CN113151203B - 一种生物催化合成香兰素的单加氧酶的突变体及应用 - Google Patents
一种生物催化合成香兰素的单加氧酶的突变体及应用 Download PDFInfo
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Abstract
本发明涉及一种生物催化合成香兰素的单加氧酶的突变体及应用，所述突变体包括如SEQ ID NO.2所示氨基酸序列，其中第122位，第168位，第182位，第214位，第281位，第299位，第370位，第435位中至少有一个突变。本发明通过定向进化提高酶活力的异丁香酚单加氧酶(ISO)突变体，提高了异丁香酚单加氧酶催化效率。
Description
技术领域
本发明属于生物技术领域，特别涉及一种生物催化合成香兰素的单加氧酶的突变体及应用。
背景技术
香兰素(Vanillin)，又名香草醛，为一种广泛使用的可食用香料，可在香荚兰的种子中找到，也可以人工合成，有浓烈奶香气息。香兰素是重要的食用香料之一，是食用调香剂，具有香荚兰豆香气及浓郁的奶香，是食品添加剂行业中不可缺少的重要原料，广泛运用在各种需要增加奶香气息的调香食品中，如蛋糕、冷饮、巧克力、糖果、饼干、方便面、面包以及烟草、调香酒类、牙膏、肥皂、香水、化妆品、冰淇淋、饮料以及日用化妆品中起增香和定香作用。还可用于香皂、牙膏、橡胶、塑料、医药品。
目前香兰素的合成主要有化学法，微生物转化法和生物催化法。其中化学法合成香兰素的过程中用到高温高压，强酸或强碱以及有毒试剂，对环境产生污染。而微生物转发法的产量比较低，发酵过程产生大量的废液。生物催化法生产香兰素高效环保，反应条件温和。但是生产香兰素的异丁香酚单加氧酶稳定性差，活力低，制约了香兰素的工业化生产。专利
CN106754802A对来自Pseudomonas putida IE27的异丁香酚单加氧酶进行氨基酸突变，努力提高该酶的活力，但是活力提高仅仅2倍。
发明内容
本发明所要解决的技术问题在于，针对现有异丁香酚单加氧酶的稳定性差和活力低的难题，提供一种生物催化合成香兰素的单加氧酶的突变体及应用。
本发明的一种异丁香酚单加氧酶突变体，所述突变体包括如SEQ ID NO.2(异丁香酚单加氧酶氨基酸序列)所示氨基酸序列，其中第122位，第168位，第182位，第214位，第281位，第299位，第370位，第435位中至少有一个突变。
优选地，所述第122位氨基酸的丝氨酸Ser突变为异亮氨酸Ile、亮氨酸Leu或甲硫氨酸Met；或者第168位的脯氨酸突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为谷氨酰胺Gln、亮氨酸Leu、苯丙氨酸Phe、色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为苏氨酸Thr、苯丙氨酸Phe、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第281位的苯丙氨酸Phe突变为甲硫氨酸Met；或者第299位的谷氨酸突变为异亮氨酸Ile或天冬氨酸Asp；或者370位的天冬氨酸Asp突变为苏氨酸Thr、苏氨酸Thr、甘氨酸Gly、丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为缬氨酸Val、谷氨酰胺Gln、精氨酸Arg或甲硫氨酸Met。
进一步优选地，所述第122位氨基酸的丝氨酸Ser突变为异亮氨酸Ile、亮氨酸Leu或甲硫氨酸Met；或者第168位的脯氨酸突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为亮氨酸Leu、苯丙氨酸Phe、色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第281位的苯丙氨酸Phe突变为甲硫氨酸Met；或者第299位的谷氨酸突变为天冬氨酸Asp；或者370位的天冬氨酸Asp突变为甘氨酸Gly、丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为谷氨酰胺Gln、精氨酸Arg或甲硫氨酸Met。
再优选地，所述第122位氨基酸的丝氨酸Ser突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为组氨酸His、或精氨酸Arg；或者第281位的苯丙氨酸Phe突变为甲硫氨酸Met；或者370位的天冬氨酸Asp突变为丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为精氨酸Arg或甲硫氨酸Met。
更优选地，所述第122位氨基酸的丝氨酸Ser突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为精氨酸Arg；或者370位的天冬氨酸Asp突变为半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为甲硫氨酸Met。
最优选地，所述第182位的丝氨酸Ser突变为精氨酸Arg；或者370位的天冬氨酸Asp突变为色氨酸Trp、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met。
本发明的一种所述异丁香酚单加氧酶突变体在生物催化合成香兰素中的应用。
有益效果
本发明中通过对异丁香酚单加氧酶序列进行突变，进过筛选，该系列突变体能够高效催化异丁香酚生成香兰素，其具有比模板高出2-4倍的活力。
附图说明
图1为筛选出的比亲本活力高的蛋白突变体；
图2为异丁香酚单加氧酶与异丁香酚相互作用的关键位点；
图3为异丁香酚分子周围6埃的氨基酸。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例中所用的主要试剂中，所用的限制性内切酶、DNA连接酶、高保真聚合酶购自NEB公司，质粒抽提试剂盒、DNA凝胶回收试剂盒购自上海生工生物工程公司。大肠杆菌表达宿主BL21(DE3)和表达载体pET28a购自Merck公司，其余所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中，未特别说明的各种分子生物学操作，包括PCR条件，DNA酶切条件，DNA连接条件，感受态细胞制备和转化方法，DNA回收纯化等操作，按照购买的酶或试剂盒的说明书操作，或者按照《分子克隆实验指南》操作。
实施例1
野生型异丁香酚单加氧酶ISO基因序列的设计、合成及表达载体的构建：
根据GenBank数据库中Pseudomonas putida异丁香酚单加氧酶的编码序列(GenBank:AB291707)，委托苏州金唯智生物科技有限公司合成基因片段，命名为ISO基因(如SEQ ID NO.1所示)，其对应的氨基酸序列(如SEQ ID NO.2所示)为：
MATFDRNDPQLAGTMFPTRIEANVFDLEIEGEIPRAINGSFFRNTPEPQVTTQPFHTFIDGDGLASAFHFEDGQVDFVSRWVCTPRFEAERSARKSLFGMYRNPFTDDPSVEGIDRTVANTSIITHHGKVLAAKEDGLPYELDPQTLETRGRYDYKGQVTSHTHTAHPKFDPQTGEMLLFGSAAKGERTLDMAYYIVDRYGKVTHETWFKQPYGAFMHDFAVTRNWSIFPIMPATNSLERLKAKQPIYMWEPERGSYIGVLPRRGQGKDIRWFRAPALWVFHVVNAWEEGNRILIDLMESEILPFPFPNSQNLPFDPSKAVPRLTRWEIDLNSGNDEMKRTQLHEYFAEMPIMDFRFALQDHRYAYMGVDDPRRPLAHQQAEKIFAYNSLGVWDNHRKDYELWFTGKMSAAQEPAFVPRSPDAPEGDGYLLSVVGRLDEDRSDLVILDTQCLAAGPVATVKLPFRLRAALHGCWQSKN
为了便于后续克隆，基因片段5'端增加gagctc两个碱基，形成SacI酶切位点，3’端增加ctcgag六个碱基，形成XhoI位点。
将合成的ISO基因和pBBR1MCS-2载体分别用SacI和XhoI进行双酶切，分别用DNA凝胶试剂盒(购自上海生工生物工程公司)回收片段，用T4 DNA连接酶连接后，转化大肠杆菌DH5a，用卡那霉素抗性LB平板筛选，得到克隆子。
挑取10个克隆子，用质粒提取试剂盒(购自上海生工生物工程公司)抽提质粒后，由苏州金唯智生物科技有限公司测序，选择正确插入载体且无突变的克隆子，得到异丁香酚单加氧酶基因表达载体，命名pBBR1MCS-2-iso。
实施例2
ISO野生型酶的表达及制备：
将实施例1中得到的表达载体pBBR1MCS-2-iso转化到大肠杆菌BL21(DE3)中，得到能表达异丁香酚单加氧酶的大肠杆菌基因工程菌pBBR1MCS-2-iso/BL21(DE3)。
将工程菌pBBR1MCS-2-iso/BL21(DE3)按常规方法在摇瓶中发酵培养，当发酵液OD600达到0.6-0.8时，加入终浓度为1mM的异丙基硫代半乳糖苷(IPTG)诱导，降温到20℃继续培养20小时左右，4000转离心20分钟，收集菌体，用50mM pH7.4的PBS缓冲液洗两次，得到含有异丁香酚单加氧酶的菌体，用于后续催化研究和酶活性测定。
实施例3
基因的定点突变：
通过序列比对选择与鞘氨醇单胞菌来源的lignostilbene dioxygenase(LsdA)序列最相近，以该三维结构为模板利用Swiss-model同源建模，利用Discovery Studio软件将异丁香酚与ISO进行分子对接，得到酶与底物相互作用的关键位点(如图2所示)。分析异丁香酚分子周围6埃的氨基酸(如图3所示)。选择了40个氨基酸位点做定点饱和突变，并检测催化活性的变化。
所述的突变位点，其中第13，46，101，102，113,117，121,122,134，135,143，167,168，181,182，214,216，219,232，235，248，257，274，279，281,299，301,303，304,305,306,307,308，349,350，351,370，387,435位，指的是将原位点的氨基酸分别突变为其他19种氨基酸；
根据选择的位点设计简并引物引物如下:
N＝A,T,C,G；K＝G,T；M＝A,C(A,T,C,G为核苷酸碱基)
PCR扩增条件为:95℃30s,95℃15s,60℃30s,68℃5min,30个循环，68℃10min。
PCR结束后，将40个PCR产物分别吸取10uL混合后加入10uL限制性内切酶DpnI除掉模板质粒，DNA凝胶回收试剂盒回收后，电击转化大肠杆菌BL21(DE3)感受态细胞，在卡那霉素抗性平板上涂平板，37℃培养过夜。第二天使用灭菌牙签将单菌落挑取到装有灭菌LB培养基(卡那霉素：50ug/mL)的96浅孔板中，其中D4-D8为野生型异丁香酚单加氧酶，共20块96孔板。37℃，400rpm培养16h后，使用96孔移液器加入至终浓度10％的灭菌甘油，吹打均匀后-80℃待用。
实施例4
ISO单点突变体的转化率测定:
将实施例4获得的文库菌种转接至装有灭菌TB培养基(卡那霉素：50ug/mL)的96深孔板中37℃培养4h，待OD＝0.6-0.8时，加入IPTG至终浓度0.1mM，培养温度调整到20℃，继续培养24h。深孔板4℃4000rpm离心20min，倒掉上清培养基，获得含有异丁香酚单加氧酶突变体的细胞。
配制反应液：在每一个孔中加入200uL甘氨酸/NaOH(pH＝10)缓冲液，异丁香酚：4mg DMSO：12uL，30℃反应过夜。反应结束后，使用96孔移液工作站每孔加入1mL无水乙醇，混匀后，4000rpm室温离心20分钟。将上清转移至96孔浅孔板使用HPLC进行样品分析。
HPLC检测条件：Lichrospher 100RP-18色谱柱(250mm*4mm*5um)，检测波长280nm，检测温度30℃，检测流速1ml/min，流动相A为含有13:7的甲醇；流动相B为0.01％(v/v醋酸水溶液)；流动相B为A:甲醇＝70：30。其中香兰素和异丁香酚的出峰时间为3min和9min。
根据测定得到的香兰素峰面积计算转化率，转化率计算方式如下：
Con＝Avan/(Avan+Aiso)*100％，其中Avan为香兰素的峰面积，Aiso为异丁香酚的峰面积
选取转化率高于亲本WT的突变点并进行测序，结果如图1所示。
表1为比亲本活力高的蛋白突变体
实施例5
表达异丁香酚单加氧酶突变体ISO(D370R或M298K)的重组菌的诱导表达:
将表达异丁香酚单加氧酶突变体ISO(D370R)的重组菌和表达异丁香酚单加氧酶的重组菌分别以1％的比例接种于LB培养基(含有蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，卡那霉素5ug/mL),220rpm,37℃培养至OD＝0.6-0.8后，加入IPTG至终浓度0.1mM，20℃继续培养24h，6000rpm离心后收集诱导表达菌体，用pH 7.0的PBS缓冲液清洗两次，收集ISO(D370R)重组菌体和ISO重组菌体。
实施例6
表达ISO(WT,D370R或M298K)的重组菌的功能验证
反应底物溶液组分：异丁香酚40mg，2mL甘氨酸/NaOH缓冲液pH10.0，120uL DMSO.向上述底物溶液中分别加入ISO(D370R)重组菌体，ISO重组菌体和ISO(或M298K)，且每种菌体的浓度均为20mg/mL；250rpm,30℃催化反应18h，得到反应产物。吸取100uL反应液，再加入1mL无水乙醇，颠倒混匀后12000rpm离心2min’，上清经过0.22um过滤膜过滤后进行HPLC分析。
结果如下：
根据测定得到的香兰素峰面积计算转化率，转化率计算方式如下：
Con＝Avan/(Avan+Aiso)*100％，其中Avan为香兰素的峰面积，Aiso为异丁香酚的峰面积
ISO(D370R)催化异丁香酚单加氧酶生成香兰素的转化率达到82.17％；ISO催化异丁香酚单加氧酶生成香兰素的转化率达到18.4％；ISO(M298K，来自于CN106754802A)催化异丁香酚单加氧酶生成香兰素的转化率达到33.75％，上述结果表明ISO(D370R)的催化活性是ISO的4.46倍，是ISO(M298K)催化活性的2.43倍。
SEQ ID NO.1:
TCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGGCTACCTTCGACCGTAACGACCCGCAGCTGGCTGGTACCATGTTCCCGACCCGTATCGAAGCTAACGTTTTCGACCTGGAAATCGAAGGTGAAATCCCGCGTGCTATCAACGGTTCTTTCTTCCGTAACACCCCGGAACCGCAGGTTACCACCCAGCCGTTCCACACCTTCATCGACGGTGACGGTCTGGCTTCTGCTTTCCACTTCGAAGACGGTCAGGTTGACTTCGTTTCTCGTTGGGTTTGCACCCCGCGTTTCGAAGCTGAACGTTCTGCTCGTAAATCTCTGTTCGGTATGTACCGTAACCCGTTCACCGACGACCCGTCTGTTGAAGGTATCGACCGTACCGTTGCTAACACCTCTATCATCACCCACCACGGTAAAGTTCTGGCTGCTAAAGAAGACGGTCTGCCGTACGAACTGGACCCGCAGACCCTGGAAACCCGTGGTCGTTACGACTACAAAGGTCAGGTTACCTCTCACACCCACACCGCTCACCCGAAATTCGACCCGCAGACCGGTGAAATGCTGCTGTTCGGTTCTGCTGCTAAAGGTGAACGTACCCTGGACATGGCTTACTACATCGTTGACCGTTACGGTAAAGTTACCCACGAAACCTGGTTCAAACAGCCGTACGGTGCTTTCATGCACGACTTCGCTGTTACCCGTAACTGGTCTATCTTCCCGATCATGCCGGCTACCAACTCTCTGGAACGTCTGAAAGCTAAACAGCCGATCTACATGTGGGAACCGGAACGTGGTTCTTACATCGGTGTTCTGCCGCGTCGTGGTCAGGGTAAAGACATCCGTTGGTTCCGTGCTCCGGCTCTGTGGGTTTTCCACGTTGTTAACGCTTGGGAAGAAGGTAACCGTATCCTGATCGACCTGATGGAATCTGAAATCCTGCCGTTCCCGTTCCCGAACTCTCAGAACCTGCCGTTCGACCCGTCTAAAGCTGTTCCGCGTCTGACCCGTTGGGAAATCGACCTGAACTCTGGTAACGACGAAATGAAACGTACCCAGCTGCACGAATACTTCGCTGAAATGCCGATCATGGACTTCCGTTTCGCTCTGCAGGACCACCGTTACGCTTACATGGGTGTTGACGACCCGCGTCGTCCGCTGGCTCACCAGCAGGCTGAAAAAATCTTCGCTTACAACTCTCTGGGTGTTTGGGACAACCACCGTAAAGACTACGAACTGTGGTTCACCGGTAAAATGTCTGCTGCTCAGGAACCGGCTTTCGTTCCGCGTTCTCCGGACGCTCCGGAAGGTGACGGTTACCTGCTGTCTGTTGTTGGTCGTCTGGACGAAGACCGTTCTGACCTGGTTATCCTGGACACCCAGTGCCTGGCTGCTGGTCCGGTTGCTACCGTTAAACTGCCGTTCCGTCTGCGTGCTGCTCTGCACGGTTGCTGGCAGTCTAAAAACTAA；
SEQ ID NO.2：(异丁香酚单加氧酶氨基酸序列)
MATFDRNDPQLAGTMFPTRIEANVFDLEIEGEIPRAINGSFFRNTPEPQVTTQPFHTFIDGDGLASAFHFEDGQVDFVSRWVCTPRFEAERSARKSLFGMYRNPFTDDPSVEGIDRTVANTSIITHHGKVLAAKEDGLPYELDPQTLETRGRYDYKGQVTSHTHTAHPKFDPQTGEMLLFGSAAKGERTLDMAYYIVDRYGKVTHETWFKQPYGAFMHDFAVTRNWSIFPIMPATNSLERLKAKQPIYMWEPERGSYIGVLPRRGQGKDIRWFRAPALWVFHVVNAWEEGNRILIDLMESEILPFPFPNSQNLPFDPSKAVPRLTRWEIDLNSGNDEMKRTQLHEYFAEMPIMDFRFALQDHRYAYMGVDDPRRPLAHQQAEKIFAYNSLGVWDNHRKDYELWFTGKMSAAQEPAFVPRSPDAPEGDGYLLSVVGRLDEDRSDLVILDTQCLAAGPVATVKLPFRLRAALHGCWQSKN*
序列表
<110> 上海必诺检测技术服务有限公司
2, 1
<120> 一种生物催化合成香兰素的单加氧酶的突变体及应用
<130> 1
<160> 78
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1600
<212> DNA
<213> 人工序列()
<400> 1
tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg 60
gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt 120
ggaattgtga gcggataaca atttcacaca ggaaacagta ttcatggcta ccttcgaccg 180
taacgacccg cagctggctg gtaccatgtt cccgacccgt atcgaagcta acgttttcga 240
cctggaaatc gaaggtgaaa tcccgcgtgc tatcaacggt tctttcttcc gtaacacccc 300
ggaaccgcag gttaccaccc agccgttcca caccttcatc gacggtgacg gtctggcttc 360
tgctttccac ttcgaagacg gtcaggttga cttcgtttct cgttgggttt gcaccccgcg 420
tttcgaagct gaacgttctg ctcgtaaatc tctgttcggt atgtaccgta acccgttcac 480
cgacgacccg tctgttgaag gtatcgaccg taccgttgct aacacctcta tcatcaccca 540
ccacggtaaa gttctggctg ctaaagaaga cggtctgccg tacgaactgg acccgcagac 600
cctggaaacc cgtggtcgtt acgactacaa aggtcaggtt acctctcaca cccacaccgc 660
tcacccgaaa ttcgacccgc agaccggtga aatgctgctg ttcggttctg ctgctaaagg 720
tgaacgtacc ctggacatgg cttactacat cgttgaccgt tacggtaaag ttacccacga 780
aacctggttc aaacagccgt acggtgcttt catgcacgac ttcgctgtta cccgtaactg 840
gtctatcttc ccgatcatgc cggctaccaa ctctctggaa cgtctgaaag ctaaacagcc 900
gatctacatg tgggaaccgg aacgtggttc ttacatcggt gttctgccgc gtcgtggtca 960
gggtaaagac atccgttggt tccgtgctcc ggctctgtgg gttttccacg ttgttaacgc 1020
ttgggaagaa ggtaaccgta tcctgatcga cctgatggaa tctgaaatcc tgccgttccc 1080
gttcccgaac tctcagaacc tgccgttcga cccgtctaaa gctgttccgc gtctgacccg 1140
ttgggaaatc gacctgaact ctggtaacga cgaaatgaaa cgtacccagc tgcacgaata 1200
cttcgctgaa atgccgatca tggacttccg tttcgctctg caggaccacc gttacgctta 1260
catgggtgtt gacgacccgc gtcgtccgct ggctcaccag caggctgaaa aaatcttcgc 1320
ttacaactct ctgggtgttt gggacaacca ccgtaaagac tacgaactgt ggttcaccgg 1380
taaaatgtct gctgctcagg aaccggcttt cgttccgcgt tctccggacg ctccggaagg 1440
tgacggttac ctgctgtctg ttgttggtcg tctggacgaa gaccgttctg acctggttat 1500
cctggacacc cagtgcctgg ctgctggtcc ggttgctacc gttaaactgc cgttccgtct 1560
gcgtgctgct ctgcacggtt gctggcagtc taaaaactaa 1600
<210> 2
<211> 478
<212> PRT
<213> 人工序列()
<400> 2
Met Ala Thr Phe Asp Arg Asn Asp Pro Gln Leu Ala Gly Thr Met Phe
1 5 10 15
Pro Thr Arg Ile Glu Ala Asn Val Phe Asp Leu Glu Ile Glu Gly Glu
20 25 30
Ile Pro Arg Ala Ile Asn Gly Ser Phe Phe Arg Asn Thr Pro Glu Pro
35 40 45
Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile Asp Gly Asp Gly Leu
50 55 60
Ala Ser Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser Arg
65 70 75 80
Trp Val Cys Thr Pro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser
85 90 95
Leu Phe Gly Met Tyr Arg Asn Pro Phe Thr Asp Asp Pro Ser Val Glu
100 105 110
Gly Ile Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Thr His His Gly
115 120 125
Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro
130 135 140
Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln Val Thr
145 150 155 160
Ser His Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu
165 170 175
Met Leu Leu Phe Gly Ser Ala Ala Lys Gly Glu Arg Thr Leu Asp Met
180 185 190
Ala Tyr Tyr Ile Val Asp Arg Tyr Gly Lys Val Thr His Glu Thr Trp
195 200 205
Phe Lys Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Val Thr Arg
210 215 220
Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Glu Arg
225 230 235 240
Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Arg Gly Ser
245 250 255
Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Lys Asp Ile Arg Trp
260 265 270
Phe Arg Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu
275 280 285
Glu Gly Asn Arg Ile Leu Ile Asp Leu Met Glu Ser Glu Ile Leu Pro
290 295 300
Phe Pro Phe Pro Asn Ser Gln Asn Leu Pro Phe Asp Pro Ser Lys Ala
305 310 315 320
Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asn Ser Gly Asn Asp
325 330 335
Glu Met Lys Arg Thr Gln Leu His Glu Tyr Phe Ala Glu Met Pro Ile
340 345 350
Met Asp Phe Arg Phe Ala Leu Gln Asp His Arg Tyr Ala Tyr Met Gly
355 360 365
Val Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala Glu Lys Ile
370 375 380
Phe Ala Tyr Asn Ser Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr
385 390 395 400
Glu Leu Trp Phe Thr Gly Lys Met Ser Ala Ala Gln Glu Pro Ala Phe
405 410 415
Val Pro Arg Ser Pro Asp Ala Pro Glu Gly Asp Gly Tyr Leu Leu Ser
420 425 430
Val Val Gly Arg Leu Asp Glu Asp Arg Ser Asp Leu Val Ile Leu Asp
435 440 445
Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys Leu Pro Phe
450 455 460
Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn
465 470 475
<210> 3
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 3
gtaacgaccc gcagctggct nnkaccatgt tcccgacccg tatc 44
<210> 4
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 4
gatacgggtc gggaacatgg tmnnagccag ctgcgggtcg ttac 44
<210> 5
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 5
gttctttctt ccgtaacnnk ccggaaccgc aggttaccac 40
<210> 6
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 6
gtggtaacct gcggttcmnn ggtgttacgg aagaaagaac 40
<210> 7
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 7
taaatctctg ttcggtatgn nkcgtaaccc gttcaccgac 40
<210> 8
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 8
gtcggtgaac gggttacgmn ncataccgaa cagagattta 40
<210> 9
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 9
atctctgttc ggtatgtacn nkaacccgtt caccgacgac ccg 43
<210> 10
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 10
cgggtcgtcg gtgaacgggt tmnngtacat accgaacaga gat 43
<210> 11
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 11
gacgacccgt ctgttgaann katcgaccgt accgttgcta ac 42
<210> 12
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 12
gttagcaacg gtacggtcga tmnnttcaac agacgggtcg tc 42
<210> 13
<211> 38
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 13
ttgaaggtat cgaccgtnnk gttgctaaca cctctatc 38
<210> 14
<211> 38
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 14
gatagaggtg ttagcaacmn nacggtcgat accttcaa 38
<210> 15
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 15
ccgtaccgtt gctaacnnkt ctatcatcac ccaccacgg 39
<210> 16
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 16
ccgtggtggg tgatgataga mnngttagca acggtacgg 39
<210> 17
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 17
ccgtaccgtt gctaacaccn nkatcatcac ccaccacggt aaag 44
<210> 18
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<400> 18
ctttaccgtg gtgggtgatg atmnnggtgt tagcaacggt acgg 44
<210> 19
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 19
cggtaaagtt ctggctgctn nkgaagacgg tctgccgtac g 41
<210> 20
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 20
cgtacggcag accgtcttcm nnagcagcca gaactttacc g 41
<210> 21
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 21
gtaaagttct ggctgctaaa nnkgacggtc tgccgtacga ac 42
<210> 22
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 22
gttcgtacgg cagaccgtcm nntttagcag ccagaacttt ac 42
<210> 23
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 23
tctcacaccc acaccgctnn kccgaaattc gacccgcag 39
<210> 24
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 24
ctgcgggtcg aatttcggmn nagcggtgtg ggtgtgaga 39
<210> 25
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 25
cacacccaca ccgctcacnn kaaattcgac ccgcagaccg g 41
<210> 26
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 26
ccggtctgcg ggtcgaattt mnngtgagcg gtgtgggtgt g 41
<210> 27
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 27
cggtgaaatg ctgctgttcn nktctgctgc taaaggtgaa cg 42
<210> 28
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 28
cgttcacctt tagcagcaga mnngaacagc agcatttcac cg 42
<210> 29
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 29
gaaatgctgc tgttcggtnn kgctgctaaa ggtgaacgta c 41
<210> 30
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 30
gtacgttcac ctttagcagc mnnaccgaac agcagcattt c 41
<210> 31
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 31
cctggttcaa acagccgtac nnkgctttca tgcacgactt c 41
<210> 32
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 32
gaagtcgtgc atgaaagcmn ngtacggctg tttgaaccag g 41
<210> 33
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 33
aaacagccgt acggtgctnn katgcacgac ttcgctgtta c 41
<210> 34
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 34
gtaacagcga agtcgtgcat mnnagcaccg tacggctgtt t 41
<210> 35
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 35
gtacggtgct ttcatgcacn nkttcgctgt tacccgtaac tgg 43
<210> 36
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 36
ccagttacgg gtaacagcga amnngtgcat gaaagcaccg tac 43
<210> 37
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 37
ctggtctatc ttcccgatcn nkccggctac caactctctg gaac 44
<210> 38
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<400> 38
gttccagaga gttggtagcc ggmnngatcg ggaagataga ccag 44
<210> 39
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 39
cttcccgatc atgccggctn nkaactctct ggaacgtctg aaag 44
<210> 40
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<400> 40
ctttcagacg ttccagagag ttmnnagccg gcatgatcgg gaag 44
<210> 41
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 41
ctgaaagcta aacagccgat cnnkatgtgg gaaccggaac gtg 43
<210> 42
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 42
cacgttccgg ttcccacatm nngatcggct gtttagcttt cag 43
<210> 43
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 43
gggaaccgga acgtggttct nnkatcggtg ttctgccgcg tc 42
<210> 44
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 44
gacgcggcag aacaccgatm nnagaaccac gttccggttc cc 42
<210> 45
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 45
taaagacatc cgttggttcn nkgctccggc tctgtgggtt ttc 43
<210> 46
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 46
gaaaacccac agagccggag cmnngaacca acggatgtct tta 43
<210> 47
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 47
gttccgtgct ccggctctgn nkgttttcca cgttgttaac gc 42
<210> 48
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 48
gcgttaacaa cgtggaaaac mnncagagcc ggagcacgga ac 42
<210> 49
<211> 45
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 49
gtgctccggc tctgtgggtt nnkcacgttg ttaacgcttg ggaag 45
<210> 50
<211> 45
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (24)..(25)
<223> n is a, c, g, or t
<400> 50
cttcccaagc gttaacaacg tgmnnaaccc acagagccgg agcac 45
<210> 51
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 51
gtatcctgat cgacctgatg nnktctgaaa tcctgccgtt cccg 44
<210> 52
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 52
cgggaacggc aggatttcag amnncatcag gtcgatcagg atac 44
<210> 53
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 53
gatcgacctg atggaatctn nkatcctgcc gttcccgttc c 41
<210> 54
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 54
ggaacgggaa cggcaggatm nnagattcca tcaggtcgat c 41
<210> 55
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 55
cctgatggaa tctgaaatcn nkccgttccc gttcccgaac tc 42
<210> 56
<211> 42
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 56
gagttcggga acgggaacgg mnngatttca gattccatca gg 42
<210> 57
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 57
gatggaatct gaaatcctgn nkttcccgtt cccgaactct c 41
<210> 58
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 58
gagagttcgg gaacgggaam nncaggattt cagattccat c 41
<210> 59
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 59
tggaatctga aatcctgccg nnkccgttcc cgaactctca gaac 44
<210> 60
<211> 44
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 60
gttctgagag ttcgggaacg gmnncggcag gatttcagat tcca 44
<210> 61
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 61
ctgaaatcct gccgttcnnk ttcccgaact ctcagaacc 39
<210> 62
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 62
ggttctgaga gttcgggaam nngaacggca ggatttcag 39
<210> 63
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 63
ctgaaatcct gccgttcccg nnkccgaact ctcagaacct g 41
<210> 64
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 64
caggttctga gagttcggmn ncgggaacgg caggatttca g 41
<210> 65
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 65
atcctgccgt tcccgttcnn kaactctcag aacctgccg 39
<210> 66
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 66
cggcaggttc tgagagttmn ngaacgggaa cggcaggat 39
<210> 67
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 67
ctgcacgaat acttcgctnn katgccgatc atggacttcc g 41
<210> 68
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
<400> 68
cggaagtcca tgatcggcat mnnagcgaag tattcgtgca g 41
<210> 69
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 69
cacgaatact tcgctgaann kccgatcatg gacttccgtt 40
<210> 70
<211> 40
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (21)..(22)
<223> n is a, c, g, or t
<400> 70
aacggaagtc catgatcggm nnttcagcga agtattcgtg 40
<210> 71
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 71
gaatacttcg ctgaaatgnn katcatggac ttccgtttc 39
<210> 72
<211> 39
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 72
gaaacggaag tccatgatmn ncatttcagc gaagtattc 39
<210> 73
<211> 38
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 73
acgcttacat gggtgttnnk gacccgcgtc gtccgctg 38
<210> 74
<211> 38
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 74
cagcggacga cgcgggtcmn naacacccat gtaagcgt 38
<210> 75
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<400> 75
ggctgaaaaa atcttcgctn nkaactctct gggtgtttgg gac 43
<210> 76
<211> 43
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (23)..(24)
<223> n is a, c, g, or t
<400> 76
gtcccaaaca cccagagagt tmnnagcgaa gattttttca gcc 43
<210> 77
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<400> 77
tacctgctgt ctgttgttnn kcgtctggac gaagaccgtt c 41
<210> 78
<211> 41
<212> DNA
<213> 人工序列()
<220>
<221> misc_feature
<222> (22)..(23)
<223> n is a, c, g, or t
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gaacggtctt cgtccagacg mnnaacaaca gacagcaggt a 41
Claims (6)
1.一种异丁香酚单加氧酶突变体，其特征在于，所述突变体具有如SEQ ID NO.2所示氨基酸序列，并且具有选择如SEQ ID NO.2所示氨基酸序列的第122位，第168位，第182位，第214位，第281位，第299位，第370位，第435位中的一个突变；
所述第122位氨基酸的丝氨酸Ser突变为异亮氨酸Ile、亮氨酸Leu或甲硫氨酸Met；或者第168位的脯氨酸突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为谷氨酰胺Gln、亮氨酸Leu、苯丙氨酸Phe、色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为苏氨酸Thr、苯丙氨酸Phe、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第281位的苯丙氨酸Phe突变为甲硫氨酸Met；或者第299位的谷氨酸突变为异亮氨酸Ile或天冬氨酸Asp；或者370位的天冬氨酸Asp突变为苏氨酸Thr、甘氨酸Gly、丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr、甲硫氨酸Met或精氨酸Arg；或者第435位的甘氨酸Gly突变为缬氨酸Val、谷氨酰胺Gln、精氨酸Arg或甲硫氨酸Met。
2.根据权利要求1所示突变体，其特征在于，所述第182位的丝氨酸Ser突变为亮氨酸Leu、苯丙氨酸Phe、色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第299位的谷氨酸突变为天冬氨酸Asp；或者370位的天冬氨酸Asp突变为甘氨酸Gly、丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为谷氨酰胺Gln、精氨酸Arg或甲硫氨酸Met。
3.根据权利要求2所述突变体，其特征在于，第122位氨基酸的丝氨酸Ser突变为甲硫氨酸Met；或者第182位的丝氨酸Ser突变为色氨酸Trp、组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为组氨酸His、或精氨酸Arg；或者370位的天冬氨酸Asp突变为丝氨酸Ser、丙氨酸Ala、赖氨酸Lys、天冬氨酸Asp、半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为精氨酸Arg或甲硫氨酸Met。
4.根据权利要求3所述突变体，其特征在于，所述第182位的丝氨酸Ser突变为组氨酸His、甲硫氨酸Met或精氨酸Arg；或者第214位的甘氨酸Gly突变为精氨酸Arg；或者370位的天冬氨酸Asp突变为半胱氨酸Cys、色氨酸Trp、谷氨酰胺Gln、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met；或者第435位的甘氨酸Gly突变为甲硫氨酸Met。
5.根据权利要求4所述突变体，其特征在于，所述第182位的丝氨酸Ser突变为精氨酸Arg；或者370位的天冬氨酸Asp突变为色氨酸Trp、组氨酸His、苯丙氨酸Phe、亮氨酸Leu、酪氨酸Tyr或甲硫氨酸Met。
6.一种权利要求1所述异丁香酚单加氧酶突变体在生物催化异丁香酚合成香兰素中的应用。
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