CN109370995A - 锰过氧化物酶CsMnP及其编码基因和应用 - Google Patents
锰过氧化物酶CsMnP及其编码基因和应用 Download PDFInfo
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- CN109370995A CN109370995A CN201811507518.9A CN201811507518A CN109370995A CN 109370995 A CN109370995 A CN 109370995A CN 201811507518 A CN201811507518 A CN 201811507518A CN 109370995 A CN109370995 A CN 109370995A
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- Prior art keywords
- csmnp
- ala
- manganese peroxidase
- asp
- gly
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Abstract
本发明属于农业生物技术领域，具体涉及锰过氧化物酶CsMnP及其编码基因和应用。发明提供了一种锰过氧化物酶CsMnP，其氨基酸序列如SEQ ID No.1或SEQ ID No.3所示。本发明的锰过氧化物酶能有效的降解不同结构类型的霉菌毒素，可作为一种新型的酶制剂，广泛用于食品和饲料霉菌毒素脱毒领域。
Description
技术领域
本发明属于农业生物技术领域，具体涉及锰过氧化物酶CsMnP及其编码基因和应用。
背景技术
霉菌毒素是真菌次级代谢产物，对家畜、家禽和人类的健康有害。常见的霉菌毒素包括黄曲霉毒素、玉米赤霉烯酮、呕吐毒素(脱氧雪腐镰刀菌烯醇)、桔霉素、赭曲霉毒素、伏马菌素、展青霉素和单端孢霉菌毒素，可以分为具环状结构和不具环状结构的两类毒素。大多数霉菌毒素，如黄曲霉毒素和玉米赤霉烯酮，属于具环状结构的亚组中，通常通过真菌聚酮途径进行合成。例如，黄曲霉毒素(aflatoxin)B1由黄曲霉(Aspergillus flavus)产生，具有一个核心的香豆素环和两侧的五元碳环和两个并列的二氢呋喃环，其是很强的肝脏致癌物。此外，玉米赤霉烯酮的结构是间二羟基苯甲酸酚内酯，伏马菌素则具有22碳对氨基苯酚线性骨架，侧链为两个丙三酸。
物理吸附(或灭活)以及生物转化是将食物和饲料中的霉菌毒素脱毒的两种主要方式。除此之外，使用微生物，特别是通过微生物产生的酶，进行霉菌毒素的脱毒是一种新兴的手段。现有技术中漆酶、泛解酸内酯水解酶、过氧化物酶以及一些尚不能分类的酶可通过氧化或水解机理降解黄曲霉毒素和玉米赤霉烯酮。来自于木质纤维素降解菌的锰过氧化物酶(MnP)是一组参与木质素氧化降解的酶。现有技术中公开的少数几个微生物产酶均只能特异性的降解黄曲霉毒素、玉米赤霉烯酮等一到两种霉菌毒素，因此，在实际应用中受到极大的限制。
发明内容
本发明的目的在于提供一种锰过氧化物酶CsMnP。
本发明的再一目的在于提供上述锰过氧化物酶CsMnP的编码基因。
本发明的再一目的在于提供含有上述编码基因的重组表达载体。
本发明的再一目的在于提供含有上述编码基因的重组菌株。
本发明的再一目的在于提供上述锰过氧化物酶CsMnP的制备方法。
本发明的再一目的在于提供上述锰过氧化物酶CsMnP的应用。
本发明的野生型的锰过氧化物酶CsMnP，其氨基酸序列如SEQ ID No.1所示：
MAFTSFVALAALVGIASAAPTTICPDGTRVSNHACCAFIPLAEDLQKTIFMNDCGEDAHEVIRLTFHDAVAISRKLGPKAGGGADGSMLLFPTVEPNFSANNGIDDSVNNLIPFMARHPTVSAGDLVQFAGAVALSNCPGAPRLEFLAGRPNHTIAAIDGLIPEPQDDVTKILERFDDAGGFTPFEVVSLLASHTVARADKVDETIDAAPFDSTPFTFDTQVFLEVLLKGVGFPGTDNNTGEVASPLPKGSGNDTGEMRLQSDFALARDPRTACFWQGFVDEQEFMAESFKAAMAKLAILGHNRASLTDCSDVVPIPRPAVKKPASFPATTGPKDLELTCRAERFPTLTVDRGAVQALIPHCSNGGQDCPSVQFDGPA
其中，该酶基因编码379个氨基酸，N端18个氨基酸为其信号肽序列，信号肽的序列如SEQ ID No.2所示：
MAFTSFVALAALVGIASA
因此，成熟的锰过氧化物酶CsMnP的理论分子量为38.3kDa，其氨基酸序列如SEQID No.3所示：
APTTICPDGTRVSNHACCAFIPLAEDLQKTIFMNDCGEDAHEVIRLTFHDAVAISRKLGPKAGGGADGSMLLFPTVEPNFSANNGIDDSVNNLIPFMARHPTVSAGDLVQFAGAVALSNCPGAPRLEFLAGRPNHTIAAIDGLIPEPQDDVTKILERFDDAGGFTPFEVVSLLASHTVARADKVDETIDAAPFDSTPFTFDTQVFLEVLLKGVGFPGTDNNTGEVASPLPKGSGNDTGEMRLQSDFALARDPRTACFWQGFVDEQEFMAESFKAAMAKLAILGHNRASLTDCSDVVPIPRPAVKKPASFPATTGPKDLELTCRAERFPTLTVDRGAVQALIPHCSNGGQDCPSVQFDGPA
本发明还提供了编码上述锰过氧化物酶CsMnP的基因序列，其基因组序列如SEQID No.4所示：
Atggcctttaccagctttgttgcactggcagcactggttggtattgcaagcgcagcaccgaccaccatttgtccggatggcacccgtgttagcaatcatgcatgttgtgcatttattccgctggcagaagatctgcagaaaaccatttttatgaacgattgtggtgaagatgcccatgaagttattcgtctgacctttcatgatgcagttgcaattagccgtaaactgggtccgaaagccggtggtggtgcagatggtagcatgctgctgtttccgaccgttgaaccgaattttagcgcaaataatggcattgatgatagcgtgaataacctgattccgtttatggcacgtcatccgaccgtgagtgccggtgatctggttcagtttgccggtgcagttgccctgagcaattgtccgggtgcaccgcgtctggaatttctggcaggtcgtccgaatcataccattgcagcaattgatggtctgattccggaaccgcaggatgatgttaccaaaattctggaacgttttgatgatgccggtggttttaccccgtttgaagttgttagcctgctggcaagccataccgttgcacgtgccgataaagttgatgaaaccattgatgcagcaccgtttgatagcaccccgtttacctttgatacccaggtttttctggaagttctgctgaaaggtgttggttttccgggtacagataataacaccggtgaagttgcaagtccgctgccgaaaggtagcggtaatgataccggtgaaatgcgtctgcagagcgattttgccctggcacgcgatccgcgtaccgcatgtttttggcagggttttgttgatgaacaagaatttatggccgagtcctttaaagcagcaatggcaaaactggcaattctgggtcataatcgtgcaagcctgaccgattgtagtgatgttgttccgattccgcgtccggcagttaaaaaaccggcaagttttccggcaaccaccggtcctaaagatctggaactgacctgtcgtgcagaacgttttccgaccctgaccgttgatcgtggtgcagttcaggcactgattccgcattgtagcaatggtggccaggattgtccgagcgtgcagtttgatggtccggcataa
锰过氧化物酶CsMnP编码基因序列全长1137bp。其中，信号肽的碱基序列如SEQ IDNo.5所示：
Atggcctttaccagctttgttgcactggcagcactggttggtattgcaagcgca
成熟的锰过氧化物酶CsMnP的cDNA(去信号肽)序列如SEQ ID No.6所示：
Gcaccgaccaccatttgtccggatggcacccgtgttagcaatcatgcatgttgtgcatttattccgctggcagaagatctgcagaaaaccatttttatgaacgattgtggtgaagatgcccatgaagttattcgtctgacctttcatgatgcagttgcaattagccgtaaactgggtccgaaagccggtggtggtgcagatggtagcatgctgctgtttccgaccgttgaaccgaattttagcgcaaataatggcattgatgatagcgtgaataacctgattccgtttatggcacgtcatccgaccgtgagtgccggtgatctggttcagtttgccggtgcagttgccctgagcaattgtccgggtgcaccgcgtctggaatttctggcaggtcgtccgaatcataccattgcagcaattgatggtctgattccggaaccgcaggatgatgttaccaaaattctggaacgttttgatgatgccggtggttttaccccgtttgaagttgttagcctgctggcaagccataccgttgcacgtgccgataaagttgatgaaaccattgatgcagcaccgtttgatagcaccccgtttacctttgatacccaggtttttctggaagttctgctgaaaggtgttggttttccgggtacagataataacaccggtgaagttgcaagtccgctgccgaaaggtagcggtaatgataccggtgaaatgcgtctgcagagcgattttgccctggcacgcgatccgcgtaccgcatgtttttggcagggttttgttgatgaacaagaatttatggccgagtcctttaaagcagcaatggcaaaactggcaattctgggtcataatcgtgcaagcctgaccgattgtagtgatgttgttccgattccgcgtccggcagttaaaaaaccggcaagttttccggcaaccaccggtcctaaagatctggaactgacctgtcgtgcagaacgttttccgaccctgaccgttgatcgtggtgcagttcaggcactgattccgcattgtagcaatggtggccaggattgtccgagcgtgcagtttgatggtccggcataa
本发明还提供了包含上述锰过氧化物酶基因CsMnP的重组载体，优选为pCold I-CsMnP。将本发明的锰过氧化物酶基因插入到表达载体合适的限制性酶切位点之间，使其核苷酸序列可操作的与表达调控序列相连接。作为本发明的一个最优选的实施方案，将本发明的锰过氧化物酶基因CsMnP插入到质粒pCold I上的NdeI-BamHI限制性酶切位点之间，使该核苷酸序列位于CSPA启动子的下游并受其调控，得到重组大肠杆菌表达质粒pCold I-CsMnP。
本发明还提供了包含上述锰过氧化物酶基因CsMnP的重组菌株，优选为重组大肠杆菌菌株BL21(DE3)/CsMnP。
本发明还提供了一种制备锰过氧化物酶CsMnP的方法，包括以下步骤：
(1)用上述的重组载体转化宿主细胞，得重组菌株；
(2)培养重组菌株，诱导重组锰过氧化物酶表达了；
(3)纯化锰过氧化物酶CsMnP。
本发明提供了上述锰过氧化物酶CsMnP的应用，尤其是在降解霉菌毒素脱毒方面，其对黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素、伏马菌素B1具有良好的降解率。
附图说明
图1显示重组锰过氧化物酶CsMnP对黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素、伏马菌素B1的降解率；
图2显示重组锰过氧化物酶CsMnP降解黄曲霉毒素B1的HPLC分析结果；
图3显示重组锰过氧化物酶CsMnP降解玉米赤霉烯酮的HPLC分析结果；
图4显示重组锰过氧化物酶CsMnP降解呕吐毒素的HPLC分析结果；
图5显示重组锰过氧化物酶CsMnP降解伏马菌素B1的HPLC分析结果。
具体实施方式
试验材料和试剂
1、基因及载体：大肠杆菌表达载体pCold I及菌株BL21(DE3)；
2、酶类及其它生化试剂：内切酶、重组酶、黄曲霉毒素B1、玉米赤霉烯酮和呕吐毒素、伏马菌素。
3、培养基：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锰过氧化物酶CsMnP编码基因的克隆
本发明目的基因来源于多孔菌科真菌虫拟蜡菌(Ceriporiopsissubvermispora)。
设计特异性引物：
CsMnP-F:5'-CATATCGAAGGTAGGCATATGGCACCGACCACCAT-3'；
CsMnP-R:5'-GACAAGCTTGAATTCGGATCCTGCCGGACCATCAA-3'。
以带有目的基因的质粒为模板进行PCR扩增。在1％琼脂糖凝胶上电泳，切胶得到目的片段，将该片段回收后与NdeI-BamHI双酶切的pCold I载体通过同源重组的方法相连，转化TransI克隆宿主，测序验证，得到氧化物酶CsMnP编码基因。
实施例2重组锰过氧化物酶CsMnP的制备
将获得的含有锰过氧化物酶基因CsMnP的重组大肠杆菌表达质粒pCold I-CsMnP转化大肠杆菌BL21(DE3)，获得重组大肠杆菌菌株BL21(DE3)/CsMnP。
以同样的方法构建含信号肽序列的锰过氧化物酶基因CsMnP的重组表达载体。
取含有重组质粒的BL21(DE3)/CsMnP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2h(OD590≈0.5)。冰水浴处理后于10℃ 220rpm培养30分钟后加入60μL浓度1M的诱导剂IPTG，于10℃220rpm继续培养6h。之后9h培养过程中每小时加入33μL浓度1M的CaCl2，330μL浓度10g/L的Hemin(Hemin溶于0.1M NaOH中)。10℃ 220rpm继续培养10h后离心收集菌体。采取超声法法裂解菌体，利用镍柱进行亲和层析纯化获得单一的CsMnP蛋白。
实施例3重组锰过氧化物酶CsMnP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：70μL丙二酸缓冲液(0.2M，pH 5.0)，20μL黄曲霉毒素B1溶液(50mg/L)，5μL硫酸锰(40mM)，100μL锰过氧化酶(1000U/L)，5μL过氧化氢(4mM)。以未加入锰过氧化物酶的体系作为对照，反应体系设3个重复。反应在30℃下进行，48h后加入三倍体积的甲醇终止反应，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，可见大部分黄曲霉毒素已被降解，降解率为80.8％。
实施例4重组锰过氧化物酶CsMnP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：70μL丙二酸缓冲液(0.2M，pH 5.0)，20μL玉米赤霉烯酮溶液(50mg/L)，5μL硫酸锰(40mM)，100μL锰过氧化酶(1000U/L)，5μL过氧化氢(4mM)。以未加入锰过氧化物酶的体系作为对照，反应体系设3个重复。反应在30℃下进行，48h后加入三倍体积的甲醇终止反应，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，可见大部分玉米赤霉烯酮已被降解，降解率为78.9％。
实施例5重组锰过氧化物酶CsMnP降解呕吐毒素
将呕吐毒素溶解到甲醇中配制成100mg/L的母液，按如下反应体系：70μL丙二酸缓冲液(0.2M，pH 5.0)，20μL呕吐毒素溶液(100mg/L)，5μL硫酸锰(40mM)，100μL锰过氧化酶(1000U/L)，5μL过氧化氢(4mM)。以未加入锰过氧化物酶的体系作为对照，反应体系设3个重复。反应在30℃下进行，48h后加入三倍体积的甲醇终止反应，采用高效液相色谱(HPLC)分析呕吐毒素的降解率。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(水)，流动相B(甲醇)；梯度洗脱条件20％B洗脱20分钟，20％-100％B洗脱1分钟，100％B洗脱6分钟；检测波长220nm。
结果如图1、图4所示，可见大部分呕吐毒素已被降解，降解率为86.3％。
实施例6重组锰过氧化物酶CsMnP降解伏马菌素B1
将伏马菌素B1溶解到DMSO中配制成100mg/L的母液，按如下反应体系：70μL丙二酸缓冲液(0.2M，pH 5.0)，20μL伏马菌素B1溶液(100mg/L)，5μL硫酸锰(40mM)，100μL锰过氧化酶(1000U/L)，5μL过氧化氢(4mM)。以未加入锰过氧化物酶的体系作为对照，反应体系设3个重复。反应在30℃下进行，48h后加入三倍体积的甲醇终止反应，采用高效液相色谱(HPLC)-质谱(MS)联用分析伏马菌素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(4.6X 250,5um)，流动相A(0.1％甲酸的水)，流动相B(乙腈:甲醇1:1)；梯度洗脱条件30％-100％B梯度洗脱10分钟，30％B洗脱18分钟，100％B洗脱2分钟；质谱为SCIEX Triple TOF分析系统，质谱条件为CE：35V±15V，Ion source gas：50，TEM 500℃，ISVF 5500V，sean：m/z 100-1000(目标物721)。
结果如图1、图5所示，可见部分伏马菌素已被降解，降解率为21.3％。
序列表
<110> 中国农业科学院饲料研究所
<120> 锰过氧化物酶CsMnP及其编码基因和应用
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<211> 378
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<213> 多孔菌科真菌虫拟蜡菌(Ceriporiopsis subvermispora)
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Met Ala Phe Thr Ser Phe Val Ala Leu Ala Ala Leu Val Gly Ile Ala
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Ser Ala Ala Pro Thr Thr Ile Cys Pro Asp Gly Thr Arg Val Ser Asn
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His Ala Cys Cys Ala Phe Ile Pro Leu Ala Glu Asp Leu Gln Lys Thr
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Ile Phe Met Asn Asp Cys Gly Glu Asp Ala His Glu Val Ile Arg Leu
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Thr Phe His Asp Ala Val Ala Ile Ser Arg Lys Leu Gly Pro Lys Ala
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Pro Phe Met Ala Arg His Pro Thr Val Ser Ala Gly Asp Leu Val Gln
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Glu Phe Leu Ala Gly Arg Pro Asn His Thr Ile Ala Ala Ile Asp Gly
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Leu Ile Pro Glu Pro Gln Asp Asp Val Thr Lys Ile Leu Glu Arg Phe
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Asp Asp Ala Gly Gly Phe Thr Pro Phe Glu Val Val Ser Leu Leu Ala
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Glu Val Leu Leu Lys Gly Val Gly Phe Pro Gly Thr Asp Asn Asn Thr
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aaagccggtg gtggtgcaga tggtagcatg ctgctgtttc cgaccgttga accgaatttt 240
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gatggtctga ttccggaacc gcaggatgat gttaccaaaa ttctggaacg ttttgatgat 480
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gatacccagg tttttctgga agttctgctg aaaggtgttg gttttccggg tacagataat 660
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cgtctgcaga gcgattttgc cctggcacgc gatccgcgta ccgcatgttt ttggcagggt 780
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attctgggtc ataatcgtgc aagcctgacc gattgtagtg atgttgttcc gattccgcgt 900
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acctgtcgtg cagaacgttt tccgaccctg accgttgatc gtggtgcagt tcaggcactg 1020
attccgcatt gtagcaatgg tggccaggat tgtccgagcg tgcagtttga tggtccggca 1080
taa 1083
Claims (8)
1.锰过氧化物酶CsMnP，其特征在于，其氨基酸序列如SEQ ID No.1或SEQ ID No.3所示。
2.锰过氧化物酶CsMnP基因，其特征在于，其编码权利要求1所述的锰过氧化物酶CsMnP。
3.根据权利要求2所述的锰过氧化物酶CsMnP基因，其特征在于，其核苷酸序列如SEQID No.4或SEQ ID No.6所示。
4.包含权利要求2所述的锰过氧化物酶CsMnP基因的重组表达载体。
5.包含权利要求2所述的锰过氧化物酶CsMnP基因的重组菌株。
6.制备权利要求1所述的锰过氧化物酶CsMnP的方法，其特征在于，所述方法包括以下步骤：
(1)用包含编码锰过氧化物酶CsMnP基因的重组表达载体转化宿主细胞，得到重组菌株；
(2)培养所述重组菌株，诱导锰过氧化物酶CsMnP表达；
(3)分离纯化所述锰过氧化物酶CsMnP。
7.权利要求1所述的锰过氧化物酶CsMnP的应用。
8.权利要求1所述的锰过氧化物酶CsMnP在霉菌毒素脱毒方面的应用。
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| CN113897378A (zh) * | 2021-08-20 | 2022-01-07 | 江南大学 | 一种白腐真菌锰过氧化物酶在聚乙烯降解中的应用 |
| CN117814431A (zh) * | 2024-01-17 | 2024-04-05 | 中国农业科学院北京畜牧兽医研究所 | 一种靶向降解饲料中的呕吐毒素的方法以及融合酶 |
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| CN113897378A (zh) * | 2021-08-20 | 2022-01-07 | 江南大学 | 一种白腐真菌锰过氧化物酶在聚乙烯降解中的应用 |
| CN113897378B (zh) * | 2021-08-20 | 2023-10-03 | 江南大学 | 一种白腐真菌锰过氧化物酶在聚乙烯降解中的应用 |
| CN117814431A (zh) * | 2024-01-17 | 2024-04-05 | 中国农业科学院北京畜牧兽医研究所 | 一种靶向降解饲料中的呕吐毒素的方法以及融合酶 |
| CN117814431B (zh) * | 2024-01-17 | 2025-10-14 | 中国农业科学院北京畜牧兽医研究所 | 一种靶向降解饲料中的呕吐毒素的方法以及融合酶 |
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