CN110292105B - 提高漆酶对真菌毒素降解率的黄瓜介体及其应用 - Google Patents
提高漆酶对真菌毒素降解率的黄瓜介体及其应用 Download PDFInfo
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- CN110292105B CN110292105B CN201910484512.2A CN201910484512A CN110292105B CN 110292105 B CN110292105 B CN 110292105B CN 201910484512 A CN201910484512 A CN 201910484512A CN 110292105 B CN110292105 B CN 110292105B
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- cucumber
- laccase
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Abstract
本发明属于农业生物领域，具体涉及提高漆酶对真菌毒素降解率的黄瓜介体及其应用。本发明提供了一种可用于真菌毒素降解的漆酶介体，其包括黄瓜水提物。本发明的介体能够协助漆酶有效地降解不同结构类型的真菌毒素，广泛用于食品和饲料真菌毒素脱毒领域。
Description
技术领域
本发明属于农业生物技术领域，具体涉及提高漆酶对真菌毒素降解率的黄瓜介体及其应用。
背景技术
真菌毒素是真菌产生的次级代谢产物，主要污染储存的粮油食品和饲料，严重危害人畜健康。根据其结构特征，真菌毒素可分为芳香环和非芳香环两大类，芳香环类包括黄曲霉毒素、玉米赤霉烯酮、桔霉素、赭曲霉素、棒曲霉素和单端孢霉烯族毒素等；非芳香环类仅包括伏马毒素，其中黄曲霉毒素、玉米赤霉烯酮是最为常见和危害最大的真菌毒素。
目前，对真菌毒素污染的饲料的脱毒方法主要包括物理法、化学法、吸附法和生物法等。物理和化学脱毒法存在操作困难、效果不稳定、营养成分损失大以及影响饲料适口性等缺点。吸附法虽简单易行，但存在用量大、不够经济，容易引起二次污染等缺点。微生物脱毒法具有作用条件温和，对原料的感官性状、适口性等影响极小、增加原料营养价值等优点，被认为是最佳脱毒方法。生物脱毒作用主要是指降解酶通过酶促反应将毒素转化成低毒或者无毒产物，其中降解酶包括氧化酶如漆酶、锰过氧化物酶和水解酶(如酯酶)等。
生物脱毒技术实现规模化应用的过程中，寻找和筛选能降解真菌毒素的菌株，并对其所产胞外降解酶进行特性研究及对降解酶基因进行克隆和表达，是真菌毒素生物降解研究领域重要的突破点和发展方向。现有漆酶对真菌毒素的降解率普遍较低，因此，如何有效提高漆酶对真菌毒素的降解率是生物脱毒技术的关键所在。
研究表明，漆酶在介体的帮助下能氧化多种难降解的底物。漆酶介体是一类低分子量、低氧化还原电势的化合物，易得失电子，能在漆酶的作用下形成活性高且稳定的中间体，并作用于底物，使其被氧化。目前，常用介体可分为3类：1)合成介体，如：2,2-联氨-二(3-乙基-苯并噻唑-6-磺酸)(ABTS)、紫脲酸(VIO)、1-羟基苯并三唑(HBT)、2,2’,6,6’-四甲基呱啶氧化物(TEMPO)等；2)天然介体，如：丁香醛(Syringaldehyde，SA)、乙酰丁香酮(Acetosyringone，AS)、乙酰香草酮(Acetovanillone)、对香豆酸(p-coumaric acid)等；3)其他类介体，如多金属氧酸盐(Polyoxometalates，POM)等。但合成介体存在以下缺点：1)稳定性较差；2)价格昂贵；3)有潜在毒性，有些介体氧化后会产生某些毒副产物使酶失活；4)大多数合成介体被氧化后不能再生。上述缺点限制了合成介体的应用。而天然介体不仅来源易、花费少而且对环境友好，更有经济价值，因此获得了越来越多的关注。
发明内容
本发明的目的在于提供一种提高漆酶对真菌毒素降解率的黄瓜介体。
本发明的再一目的为提供上述介体的应用。
本发明再一目的在于提供提高漆酶对真菌毒素的降解率的方法。
本发明的再一目的在于提供真菌毒素降解剂。
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，所述介体包括黄瓜水提物。
其中，漆酶BsCotA氨基酸序列如SEQ ID No.1所示：
MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINASNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPVLLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，所述黄瓜水提物由包括以下步骤的方法制备得到：
(1)粉粹黄瓜；
(2)以水为提取剂提取黄瓜，料液比为33-35g/L，调节pH 8.7-9.3，超声提取；
(3)将步骤(2)提取得到的上清液进行旋转蒸发；
(4)溶解步骤(3)旋转蒸发后产物，进行冷冻干燥，即得黄瓜水提物。
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，步骤(2)中，在温度45-55℃，功率600-800W条件下，超声提取20-25min。
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，步骤(3)中，在温度45-55℃，转速50-60rpm条件下，进行旋转蒸发。
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，所述介体为黄瓜水提物的三羟甲基氨基甲烷缓冲溶液，缓冲溶液的浓度为50mM、pH为7.0。
根据本发明具体实施方式的提高漆酶对真菌毒素降解率的黄瓜介体，其中，所述真菌毒素包括黄曲霉毒素B1或玉米赤霉烯酮。
根据本发明具体实施方式的显著提高漆酶对真菌毒素降解率的方法，所述方法包括将漆酶置于上述介体中降解真菌毒素的步骤。
根据本发明具体实施方式的真菌毒素降解剂，其包括上述介体和漆酶。
本发明的介体能够高效降解真菌毒素、成本低、适用范围广，可广泛用于饲料毒素降解酶领域。
附图说明
图1显示漆酶-黄瓜水提物体系对黄曲霉毒素B1和玉米赤霉烯酮的降解作用；
图2显示漆酶-黄瓜水提物体系降解黄曲霉毒素B1的HPLC分析结果；
图3显示漆酶-黄瓜水提物体系降解玉米赤霉烯酮的HPLC分析结果。
具体实施方式
试验材料和试剂
1、菌株：产枯草芽孢杆菌来源漆酶BsCotA的大肠杆菌工程菌株。
2、生化试剂：黄曲霉毒素B1、玉米赤霉烯酮、丁香酸甲酯；色谱纯乙腈、三氟乙酸、Tris。
3、培养基：
(1)大肠杆菌培养基LB(1％蛋白胨、0.5％酵母提取物、1％NaCl,pH7.0)
实施例1制备重组漆酶BsCotA
取含有重组质粒的BL21(DE3)/BsCotA大肠杆菌工程菌株，接种于50mL LB培养液中，37℃，220rpm振荡培养12h后，按2％比例转接于300mL LB培养基中，37℃220rpm振荡培养约3h(OD600≈0.6)，加入终浓度1mM的诱导剂IPTG，于16℃诱导15h后，离心收集菌体。将菌体重悬于磷酸氢二钠-柠檬酸缓冲液(20mM，pH 7.5)中。采取超声破碎法裂解菌体。将破碎后的菌体碎片离心去除，利用Ni亲和层析柱进行纯化，收集电泳纯的洗脱组分并透析至Tris-HCl蛋白储存液(50mM Tris-HCl，pH7.4，150mM NaCl)中。
实施例2制备黄瓜水提物介体
2.1将黄瓜切片后于40℃烘箱烘干4h。将干片加入粉碎机中进行粉碎，1min/次，共两次。以纯水作为提取剂，料液比33g/L，调节pH8.7左右，温度45℃，功率600W，超声提取20min，离心弃沉淀。将上清转移至圆底烧瓶中，温度45℃，转速50rpm，旋转蒸发。向烧瓶中加入约20mL蒸馏水，混匀后倒入两个50mL离心管中。置于-80℃冷冻2h。冷冻干燥，获得黄瓜水提物。
配制50mg/mL的黄瓜水提物溶液，室温颠倒混匀1h，12000rpm离心5分钟，弃沉淀。
2.2将黄瓜切片后于40℃烘箱烘干4h。将干片加入粉碎机中进行粉碎，1min/次，共两次。以纯水作为提取剂，料液比33g/L，调节pH8.7左右，温度45℃，功率600W，超声提取20min，离心弃沉淀。将上清转移至圆底烧瓶中，温度45℃，转速50rpm，旋转蒸发。向烧瓶中加入约20mL蒸馏水，混匀后倒入两个50mL离心管中。置于-80℃冷冻2h。冷冻干燥，获得黄瓜水提物。
配制50mg/mL的黄瓜水提物溶液，室温颠倒混匀1h，12000rpm离心5分钟，弃沉淀。
2.3将黄瓜切片后于40℃烘箱烘干4h。将干片加入粉碎机中进行粉碎，1min/次，共两次。以纯水作为提取剂，料液比33g/L，调节pH8.7左右，温度45℃，功率600W，超声提取20min，离心弃沉淀。将上清转移至圆底烧瓶中，温度45℃，转速50rpm，旋转蒸发。向烧瓶中加入约20mL蒸馏水，混匀后倒入两个50mL离心管中。置于-80℃冷冻2h。冷冻干燥，获得黄瓜水提物。
配制50mg/mL的黄瓜水提物水溶液，室温颠倒混匀1h，12000rpm离心5分钟，弃沉淀。
实施例3 BsCotA-黄瓜水提物介体体系降解黄曲霉毒素B1
将黄曲霉毒素B1溶解到二甲基亚砜中配制成50mg/L的母液，按如下反应体系：50mM Tris-HCl(pH 7.0)，20μL黄曲霉毒素B1溶液(50mg/L)，20μL黄瓜水提物水溶液(50mg/mL)，20μL BsCotA(300U/L)。以未加入漆酶BsCotA的体系作为对照，反应体系设3个重复。反应在30℃下进行，10h后加入三倍体积的乙腈终止反应，采用高效液相色谱(HPLC)分析黄曲霉毒素B1的降解率。液相色谱为岛津Nexera UHPLC高效液相色谱分析系统，色谱分离柱为Zorbax SB-C18(4.6×250mm,5μm)，流动相A(0.06％TFA的水)，流动相B(0.05％TFA的乙腈)；梯度洗脱条件0％B洗脱4分钟，0％-100％B洗脱15分钟，100％B洗脱6分钟；检测波长365nm。
结果如图1、图2所示，部分黄曲霉毒素B1已被降解，降解率为30.5％。
漆酶在不添加介体的水和Tris-HCl缓冲液降解体系中，对黄曲霉毒素B1降解率分别为2.5％、8.3％，而添加了黄瓜水提物作为介体的体系中，黄曲霉毒素B1的降解率提高了约4-15倍。因此，添加了黄瓜水提物介体的体系显著提高了漆酶对黄曲霉毒素B1降解率。
实施例4 BsCotA-黄瓜水提物介体体系降解玉米赤霉烯酮
将玉米赤霉烯酮溶解到二甲基亚砜中配制成50mg/L的母液，按如下反应体系：50mM Tris-HCl(pH 7.0)，20μL黄曲霉毒素B1溶液(50mg/L)，20μL黄瓜水提物水溶液(50mg/mL)，20μL BsCotA(300U/L)。以未加入漆酶BsCotA的体系作为对照，反应体系设3个重复。反应在30℃下进行，10h后加入三倍体积的乙腈终止反应，采用高效液相色谱(HPLC)分析玉米赤霉烯酮的降解率。液相色谱为岛津Nexera UHPLC高效液相色谱分析系统，色谱分离柱为Zorbax SB-C18(4.6×250mm,5μm)，流动相A(0.06％TFA的水)，流动相B(0.05％TFA的乙腈)；梯度洗脱条件0％B洗脱4分钟，0％-100％B洗脱15分钟，100％B洗脱6分钟；检测波长316nm。
结果如图1、图3所示，部分玉米赤霉烯酮已被降解，降解率为26.4％。
漆酶在不添加介体的水和Tris-HCl缓冲液降解体系中，对玉米赤霉烯酮降解率分别为1.7％、2.6％，而添加了黄瓜水提物作为介体的体系中，玉米赤霉烯酮的降解率提高了约10-15倍。因此，添加了黄瓜水提物介体的体系显著提高了漆酶对玉米赤霉烯酮降解率。
序列表
<110> 中国农业科学院饲料研究所
<120> 提高漆酶对真菌毒素降解率的黄瓜介体及其应用
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<170> SIPOSequenceListing 1.0
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<213> 枯草芽孢杆菌(Bacillus subtilis)
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Claims (5)
1.提高漆酶对真菌毒素降解率的方法，其特征在于，所述方法包括将漆酶置于介体中降解真菌毒素的步骤，其中，所述真菌毒素为黄曲霉毒素B1或玉米赤霉烯酮，所述漆酶的氨基酸序列如SEQ ID No.1所示，所述介体为黄瓜水提物，所述黄瓜水提物由包括以下步骤的方法制备得到：
（1）粉粹黄瓜；
（2）以水为提取剂提取黄瓜，料液比为33-35g/L，调节pH 8.7 - 9.3，超声提取；
（3）将步骤（2）提取得到的上清液进行旋转蒸发；
（4）溶解步骤（3）旋转蒸发后产物，进行冷冻干燥，即得黄瓜水提物。
2.根据权利要求1所述的提高漆酶对真菌毒素降解率的方法，其特征在于，步骤（2）中，在温度45-55 ℃，功率600-800 W条件下，超声提取20-25 min。
3.根据权利要求1所述的提高漆酶对真菌毒素降解率的方法，其特征在于，步骤（3）中，在温度45-55℃，转速50-60 rpm条件下，进行旋转蒸发。
4.根据权利要求1所述的提高漆酶对真菌毒素降解率的方法，其特征在于，所述介体为黄瓜水提物的三羟甲基氨基甲烷缓冲溶液，缓冲溶液的浓度为50 mM、pH为7.0。
5.真菌毒素降解菌剂，其特征在于，包括介体和漆酶，所述真菌毒素为黄曲霉毒素B1或玉米赤霉烯酮，其中，所述漆酶的氨基酸序列如SEQ ID No.1所示，所述介体为黄瓜水提物，所述黄瓜水提物由包括以下步骤的方法制备得到：
（1）粉粹黄瓜；
（2）以水为提取剂提取黄瓜，料液比为33-35g/L，调节pH 8.7 - 9.3，超声提取；
（3）将步骤（2）提取得到的上清液进行旋转蒸发；
（4）溶解步骤（3）旋转蒸发后产物，进行冷冻干燥，即得黄瓜水提物。
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| CN108126083A (zh) * | 2017-12-21 | 2018-06-08 | 解清华 | 一种治疗肩周炎药膏的制备方法 |
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