CN114958933B - 一种利用黑芥子酶Emyr制备莱菔素的方法 - Google Patents
一种利用黑芥子酶Emyr制备莱菔素的方法 Download PDFInfo
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- CN114958933B CN114958933B CN202210469833.7A CN202210469833A CN114958933B CN 114958933 B CN114958933 B CN 114958933B CN 202210469833 A CN202210469833 A CN 202210469833A CN 114958933 B CN114958933 B CN 114958933B
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- myrosinase
- radish
- emyr
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- C12Y—ENZYMES
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Abstract
本发明公开了一种利用黑芥子酶Emyr制备莱菔素的方法，属于功能酶技术领域，具体制备方法为：采用黑芥子酶Emyr降解莱菔苷，制备得到莱菔素；所述黑芥子酶Emyr，其氨基酸序列如SEQ ID NO.1所示。所述降解的具体方式为：将黑芥子酶Emyr加入到含有莱菔苷的溶液中，在25～40℃、pH 6.0～8.0条件下反应30分钟～120小时。本发明还公开了黑芥子酶Emyr在降解莱菔苷中的应用，在制备莱菔素中的应用。本发明的制备莱菔素的方法，采用稳定性好、酶活高的黑芥子酶Emyr降解莱菔苷，对实现莱菔素的大批量制备具有重要意义。
Description
技术领域
本发明涉及一种利用黑芥子酶Emyr制备莱菔素的方法，以及黑芥子酶Emyr在降解莱菔苷、制备莱菔素中的应用，属于功能酶技术领域。
背景技术
异硫氰酸酯最初是在十字花科植物中发现的一类具有强大生物活性的物质，具有广阔的应用前景。莱菔素(sulforaphene)是其中的一种，并且当前的研究已经证明莱菔素在抗炎、抗癌、抑制肥胖、改善心血管疾病和神经性疾病等方面具有强大的功效，是一种十分有潜力的活性物质。但是由于其在十字花科植物中含量很低，难以直接提取，一般需要酶解制备，这涉及到一个关键的水解酶——黑芥子酶。目前莱菔素的酶解制备方法主要有两种：一是利用内源酶，在十字花科植物中存在的物质主要是硫代葡萄糖苷，与黑芥子酶是不接触的，只有当植物受损、细胞破裂时，黑芥子酶才会释放出来催化硫代葡萄糖苷水解生成莱菔素，该方法存在酶活效率低、产率低的缺陷。二是添加外源酶，从植物中提取黑芥子酶或者直接添加黑芥子酶制剂，催化硫代葡萄糖苷水解制备，但其步骤繁琐，成本高。因此，便捷的获得大量的黑芥子酶制剂是实现莱菔素大批量生产的关键。
目前，根据来源不同，黑芥子酶可以分为植物源黑芥子酶、蚜虫源黑芥子酶、微生物源黑芥子酶。植物源黑芥子酶已在许多宿主中进行异源表达，包括毕赤酵母、酿酒酵母等，但是存在酶活低、反应时间长的问题，在实际生产上并不具备优势。蚜虫源的黑芥子酶异源表达困难，难以进行大批量酶制剂的获得。微生物来源的黑芥子酶进行异源表达的研究较少，只有Catherine等人定位了肠道微生物Bacteroides thetaiotaomicron中的操纵子BT2156-BT2160，但其单个酶蛋白并不具有黑芥子酶活性。
发明内容
针对上述现有技术，本发明提供了一种制备莱菔素的方法。本发明通过挖掘新的黑芥子酶基因，实现其异源表达，对其进行了酶学性质的研究，利用该酶催化萝卜种子进行莱菔素的制备，为其实际应用奠定了理论基础。
本发明是通过以下技术方案实现的：
一种利用黑芥子酶Emyr制备莱菔素的方法：采用黑芥子酶Emyr降解莱菔苷(glucoraphenin)，制备得到莱菔素；所述黑芥子酶Emyr，其氨基酸序列如SEQ ID NO.1所示。所述降解的具体方式为：将黑芥子酶Emyr加入到含有莱菔苷的溶液中，在25～40℃、pH6.0～8.0条件下反应30分钟～120小时。
进一步地，所述含有莱菔苷的溶液为萝卜种子溶液，是由粉碎的萝卜种子和水按重量体积比1g:8～12ml组成的，其中莱菔苷的浓度约为4.5μmol/mL。
进一步地，所述黑芥子酶Emyr的添加量为40～50U/g萝卜种子。
进一步地，向含有莱菔苷的溶液中加入终浓度为1～10mmol/L的镁离子，以增强酶活。所述镁离子是以氯化镁的形式加入的。
进一步地，所述温度为35℃。
进一步地，所述pH为6.0。
进一步地，所述反应时间为80～120小时。
更进一步地，反应条件为：在30℃、pH 6.0或7.0条件下反应104～118小时。
黑芥子酶Emyr在降解莱菔苷中的应用，在制备莱菔素中的应用，所述黑芥子酶Emyr氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1：
MQNIPQPELGHTSAPLLSQDGYQFKDLNRDGKLNQYEDWRLSSAQRAEDLTRRMTLKEKAGLMMHGTGPVSGNNFGNGDVYDLDAAKKMIVDAHINSILTRLGGKEPRRLAEQNNKLQEIAESARLGIPVTVSTDPRNSYQALAGISNPAGKFSQWPEPIGIGAAGSESLARAFASKIGQEYRAVGITEALSPQADIATEPRWARISGTFGEDPELARKLVRGYITGMQNGTQGLNPQGVAAVVKHWVGYGAAEDGWDGHNAYGKNVVFKTNNLEEHIVPFKGAFESQVAAVMPTYSVLKGVSLNGKSPEPVAAGYSHFLLTDLLRGQYNFKGVIISDWLITNDCDDECIHGAPGGKKPVTGGMPWGVESLTQEQRFVKAVQAGIDQFGGVTDSDIITGAVEKDLISESRINQSAQRILLQKFELGLFEQPYVNAADAEKIVGRGETQKEANQAQMQSLVLLQNKNILPLKPGTRVWLYGADAAEAKKAGLAVVDRPEDAEVAVMRTSAPFEQPHYNYFFGRRHHEGSLEYKADNDVMKTLSDVARKVPVVMTMYMERPAVLTGVTDKTQAFIANFGLSDEVLFSRLISDASYSGRLPFALPASMDAVLKQDPSVPGDLEAPLYALGFGLSRLE。
上述黑芥子酶Emyr的编码基因，其核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2：
5’-ATGCAGAACATCCCTCAGCCAGAACTGGGTCACACCTCCGCTCCTCTGCTGTCTCAGGATGGTTACCAGTTCAAAGACCTGAACCGTGATGGTAAACTGAACCAGTACGAAGATTGGCGTCTGTCCTCTGCTCAGCGTGCTGAAGATCTGACCCGTCGTATGACTCTGAAAGAAAAAGCTGGTCTGATGATGCACGGTACCGGCCCAGTTTCCGGTAACAACTTCGGTAACGGCGATGTTTACGATCTGGATGCAGCAAAAAAGATGATCGTTGATGCTCACATCAACTCTATCCTGACTCGTCTGGGTGGTAAAGAACCACGTCGTCTGGCTGAACAGAACAACAAACTGCAGGAAATCGCTGAATCCGCTCGTCTGGGTATTCCAGTTACCGTTTCTACCGACCCACGTAACAGCTACCAGGCACTGGCCGGTATCTCTAACCCAGCTGGCAAATTTTCTCAGTGGCCTGAACCAATCGGTATCGGTGCAGCAGGTTCTGAATCTCTGGCTCGTGCTTTTGCATCTAAAATCGGTCAGGAATACCGTGCTGTGGGTATCACCGAAGCACTGTCTCCACAGGCTGATATCGCTACCGAACCACGTTGGGCACGTATCTCCGGTACTTTCGGCGAAGATCCTGAACTGGCTCGTAAACTGGTTCGTGGCTACATCACCGGCATGCAGAACGGTACCCAGGGTCTCAACCCACAGGGTGTGGCTGCCGTTGTTAAACATTGGGTTGGTTACGGCGCTGCTGAAGATGGTTGGGATGGCCACAACGCTTATGGTAAAAACGTTGTTTTCAAAACCAACAACCTGGAAGAACACATCGTTCCTTTCAAAGGTGCTTTCGAATCTCAGGTTGCTGCCGTAATGCCAACCTATTCTGTACTGAAAGGTGTGTCCCTGAACGGTAAATCCCCTGAACCAGTTGCTGCTGGTTACTCTCACTTCCTGCTGACTGATCTGCTGCGTGGTCAGTACAACTTCAAAGGTGTAATTATCTCTGACTGGCTGATCACTAACGACTGTGATGACGAATGCATCCACGGTGCTCCAGGTGGTAAAAAACCAGTTACCGGCGGTATGCCGTGGGGCGTTGAATCGCTGACCCAGGAACAGCGTTTCGTGAAAGCAGTTCAGGCAGGTATCGATCAGTTCGGTGGTGTTACCGACTCTGATATCATTACTGGTGCTGTTGAAAAAGACCTGATTTCTGAATCTCGTATCAACCAGTCTGCTCAGCGTATCCTGCTGCAGAAATTCGAACTGGGTCTGTTTGAACAGCCATACGTTAATGCTGCCGATGCTGAAAAAATCGTTGGTCGTGGTGAAACCCAGAAAGAAGCTAACCAGGCTCAGATGCAGTCCCTGGTTCTGCTGCAGAACAAAAACATTCTGCCACTGAAACCAGGCACCCGTGTTTGGCTGTATGGTGCAGACGCTGCTGAAGCTAAAAAAGCAGGTCTGGCTGTTGTTGACCGTCCTGAAGATGCTGAAGTTGCCGTTATGCGTACTTCCGCACCATTCGAACAGCCACACTACAACTACTTCTTCGGCCGTCGTCACCACGAAGGTTCTCTGGAATACAAAGCTGATAACGATGTTATGAAAACCCTGTCCGATGTTGCTCGTAAAGTTCCTGTGGTAATGACCATGTACATGGAACGTCCAGCAGTTCTGACCGGTGTTACTGATAAAACCCAGGCTTTCATCGCTAACTTCGGCCTGTCTGACGAAGTTCTGTTCTCTCGTCTGATCTCCGACGCTTCCTACTCTGGTCGTCTGCCTTTCGCACTGCCAGCATCTATGGACGCTGTGCTGAAACAGGATCCATCCGTTCCTGGTGACCTGGAAGCTCCACTGTATGCACTGGGTTTCGGTCTGTCTCGTCTCGAG-3’。
一种重组表达载体，其携带有上述黑芥子酶Emyr的编码基因。
一种表达黑芥子酶Emyr的重组工程菌，其基因组中包含有上述黑芥子酶Rmyr的编码基因或上述重组表达载体，能够表达黑芥子酶Emyr。可通过转化/转染上述重组表达载体制备得到。
进一步地，所述重组工程菌的宿主为大肠杆菌。
所述重组表达载体、重组工程菌在制备黑芥子酶Emyr中的应用。
一种酶制剂，包含有上述黑芥子酶Emyr。
所述酶制剂在降解莱菔苷中的应用，在制备莱菔素中的应用。
本发明的黑芥子酶Emyr，来源于微生物Enterobacteriaceae，是一种比较少见的来源于肠道微生物的黑芥子酶，最适反应温度35℃，最适反应pH 6.0，在25～40℃下均具有较高的酶活力。此外，通过对其稳定性测试发现，该酶较稳定，在30℃孵育118小时后酶活仍保留50％以上；在pH 6.0/pH 7.0、4℃的条件下存放104小时酶活仍保留50％以上，是一个比较稳定的酶。本发明通过发酵、冻干获得酶制剂，并在体外验证了其产物制备能力，通过液相色谱和质谱对产物进行了鉴定，同时优化了产物莱菔素制备的最适pH，完成其生产制备中条件的摸索。本发明的制备莱菔素的方法，采用稳定性好、酶活高的黑芥子酶Emyr降解莱菔苷，对实现莱菔素的大批量制备具有重要意义。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1：本发明的黑芥子酶纯化后的纯酶SDS-PAGE电泳图，其中，M为标准蛋白Marker；1为粗酶液；2为50mM咪唑溶液洗脱下来的目的蛋白。
图2：温度变化对相对酶活的影响示意图。
图3：pH变化对相对酶活的影响示意图。
图4：本发明的黑芥子酶在不同温度下放置不同时间残留酶活变化图。
图5：本发明的黑芥子酶在4℃、不同pH下放置不同时间残留酶活变化图。
图6：本发明的黑芥子酶在不同金属离子和化学试剂下相对酶活的变化示意图。
图7：本发明的黑芥子酶水解萝卜种子制备产物的质谱鉴定图。
图8：本发明的黑芥子酶制备莱菔素时pH对产物生成的影响示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。
实施例1黑芥子酶Emyr的编码基因的克隆
本发明的黑芥子酶Emyr的编码基因为全基因合成所得，是在NCBI文库中挖掘到的(目前黑芥子酶的来源主要是植物、动物还有微生物来源，植物和动物来源的黑芥子酶在克隆表达的时候存在物种差异，往往由于蛋白的修饰等问题难有酶活；而实验证明很多肠道微生物来源的细菌有黑芥子酶活性，并且肠道微生物来源的黑芥子酶在异源表达的时候比植物来源的黑芥子酶更具有优势，更有可能实现在大肠杆菌中的异源表达，所以本发明也挖掘了肠道来源的基因，这样更有可能找到潜在的黑芥子酶基因)，来源于肠道微生物Enterobacteriaceae，序列号为WP_008786844.1，该片段包含了1902个碱基序列，如SEQ IDNO.2所示，编码634个氨基酸序列，如SEQ ID NO.1所示。本发明是首次将该酶表达、纯化并进行相关研究。
以合成的基因为模板，在黑芥子酶基因的上、下游设计用于无缝连接的引物，进行PCR扩增基因片段。
引物的序列如下所示：
上游引物：5’-GATATACCATGCAGAACATCCCTCAGCCAG-3’，如SEQ ID NO.3所示；
下游引物：5’-GGTGGTGCTCGAGACGAGACAGAC-3’，如SEQ ID NO.4所示。
PCR反应体系为：2×PCR Buffer 25μl，dNTP 10μl，引物各1.5μl，模板1μl，KOD Fx酶1μl，无菌水10μl，总体系50μl。
PCR反应条件为：94℃预变性5min，95℃变性20s，60℃退火30s，72℃延伸120s，反应30个循环，72℃后延伸10min。
琼脂糖凝胶电泳后回收1902bp的PCR产物片段，即为目的基因片段。
实施例2重组表达载体的构建
目的基因片段与pET-28a克隆载体采用无缝克隆技术进行连接，将连接产物转入E.coli DH5α感受态细胞，涂布于含有50μg/mL卡那霉素的LB培养基固体平板上，37℃温箱中培养15小时后，挑取单克隆至含有50μg/mL卡那霉素的LB液体培养基，37℃、220rpm摇床培养12小时，阳性验证后测序，并命名为pET28a-Emyr。
实施例3重组质粒及工程菌的构建
提取测序正确的重组质粒，并转化至宿主E.coli BL21感受态细胞中，在硫酸卡那霉素抗性平板上长出的单菌落，即为构建好的工程菌。
实施例4利用大肠杆菌工程菌制备黑芥子酶Emyr
挑取大肠杆菌重组菌株(实施例3中的单菌落)，接种在5ml含有硫酸卡那霉素的LB液体培养基中，37℃，220rpm培养12h后按1％的接种量接入含有硫酸卡那霉的ZYP-5052培养基，20℃、200rpm培养48h，自诱导表达黑芥子酶。培养液4℃，8000g离心10min，收集菌体，细胞重悬于50mM、pH 7.0的Tirs-HCl缓冲液中，超声破碎30min后12000g离心15min，上清液即为粗酶液。粗酶液使用Ni-NTA柱进行亲和层析纯化，使用平衡缓冲液(500mM NaCl，50mMTris-HCl)平衡柱子，然后用20mM咪唑溶液(20mM咪唑，500mM NaCl，50mM Tris-HCl)洗脱结合力弱的杂蛋白，50mM咪唑溶液(50mM咪唑，500mM NaCl，50mM Tris-HCl)洗脱目的蛋白，得到洗脱液，浓缩至蛋白浓度为0.16mg/mL(使用Bradford法测定酶液中的蛋白浓度)，得到酶液。对得到的酶液进行SDS-PAGE检测，核对条带是否单一、大小是否准确，结果如图1所示，由图可见，得到了大小为68.8KDa的条带，与预测相一致，证明该目的蛋白即为SEQ ID NO.1所示的蛋白。同时采用DNS验证所获蛋白是否具有酶活(结果见实施例5)。
实施例5黑芥子酶Emyr的酶活测定
黑芥子酶Emyr活性初步测定方法为：将80μL酶液(指实施例4得到的酶液，下同)加入到120μL的浓度为0.5％(w/v，g/ml)的黑芥子苷溶液(是用pH 7.0的磷酸盐缓冲溶液配制的，下同)中，在40℃下反应90min，反应结束后吸取100μL上清液加入300μL的DNS试剂中沸水浴5min进行显色，在OD540nm下检测其吸光度。酶活力定义为在标准条件下每mg酶在每min产生葡萄糖的量(μmol)。在此条件下，黑芥子酶Emyr的活力达到了1.3U/mg。
实施例6测定黑芥子酶Emyr的最适反应条件
将80μL酶液加入到120μL的浓度为0.5％(w/v，g/ml)的黑芥子苷溶液中，分别在25℃、30℃、35℃、40℃、45℃、50℃下反应1h测定最适温度。在40℃下，选用pH为3.0～10.0的缓冲液作为酶反应的不同测定pH缓冲液，根据黑芥子酶的酶活力，测定黑芥子酶的最适pH。
结果如图2，图3所示，黑芥子酶Emyr的最适反应温度为35℃，最适pH为6.0，同时其最适温度测定实验结果显示黑芥子酶Emyr在25～40℃酶活均比较高，表明该酶具有一定的嗜冷性。
实施例7测定黑芥子酶Emyr在不同温度下的稳定性
在30℃、35℃、40℃、45℃的温度下对酶液进行温育，于不同时间在最适条件下(温度为35℃，pH为6.0)测定其残留酶活，得到其温度稳定性。结果如图4所示，黑芥子酶Emyr在30℃下放置118h，酶活仍可保留50％以上，酶活稳定性较好。
实施例8测定黑芥子酶Emyr在不同pH下的稳定性
80μL酶液分别与120μL的pH 6.0、pH 7.0、pH 8.0的磷酸盐缓冲液混合，并于4℃中孵育，于不同时间在最适温度下测定其残留酶活，得到其pH稳定性。结果如图5所示，黑芥子酶Emyr在pH 6.0、7.0的缓冲液中保留104h，酶活也能保留50％以上，说明该酶的稳定性较好。
实施例9金属离子和化学试剂对黑芥子酶Emyr的活性影响
将80μL酶液加入到120μL的浓度为0.5％(w/v，g/ml)的黑芥子苷溶液(pH 6.0)中，加入不同的金属离子和化学试剂使其终浓度分别达到1mM和10mM，35℃条件下反应30min。反应结束后测定酶活。结果如图6所示，大部分的试剂在低浓度和高浓度下都不能使酶活提高，但是Mg2+不论是低浓度还是高浓度下都可以使酶活提高。
实施例10制备酶制剂
实施例4制备的酶液，真空冷冻干燥机进行冻干，获得可长期保存的酶粉。
实施例11黑芥子酶Emyr制备产物的鉴定
将萝卜种子高温处理进行酶的灭活，之后粉碎。以粉碎的萝卜种子为底物，按照1:10(m/v，g/ml)的体系溶解于水中，得萝卜种子溶液。将80μL酶液加入到120μL的萝卜种子溶液中，在35℃、150rpm的条件下进行产物的制备，反应时间30min，反应结束后将样品用两倍体积的乙酸乙酯萃取，结束后进行旋干，并用和样品同等体积的乙腈复溶，之后液相检测产物出峰时间并用质谱进行进一步的鉴定。结果如图7所示，通过质谱的结果，可以得出黑芥子酶Emyr成功催化萝卜种子中的莱菔苷水解生成了产物莱菔素(Mr＝176.0199)，可以应用于莱菔素的制备。
实施例12黑芥子酶Emyr制备产物的pH优化
实施例11的萝卜种子溶液，用盐酸和氢氧化钠溶液调节pH，得到pH分别为3、4、5、6、7的萝卜种子溶液，加入酶液(酶液与萝卜种子溶液的体积比为2:3)，在35℃、150rpm的条件下进行产物的制备，反应时间30min，之后用液相色谱检测不同pH下产物的生成量。结果如图8所示，黑芥子酶Emyr制备莱菔素的最佳pH为6.0，这与其最适pH一致。
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
序列表
<110> 中国海洋大学
<120> 一种利用黑芥子酶Emyr制备莱菔素的方法
<141> 2022-04-30
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 634
<212> PRT
<213> Enterobacteriaceae
<400> 1
Met Gln Asn Ile Pro Gln Pro Glu Leu Gly His Thr Ser Ala Pro Leu
1 5 10 15
Leu Ser Gln Asp Gly Tyr Gln Phe Lys Asp Leu Asn Arg Asp Gly Lys
20 25 30
Leu Asn Gln Tyr Glu Asp Trp Arg Leu Ser Ser Ala Gln Arg Ala Glu
35 40 45
Asp Leu Thr Arg Arg Met Thr Leu Lys Glu Lys Ala Gly Leu Met Met
50 55 60
His Gly Thr Gly Pro Val Ser Gly Asn Asn Phe Gly Asn Gly Asp Val
65 70 75 80
Tyr Asp Leu Asp Ala Ala Lys Lys Met Ile Val Asp Ala His Ile Asn
85 90 95
Ser Ile Leu Thr Arg Leu Gly Gly Lys Glu Pro Arg Arg Leu Ala Glu
100 105 110
Gln Asn Asn Lys Leu Gln Glu Ile Ala Glu Ser Ala Arg Leu Gly Ile
115 120 125
Pro Val Thr Val Ser Thr Asp Pro Arg Asn Ser Tyr Gln Ala Leu Ala
130 135 140
Gly Ile Ser Asn Pro Ala Gly Lys Phe Ser Gln Trp Pro Glu Pro Ile
145 150 155 160
Gly Ile Gly Ala Ala Gly Ser Glu Ser Leu Ala Arg Ala Phe Ala Ser
165 170 175
Lys Ile Gly Gln Glu Tyr Arg Ala Val Gly Ile Thr Glu Ala Leu Ser
180 185 190
Pro Gln Ala Asp Ile Ala Thr Glu Pro Arg Trp Ala Arg Ile Ser Gly
195 200 205
Thr Phe Gly Glu Asp Pro Glu Leu Ala Arg Lys Leu Val Arg Gly Tyr
210 215 220
Ile Thr Gly Met Gln Asn Gly Thr Gln Gly Leu Asn Pro Gln Gly Val
225 230 235 240
Ala Ala Val Val Lys His Trp Val Gly Tyr Gly Ala Ala Glu Asp Gly
245 250 255
Trp Asp Gly His Asn Ala Tyr Gly Lys Asn Val Val Phe Lys Thr Asn
260 265 270
Asn Leu Glu Glu His Ile Val Pro Phe Lys Gly Ala Phe Glu Ser Gln
275 280 285
Val Ala Ala Val Met Pro Thr Tyr Ser Val Leu Lys Gly Val Ser Leu
290 295 300
Asn Gly Lys Ser Pro Glu Pro Val Ala Ala Gly Tyr Ser His Phe Leu
305 310 315 320
Leu Thr Asp Leu Leu Arg Gly Gln Tyr Asn Phe Lys Gly Val Ile Ile
325 330 335
Ser Asp Trp Leu Ile Thr Asn Asp Cys Asp Asp Glu Cys Ile His Gly
340 345 350
Ala Pro Gly Gly Lys Lys Pro Val Thr Gly Gly Met Pro Trp Gly Val
355 360 365
Glu Ser Leu Thr Gln Glu Gln Arg Phe Val Lys Ala Val Gln Ala Gly
370 375 380
Ile Asp Gln Phe Gly Gly Val Thr Asp Ser Asp Ile Ile Thr Gly Ala
385 390 395 400
Val Glu Lys Asp Leu Ile Ser Glu Ser Arg Ile Asn Gln Ser Ala Gln
405 410 415
Arg Ile Leu Leu Gln Lys Phe Glu Leu Gly Leu Phe Glu Gln Pro Tyr
420 425 430
Val Asn Ala Ala Asp Ala Glu Lys Ile Val Gly Arg Gly Glu Thr Gln
435 440 445
Lys Glu Ala Asn Gln Ala Gln Met Gln Ser Leu Val Leu Leu Gln Asn
450 455 460
Lys Asn Ile Leu Pro Leu Lys Pro Gly Thr Arg Val Trp Leu Tyr Gly
465 470 475 480
Ala Asp Ala Ala Glu Ala Lys Lys Ala Gly Leu Ala Val Val Asp Arg
485 490 495
Pro Glu Asp Ala Glu Val Ala Val Met Arg Thr Ser Ala Pro Phe Glu
500 505 510
Gln Pro His Tyr Asn Tyr Phe Phe Gly Arg Arg His His Glu Gly Ser
515 520 525
Leu Glu Tyr Lys Ala Asp Asn Asp Val Met Lys Thr Leu Ser Asp Val
530 535 540
Ala Arg Lys Val Pro Val Val Met Thr Met Tyr Met Glu Arg Pro Ala
545 550 555 560
Val Leu Thr Gly Val Thr Asp Lys Thr Gln Ala Phe Ile Ala Asn Phe
565 570 575
Gly Leu Ser Asp Glu Val Leu Phe Ser Arg Leu Ile Ser Asp Ala Ser
580 585 590
Tyr Ser Gly Arg Leu Pro Phe Ala Leu Pro Ala Ser Met Asp Ala Val
595 600 605
Leu Lys Gln Asp Pro Ser Val Pro Gly Asp Leu Glu Ala Pro Leu Tyr
610 615 620
Ala Leu Gly Phe Gly Leu Ser Arg Leu Glu
625 630
<210> 2
<211> 1902
<212> DNA
<213> Enterobacteriaceae
<400> 2
atgcagaaca tccctcagcc agaactgggt cacacctccg ctcctctgct gtctcaggat 60
ggttaccagt tcaaagacct gaaccgtgat ggtaaactga accagtacga agattggcgt 120
ctgtcctctg ctcagcgtgc tgaagatctg acccgtcgta tgactctgaa agaaaaagct 180
ggtctgatga tgcacggtac cggcccagtt tccggtaaca acttcggtaa cggcgatgtt 240
tacgatctgg atgcagcaaa aaagatgatc gttgatgctc acatcaactc tatcctgact 300
cgtctgggtg gtaaagaacc acgtcgtctg gctgaacaga acaacaaact gcaggaaatc 360
gctgaatccg ctcgtctggg tattccagtt accgtttcta ccgacccacg taacagctac 420
caggcactgg ccggtatctc taacccagct ggcaaatttt ctcagtggcc tgaaccaatc 480
ggtatcggtg cagcaggttc tgaatctctg gctcgtgctt ttgcatctaa aatcggtcag 540
gaataccgtg ctgtgggtat caccgaagca ctgtctccac aggctgatat cgctaccgaa 600
ccacgttggg cacgtatctc cggtactttc ggcgaagatc ctgaactggc tcgtaaactg 660
gttcgtggct acatcaccgg catgcagaac ggtacccagg gtctcaaccc acagggtgtg 720
gctgccgttg ttaaacattg ggttggttac ggcgctgctg aagatggttg ggatggccac 780
aacgcttatg gtaaaaacgt tgttttcaaa accaacaacc tggaagaaca catcgttcct 840
ttcaaaggtg ctttcgaatc tcaggttgct gccgtaatgc caacctattc tgtactgaaa 900
ggtgtgtccc tgaacggtaa atcccctgaa ccagttgctg ctggttactc tcacttcctg 960
ctgactgatc tgctgcgtgg tcagtacaac ttcaaaggtg taattatctc tgactggctg 1020
atcactaacg actgtgatga cgaatgcatc cacggtgctc caggtggtaa aaaaccagtt 1080
accggcggta tgccgtgggg cgttgaatcg ctgacccagg aacagcgttt cgtgaaagca 1140
gttcaggcag gtatcgatca gttcggtggt gttaccgact ctgatatcat tactggtgct 1200
gttgaaaaag acctgatttc tgaatctcgt atcaaccagt ctgctcagcg tatcctgctg 1260
cagaaattcg aactgggtct gtttgaacag ccatacgtta atgctgccga tgctgaaaaa 1320
atcgttggtc gtggtgaaac ccagaaagaa gctaaccagg ctcagatgca gtccctggtt 1380
ctgctgcaga acaaaaacat tctgccactg aaaccaggca cccgtgtttg gctgtatggt 1440
gcagacgctg ctgaagctaa aaaagcaggt ctggctgttg ttgaccgtcc tgaagatgct 1500
gaagttgccg ttatgcgtac ttccgcacca ttcgaacagc cacactacaa ctacttcttc 1560
ggccgtcgtc accacgaagg ttctctggaa tacaaagctg ataacgatgt tatgaaaacc 1620
ctgtccgatg ttgctcgtaa agttcctgtg gtaatgacca tgtacatgga acgtccagca 1680
gttctgaccg gtgttactga taaaacccag gctttcatcg ctaacttcgg cctgtctgac 1740
gaagttctgt tctctcgtct gatctccgac gcttcctact ctggtcgtct gcctttcgca 1800
ctgccagcat ctatggacgc tgtgctgaaa caggatccat ccgttcctgg tgacctggaa 1860
gctccactgt atgcactggg tttcggtctg tctcgtctcg ag 1902
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 3
gatataccat gcagaacatc cctcagccag 30
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 4
ggtggtgctc gagacgagac agac 24
Claims (1)
1.一种利用黑芥子酶Emyr制备莱菔素的方法，其特征在于：采用黑芥子酶Emyr降解莱菔苷，制备得到莱菔素；所述黑芥子酶Emyr的氨基酸序列如SEQ ID NO.1所示；所述降解的具体方式为：将黑芥子酶Emyr加入到含有莱菔苷的溶液中，在35℃、pH 6.0条件下反应30分钟；
所述含有莱菔苷的溶液为萝卜种子溶液，是由粉碎的萝卜种子和水按重量体积比1 g:8～12 ml组成的；
所述黑芥子酶Emyr的添加量为40～50 U/g萝卜种子；
所述含有莱菔苷的溶液中莱菔苷的浓度为4.5 μmol/mL。
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