CN113736763A - 黑芥子酶Rmyr及其在制备萝卜硫素、莱菔素中的应用 - Google Patents
黑芥子酶Rmyr及其在制备萝卜硫素、莱菔素中的应用 Download PDFInfo
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- CN113736763A CN113736763A CN202111189552.8A CN202111189552A CN113736763A CN 113736763 A CN113736763 A CN 113736763A CN 202111189552 A CN202111189552 A CN 202111189552A CN 113736763 A CN113736763 A CN 113736763A
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- Prior art keywords
- myrosinase
- sulforaphane
- glucosinolate
- ala
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- 108010058651 thioglucosidase Proteins 0.000 title claims abstract description 76
- SUVMJBTUFCVSAD-JTQLQIEISA-N 4-Methylsulfinylbutyl isothiocyanate Natural products C[S@](=O)CCCCN=C=S SUVMJBTUFCVSAD-JTQLQIEISA-N 0.000 title claims abstract description 56
- 235000015487 sulforaphane Nutrition 0.000 title claims abstract description 56
- 229960005559 sulforaphane Drugs 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 125000004383 glucosinolate group Chemical group 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
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- GMMLNKINDDUDCF-JRWRFYLSSA-N [(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1e)-5-[(r)-methylsulfinyl]-n-sulfooxypentanimidothioate Chemical group C[S@@](=O)CCCC\C(=N/OS(O)(=O)=O)S[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GMMLNKINDDUDCF-JRWRFYLSSA-N 0.000 claims abstract description 11
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- 241001465754 Metazoa Species 0.000 description 2
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 2
- 241000592805 Rahnella inusitata Species 0.000 description 2
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- 238000010367 cloning Methods 0.000 description 2
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Abstract
本发明公开了一种黑芥子酶Rmyr,其氨基酸序列如SEQ ID NO.1所示。编码黑芥子酶Rmyr的基因,其核苷酸序列如SEQ ID NO.2所示。所述黑芥子酶Rmyr在降解硫代葡萄糖苷中的应用和/或在制备异硫氰酸酯中的应用;所述硫代葡萄糖苷选自萝卜硫苷、莱菔苷;所述异硫氰酸酯选自萝卜硫素、莱菔素。本发明还公开了一种降解硫代葡萄糖苷/制备异硫氰酸酯的方法:采用黑芥子酶Rmyr降解硫代葡萄糖苷。本发明还公开了包含黑芥子酶Rmyr基因的重组表达载体和重组工程菌。本发明的黑芥子酶Rmyr属于GH3家族,能够降解硫代葡萄糖苷底物,稳定性高,能被抗坏血酸激活,底物转化率高。本发明为实现萝卜硫素和莱菔素的大批量制备奠定了基础。
Description
技术领域
本发明涉及黑芥子酶Rmyr,其编码基因,以及其在降解硫代葡萄糖苷、制备异硫氰酸酯中的应用,属于功能酶技术领域。
背景技术
萝卜硫素和莱菔素是植物中存在的一种天然的生物活性物质,属于异硫氰酸酯化合物,具有强大的药效,如预防心血管疾病、抑制肥胖、预防糖尿病和阿尔兹海默症等,尤其是广谱的抗癌功效引起了人们的广泛关注,包括乳腺癌、前列腺癌、胃癌、肝癌等。但是萝卜硫素和莱菔素在天然植物中的含量很低,没有办法直接提取,其低含量也抑制了应用。
目前萝卜硫素和莱菔素的制备主要有三种方法:一是利用内源酶,当十字花科植物被破坏时,其自身的黑芥子酶释放出来并催化硫代葡萄糖苷水解制备萝卜硫素和莱菔素,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,步骤繁琐,成本高。三是生物合成的方法,在微生物中实现萝卜硫素和莱菔素的制备,效率很低,且产物的分离纯化复杂。因此,便捷的获得大量的黑芥子酶制剂是实现萝卜硫素和莱菔素大批量生产的关键。
发明内容
针对上述现有技术,本发明提供了一种黑芥子酶Rmyr,其编码基因,以及其在降解硫代葡萄糖苷、制备异硫氰酸酯中的应用。本发明通过挖掘新的黑芥子酶基因,实现其异源表达并获得大量的酶制剂,并利用该酶制剂在体外实现了萝卜硫素和莱菔素的小批量制备,为其大批量生产奠定了基础。
本发明是通过以下技术方案实现的:
一种黑芥子酶Rmyr,其氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1:
MDNTQPELSQREVTLLTVDGLQFKDLNHSGKLEPYEDWRLTPQERAADLVKRMTLEEKAGVMMHGSAPTANSPIGAGTHYDMAAARKMIEGAKVNSLITRLSAEDPAVMAEENNKLQQIAETSRLGIPVTISSDPRNSFEYLIGASTSSGKFTQWPETLGLAAIGNEKVTRRYADIVRQEYLAVGIREALSPQADLATEPRWARISGTFGEDPTRVHHMVRGYVEGMQNGADGLNSGSVISVVKHWVGYGAAENGFDSHNVYGKNAVFPGNNLKEHIYPFTGAFEANVASVMPTYSILKNVSIEGKPLEQAGAGFSHQLLTDILRGQYGFKGVILSDWLITSTCDDVCTHGTPEGKEPVPGGMSWGVENLTPQQRFVKAVKAGVDQFGGVTDSQLLVSAVKEKQLTEERLNESVIRILEQKFQTGLFENPYVDVQKAVQTVGRADWQKEADAAQGHSLVLLQNTGDLLPLKKGQKIWLYGIAPKAAEAAGFTVVDSPEKADVALIRAQTPYEKLHQAWFFGKRHHEGSLEFTGDNADYQAIVNASKHVPTVVTVYLDRPAILSNVKDKAKAIVGNFGVSDAVLFTRLTSGEAFTGKLPFELPSSMEAVLKQQSDMPHDSESPLFDIGFGLARLE。
一种编码上述黑芥子酶Rmyr的基因,其核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2:
5’-ATGGATAACACCCAGCCAGAACTGTCTCAGCGTGAAGTTACTCTGCTGACTGTTGACGGTCTGCAGTTCAAAGACCTGAACCACTCTGGTAAACTGGAACCGTATGAAGATTGGCGTCTGACCCCACAGGAACGTGCTGCTGATCTGGTTAAACGTATGACCCTTGAAGAAAAAGCTGGCGTAATGATGCACGGCTCTGCACCTACCGCTAACTCCCCAATCGGTGCAGGTACCCACTACGATATGGCTGCTGCTCGTAAAATGATTGAAGGTGCTAAAGTTAACTCTCTGATCACCCGTCTGTCTGCAGAAGATCCAGCCGTTATGGCTGAAGAAAACAACAAACTGCAGCAGATCGCTGAAACTTCTCGTCTGGGCATCCCTGTTACCATCTCTTCTGACCCACGTAACTCTTTCGAATACCTGATCGGCGCATCTACTTCTTCTGGTAAATTCACCCAGTGGCCAGAAACCCTGGGTCTGGCAGCTATCGGTAACGAAAAAGTTACCCGTCGTTACGCTGATATCGTTCGTCAGGAATATCTGGCCGTTGGTATCCGTGAAGCACTGTCTCCTCAGGCTGATCTGGCAACTGAACCTCGTTGGGCTCGTATCTCTGGTACATTCGGTGAAGATCCTACGCGTGTTCACCACATGGTTCGTGGTTACGTAGAAGGCATGCAGAACGGTGCTGATGGACTGAACTCCGGTTCTGTGATTTCCGTTGTTAAACACTGGGTTGGTTACGGCGCTGCTGAAAACGGTTTCGATTCTCACAACGTTTACGGTAAAAACGCCGTATTCCCAGGTAACAACCTGAAAGAACACATCTACCCTTTCACCGGTGCATTCGAAGCCAACGTTGCTTCCGTGATGCCTACCTACTCTATTCTGAAAAACGTTTCCATTGAAGGCAAACCTCTGGAACAGGCTGGTGCTGGCTTCTCTCACCAGCTGCTGACTGATATCCTGCGTGGTCAGTACGGTTTCAAAGGTGTAATCCTGTCTGATTGGTTGATCACCTCTACTTGCGATGACGTATGCACCCACGGCACCCCTGAGGGTAAAGAACCAGTTCCGGGTGGCATGTCTTGGGGCGTAGAAAACCTGACCCCTCAGCAGCGTTTCGTTAAAGCAGTGAAAGCTGGTGTGGATCAGTTCGGTGGTGTGACCGATTCTCAGCTGCTGGTTAGCGCAGTTAAAGAAAAACAGCTGACCGAAGAACGCCTGAACGAATCCGTGATTCGTATCCTGGAACAGAAATTCCAGACCGGTCTGTTTGAAAACCCATACGTTGACGTGCAGAAAGCTGTTCAGACCGTTGGCCGTGCTGATTGGCAGAAAGAAGCTGACGCTGCTCAGGGCCACTCCCTGGTTCTGCTGCAGAACACAGGCGATCTGCTGCCACTGAAAAAAGGCCAGAAAATCTGGCTCTACGGCATCGCACCAAAAGCTGCTGAAGCAGCCGGTTTCACCGTGGTTGACTCCCCAGAAAAAGCTGATGTTGCTCTGATCCGTGCTCAGACCCCTTACGAAAAACTGCACCAGGCTTGGTTCTTCGGTAAACGTCACCACGAAGGTTCCCTGGAATTCACCGGCGATAACGCAGACTATCAGGCTATCGTTAACGCCAGCAAACACGTTCCGACCGTGGTTACCGTTTATCTGGACCGTCCAGCTATCCTGTCTAACGTTAAAGATAAAGCAAAAGCTATCGTTGGTAACTTCGGCGTTTCTGACGCAGTTCTGTTCACCCGTCTGACCTCTGGTGAAGCATTCACCGGTAAACTGCCATTCGAACTGCCGTCCTCTATGGAAGCTGTGCTGAAACAGCAGTCTGACATGCCACACGACTCTGAATCTCCACTGTTCGACATCGGTTTCGGTCTGGCTCGTCTCGAG-3’。
所述黑芥子酶Rmyr在降解硫代葡萄糖苷中的应用,在制备异硫氰酸酯中的应用。所述硫代葡萄糖苷选自萝卜硫苷(glucoraphanin)、莱菔苷(glucoraphenin)。所述异硫氰酸酯选自萝卜硫素(sulforaphane)、莱菔素(sulforaphene)。所述萝卜硫苷存在于西兰花种子中。所述莱菔苷存在于萝卜种子中。
一种降解硫代葡萄糖苷/制备异硫氰酸酯的方法;采用上述黑芥子酶Rmyr降解硫代葡萄糖苷。
进一步地,降解条件为:最适温度为40℃,最适pH为7.0。
一种重组表达载体,其携带有上述编码黑芥子酶Rmyr的基因。
一种表达黑芥子酶Rmyr的重组工程菌,其基因组中包含有上述编码黑芥子酶Rmyr的基因或上述重组表达载体,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Rmyr中的应用。
一种酶制剂,包含有上述黑芥子酶Rmyr。
所述酶制剂在降解硫代葡萄糖苷中的应用,在制备异硫氰酸酯中的应用。
本发明的黑芥子酶Rmyr,是一种比较少见的属于糖苷水解酶第3家族(GH3)的黑芥子酶,其在25~45℃下均具有较高的酶活力,可以在较广的温度范围内实现工业应用。此外,通过对其稳定性测试发现,该酶较稳定,在30℃孵育12天酶活仍保留50%;在pH 7、4℃的条件下存放12天酶活仍保留50%以上,是一个比较稳定的酶。该酶能被抗坏血酸激活,在10mM的浓度下酶活提高了7.44倍。另外,通过对其底物催化在体外制备了萝卜硫素和莱菔素,通过增加加酶量最终在10min得到了最高产量,分别是53.92μmol/g种子和28.94μmol/g种子,底物转化率分别达到了92.48%和97.84%,其生产强度(单位时间催化单位种子产生萝卜硫素和莱菔素的量)分别达到了5.39μmol/g·min和2.89μmol/g·min,其中莱菔素的生产强度是目前已知最高的,萝卜硫素的生产强度仅次于Wu Yuanfeng等人实验中的6.42μmol/g·min,是一种比较理想的工业用酶,为实现萝卜硫素和莱菔素的大批量制备奠定了基础。
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为空载的破碎液;2为含有黑芥子酶基因的破碎液;3为20mM咪唑溶液洗脱下来的目的蛋白;4为50mM咪唑溶液洗脱下来的目的蛋白。
图2:温度和pH变化对相对酶活的影响示意图。
图3:金属离子和化学试剂对相对酶活的影响示意图。
图4:抗坏血酸对相对酶活的影响示意图。
图5:本发明的黑芥子酶底物特异性的分析图。
图6:本发明的黑芥子酶制备萝卜硫素和莱菔素的分析图,其中,A、萝卜硫素;B、莱菔素。
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。本领域技术人员能够理解,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黑芥子酶基因Rmyr的克隆
本发明的黑芥子酶基因Rmyr为全基因合成所得,是在NCBI文库中挖掘的(目前黑芥子酶的来源主要是植物、动物还有微生物来源,植物和动物的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Rmyrella Inusitata,序列号为WP_112168067.1。该片段包含了1902个碱基序列,如SEQ ID NO.2所示,编码634个氨基酸序列,如SEQ ID NO.1所示。根据序列比对和进化树分析,发现该黑芥子酶Rmyr属于糖苷水解酶第3家族(GH3)。本发明是首次将该酶表达、纯化并进行相关研究,并发现该酶具有独特的特性和优势。
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Rmyr基因片段。
引物的序列如下所示:
上游引物:5’-ATGGATAACACCCAGCCAGAACTGTCTCAG-3’,如SEQ ID NO.3所示;
下游引物:5’-CTCGAGACGAGCCAGACCGAAACCGAT-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。
实施例2黑芥子酶基因的表达载体构建
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/m L卡那霉素的(LB)培养基固体平板上。37℃温箱中培养12-16h后,挑取单克隆至含有50μg/m L卡那霉素LB液体培养基,转速220rpm的37℃摇床培养过夜,阳性验证后测序,并命名为pET28a-Rmyr。
实施例3黑芥子酶基因的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,构建好的工程菌在硫酸卡那霉素抗性平板上长出。
实施例4利用大肠杆菌工程菌制备重组黑芥子酶
挑取大肠杆菌重组菌株接种在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。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,50mM Tris-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,将得到的溶液进行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69.0KDa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,使用Bradford法测定蛋白浓度,结果为0.16mg/mL。
实施例5黑芥子酶比酶活测定
黑芥子酶Rmyr活性的标准测定方法为:活10μL酶液加入90μL pH 7.0 0.5%(w/v)黑芥子苷,在40℃下反应15min,反应结束后加入300μL的DNS试剂沸水浴5min进行显色,在OD540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)。经测定,纯化后的黑芥子酶活力可达12.73U/mg。
实施例6测定重组黑芥子酶的最适反应条件和稳定性
反应条件为:选取25℃、30℃、35℃、40℃、45℃、50℃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,确定黑芥子酶的最适pH。在30℃、35℃、40℃、45℃的温度下进行温育,于不同时间在最适条件下(温度为40℃,pH为7)测定其残留酶活,得到其温度稳定性。分别与pH 6、pH7、pH 8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2所示,重组黑芥子酶的最适反应温度为40℃,最适pH为7,同时其最适温度测定实验结果显示该重组黑芥子酶在25~45℃酶活均比较高。重组黑芥子酶在30℃下放置12天,酶活仍可保留50%以上;在pH7的缓冲液中保留12天,酶活也能保留50%以上,说明酶活稳定性较好。
实施例7测定金属离子和化学试剂对重组黑芥子酶的活性影响
选择黑芥子苷作为底物,按照1:9的体系将酶和底物混合,并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,置于40℃,pH 7.0的条件下反应30min。反应结束后,用DNS进行酶活的测定。结果如图3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Mn2+不论是低浓度还是高浓度下都可以使酶活提高。
实施例8测定抗坏血酸对重组黑芥子酶的活性影响
选择黑芥子苷作为底物,按照1:9的体系将酶和底物混合,并添加不同浓度的抗坏血酸,置于40℃,pH 7.0的条件下反应30min。反应结束后,用DNS进行酶活的测定。结果如图4所示,在0~10mM的浓度范围内,随着抗坏血酸的浓度增加,黑芥子酶的活力不断增强,最终在10mM的抗坏血酸浓度下黑芥子酶活力提高了7.44倍,说明抗坏血酸对黑芥子酶Rmyr的活力影响巨大。
实施例9测定重组黑芥子酶的底物特异性
选择黑芥子苷、萝卜硫苷和莱菔苷作为底物,按照1:9的体系将酶和底物混合,底物浓度从0开始不断增加,置于40℃,pH 7.0的条件下反应15min。反应结束后,用DNS进行酶活的测定,用Bradford法进行蛋白浓度的测定。每组设置三个平行。实验结束后,用origin软件进行米氏方程曲线的拟合。结果如图5所示,黑芥子酶Rmyr对底物黑芥子苷的催化活性最强,其次是莱菔苷,催化活力最差的是萝卜硫苷。
实施例10利用重组黑芥子酶制备酶制剂
利用实施例4制备的重组黑芥子酶制备酶制剂:发酵破碎后的溶液纯化后,用缓冲液置换咪唑,冻干后保存酶粉。
实施例11测定重组黑芥子酶在制备异硫氰酸酯中的应用
分别以粉碎的西兰花种子和萝卜种子为底物,按照1:10(m/v)的体系溶解于水中,添加不同酶活力的酶制剂在40℃、150rpm的条件下进行产物的制备,分别于5min、10min、15min、20min进行取样,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,之后液相检测产物的产量。结果如图6所示,无论是萝卜硫素还是莱菔素,随着加酶量的不断增加均在10min时达到最大产量,分别为9.56mg/g和5.07mg/g,底物的转化效率分别达到了92.48%和97.84%,生产强度分别达到了5.39μmol/g·min和2.89μmol/g·min。目前的萝卜硫素和莱菔素的制备工艺,底物转化效率不清或者转化效率不够高,如Cai等人的研究中由西兰花制备萝卜硫素的底物转化率只有57%左右,所以黑芥子酶Rmyr在生产时具有一定的优势。
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> 黑芥子酶Rmyr及其在制备萝卜硫素、莱菔素中的应用
<141> 2021-09-09
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 634
<212> PRT
<213> Rahnella Inusitata
<400> 1
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<210> 2
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Claims (10)
1.一种黑芥子酶Rmyr,其特征在于:其氨基酸序列如SEQ ID NO.1所示。
2.一种编码权利要求1所述的黑芥子酶Rmyr的基因,其特征在于:其核苷酸序列如SEQID NO.2所示。
3.权利要求1所述的黑芥子酶Rmyr在降解硫代葡萄糖苷中的应用和/或在制备异硫氰酸酯中的应用;所述硫代葡萄糖苷选自萝卜硫苷、莱菔苷;所述异硫氰酸酯选自萝卜硫素、莱菔素。
4.一种降解硫代葡萄糖苷/制备异硫氰酸酯的方法,其特征在于:采用权利要求1所述的黑芥子酶Rmyr降解硫代葡萄糖苷;所述硫代葡萄糖苷选自萝卜硫苷、莱菔苷;所述异硫氰酸酯选自萝卜硫素、莱菔素。
5.根据权利要求4所述的方法,其特征在于:降解条件为:温度40℃,pH 7.0。
6.一种重组表达载体,其特征在于:所述重组表达载体携带有权利要求2所述的编码黑芥子酶Rmyr的基因。
7.一种表达黑芥子酶Rmyr的重组工程菌,其特征在于:基因组中包含有权利要求2所述的编码黑芥子酶Rmyr的基因或权利要求6所述的重组表达载体。
8.权利要求6所述的重组表达载体、权利要求7所述的重组工程菌在制备黑芥子酶中的应用。
9.一种酶制剂,其特征在于:包含有黑芥子酶Rmyr,黑芥子酶Rmyr的氨基酸序列如SEQID NO.1所示。
10.权利要求9所述的酶制剂在降解硫代葡萄糖苷中的应用和/或在制备异硫氰酸酯中的应用;所述硫代葡萄糖苷选自萝卜硫苷、莱菔苷;所述异硫氰酸酯选自萝卜硫素、莱菔素。
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