IDNo.1或2所示的氨基酸序列；或
(Ⅱ)、如(Ⅰ)所述的氨基酸序列经取代、缺失或添加一个或两个氨基酸残基获得的氨基酸序列，且与(Ⅰ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
(III)、与(Ⅰ)或(Ⅱ)所述序列具有至少90％序列一致性，优选95％序列一致性，且与(Ⅰ)所示的氨基酸序列功能相同或相似的氨基酸序列。
本发明还提供了所述的酶在真菌毒素生物脱毒中的应用。
本发明还提供了编码所述酶的核苷酸，具有：
(Ⅰ)、如SEQ IDNo.3或4所示的核苷酸序列；或
(Ⅱ)、如SEQ ID No.3或4所示的核苷酸序列的互补核苷酸序列；或
(Ⅲ)、与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的核苷酸序列；或
(Ⅳ)、与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列经取代、缺失或添加一个或两个核苷酸序列获得的核苷酸序列，且与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(V)、与(Ⅰ)、(Ⅱ)、(Ⅲ)或(Ⅳ)所述核苷酸序列具有至少90％序列一致性的核苷酸序列，优选95％序列一致性的核苷酸序列。
此外，本发明还提供了一种制备所述酶的方法，包括以下步骤：
步骤1、称取10g大豆种皮或粉碎的大豆溶于200mL pH 7.0的磷酸盐缓冲液中，37℃下180r/min震荡提取1～4h，离心取上清液，在冰水浴中缓慢加入硫酸铵粉末至60～80％饱和度，4℃层析柜中静置过夜，离心取析出的蛋白质沉淀，溶解于10mM Tris-HCl中得粗酶液；
步骤2、先用强阴离子交换层析柱ENrich TM Q对步骤1制备的粗酶液进行层析得到不同组分的蛋白，不同组分的蛋白分别与真菌毒素反应验证其对毒素的降解活性，将对真菌毒素有降解活性的蛋白组分再进行分子筛ENrich TM SEC 70层析得到不同组分的蛋白，不同组分的蛋白再分别与真菌毒素反应验证其对毒素的降解活性，再将分子筛层析后对真菌毒素有降解活性的蛋白进行SDS-PAGE电泳确定蛋白纯度，获得酶；
步骤3、质谱鉴定。
本发明还提供了所述的酶或所述方法制得的酶偶联葡萄糖氧化酶GOD和葡萄糖在真菌毒素脱毒上的应用。
此外，本发明还提供了一种复合酶，将所述的酶或所述方法制得的酶与葡萄糖氧化酶GOD、葡萄糖、淀粉和三氧化二铁按20～40：10～20：10～20：10～25：5～10比例混合。
本发明还提供了一种真菌毒素生物降解剂，包括所述的酶、所述方法制得的酶或所述的复合酶，以及生理上可接受的配伍载体。
在本发明的一些具体实施方案中，所述生理上可接受的配伍载体包括但不限于稻壳、米糠、麦芽糖糊精、环糊精、麦麸、淀粉、膨润土、寡糖或酵母细胞壁中的一种或多种。
本发明还提供了所述的复合酶或所述的真菌毒素生物降解剂在真菌毒素脱毒中的应用。
在本发明的一些具体实施方案中，所述的真菌毒素包括但不限于玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、黄曲霉毒素M2、交链孢毒素、呕吐毒素、T-2毒素、赭曲霉毒素、烟曲霉毒素、展青霉素、杂色曲霉素，优选为玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素B1、黄曲霉毒素M1或交链孢毒素。
本发明的优点和有益效果包括但不限于：
本发明提供了一种来源于大豆具有降解真菌毒素功能的酶(SHP)的制备方法及其编码基因和应用，尤其涉及其在降解真菌毒素方面的应用，试验证实该酶能够高效降解玉米赤霉烯酮、玉米赤霉烯醇和玉米赤霉醇、交链孢毒素等真菌毒素。本发明第一次报道来源于大豆的酶能够降解多种真菌毒素(玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、黄曲霉毒素M2、交链孢毒素、呕吐毒素、T-2毒素、赭曲霉毒素、烟曲霉毒素、展青霉素、杂色曲霉素)，且该酶催化效率高、作用范围广、转化效率快，在饲料和食品工业有非常广阔的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示酶(SHP)在不同温度条件下降解玉米赤霉烯酮ZEN情况；
图2示酶(SHP)在不同pH条件下降解玉米赤霉烯酮ZEN情况；
图3示酶(SHP)对AFB1的降解情况；
图4示酶(SHP)对交链孢毒素AOH的降解情况；
图5示酶(SHP)的分离纯化图；
图6示酶(SHP)的电泳及全波长扫描图；
图7示酶(SHP)联合葡萄糖氧化酶GOD降解玉米赤霉烯酮ZEN。
具体实施方式
本发明公开了一种酶及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供一种来源于大豆的酶的新功能，其可以降解真菌毒素玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素、交链孢毒素等。
本发明的另一目的在于提供上述来源大豆具有降解真菌毒素功能的酶(SHP)的应用，主要指将其应用于畜牧和食品领域对饲料和食品中的真菌毒素进行脱毒。
本发明的另一目的在于提供一种来源于大豆具有降解真菌毒素功能的酶(SHP)的制备方法。主要步骤如下：称取一定量的大豆种皮或粉碎的大豆溶于磷酸盐缓冲液中，震荡提取，离心取上清液，加入硫酸铵静置过夜，离心取析出的蛋白质沉淀，溶解于缓冲液中，透析过夜；再用离子交换层析和分子筛层析分离纯化SHP，每步层析得到不同组分的蛋白与真菌毒素反应验证其对毒素的降解活性，将对真菌毒素有降解活性的蛋白组分进行SDS-PAGE电泳确定蛋白纯度；再对纯化的SHP进行质谱鉴定。
本发明的另一目的在于提供一种含有上述来源于大豆具有降解真菌毒素功能的酶(SHP)偶联葡萄糖氧化酶GOD和葡萄糖在真菌毒素脱毒中的应用。
本发明的另一目的在于提供一种含有上述酶(SHP)的复合酶及其制备方法。
本发明的另一目的在于提供一种含有上述酶(SHP)的真菌毒素生物降解剂及其制备方法。
本发明的另一目的在于提供上述含有SHP的复合酶和真菌毒素生物降解剂在饲料和食品中真菌毒素脱毒中的应用。
本发明的另一目的在于提供上述具有降解真菌毒素功能的酶(SHP)的氨基酸序列及其编码基因。
具有降解真菌毒素功能的酶(SHP)氨基酸序列可以为：
SEQ ID No.1或SEQ ID No.2所示序列，其中SEQ ID No.1含有信号肽，SEQ IDNo.2不含信号肽；
或者SEQ ID No.1或SEQ ID No.2所示序列中的一个或两个氨基酸残基被取代和/或缺失和/或插入；
或者与SEQ ID No.1或SEQ ID No.2所示的氨基酸序列具有至少90％以上的序列一致性，优选95％序列一致性，并具有酶(SHP)的功能的序列。
本发明还提供了编码所述具有降解真菌毒素功能的酶(SHP)的核苷酸序列为：
SEQ ID No.3或SEQ ID No.4所示序列；
或者SEQ ID No.3或SEQ ID No.4所示序列中的一个或两个核苷酸残基被取代和/或缺失和/或插入；
或者与SEQ ID No.3或SEQ ID No.4所示的核苷酸序列具有至少90％以上的序列一致性，优选95％序列一致性的核苷酸序列。
SEQ ID No.1：
MGSMRLLVVALLCAFAMHAGFSVSYAQLTPTFYRETCPNLFPIVFGVIFDASFTDPRIGASLMRLHFHDCFVQGCDGSVLLNNTDTIESEQDALPNINSIRGLDVVNDIKTAVENSCPDTVSCADILAIAAEIASVLGGGPGWPVPLGRRDSLTANRTLANQNLPAPFFNLTQLKASFAVQGLNTLDLVTLSGGHTFGRARCSTFINRLYNFSNTGNPDPTLNTTYLEVLRARCPQNATGDNLTNLDLSTPDQFDNRYYSNLLQLNGLLQSDQELFSTPGADTIPIVNSFSSNQNTFFSNFRVSMIKMGNIGVLTGDEGEIRLQCNFVNGDSFGLASVASKDAKQKLVAQSK
SEQ ID No.2：
QLTPTFYRETCPNLFPIVFGVIFDASFTDPRIGASLMRLHFHDCFVQGCDGSVLLNNTDTIESEQDALPNINSIRGLDVVNDIKTAVENSCPDTVSCADILAIAAEIASVLGGGPGWPVPLGRRDSLTANRTLANQNLPAPFFNLTQLKASFAVQGLNTLDLVTLSGGHTFGRARCSTFINRLYNFSNTGNPDPTLNTTYLEVLRARCPQNATGDNLTNLDLSTPDQFDNRYYSNLLQLNGLLQSDQELFSTPGADTIPIVNSFSSNQNTFFSNFRVSMIKMGNIGVLTGDEGEIRLQCNFVNGDSFGLASVASKDAKQKLVAQSK
SEQ ID No.3：
ATGGGTTCCATGCGTCTATTAGTAGTGGCATTGTTGTGTGCATTTGCTATGCATGCAGGTTTTTCAGTCTCTTATGCTCAGCTTACTCCTACGTTCTACAGAGAAACATGTCCAAATCTGTTCCCTATTGTGTTTGGAGTAATCTTCGATGCTTCTTTCACCGATCCCCGAATCGGGGCCAGTCTCATGAGGCTTCATTTTCATGATTGCTTTGTTCAAGGTTGTGATGGATCAGTTTTGCTGAACAACACTGATACAATAGAAAGCGAGCAAGATGCACTTCCAAATATCAACTCAATAAGAGGATTGGACGTTGTCAATGACATCAAGACAGCGGTGGAAAATAGTTGTCCAGACACAGTTTCTTGTGCTGATATTCTTGCTATTGCAGCTGAAATAGCTTCTGTTCTGGGAGGAGGTCCAGGATGGCCAGTTCCATTAGGAAGAAGGGACAGCTTAACAGCAAACCGAACCCTTGCAAATCAAAACCTTCCAGCACCTTTCTTCAACCTCACTCAACTTAAAGCTTCCTTTGCTGTTCAAGGTCTCAACACCCTTGATTTAGTTACACTCTCAGGTGGTCATACGTTTGGAAGAGCTCGGTGCAGTACATTCATAAACCGATTATACAACTTCAGCAACACTGGAAACCCTGATCCAACTCTGAACACAACATACTTAGAAGTATTGCGTGCAAGATGCCCCCAGAATGCAACTGGGGATAACCTCACCAATTTGGACCTGAGCACACCTGATCAATTTGACAACAGATACTACTCCAATCTTCTGCAGCTCAATGGCTTACTTCAGAGTGACCAAGAACTTTTCTCCACTCCTGGTGCTGATACCATTCCCATTGTCAATAGCTTCAGCAGTAACCAGAATACTTTCTTTTCCAACTTTAGAGTTTCAATGATAAAAATGGGTAATATTGGAGTGCTGACTGGGGATGAAGGAGAAATTCGCTTGCAATGTAATTTTGTGAATGGAGACTCGTTTGGATTAGCTAGTGTGGCGTCCAAAGATGCTAAACAAAAGCTTGTTGCTCAATCTAAATAAACCAATAATTAATGGGGATGTGCATGCTAGCTAGCATGTAAAGGCAAATTAGGTTGTAAACCTCTTTGCTAGCTATATTGAAATAAACCAAAGGAGTAGTGTGCATGTCAATTCGATTTTGCCATGTACCTCTTGGAATATTATGTAATAATTATTTGAATCTCTTTAAGGTACTTAATTAATCA
SEQ ID No.4：
CAGCTTACTCCTACGTTCTACAGAGAAACATGTCCAAATCTGTTCCCTATTGTGTTTGGAGTAATCTTCGATGCTTCTTTCACCGATCCCCGAATCGGGGCCAGTCTCATGAGGCTTCATTTTCATGATTGCTTTGTTCAAGGTTGTGATGGATCAGTTTTGCTGAACAACACTGATACAATAGAAAGCGAGCAAGATGCACTTCCAAATATCAACTCAATAAGAGGATTGGACGTTGTCAATGACATCAAGACAGCGGTGGAAAATAGTTGTCCAGACACAGTTTCTTGTGCTGATATTCTTGCTATTGCAGCTGAAATAGCTTCTGTTCTGGGAGGAGGTCCAGGATGGCCAGTTCCATTAGGAAGAAGGGACAGCTTAACAGCAAACCGAACCCTTGCAAATCAAAACCTTCCAGCACCTTTCTTCAACCTCACTCAACTTAAAGCTTCCTTTGCTGTTCAAGGTCTCAACACCCTTGATTTAGTTACACTCTCAGGTGGTCATACGTTTGGAAGAGCTCGGTGCAGTACATTCATAAACCGATTATACAACTTCAGCAACACTGGAAACCCTGATCCAACTCTGAACACAACATACTTAGAAGTATTGCGTGCAAGATGCCCCCAGAATGCAACTGGGGATAACCTCACCAATTTGGACCTGAGCACACCTGATCAATTTGACAACAGATACTACTCCAATCTTCTGCAGCTCAATGGCTTACTTCAGAGTGACCAAGAACTTTTCTCCACTCCTGGTGCTGATACCATTCCCATTGTCAATAGCTTCAGCAGTAACCAGAATACTTTCTTTTCCAACTTTAGAGTTTCAATGATAAAAATGGGTAATATTGGAGTGCTGACTGGGGATGAAGGAGAAATTCGCTTGCAATGTAATTTTGTGAATGGAGACTCGTTTGGATTAGCTAGTGTGGCGTCCAAAGATGCTAAACAAAAGCTTGTTGCTCAATCTAAATAAACCAATAATTAATGGGGATGTGCATGCTAGCTAGCATGTAAAGGCAAATTAGGTTGTAAACCTCTTTGCTAGCTATATTGAAATAAACCAAAGGAGTAGTGTGCATGTCAATTCGATTTTGCCATGTACCTCTTGGAATATTATGTAATAATTATTTGAATCTCTTTAAGGTACTTAATTAATCA
本发明提供的酶及其制备方法、应用中，所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1来源于大豆具有降解真菌毒素功能的酶(SHP)在不同温度条件下降解玉米赤霉烯酮ZEN情况
将玉米赤霉烯酮(ZEN)溶解到甲醇中配成1000μg/mL储备液，在1mL磷酸钠缓冲液(0.1M，pH8.0)体系中进行降解反应，SHP含量为2U/mL，ZEN浓度为10μg/mL，反应体系中H2O2含量为100μM，以未加入SHP蛋白的体系作为对照；分别在27、32、37、42、47、52和57℃下反应1h，加入1mL甲醇终止反应，采用高效液相色谱检测ZEN含量。
高效液相色谱检测ZEN的色谱条件为：色谱柱：Agilent C18色谱柱，4.6mm×150mm×5μm；流动相：乙腈-水-甲醇(46：46：8)；流速：1mL/min；泵压：45bar；进样量：20μL；荧光检测器检测波长：λex＝274nm，λem＝440nm；采集时间：18分钟。
结果如图1所示，SHP降解ZEN最适温度为57℃，在最适温度条件下SHP与ZEN(10μg/mL)反应1h对ZEN的降解率为90％。
实施例2来源于大豆具有降解真菌毒素功能的酶(SHP)在不同pH条件下降解玉米赤霉烯酮ZEN情况
为测试在不同pH条件下酶(SHP)降解ZEN活性，在1mL不同pH值的缓冲液(pH4-6，0.1M柠檬酸钠缓冲液；pH7-8，0.1M磷酸钠缓冲液；pH9-10，0.1M甘氨酸-NaOH缓冲液)中进行降解反应，反应体系中H2O2含量为100μM，酶(SHP)浓度为2U/mL，ZEN浓度为10μg/mL；以未加入SHP的体系作为对照；在42℃下反应1h，加入1mL甲醇终止反应，采用高效液相色谱检测ZEN含量。
结果如图2所示，SHP降解ZEN最适pH为8，在最适pH条件下SHP与ZEN(10μg/mL)在42℃下反应1h对ZEN的降解率为88％。
实施例3来源于大豆具有降解真菌毒素功能的酶(SHP)对黄曲霉素毒素B1(AFB1)的降解情况
为测试在不同pH条件下酶(SHP)降解AFB1活性，在1mL不同pH值的缓冲液(pH3-6，0.1M柠檬酸钠缓冲液；pH7-8，0.1M磷酸钠缓冲液；pH9-10，0.1M甘氨酸-NaOH缓冲液)中进行降解反应，反应体系中H2O2含量为100μM，酶(SHP)浓度为2U/mL，AFB1浓度为1μg/mL，ABTS含量为1mM和5mM；以未加入SHP的体系作为对照；在42℃下反应1h后，加入1mL甲醇终止反应，采用高效液相色谱检测AFB1含量。
结果如图3所示，pH4，5和6条件下AFB1降解率分别为22％，27％和40％；pH 7，8，9和10条件下AFB1降解率分别为70％，78％，75％和80％。
实施例4来源于大豆具有降解真菌毒素功能的酶(SHP)对交链孢毒素AOH的降解情况
为测试在不同pH条件下酶(SHP)降解AOH活性，在1mL不同pH值的缓冲液(pH3-6，0.1M柠檬酸钠缓冲液；pH7-8，0.1M磷酸钠缓冲液；pH9-10，0.1M甘氨酸-NaOH缓冲液)中进行降解反应，反应体系中H2O2含量为100μM，酶(SHP)浓度为2U/mL，AOH浓度为1μg/mL；以未加入SHP的体系作为对照；在42℃下反应1h后，加入1mL甲醇终止反应，采用高效液相色谱检测AOH含量。
高效液相色谱检测AOH的色谱条件为：色谱柱：Agilent C18色谱柱，4.6mm×150mm×5μm；流动相：甲醇：水：冰乙酸(59：40：1)；流速：1mL/min；泵压：45bar；进样量：20μL；荧光检测器检测波长：λex＝345nm，λem＝421nm。
结果如图4所示，pH 4-8条件下酶(SHP)能够降解AOH，降解率平均值为90％。
实施例5来源于大豆具有降解真菌毒素功能的酶(SHP)的制备
称取10g大豆种皮溶于200mLpH 7.0的磷酸盐缓冲液中，37℃下180r/min震荡提取4h，离心取上清液，在冰水浴中缓慢加入硫酸铵粉末至80％饱和度，4℃层析柜中静置过夜，离心取析出的蛋白质沉淀，溶解于10mM Tris-HCl中得粗酶液，透析过夜.
先用强阴离子交换层析柱ENrich TM Q对制备的粗酶液进行层析得到不同组分的蛋白，不同组分的蛋白分别与真菌毒素反应验证其对毒素的降解活性，将对真菌毒素有降解活性的蛋白组分再进行分子筛ENrich TM SEC 70层析得到不同组分的蛋白，不同组分的蛋白再分别与真菌毒素反应验证其对毒素的降解活性，再将分子筛层析后对真菌毒素有降解活性的蛋白进行SDS-PAGE电泳确定蛋白纯度，获得纯酶(如图5)。
通过质谱技术对SDS-PAGE电泳所得的比较纯的酶进行鉴定。
试验结果表明，强阴离子交换层析色谱图如图5A所示，SHP主要分布于洗脱峰1中，分子筛层析色谱图如图5B所示，SHP主要分布于洗脱峰2中。纯化的SHP经过SDS-PAGE电泳可见分子量为43kDa的单一条带，纯化的SHP在401、501和632nm处有三个吸收峰(图6)；另外，纯化的SHP经质谱鉴定可以获得8条特征性肽段，如表1所示；经计算得知大豆种皮提取液中的SHP经三步纯化后得率为10.2％，纯化倍数为20.2倍(见表2)。
表1纯化的SHP经质谱鉴定获得的特征性肽段
表2 SHP的纯化步骤及得率
实施例6来源于大豆具有降解真菌毒素功能的酶(SHP)偶联葡萄糖氧化酶GOD和葡萄糖降解玉米赤霉烯酮ZEN
为测试实施例5制得的来源于大豆具有降解真菌毒素功能的酶(SHP)偶联不同浓度葡萄糖氧化酶GOD及葡萄糖降解ZEN的作用效果，采用实施例2所用的1mL磷酸钠缓冲液(0.1M，pH8.0)反应体系，体系中葡萄糖氧化酶的含量分别为0.0125、0.025、0.05、0.1、0.2、0.4和0.5U/mL，葡萄糖含量为分别为50，100和200μM，SHP蛋白浓度为2U/mL，ZEN浓度为10μg/mL，以未加入SHP蛋白的体系作为对照；在42℃下分别反应1h，加入1mL甲醇终止反应，采用高效液相色谱检测ZEN含量。
结果如图7所示，实施例5制得的酶(SHP)可以偶联葡萄糖氧化酶GOD，以葡萄糖为底物氧化ZEN。随着反应体系中GOD含量增加，产生的过氧化氢含量增加，ZEN降解率增加。随着体系中葡萄糖含量增加，ZEN降解率增加，当体系中葡萄糖含量为100μM时，ZEN降解率达到最大值，进一步增加体系中葡萄糖含量不能进一步提高ZEN降解率。
实施例7一种降解真菌毒素的复合酶及其制备方法
一种降解真菌毒素的复合酶由实施例5制得的来源于大豆具有降解真菌毒素功能的酶(SHP)、葡萄糖氧化酶GOD、葡萄糖、淀粉和三氧化二铁组成。制备方法为，按重量百分比将40份SHP与20份葡萄糖氧化酶GOD、10份葡萄糖、25份淀粉和5份三氧化二铁均匀混合配制而成。
实施例8一种降解真菌毒素的复合酶及其制备方法
一种降解真菌毒素的复合酶由实施例5制得的来源于大豆具有降解真菌毒素功能的酶(SHP)、葡萄糖氧化酶GOD、葡萄糖、淀粉和三氧化二铁组成。制备方法为，按重量百分比将30份SHP与15份葡萄糖氧化酶GOD、15份葡萄糖、45份淀粉和5份三氧化二铁均匀混合配制而成。
实施例9一种真菌毒素生物降解剂及其制备方法
一种真菌毒素生物降解剂由实施例5制得的来源于大豆具有降解真菌毒素功能的酶(SHP)、实施例7或实施例8制得的复合酶，及载体组成。制备方法为，按重量百分比将20份实施例5制得的SHP与10份稻米、30份米糠、20分寡糖和20份麦芽糖糊精均匀混合配制而成，得到真菌毒素生物降解剂。
实施例10一种真菌毒素生物降解剂及其制备方法
一种真菌毒素生物降解剂由实施例5制得的来源于大豆具有降解真菌毒素功能的酶(SHP)、实施例7或实施例8制得的复合酶，及载体组成。制备方法为，按重量百分比将40份实施例8制得的一种复合酶与10份环糊精、20份麦麸、10份淀粉、10份蒙脱石和10份酵母细胞壁均匀混合配制而成，得到真菌毒素生物降解剂。
实施例11来源于大豆具有降解真菌毒素功能的酶(SHP)催化降解玉米赤霉烯醇、玉米赤霉醇、AFB2、AFM1、AFM2、AFG1和AFG2的效率
将α-玉米赤霉烯醇、α-玉米赤霉醇、AFB2、AFM1、AFM2、AFG1和AFG2分别溶解到二甲基亚砜中，按如下反应体系进行试验：在1mL不同pH值的缓冲液(pH4和6，0.1M柠檬酸钠缓冲液；pH8，0.1M磷酸钠缓冲液)中进行降解反应，反应体系中H2O2含量为100μM，实施例5制得的酶(SHP)蛋白浓度为100μg/mL，玉米赤霉烯醇或玉米赤霉醇浓度为10μg/mL，AFB2、AFM1、AFM2、AFG1或AFG2浓度为2μg/mL，ABTS含量为5mM；以未加入酶(SHP)蛋白的体系作为对照；在42℃下反应12h后，加入1mL甲醇终止反应，采用高效液相色谱检测各毒素含量。酶(SHP)催化降解玉米赤霉烯醇、玉米赤霉醇、AFB2、AFM1、AFM2、AFG1和AFG2的效率见表3。
表3酶(SHP)催化降解不同毒素的效率
实施例12降解真菌毒素的复合酶对饲料中ZEN的脱毒效果
将实施例7或实施例8所述降解真菌毒素的复合酶处理含有ZEN的饲料(ZEN＝4ppm)按0.1％比例混合，在体外模拟动物胃肠液中消化24h，用于降解饲料中的ZEN。
模拟胃液：称取1g含有ZEN的饲料，再添加1mg酶制剂，放入100mL锥形瓶中，加入25mL PBS(0.1MpH6.0)，调pH到6.8，混匀。加入1mL配制好的淀粉酶溶液，39℃，150r/min消化2h。加10mL 0.2M HCl，用1M HCl或1MNaOH溶液调pH至2.0，加1mL新鲜配制的酸性蛋白酶(50000U/g)，混匀，用parafilm封口，于39℃恒温摇床培养4h(150r/min)。
模拟小肠液：模拟胃液孵育4h以后，再加入5mL 0.6MNaOH溶液，用1M HCl或1MNaOH将pH调到6.8后加入新鲜配制的肠道外源酶混悬液(蛋白酶：淀粉酶：脂肪酶＝3:2:1)，用parafilm封口，于38℃恒温摇床分别培养20h(150r/min)。
反应结束后测定复合酶对饲料中ZEN的降解率为96.1％。
实施例13真菌毒素生物降解剂处理含有AFB1的玉米
将实施例9或实施例10所述真菌毒素生物降解剂处理含有AFB1的玉米(AFB1＝1ppm)，按0.1％比例混合，在体外模拟动物胃肠液中消化24h，用于降解玉米中的AFB1。
模拟胃液：称取2g含有AFB1的玉米，2mg生物降解剂，放入100mL锥形瓶中，加入25mL PBS(0.1MpH6.0),调pH到6.8，混匀。加入1mL配制好的淀粉酶溶液，39℃，150r/min消化2h。加10mL 0.2M HCl，用1M HCl或1MNaOH溶液调pH至2.0，加1mL新鲜配制的酸性蛋白酶(50000U/g)，混匀，用parafilm封口，于39℃恒温摇床培养4h(150r/min)。
模拟小肠液：模拟胃液孵育4h以后，再加入5mL 0.6MNaOH溶液，用1M HCl或1MNaOH将pH调到6.8后加入新鲜配制的肠道外源酶混悬液(蛋白酶：淀粉酶：脂肪酶＝3:2:1)，用parafilm封口，于39℃恒温摇床分别培养20h(150r/min)。
反应结束后测定真菌毒素生物降解剂对玉米中AFB1的降解率为85.63％。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国农业大学
<120> 一种酶及其制备方法和应用
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aatcaaaacc ttccagcacc tttcttcaac ctcactcaac ttaaagcttc ctttgctgtt 540
caaggtctca acacccttga tttagttaca ctctcaggtg gtcatacgtt tggaagagct 600
cggtgcagta cattcataaa ccgattatac aacttcagca acactggaaa ccctgatcca 660
actctgaaca caacatactt agaagtattg cgtgcaagat gcccccagaa tgcaactggg 720
gataacctca ccaatttgga cctgagcaca cctgatcaat ttgacaacag atactactcc 780
aatcttctgc agctcaatgg cttacttcag agtgaccaag aacttttctc cactcctggt 840
gctgatacca ttcccattgt caatagcttc agcagtaacc agaatacttt cttttccaac 900
tttagagttt caatgataaa aatgggtaat attggagtgc tgactgggga tgaaggagaa 960
attcgcttgc aatgtaattt tgtgaatgga gactcgtttg gattagctag tgtggcgtcc 1020
aaagatgcta aacaaaagct tgttgctcaa tctaaataaa ccaataatta atggggatgt 1080
gcatgctagc tagcatgtaa aggcaaatta ggttgtaaac ctctttgcta gctatattga 1140
aataaaccaa aggagtagtg tgcatgtcaa ttcgattttg ccatgtacct cttggaatat 1200
tatgtaataa ttatttgaat ctctttaagg tacttaatta atca 1244
<210> 4
<211> 1166
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cagcttactc ctacgttcta cagagaaaca tgtccaaatc tgttccctat tgtgtttgga 60
gtaatcttcg atgcttcttt caccgatccc cgaatcgggg ccagtctcat gaggcttcat 120
tttcatgatt gctttgttca aggttgtgat ggatcagttt tgctgaacaa cactgataca 180
atagaaagcg agcaagatgc acttccaaat atcaactcaa taagaggatt ggacgttgtc 240
aatgacatca agacagcggt ggaaaatagt tgtccagaca cagtttcttg tgctgatatt 300
cttgctattg cagctgaaat agcttctgtt ctgggaggag gtccaggatg gccagttcca 360
ttaggaagaa gggacagctt aacagcaaac cgaacccttg caaatcaaaa ccttccagca 420
cctttcttca acctcactca acttaaagct tcctttgctg ttcaaggtct caacaccctt 480
gatttagtta cactctcagg tggtcatacg tttggaagag ctcggtgcag tacattcata 540
aaccgattat acaacttcag caacactgga aaccctgatc caactctgaa cacaacatac 600
ttagaagtat tgcgtgcaag atgcccccag aatgcaactg gggataacct caccaatttg 660
gacctgagca cacctgatca atttgacaac agatactact ccaatcttct gcagctcaat 720
ggcttacttc agagtgacca agaacttttc tccactcctg gtgctgatac cattcccatt 780
gtcaatagct tcagcagtaa ccagaatact ttcttttcca actttagagt ttcaatgata 840
aaaatgggta atattggagt gctgactggg gatgaaggag aaattcgctt gcaatgtaat 900
tttgtgaatg gagactcgtt tggattagct agtgtggcgt ccaaagatgc taaacaaaag 960
cttgttgctc aatctaaata aaccaataat taatggggat gtgcatgcta gctagcatgt 1020
aaaggcaaat taggttgtaa acctctttgc tagctatatt gaaataaacc aaaggagtag 1080
tgtgcatgtc aattcgattt tgccatgtac ctcttggaat attatgtaat aattatttga 1140
atctctttaa ggtacttaat taatca 1166
<210> 5
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Ser Phe Ala Val Gln Gly Leu Asn Thr Leu Asp Leu Val Thr Leu
1 5 10 15
Ser Gly Gly His Thr Phe Gly Arg
20
<210> 6
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Cys Pro Gln Asn Ala Thr Gly Asp Asn Leu Thr Asn Leu Asp Leu Ser
1 5 10 15
Thr Pro Asp Gln Phe Asp Asn Arg
20
<210> 7
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ala Arg Cys Ser Thr Phe Ile Asn Arg
1 5
<210> 8
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Glu Thr Cys Pro Asn Leu Phe Pro Ile Val Phe Gly Val Ile Phe Asp
1 5 10 15
Ala Ser Phe Thr Asp Pro Arg
20
<210> 9
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ile Gly Ala Ser Leu Met Arg
1 5
<210> 10
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gly Leu Asp Val Val Asn Asp Ile Lys
1 5
<210> 11
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Arg Asp Ser Leu Thr Ala Asn Arg
1 5
<210> 12
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Val Ser Met Ile Lys Met Gly Asn Ile Gly Val Leu Thr Gly Asp Glu
1 5 10 15
Gly Glu Ile Arg
20
Claims (5)
1.一种酶在真菌毒素生物降解中的应用，其特征在于，
所述的酶的氨基酸序列为：如SEQ ID No.1或2所示的氨基酸序列；
所述的酶的核苷酸序列为：如SEQ ID No.3或4所示的核苷酸序列；
所述的真菌毒素为：玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、黄曲霉毒素M2、交链孢毒素中的一种或几种。
2.如权利要求1所述的酶偶联葡萄糖氧化酶GOD和葡萄糖在真菌毒素脱毒上的应用，
所述的真菌毒素为：玉米赤霉烯酮、玉米赤霉烯醇、玉米赤霉醇、黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、黄曲霉毒素M2、交链孢毒素中的一种或几种。
3.如权利要求1或权利要求2所述的应用，其特征在于，将如权利要求1所述的酶与葡萄糖氧化酶GOD、葡萄糖、淀粉和三氧化二铁按20~40：10~20：10~20：10~25：5~10比例混合。
4.如权利要求1或权利要求2所述的应用，其特征在于，包括如权利要求1所述的酶，与生理上可接受的配伍载体，
所述的生理上可接受的配伍载体包括稻壳、米糠、麦麸、淀粉、膨润土、寡糖或酵母细胞壁中的一种或多种。
5.如权利要求1或权利要求2所述的应用，其特征在于，包括如权利要求1所述的酶，与生理上可接受的配伍载体，
所述的生理上可接受的配伍载体包括麦芽糖糊精和/或环糊精。
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Citations (1)
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| CN111418756A (zh) * | 2020-01-07 | 2020-07-17 | 中国农业大学 | 葡萄糖氧化酶联合过氧化物酶在霉菌毒素脱毒中的应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111418756A (zh) * | 2020-01-07 | 2020-07-17 | 中国农业大学 | 葡萄糖氧化酶联合过氧化物酶在霉菌毒素脱毒中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| NM_001251386.1;Genbank;NCBI;第1-2页 * |
| Zearalenone reduction by commercial peroxidase enzyme and peroxidases from soybean bran and rice bran;Sabrina O. Garcia等;Food Additives & Contaminants: Part A;摘要 * |
| 生物降解玉米赤霉烯酮的优势及应用前景;杜京霖;申屠婉铃;邵亮亮;房芳;汪凯骏;潘丹杰;李燕;;粮食储藏(第02期);摘要 * |
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