CN111394342B - 一种酰胺酶及其编码基因、重组载体、重组菌及应用 - Google Patents
一种酰胺酶及其编码基因、重组载体、重组菌及应用 Download PDFInfo
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- CN111394342B CN111394342B CN201910004511.3A CN201910004511A CN111394342B CN 111394342 B CN111394342 B CN 111394342B CN 201910004511 A CN201910004511 A CN 201910004511A CN 111394342 B CN111394342 B CN 111394342B
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Abstract
本发明公开了一种酰胺酶及其编码基因、重组载体、重组菌及应用，属于酶工程技术领域。本发明提供了一种酰胺酶及其编码基因，还提供了该酰胺酶的最适pH及其在水解赭曲霉毒素和粮食赭曲霉毒素脱毒中的应用。该酰胺酶在室温条件下，pH 7.3的缓冲体系中处理赭曲霉毒A 2分钟，赭曲霉毒素A的降解率即达到100％，这个降解效率在本领域是前所未有的。
Description
技术领域
本发明涉及一种酰胺酶及其编码基因、重组载体、重组菌及应用，属于酶工程技术领域。
背景技术
曲霉类真菌毒素主要由黄曲霉、寄生曲霉和赭曲霉等产毒曲霉菌株产生的一类次生代谢产物，已分离鉴定了二十多种结构类似物，其中以黄曲霉毒素B1(AFB1)和赭曲霉毒素A(OTA)毒性最大，在农业生产和食品工业中污染最为广泛。1993年世界卫生组织(WHO)癌症研究机构将黄曲霉毒素划定为Ⅰ类致癌物，将赭曲霉毒素定为ⅡB类致癌物。研究表明，赭曲霉毒素是人类巴尔干肾病的主要病因，因其具有强烈的肾毒性，以及可能的致癌、致畸和致突变性，已逐渐成为公共健康和科研领域关注的焦点。曲霉类真菌毒素污染谷物和油料作物已成为全球性问题，在畜牧业中，曲霉类真菌毒素可通过污染饲料危害动物健康，导致畜牧业生产率低下，造成严重的经济损失，并通过直接或间接(动物产品传播)途径污染食品，影响人类的身体健康。随着人们对曲霉类真菌毒素危害性认识的逐步加深，对于该类真菌毒素检测、污染防控和脱毒技术方面的研究工作也在不断深入，其与人们生活品质、身体健康和经济利益休戚相关。
现阶段常采用的赭曲霉毒素A的脱毒方法包括：物理吸附法和化学分解法，能够有效的降低或降解赭曲霉毒素A在食品及饲料中的污染浓度，但是这些脱毒方法通常仍具有很花费昂贵、脱毒难以彻底以及并且它们在处理过程中会引起食品及饲料的感官变化，且导致及其营养成分发生流失的问题变化。
生物脱毒因其成本低、安全高效等优势，近年来被认为是去除控制赭曲霉毒素A污染最有前途的方法之一，筛选生物脱毒酶、及其基因，完善脱毒酶的生物学功能，为生物解毒方法积累关键性的核心技术、生物材料及配套技术，将有效更好的促进我国生物脱毒技术的研究。酰胺酶是可以水解酰胺键的酶的统称，广泛的存在于真菌、植物、昆虫和细菌中，自发现以来一直是生物、化学和环境科学领域的研究热点。但是现有的用于降解赭曲霉素的酶的降解效率不高，或不适合工业应用，目前还没有一种赭曲霉毒素降解率高，且适合工业生产的酰胺酶。
发明内容
为解决上述问题，本发明提供一种来源于细菌，寡养单胞菌(Stenotrophomonassp.)的酰胺酶，其在原核表达系统中实现了异源高表达，易于后期的纯化和酶制剂的制备，该酶对底物OTA的降解效率极高，其商业化生产成本低。
本发明的第一个目的是提供一种酰胺酶，其氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的酰胺酶来源于寡养单胞菌(Stenotrophomonas sp.)。
进一步地，所述的酰胺酶可以是重组蛋白、天然蛋白或合成蛋白，可以是纯天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主中产生。根据重组生产方案所用的宿主，本发明的肽蛋白可以是糖基化的。
本发明的第二个目的是提供编码所述酰胺酶的基因。
进一步地，所述的基因具有如SEQ ID NO.2所示的核苷酸序列。
编码本发明所述酰胺酶的多核苷酸序列能用多种方法获得。例如，用本领域熟知的杂交技术分离多核苷酸。这些技术包括(但不局限于)：(1)用探针与基因或cDNA文库杂交以检出同源性的多核苷酸序列和(2)表达文库的活性筛选以检出具有共同结构特征的克隆的多核苷酸片段，该表达文库可以包括环境宏基因组文库或某个纯培养菌株所建的克隆文库。本发明的DNA片段序列也能用下列方法获得：(1)从基因组DNA分离双链DNA序列；(2)化学合成DNA序列以获得所述酰胺酶的双链DNA。
本发明的第三个目的是提供一种携带所述基因的重组载体。
进一步地，所述的载体为细菌质粒、噬菌体、酵母质粒、植物细胞病毒或哺乳动物细胞病毒。
本发明中，编码酰胺酶的核苷酸序列可插入到载体中，以构成含有本发明所述多核苷酸的重组载体。“载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。在本发明中适用的载体还包括但不限于：在细菌中表达的基于T7启动子的表达载体；在哺乳动物细胞中表达的pcDNA3.1载体和在昆虫细胞中表达的来源于杆状病毒的载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用于构建重组表达载体，优选pET载体系列以及其它原核表达载体系列。
本领域的技术人员熟知的方法能用于构建含编码酰胺酶的DNA序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA序列、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA的合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；噬菌体的PL启动子；真核启动子包括CMV早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是DNA表达顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白，或用于大肠杆菌的卡那霉素或氨苄青霉素等。
本发明的第四个目的是提供一种表达所述酰胺酶的重组菌。
进一步地，是以细菌、真菌、植物、昆虫或动物细胞为宿主细胞。
本发明中，编码酰胺酶的多核苷酸或含有该多核苷酸的重组载体可转化或转导入宿主细胞，以构成含有该核苷酸或重组载体的基因工程化宿主细胞。“宿主细胞”指原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表例子有：大肠杆菌，链霉菌属；细菌细胞如鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇S2或Sf9；动物细胞如CHO、COS或Bowes黑素瘤细胞等。
用本发明所述的DNA序列或含有所述DNA序列的重组载体转化宿主细胞可用本领域技术熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的本领域众所周知，可供选择的是用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，或者常规机械方法如显微注射、电穿孔、脂质体包装等。
通过常规的重组DNA技术，利用本发明的多核苷酸序列可用来表达或生产重组的酰胺酶。一般来说有以下步骤：
(1)用本发明的编码酰胺酶的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转染合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
在步骤(2)中，根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞的条件下进行培养。当宿主细胞生长在适当的细胞密度后，用合适的方法诱导选择的启动子，将细胞再培养一段时间。
在步骤(3)中，重组酶可包被于细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法包括但不限于：常规的复性处理、蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声波处理、超离心、亲和层析、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的第五个目的是提供所述的酰胺酶在降解赭曲霉毒素中的应用。
进一步地，所述的应用包括降解粮食或茶叶中的赭曲霉毒素。
本发明的第六个目的是提供一种包含所述酰胺酶的酶制剂。
本发明的有益效果是：
本发明提供了一种酰胺酶及其编码基因，还提供了该酰胺酶的最适pH及其在水解赭曲霉毒素和粮食赭曲霉毒素脱毒中的应用。该酰胺酶在室温条件下，pH 7.3的缓冲体系中处理赭曲霉毒A 2分钟，赭曲霉毒素A的降解率即达到100％，这个降解效率在本领域是前所未有的。
附图说明
图1为原核表达的酰胺酶SDS-PAGE蛋白电泳；
图2为酰胺酶对粮食(小麦粉)中赭曲霉毒素A的降解效果；
图3为酰胺酶对茯砖茶中赭曲霉毒素A的降解效果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
酰胺酶的氧化活性鉴定方法和降解AFB1活性测定方法如下：
酰胺酶活性测定方法如下：
含有pGEX-4T-1/adh117表达载体的转化株E.coli BL21(DE)3在最适条件下进行表达，取10μL表达菌液的破后上清液置于2mL离心管中，加入至490μL赭曲霉毒素A标准缓冲液(使用1*PBS配置pH 7.3)，赭曲霉毒素A的终浓度约为40μg/L，反应2min，向反应体系中加入1.5mL的乙腈停止反应。使用高效液相色谱检测OTA的残留量，实验结果表明，反应2min后，该重组酶粗酶液可以将反应体系中的底物全部降解。空白对照组为含有pGEX-4T-1空质粒的E.coli BL21(DE)3破碎后上清液，对照组破碎后粗酶液并无赭曲霉毒素A降解活性。
实施例1：酰胺酶基因的cDNA合成与克隆
菌株来源于实验室前期得到的寡养单胞菌，通过基因序列测定，分析得到酰胺酶基因的开放式阅读框核苷酸序列，设计扩增出完整编码阅读框的引物上游引物(adh117-F)(SEQ ID NO.3)：5’-CGCGGATCC ATGCCGATCCGCCGCCGC-3’；下游引物(adh117-R)(SEQ IDNO.4)：5’-CCGCTCGAGTCACTGCTTGTAGATCACCCCG-3’，在上游和下游引物上分别引入限制性内切酶位点(可视选用的载体而定，本发明中添加了BamHI和XhoI酶切位点，已用下划线标出，斜体序列为pGEX-4T-1载体下游末端同源序列)。已用下划线标出，斜体序列为pGEX-4T-1载体下游末端同源序列)，通过体外扩增技术获得编码SEQ ID NO.1所示氨基酸的酰胺酶基因，其序列如SEQ ID NO.2所示。在保证阅读框架正确的前提下构建好表达重组质粒pGEX-4T-1/adh117，再将其转入E.Coil BL21(DE3)中。序列分析得出，该ADH117酶的理论等电点6.90，理论分子量为45.6kDa。
实施例2：寡养单胞菌酰胺酶基因的异源表达
将实施例1中获得的E.Coil BL21(DE3)/pGEX-4T-1/adh117转化株在100mL含100μg/mL氨苄青霉素的LB液体培养基中摇床过夜培养。吸取1.0mL种子菌液接种于新鲜的100mLLB液体培养基(含有100μg/mL氨苄青霉素)中，37℃温度下，180r/min震荡培养。当菌液OD600达到0.6时，加入0.2mmol/mL的IPTG在16℃条件下诱导表达4h。在7000r/min的转速下冷冻离心10min，弃尽上清。用10mL的1×磷酸盐缓冲液(PBS)悬浮菌体，在冰浴条件下超声波破碎菌体。离心，并对沉淀进行包涵体复性。SDS-PAGE电泳结果显示菌体破碎后的上清液和沉淀(包涵体)中都有该酶，其(包含GST标签26kDa)表观分子量约为72kDa(图1)，与理论分子量相符。
实施例3：异源重组解毒酶在赭曲霉毒素降解中的应用
1、实验材料
酶制剂为pGEX-4T-1/adh239转化株表达后的破碎后上清液粗酶液，其他试剂均为分析纯化学试剂。
2、实验方法
取10μL pGEX-4T-1/adh117转化株表达后的破碎后上清液置于2mL离心管中，加入至490μL赭曲霉毒素A标准缓冲液(使用1*PBS配置pH 7.3)，赭曲霉毒素A的终浓度约为40μg/L，反应2min，向反应体系中加入1.5mL的乙腈停止反应。使用高效液相色谱检测OTA的残留量，实验结果表明，反应2min后，该破后上清液将OTA全部降解。
实施例4：异源重组酰胺酶在赭曲霉毒素污染粮食中脱毒的应用
1、实验材料
酶制剂为pGEX-4T-1/adh239转化株表达后的破碎后上清液粗酶液，实验材料为为小麦粉。其他试剂均为分析纯化学试剂。
2、实验方法
向50mL磷酸盐缓冲液中加入适量OTA标准储备液，混匀后立即倾注于500g已粉碎好的小麦粉样品中，搅拌均匀，使OTA的终浓度达到80.0μg/kg，于阴凉通风处晾干待用。取200mL酶制剂与200g预制备好的毒素污染小麦粉混合均匀，将配制好的样品在37℃条件下培养，于0h、1h、3h、6h取样，每个降解实验设置3个重复。同时，以200mL新鲜缓冲液与200g预制备好的毒素污染小麦粉混合均匀混合作为阴性对照处理。处理组和空白对照组每次取样20g，立即依据赭曲霉毒素标准检测方法进行提取、净化和分析。
毒素的提取与纯化。准确称取10g培养后的样品于150mL具塞三角瓶内，加入1gNaCl和20mL甲醇-水混合液(体积比4:1)，常温震荡提取30min，取下瓶塞经曹纹滤纸过滤于干净的杯子中(或在3000r/min下常温离心5min)，准确移取5.0mL上清液并加入20mL纯水稀释混匀，经玻璃纤维滤纸过滤1-2次至滤液澄清，进行免疫亲和柱纯化操作。
准确移取10.0mL(代表1.0g样品)上述澄清滤液，注入玻璃注射器中，调节压力使溶液以1-2滴/秒的流速缓慢通过免疫亲和柱，直至有部分空气通过柱体，以10mL纯水淋洗柱子(重复一次)，弃去全部流出液并使部分空气通过柱体。准确加入1.5mL甲醇洗脱，流速为1滴/秒，收集洗脱液于玻璃进样瓶中，进行HPLC-FLD检测。
酰胺酶对曲霉毒素的动态降解曲线如图2所示，实验结果表明，酰胺酶处理毒素污染的小麦粉(终浓度40μg/kg)，3h对OTA降解率接近100％。说明该酰胺酶对小麦粉中赭曲霉毒素A有非常好的降解效果。
实施例5：异源重组酰胺酶在赭曲霉毒素污染黑茶中脱毒的应用
1、实验材料
酶制剂为pGEX-4T-1/adh117转化株表达后的破碎后上清液粗酶液，实验材料为茯砖茶。其他试剂均为分析纯化学试剂。
2、实验方法
向50mL磷酸盐缓冲液中加入适量OTA标准储备液，混匀后立即倾注于500g已粉碎好的茶粉样品中，搅拌均匀，使OTA的终浓度达到80.0μg/kg，于阴凉通风处晾干待用。取200mL酶制剂与200g预制备好的毒素污染茶粉混合均匀，将配制好的样品在37℃条件下培养，于0h、1h、3h、6h取样，每个降解实验设置3个重复。同时，以200mL新鲜缓冲液与200g预制备好的毒素污染茶粉混合均匀混合作为阴性对照处理。处理组和空白对照组每次取样20g，立即依据赭曲霉毒素标准检测方法进行提取、净化和分析。
毒素的提取与纯化。准确称取10g培养后的样品于150mL具塞三角瓶内，加入1gNaCl和20mL甲醇-水混合液(体积比4:1)，常温震荡提取30min，取下瓶塞经曹纹滤纸过滤于干净的杯子中(或在3000r/min下常温离心5min)，准确移取5.0mL上清液并加入20mL纯水稀释混匀，经玻璃纤维滤纸过滤1-2次至滤液澄清，进行免疫亲和柱纯化操作。
准确移取10.0mL(代表1.0g样品)上述澄清滤液，注入玻璃注射器中，调节压力使溶液以1-2滴/秒的流速缓慢通过免疫亲和柱，直至有部分空气通过柱体，以10mL纯水淋洗柱子(重复一次)，弃去全部流出液并使部分空气通过柱体。准确加入1.5mL甲醇洗脱，流速为1滴/秒，收集洗脱液于玻璃进样瓶中，进行HPLC-FLD检测。
酰胺酶对茶粉中的赭曲霉毒素动态降解曲线如图3所示，实验结果表明，酰胺酶处理毒素污染的茶粉(终浓度40μg/kg)的6h降解率接近为100％。说明该酰胺酶对小麦粉中赭曲霉毒素A有高效的降解能力。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 安徽农业大学
<120> 一种酰胺酶及其编码基因、重组载体、重组菌及应用
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Gly Arg Ile Arg Gln Val Leu Pro Gly Thr Gly Ala Asp Ala Ala Gly
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Asp Leu His Val His Leu Gly Ser Gln Ser Ser Pro Gln Ser Tyr Ser
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Leu Val Gly Ala Pro Gly Pro Ala Glu Gly Val Val Asn Ser Val Asp
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ccgcacacgc tgctggtggc cgatggccgc atccgccagg tgctgcccgg caccggtgcc 180
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ggcttcacca gcgtgcgcga cctcggcggc gaggtcagcc cgcacctgcg cgatgcgatc 420
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ggcggccacg ccgatccgac caacggctgg aacgagcggc tcgcgcacct ggtcggcgcg 540
cccggccccg ccgaaggcgt ggtcaactcg gtggacgagg cgcggcaggc ggtgcggcag 600
cgctacaagg aaggcagcga cctgatcaag atcaccgcca ccggcggcgt gctcagctac 660
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gcgcgcgact acggcttccg cgtggccgcg catgcccacg gcaccgaggg catgaagcgc 780
gcggtgcagg ccggggtcac cagcatcgag cacggcacct acatggacga cgaggtcatg 840
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ttcggcaccg accagggcgt gggcccgcac ggcgacaacg cgcgcgaatt cgtctacatg 1080
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Claims (2)
1.酰胺酶在降解赭曲霉毒素A中的应用，其特征在于，所述的酰胺酶的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述的应用包括降解粮食、饲料或茶叶中的赭曲霉毒素A。
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