CN111088238B - 一种降解afb1的漆酶及其基因和应用 - Google Patents
一种降解afb1的漆酶及其基因和应用 Download PDFInfo
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- CN111088238B CN111088238B CN202010121822.0A CN202010121822A CN111088238B CN 111088238 B CN111088238 B CN 111088238B CN 202010121822 A CN202010121822 A CN 202010121822A CN 111088238 B CN111088238 B CN 111088238B
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Abstract
本发明提供一种降解AFB1的漆酶及其基因和应用，属于生物技术领域。所述降解AFB1的漆酶的氨基酸序列如SEQ ID NO.2所示，编码该漆酶的基因的核苷酸序列的如SEQ ID NO.1所示。该基因在酵母细胞中克隆表达，获得重组漆酶。本发明来源于Cerrena unicolor 10U的漆酶24h对AFB1的降解率将达100%，酵母细胞表达的1U的重组漆酶48h对AFB1的降解效率为20%。本发明的漆酶及重组漆酶在AFB1的降解中有巨大应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及一种降解AFB1的漆酶及其基因和应用。
技术背景
黄曲霉毒素B1（AFB1）主要是由黄曲霉（Aspergillus flavus）和寄生曲霉（Aspergillus parasiticus）产生的次级有毒代谢产物，由双呋喃环和香豆素环组成，具荧光性，在紫外线下发出蓝光，是毒性最强的黄曲霉毒素，也是已知的最强的天然化学致癌物，其中呋喃环被认为具有毒性和致癌性。目前，全球有超过50亿人面临长期接触食品中AFB1的风险，因此，如何安全有效的去除AFB1是人类面临的共同问题。
AFB1可通过物理、化学以及生物法降解，但物理方法耗时，且无法保障AFB1被完全清除；化学物质的使用虽然能显著降低AFB1浓度，但同时导致营养物质的损失，降低食品或饲料质量；直接使用微生物体降解AFB1，也会损害产品的感官特性，且降解过程的安全性无法保证。
酶具有底物特异性、有效高、环境友好的特点，被广泛应用于食品和饲料工业，所以最安全有效的方法是选用特定的酶来降解AFB1。目前，已知的可降解AFB1的纯酶包括黄曲霉素氧化酶（AFO, Armillariella tabescens）,过氧化物酶（Armoracia rusticana） ,F420H2依赖型还原酶（FDR, Mycobacterium smegmatis）,锰过氧化物酶（MnP, Pleurotus ostreatus）和漆酶（Bacillus subtilis, Trametes versicolor, Pleurotus pulmonarius）。AFO是从细胞内提取物中分离得到的唯一一种 AFB1降解酶，与AFB1有很强的亲和力, 该酶可将AFB1分子结构中的双呋喃环断裂，并且该降解过程为氧气依赖型，可同时产生过氧化氢，且过氧化氢在 AFO 的脱毒过程中起着重要作用；而源于Mycobacterium smegmatis的 F420H2依赖型还原酶（FDR）则与之相反，对AFB1的亲和力低于 AFO，但具有较高的催化活性，FDR-A 催化AFB1中的α,β-不饱和酯键，激活AFB1自发水解生成一种新的降解产物；从白腐真菌Pleurotus ostreatus 和Phanerochaete sordida中纯化的锰过氧化物酶（MnP）也能降解AFB1，其原理是 MnP 首先将AFB1转化成AFB1-8,9-环氧化合物，然后AFB1-8,9-环氧化合物再水解成为AFB1-8,9-二氢二醇。这些酶在 AFB1分子上可能有不同的靶点，不同的活性位点导致 AFB1降解产物的多样性。以上几种酶降解AFB1的机制都得到解析，但至今为止，关于漆酶降解AFB1的机制依然未知。
漆酶（EC 1.10.3.2）是含铜多酚氧化酶，能催化底物分子氧化为相应的活性自由基，同时将氧还原为水。最近研究表明漆酶能将一些环境污染物和毒素，如酚类、氧化烯烃、咔唑类、芴类、二苯并噻吩、多环芳烃等转化为无毒或毒性较低的产物。
目前,已报道的能降解AFB1的漆酶有细菌来源Bacillus subtilis (BsCotA)（Accession：AID81987.1）；真菌来源：T. versicolor (Accession：CAA77015.1，PDB code1KYA)，Pleurotus pulmonarius（Accession：AAX40733.1），Pleurotus eryngii（Accession：CAO79915.1），但可用于降解AFB1的Cerrena unicolor漆酶尚未见报道。
本专利通过纯化、转录组测序、蛋白质鉴定，克隆表达等方法，从Cerrena unicolor中获得了一种降解AFB1的漆酶及其基因。
发明内容
本发明的目的在于提供一种降解AFB1的漆酶及其基因和应用。本发明源于Cerrena unicolor的漆酶对AFB1降解效果好，应用价值巨大。
为实现上述目的，采用以下技术方案：
一种降解AFB1的漆酶，所述漆酶的氨基酸序列如SEQ ID NO.2所示。
一种编码降解AFB1的漆酶的基因，所述基因的核苷酸序列如SEQ ID NO.1所示。
一种含有编码降解AFB1的漆酶的基因的重组载体。
进一步的，一种含有编码降解AFB1的漆酶的基因的重组载体为将编码降解AFB1的漆酶的基因连接至pPICZɑ-A载体上所得的重组载体pPICZɑ-A-Lac2。
一种含有编码降解AFB1的漆酶的基因的细胞，所述细胞由含有编码降解AFB1的漆酶的基因的重组载体转化宿主细胞而得；优选的所述宿主细胞为毕氏酵母。
一种含有编码降解AFB1的漆酶的基因的细胞表达的重组漆酶。
一种降解AFB1的漆酶在降解AFB1中的应用。
一种含有编码降解AFB1的漆酶的基因的细胞表达的重组漆酶在降解AFB1中的应用。
具体为：
本发明从一色齿毛菌（Cerrena unicolor ）中分离纯化能降解AFB1的漆酶及克隆漆酶基因。
本发明通过对一色齿毛菌（Cerrena unicolor ）菌株的转录组RNA进行测序分析，得到漆酶cDNA序列，构建Cerrena unicolor漆酶库；通过对菌株Cerrena unicolor的发酵上清液进行分离纯化得到纯漆酶，对该纯漆酶进行UHPLC-MS/MS鉴定，与Cerrena unicolor漆酶库进行比对，得到降解AFB1的漆酶cDNA序列；设计特异性引物，以菌株Cerrena unicolor提取到的RNA逆转录后的cDNA为模板克隆得到该漆酶基因。所述漆酶基因包含如SEQ ID NO.1所示的核酸序列，称之为Lac2。
本发明还涉及包含SEQ ID NO.1所示核酸序列的重组载体，例如由各种本领域常用的表达载体制备的重组载体，其中，所述SEQ ID NO.1所示核酸序列不包含其来源微生物的内源性信号肽序列。将带内源性信号肽的编码序列即本发明Lac2编码序列去除信号肽，再经EcoRI 和XbaI双酶切后与EcoRI 和XbaI双酶切的pPICZɑ-A载体连接，得到酵母重组表达载体pPICZɑ-A-Lac2。
本发明制备了能够降解AFB1的漆酶，获得了优质的漆酶产品。本发明通过酶活测定，酶学性质分析，降解AFB1的分析，证明本发明的漆酶是一种具有降解AFB1能力的漆酶。
本发明的优点在于：本发明的漆酶能用于降解AFB1，24h对AFB1的降解率将达100%。目前来源于Cerrena unicolor且能降解AFB1的漆酶未见报道。本发明的漆酶及重组漆酶在AFB1的降解中有巨大应用价值。
附图说明
图1 Cerrena unicolor来源的漆酶的SDS-PAGE。M：蛋白质Marker；1：漆酶DEAE柱纯化产物。
图2 Cerrena unicolor来源的漆酶的最适pH和pH稳定性。A：pH对漆酶的活性影响；B：漆酶的pH稳定性。
图3 Cerrena unicolor来源的漆酶的最适温度和温度稳定性。A：温度对漆酶的活性影响；B：漆酶的温度稳定性。
图4 Cerrena unicolor来源的漆酶在质粒pMD18-T和pPICZɑ-A上的重组子结构图。
图5 Cerrena unicolor来源的漆酶降解AFB1。1：10U漆酶降解H2O 0h；2：10U漆酶降解AFB1 0h；3：10U漆酶降解AFB1 24h。
图6 重组漆酶降解AFB1。1：1U漆酶降解H2O 0h；2：1U漆酶降解AFB1 0h；3：1U漆酶降解AFB1 48h。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是下述的实例仅仅是本发明其中的例子而已，并不代表本发明所限定的权利保护范围，本发明的权利保护范围以权利要求书为准。
实验材料和试剂
1、菌株和载体：
大肠杆菌Top10购自Novagen公司，毕氏酵母X33及表达载体pMD18-T、pPICZɑ-A均购自Invitrogen公司（Carlsbad，CA，USA）。
Cerrena unicolor菌株：购自中国林业微生物菌种保藏管理中心（CFCC），保藏编号为CFCC NO.6884。
2、酶类及其他生化试剂：
限制性内切酶、DNA Maker、Protein Maker均购自TaKaRa公司，反转录试剂盒购自Thermo公司。
3、培养基：
（1）LB液体培养基：蛋白胨（Tryptone）10.0 g，NaCl 10.0 g，酵母提取物（YeastExtract）5.0 g，蒸馏水定容至1 L，121°C灭菌20 min。
（2）LB固体培养基是在LB液体培养基中添加2%(w/v)琼脂粉，121°C灭菌20 min。
（3）LLB液体培养基：蛋白胨（Tryptone）10.0 g，NaCl 5.0 g，酵母提取物（YeastExtract）5.0 g，蒸馏水定容至1 L，121°C灭菌20 min。
（4）LLB固体培养基是在LLB液体培养基中添加2%(w/v)琼脂粉，121°C灭菌20 min。
（5）YPD培养基：酵母粉1%(w/v)，蛋白胨2%(w/v)，121°C灭菌20 min，使用时加入2%(w/v)葡萄糖。固体培养基是在YPD液体培养基加入2%(w/v)的琼脂粉。
（6）YPM培养基：酵母粉1%(w/v)，蛋白胨2%(w/v)，121°C灭菌20 min，使用时加入2%(v/v)甲醇。
（7）PDA培养基：去皮马铃薯200g，加适量蒸馏水煮沸30min，8层纱布过滤，加入20g葡萄糖，定容至1000mL，2%琼脂粉，115°C灭菌30 min。
（8）YSJ培养基：2% v/v 甘油, 15g 蛋白胨, 6g KH2PO4, 4.14g MgSO4⋅7H2O,0.3g CaCl2, 0.18g NaCl, 0.0625g CuSO4⋅5H2O, 0.018g ZnSO4⋅7H2O, 0.015g VB1，蒸馏水定容至1 L，121°C灭菌20 min。
4、本发明中所用到的生物化学技术均为本领域中的常规技术。在以下实施例中，除非特殊说明，所有实验操作均按照以下实验手册或文献中的相关章节或部分进行，包括：[美]J.莎姆布鲁克等，分子克隆实验指南；赵永芳等，生物化学技术原理及其应用（第二版）；朱检等，生物化学实验[M]。
5、本发明中所有相关的酶活、酶活力、酶活性均是指漆酶酶活性，均采用ABTS法并按照所述的方法进行测定及计算。
实施例1 漆酶基因的获得
1、Cerrena unicolor总RNA的分离提取：
从生长Cerrena unicolor菌株的PDA平板玻璃纸上刮取菌丝，液氮研磨至粉末，称取50-100ng菌丝粉末，加1mL Trizol，研磨至融化，转至1.5mL EP管，室温静置10min，4°C10000rpm 离心5min；
取上清，加1/5体积的氯仿，上下颠倒混匀，冰上放置10min，4°C 10000rpm 离心5min；
取上清，加入等体积的酚：氯仿：异戊醇（25:24:1），颠倒混匀，4°C 10000rpm 离心5min；
取上清，加入等体积的氯仿：异戊醇（24:1），颠倒混匀，4°C 10000rpm 离心10min；重复该步骤至无白色沉淀；
取上清，加2.5倍体积的无水乙醇，混匀后-20°C沉淀20min，4°C 10000rpm 离心5min；
取沉淀，用75%乙醇洗涤一次，空气干燥3-5min，用适量含20U RNase inhibitorDEPC水溶解，获得总RNA， -80°C保藏备用。
2、 Cerrena unicolor总RNA转录组测序
将提取的Cerrena unicolor总RNA送出测序（上海生工），并将测序结果进行分析，构建Cerrena unicolor漆酶基因库（包含6条漆酶基因序列，Lac1~ Lac6）及漆酶氨基酸库（包含6条漆酶基因序列翻译后的氨基酸序列）。
3、 Cerrena unicolor漆酶的获取
Cerrena unicolor菌株用PDA平板30℃恒温培养3d，取6个直径1cm 的菌饼接种于PD种子液（50mL/100mL），于30℃震荡培养2d，得到一级种子液；8%接种量接种于PD种子液（100mL/250mL），30℃震荡培养2d，得到二级种子液；8%接种量接种于发酵产酶YSJ培养基（60mL/250mL）, 30℃震荡培养14d，12000rpm 4℃离心10min收集上清液，得到漆酶粗酶液。粗酶液用50%硫酸铵沉降，12000rpm 4℃离心10min弃沉淀，除去部分杂蛋白，再用50-90%硫酸铵沉降，弃上清，沉淀用pH8.5 Tris-HCl缓冲液溶解，并于2L pH8.5 Tris-HCl缓冲液中透析过夜，12h更换一次缓冲液，透析后酶液用0.22µm滤膜过滤后用于后续纯化。利用AKTA系统结合DEAE纤维柱进行阴离子交换纯化，流动相如下，A相：20mM pH8.5 Tris-HCl缓冲液，B相：含1M NaCl 的20mM pH8.5 Tris-HCl缓冲液。按顺序用适量体积的超纯水、100%B相、100%A相冲洗AKTA系统。系统冲洗干净后后安装DEAE纤维柱，用100%B相2mL/min冲洗DEAE纤维柱，再用A相2mL/min平衡DEAE柱，平衡后的DEAE柱用将预处理过的酶液缓慢上样。上完样后用含15%B相+85%A相洗去部分杂蛋白，再用20% B相+80%A相洗脱并收集流出液。最后的洗脱液用截流量10KDa的超滤离心管4000×g 4℃离心20min浓缩，浓缩后得到的酶液-20℃保存。
4、 Cerrena unicolor漆酶的鉴定
将纯化后的酶液进行SDS-PAGE，结果见图1。
将SDS-PAGE得到的目的蛋白凝胶带切成1mm3小块，放入1.5ml EP管中进行胶内消化，选择胰蛋白酶作为蛋白水解酶，蛋白质消化酶解步骤如下：
（1）加入600μl 脱色液（含30%ACN 的50 mM NH4HCO3）室温脱色，去除上清，重复此步骤，直到去除电泳染料；
（2）凝胶粒中加入300μl 100% ACN震荡脱水至胶粒变白，洗去ACN，并迅速干燥；
（3）加入300μl 10 mM DTT /50 mM NH4HCO3，震荡混匀至胶粒饱胀透明，并在56℃培养1 h后弃上清；
（4）凝胶粒中加入300μl 100% ACN震荡脱水至胶粒变白，洗去ACN，并迅速干燥；
（5）加入300μl 60 mM IAA /50 mM NH4HCO3, 避光震荡混匀至胶粒饱胀透明，室温下避光培养30 min；
（6）凝胶粒中加入300μl 100% ACN震荡脱水至胶粒变白，洗去ACN，并迅速干燥；
（7）加入50-80μl NH4HCO3，再加入 1-2μg胰蛋白酶，用玻棒捣碎凝胶， 37℃孵育6h以上；
（8）加入200μL含0.1%FA ACN震荡5min，吸取上清液至干净EP管；
（9）凝胶中再次加入30μL 0.1%FA震荡5min，再加入200μL含0.1%FA ACN震荡5min，吸取上清，并将两次上清合并；
（10）50℃氮气吹干，用50μL 超纯水重悬，0.22µm滤膜过滤后进行UHPLC-MS/MS鉴定肽段；
（11）UHPLC-MS/MS采用UltiMate 3000 RSLCnano/ Q ExactiveTM系统，利用Acclaim PepMapTM RSLC-C18分析柱进行肽分离，进样量为5μL。使用的两种溶剂为：溶剂A=2%ACN+98%H2O+0.04%FA， B=80%ACN+20% H2O+0.08%FA；流速设定为0.3μl/min。采用以下线性洗脱梯度进行分析分离：溶剂B在前3min保持2%，3到6min从2%升至6%，6到51min从6%缓慢升至28%，51到55min从28%升至34%，55到57min从34%快速升至95%，57到70min保持在95%，70到71min从95%快速降至2%，最后2%保持10min。
漆酶鉴定结果见表1，共鉴定到13条肽段，将13条肽段的氨基酸序列与Cerrena unicolor漆酶氨基酸库的氨基酸序列进行比对分析，与其中编号为Lac2的基因的氨基酸序列的覆盖率达48%。
表1. Cerrena unicolor漆酶鉴定结果
5、PCR获取目的基因
Cerrena unicolor漆酶经鉴定为漆酶基因库里编号为Lac2的基因所编码的，根据编号为Lac2的基因序列分别设计上下游引物P1和P2。引物P1和P2分别含有EcoRI 和XbaI酶切位点，由上海生工合成，引物序列如下：
P1：5' -CGgaattcGCAATTGGGCCCGTCTCC-3'
P2：5'-GCtctagaCACCCGTTGTATTTGGGAC-3'
以菌株Cerrena unicolor提取到的RNA逆转录后的cDNA为模板克隆得到漆酶基因。PCR程序为： 94℃预变性4 min；94℃变性30 s，55℃退火30 s，72℃延伸1.5 min，循环扩增30次；最后72℃延伸10 min。扩增结束后取PCR产物进行电泳检测，并回收凝胶中的目标基因即为漆酶基因的cDNA序列。
6、cDNA亚克隆：
制备好的漆酶基因的cDNA序列插入到载体pMD18-T上，得到重组质粒pMD18-T-Lac2（如图4A所示），用化学转化法将重组质粒pMD18-T-Lac2（转化到受体菌E.coli Top10，在含有100 mg/mL Amp的LB平板上37℃培养过夜。挑取单克隆菌落接种到2 ml含有100mg/mL Amp的LB液体培养基中，37℃ 200 rpm培养6-10 h，10000 rpm离心10 min收集菌体，提取质粒，酶切回收目的基因备用（质粒提取和胶回收分别用OMEGA公司的E.Z.N.A.Plasmid Mini Kit I和E.Z.N.A. Gel Extraction Kit试剂盒）。将所得目的基因进行DNA序列测定（上海生工），结果如SEQ ID NO.1-2所示。
由此得到的漆酶的编码序列共有1536 bp（SEQ ID NO.1），编码含22个氨基酸的信号肽分子，含490个氨基酸的成熟蛋白（SEQ ID NO.2）。根据在NCBI数据库同源性比对的结果初步确定所得到的基因片段为漆酶基因。
实施例2 酵母重组表达载体的构建
将实施例1中所得到的漆酶基因的cDNA序列和pPICZɑ-A质粒各自经过EcoRI 和XbaI双酶切的后连接，得到重组质粒pPICZɑ-A-Lac2（如图4B所示）。
将重组质粒转入E.coli Top10感受态细胞，涂布在含有25 mg/mL zeocin的LLB筛选板上过夜培养，挑取重组子到液体LLB（含 25ug/mL zeocin），用菌液PCR的方法鉴定，若为阳性重组子，送出测序，测序结果进一步证明目的基因序列和插入位点的正确性。选择插入基因正确的菌液，参照OMEGA Plasmid Mini Kit的提取步骤，提取重组质粒pPICZɑ-A-Lac2，于-20°C保存备用。
实施例3 毕氏酵母发酵生产重组漆酶
将实施例2 制备的重组质粒pPICZɑ-A-Lac2经pme I单酶切，得到线性化质粒pPICZɑ-A-Lac2。
取20 µL已线性化的重组质粒pPICZɑ-A-Lac2加入到80 µL的毕赤酵母X33感受态细胞中，轻轻吹打混匀；把混合液加入2 mm预冷的电转杯中，将电转杯放入已预热30 min的电转仪，电击一次；电击完成后，快速加入1 mL预冷的山梨醇于电转杯中轻轻吹打悬浮菌液，吸出菌液至灭菌的1.5 mL EP管中，30°C静置复苏60 min；将菌液6000 ×g离心2 min，吸取80 µL上清重悬菌体后涂布于YPD（含 100ug/mL zeocin）筛选板上；30°C倒置培养3天左右，直至转化子长出。
挑取重组质粒pPICZɑ-A-Lac2转化的毕氏酵母X33菌株阳性克隆子至 10 mL YPD液体培养基，过夜培养。按 10.0% 的添加量将菌液添加到 10 mL YPM 培养基中，30°C 、200 rpm 恒温摇床培养3天，每 24 h 添加终浓度为2.0%(v/v)的甲醇诱导表达。将菌液13000 rpm离心5 min，取上清液测定漆酶活力。
实施例4 重组漆酶的纯化
按照实施例3方法制备大量粗酶液，本发明所用的表达载体带有组氨酸标签，因此可通过镍柱亲和层析纯化异源表达的重组漆酶。将粗酶液用缓冲液A（20mM Tris-HCl，40mM咪唑，500mM NaCl, pH 8.5）稀释5倍，以1mL/min的流速加载到镍柱中，用缓冲液A洗脱杂蛋白至柱平衡，再用缓冲液B（20mM Tris-HCl，400mM咪唑，500mM NaCl，pH 8.5）以1mL/min的流速洗脱目的蛋白，收集洗脱液。
实施例5 Cerrena unicolor漆酶生化性质表征
1、漆酶的最适反应 pH 和 pH 稳定性的测定
漆酶最适反应pH测定：将实施例1（步骤3）制备的Cerrena unicolor漆酶在 45℃条件下，于不同pH（ pH 2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0 ）的底物中测酶活，以确定其最适反应 pH 。
漆酶 pH 稳定性测定：将实施例1（步骤3）制备的Cerrena unicolor漆酶在不同pH 值（pH 2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0）的缓冲液中于室温分别保温24h和48h后，取酶液于最适 pH和温度下分别测定其酶活力。结果显示漆酶最适反应 pH 为 pH2.5，在pH5-9 范围内稳定，实验结果如图2所示。
2、漆酶最适反应温度和热稳定性的测定
漆酶最适反应温度的测定：将实施例1（步骤3）制备的Cerrena unicolor漆酶在最适 pH 底物中，于不同温度（30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃）条件下反应测定酶活力，以确定其最适反应温度。
漆酶热稳定性测定：将实施例1（步骤3）制备的Cerrena unicolor漆酶在不同温度（40℃、50℃、60℃、70℃）下水浴保温处理，并在不同时间点 2h，4h，6h，8h，10h，12h 取样后迅速置于冰上，在最适 pH 和温度条件下分别测定其酶活力。结果显示漆酶最适反应温度为 50℃，在 40℃ 12h 仍可以保持 60% 以上的酶活。实验结果如图3所示。
实施例6 Cerrena unicolor漆酶降解AFB1分析
反应混合物为10µL AFB1（100µg/mL）和 10U的实施例1（步骤3）制备的Cerrena unicolor漆酶酶液，用pH7.0磷酸盐缓冲液补足至200µL，在 45℃避光孵育24 h，用二氯甲烷等体积萃取3次，50°C氮气吹干，200µL甲醇重新溶解。采用UHPLC（UltiMate 3000 TM）对漆酶降解AFB1进行分析，柱温 40°C，流动相为甲醇：乙腈：水=1：1：2，流速为 0.1 mL·min-1，进样量为 2 µL。图5中，1：以10U的Cerrena unicolor漆酶降解H2O 0h为阴性对照组，2：以10U的Cerrena unicolor漆酶降解AFB1 0h为阳性对照组，3：以10U的Cerrena unicolor漆酶降解AFB1 24h为实验组，结果如图5所示，10U的Cerrena unicolor漆酶经过24h对AFB1的降解效率将近100%。
实施例7 重组漆酶降解AFB1分析
反应混合物为10µL AFB1（100µg/mL）和 1U的实施例4制备的重组漆酶，用pH7.0磷酸盐缓冲液补足至200µL，在 45℃避光孵育48 h，用二氯甲烷等体积萃取3次，50°C氮气吹干，200µL甲醇重新溶解。采用UHPLC（UltiMate 3000 TM）对重组漆酶降解AFB1进行分析，柱温 40°C，流动相为甲醇：乙腈：水=1：1：2，流速为 0.1 mL·min-1，进样量为 2 µL。图6中，1：以1U的重组降解H2O 0h为阴性对照，2：以1U的重组降解降解AFB1 0h为阳性对照，3：以1U的重组降解降解AFB1 48h为实验组，结果如图6所示，1U的重组漆酶经过48h对AFB1的降解效率约为20%。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种降解AFB1的漆酶及其基因和应用
<130> 18
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 1536
<212> DNA
<213> 人工序列 SEQ NO.1
<400> 1
atgggattga actcggctat tccatcgctt gctatcctag ctttgtcagt cggaagctat 60
gctgcaattg ggcccgtctc cgacctgcac attgtcaaca aagaccttgc tccagatggc 120
gtacaacgtc caaccgtgct tgccggaggc acttttcctg ggacgttgat caccggtcag 180
aaaggtgaca acttccagct caatgtcatc gatgatctta ccgacgatcg gatgttgaca 240
ccaacttcca ttcattggca cggattcttc cagaagggaa ccgcttgggc cgacggtccc 300
gccttcgtaa ctcagtgccc tatcatagca gataactcct tcctgtatga cttcgacgtc 360
cccgaccagg ctggtacttt ctggtatcat agtcatttat ccacgcagta ctgtgacggt 420
ctacgtggtg ccttcgttgt gtacgatcct aacgatcccc acaaagacct atacgatgtt 480
gatgacgaga gcaccgtgat tacccttgcg gattggtacc atgttctcgc ccagaccgtt 540
gtcggcgctg ccactcctga ttctaccttg atcaacgggt taggccgttc acagaccgga 600
cccgctgatg ctgagctggc tgttatcagc gttaaacata acaaacgata ccgattccgt 660
ttggtttcga tttcgtgcga ccccaacttt accttctcca ttgatggtca taatatgact 720
gtcatcgaag tcgatggtgt caacacacga cccctgaccg ttgactctat tcaaatcttc 780
gccggacaga ggtattcctt tgtcctcaat gctaaccaac ccgacgacaa ttactggatc 840
cgtgctatgc caaacatcgg tagaaataca acaacactgg acggaaagaa tgccgctatc 900
cttcgataca agaatgcttc tgtagaagag cccaagaccg ttgggggccc cgctcaatcc 960
ccgttgaatg aagcggacct gcgtccactc gtacctgctc ctgtgcctgg aaacgctgtt 1020
ccaggtggcg cagacatcaa tcacaggctt aacttaactt tcagtaacgg ccttttcagc 1080
atcaacaatg cctccttcac taatccttcg gtccccgcct tattacaaat tctgagcggt 1140
gctcagaacg ctcaagattt acttccaacg ggtagttaca ttggccttga actaggcaag 1200
gtcgtggagc tcgttatacc tcctctggca gttggaggac cgcacccttt ccatcttcat 1260
ggccacaatt tctgggtcgt ccgtagtgca ggcagcgatg agtataactt cgacgatgct 1320
atcctcaggg acgtcgtaag cattggagca gggactgatg aagtcacaat ccgtttcgtg 1380
accgacaatc cgggcccgtg gttcctccat tgccatattg actggcattt ggaggcaggc 1440
cttgccatcg tcttcgctga gggcatcaat cagaccgctg cagccaaccc tacaccccag 1500
gcatgggatg agctttgtcc caaatacaac gggtga 1536
<210> 2
<211> 511
<212> PRT
<213> 人工序列 SEQ NO.2
<400> 2
Met Gly Leu Asn Ser Ala Ile Pro Ser Leu Ala Ile Leu Ala Leu Ser
1 5 10 15
Val Gly Ser Tyr Ala Ala Ile Gly Pro Val Ser Asp Leu His Ile Val
20 25 30
Asn Lys Asp Leu Ala Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala
35 40 45
Gly Gly Thr Phe Pro Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn
50 55 60
Phe Gln Leu Asn Val Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr
65 70 75 80
Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp
85 90 95
Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn
100 105 110
Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp
115 120 125
Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala
130 135 140
Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val
145 150 155 160
Asp Asp Glu Ser Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu
165 170 175
Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn
180 185 190
Gly Leu Gly Arg Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val
195 200 205
Ile Ser Val Lys His Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile
210 215 220
Ser Cys Asp Pro Asn Phe Thr Phe Ser Ile Asp Gly His Asn Met Thr
225 230 235 240
Val Ile Glu Val Asp Gly Val Asn Thr Arg Pro Leu Thr Val Asp Ser
245 250 255
Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn
260 265 270
Gln Pro Asp Asp Asn Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg
275 280 285
Asn Thr Thr Thr Leu Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys
290 295 300
Asn Ala Ser Val Glu Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser
305 310 315 320
Pro Leu Asn Glu Ala Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro
325 330 335
Gly Asn Ala Val Pro Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu
340 345 350
Thr Phe Ser Asn Gly Leu Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn
355 360 365
Pro Ser Val Pro Ala Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala
370 375 380
Gln Asp Leu Leu Pro Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys
385 390 395 400
Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro
405 410 415
Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Ser
420 425 430
Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile
435 440 445
Gly Ala Gly Thr Asp Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro
450 455 460
Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly
465 470 475 480
Leu Ala Ile Val Phe Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn
485 490 495
Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly
500 505 510
<210> 3
<211> 28
<212> PRT
<213> 人工序列
<400> 3
Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro
1 5 10 15
Phe His Leu His Gly His Asn Phe Trp Val Val Arg
20 25
<210> 4
<211> 37
<212> PRT
<213> 人工序列
<400> 4
Thr Val Gly Gly Pro Ala Gln Ser Pro Leu Asn Glu Ala Asp Leu Arg
1 5 10 15
Pro Leu Val Pro Ala Pro Val Pro Gly Asn Ala Val Pro Gly Gly Ala
20 25 30
Asp Ile Asn His Arg
35
<210> 5
<211> 20
<212> PRT
<213> 人工序列
<400> 5
Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val Ile Ser Val Glu
1 5 10 15
His Asn Lys Arg
20
<210> 6
<211> 19
<212> PRT
<213> 人工序列
<400> 6
Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val Ile Ser Val Glu
1 5 10 15
His Asn Lys
<210> 7
<211> 30
<212> PRT
<213> 人工序列
<400> 7
Ser Ala Gly Ser Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp
1 5 10 15
Val Val Ser Ile Gly Ala Gly Thr Asp Glu Val Thr Ile Arg
20 25 30
<210> 8
<211> 15
<212> PRT
<213> 人工序列
<400> 8
Ser Ala Gly Ser Asp Glu Tyr Asn Phe Asp Asp Ala Ile Leu Arg
1 5 10 15
<210> 9
<211> 8
<212> PRT
<213> 人工序列
<400> 9
Asn Ala Ser Val Glu Glu Pro Lys
1 5
<210> 10
<211> 6
<212> PRT
<213> 人工序列
<400> 10
Asn Ala Ala Ile Leu Arg
1 5
<210> 11
<211> 15
<212> PRT
<213> 人工序列
<400> 11
Met Leu Thr Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys
1 5 10 15
<210> 12
<211> 17
<212> PRT
<213> 人工序列
<400> 12
Tyr Ser Phe Val Leu Asn Ala Asn Gln Pro Asp Asp Asn Tyr Trp Ile
1 5 10 15
Arg
<210> 13
<211> 15
<212> PRT
<213> 人工序列
<400> 13
Met Leu Thr Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys
1 5 10 15
<210> 14
<211> 13
<212> PRT
<213> 人工序列
<400> 14
Gly Ala Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys
1 5 10
<210> 15
<211> 15
<212> PRT
<213> 人工序列
<400> 15
Asp Val Val Ser Ile Gly Ala Gly Thr Asp Glu Val Thr Ile Arg
1 5 10 15
<210> 16
<211> 41
<212> PRT
<213> 人工序列
<400> 16
Asp Leu Tyr Asp Val Asp Asp Glu Ser Thr Val Ile Thr Leu Ala Asp
1 5 10 15
Trp Tyr His Val Leu Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp
20 25 30
Ser Thr Leu Ile Asn Gly Leu Gly Arg
35 40
<210> 17
<211> 26
<212> DNA
<213> 人工序列 P1
<400> 17
cggaattcgc aattgggccc gtctcc 26
<210> 18
<211> 27
<212> DNA
<213> 人工序列 P2
<400> 18
gctctagaca cccgttgtat ttgggac 27
Claims (2)
1.一种漆酶在降解AFB1中的应用，其特征在于：所述漆酶的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的应用，其特征在于：所述漆酶的基因序列如 SEQ ID NO.1所示。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690793A (zh) * | 2012-06-15 | 2012-09-26 | 福州大学 | 一种漆酶及其基因序列 |
| CN109820132A (zh) * | 2018-12-07 | 2019-05-31 | 中国农业大学 | 细菌漆酶CotA蛋白在降解霉菌毒素中的应用 |
| CN110218709A (zh) * | 2019-06-21 | 2019-09-10 | 福州大学 | 一种耐热漆酶及其基因与应用 |
| CN110643583A (zh) * | 2019-11-06 | 2020-01-03 | 福州大学 | 一色齿毛菌来源漆酶及其基因与应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8492131B2 (en) * | 2010-05-11 | 2013-07-23 | Academia Sinica | Fungal laccases and uses thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690793A (zh) * | 2012-06-15 | 2012-09-26 | 福州大学 | 一种漆酶及其基因序列 |
| CN109820132A (zh) * | 2018-12-07 | 2019-05-31 | 中国农业大学 | 细菌漆酶CotA蛋白在降解霉菌毒素中的应用 |
| WO2020113962A1 (zh) * | 2018-12-07 | 2020-06-11 | 中国农业大学 | 细菌漆酶CotA蛋白在降解霉菌毒素中的应用 |
| CN110218709A (zh) * | 2019-06-21 | 2019-09-10 | 福州大学 | 一种耐热漆酶及其基因与应用 |
| CN110643583A (zh) * | 2019-11-06 | 2020-01-03 | 福州大学 | 一色齿毛菌来源漆酶及其基因与应用 |
Non-Patent Citations (3)
| Title |
|---|
| A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1;Zhou zhimin等;《toxins》;20200727;1-17页 * |
| 重组漆酶降解黄曲霉毒素B1分子对接分析及产物结构解析;刘英丽等;《食品科学》;20191012;119-127页 * |
| 食用菌漆酶及其对农药残留降解作用的研究进展;刘绍雄等;《食药用菌》;20180719(第04期);218-221页 * |
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