CN102220299A - 一种阿魏酸酯酶a突变体及其用途 - Google Patents
一种阿魏酸酯酶a突变体及其用途 Download PDFInfo
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Abstract
本发明属于基因工程和酶工程技术领域，具体涉及一种阿魏酸酯酶A突变体及其用途。本发明利用易错PCR对阿魏酸酯酶A母本基因随机突变，构建随机突变文库，通过高通量筛选体系筛选出12个耐热性提高的阿魏酸酯酶A突变体位点，并通过全序列合成方法将这12个突变位点整合到一条编码序列上，获得组合突变体。该组合突变体比母本的热稳定性有极大提高，显示出在生物能源、饲料、造纸、保健食品，医药等工业中潜在的应用价值。
Description
技术领域
本发明属于基因工程和酶工程技术领域，具体涉及一种阿魏酸酯酶A突变体及其用途。
背景技术
阿魏酸酯酶(EC 3.1.1.73)是能水解存在于植物细胞壁中的羟基肉桂酸类化合物、阿拉伯木聚糖与某些胶质之间的酯键，是半纤维素完全降解所需的关键酶之一。其中来源于黑曲霉的阿魏酸酯酶A是目前研究最多的阿魏酸酯酶之一，人们对其在底物作用范围，内源表达调控以及在毕赤酵母和大肠杆菌中表达方面作了大量研究。阿魏酸酯酶A在生物能源、饲料、造纸、保健食品、医药等行业具有较大应用潜力。在酶的工业应用中，热稳定性是一个重要的参数。高稳定性的酶高温下反应可以提高反应速率和反应物的溶解量，减少酶投入量等，从而降低成本(L.Viikari，et al.Thermostable Enzymes in Lignocellulose Hydrolysis.Adv Biochem Eng Biotechnol 108(2007)121-145)。蛋白质定向进化技术是体外改良蛋白质性质的重要手段，过去十年间人们成功地利用该技术改造了上百种蛋白质的性质(J.D.Bloom，F.H.Arnold.In the light of directed evolution：Pathways of adaptive protein evolution.PNAS 106(2009)9995-10000)。同时，理性设计手段也广泛应用于酶的改造中(Vincent G.H.Eiisink，et al.Rational engineering of enzyme stability.J Biotech 113(2004)105-120)。目前生产上应用的阿魏酸酯酶主要来源于黑曲霉的阿魏酸酯酶A，但是其热稳定性仍然不理想。应用现有技术，进一步改造阿魏酸酯酶A，提高其热稳定性，将推动该酶在相关领域的更广泛应用。
发明内容
本发明利用易错PCR技术对来源于黑曲霉(Aspergillus niger)的阿魏酸酯酶A进行分子改良，从而获得热稳定性提高的阿魏酸酯酶A突变酶。
为了达到以上目的，在前期已获得的母本基础上，经一轮易错PCR对母本基因随机突变，建立突变文库，通过高通量筛选体系筛选出12个耐热性提高的阿魏酸酯酶A突变体位点，并通过全序列合成方法将这12个突变位点整合到一条编码序列上，获得组合突变体(命名为：CV-FAEA)，其热稳定性得到很大提高。
本发明通过如下方式来实现：
随机突变文库的构建和筛选：我们已报道了黑曲霉(Aspergillus niger CIB423.1)中克隆的编码260个氨基酸残基的阿魏酸酯酶A编码序列(EMBL accession number：FJ430154)，并利用PoPMuSiC在线工具对其进行计算机辅助设计获得了耐热性提高了的阿魏酸酯酶A，暂命名为FAEA-D93G/S187F(见SEQ ID NO.1)，该突变体作为下述基因操作的母本(S.-B.Zhang，and Z.-L.Wu， Identification of amino acid residues responsible for increased thermostability of feruloyl esterase
采取易错PCR方法对编码母本FAEA-D93G/S187F的基因进行随机突变，以pPIC9K载体构建高效突变库，再将含有突变基因的重组质粒转入毕赤酵母(Pichia pastoris KM71)中，挑取突变子单克隆以及母本对照菌株于96孔板中培养并诱导阿魏酸酯酶A表达。将酶表达后的菌液离心，取部分上清液以2-氯对硝基苯酚为底物，反应适当时间测定酶活。同时，另取相同体积的上清液热处理一定时间，以相同的方法测定残余酶活性。然后，将残余活性高于对照的菌株转接到新的96孔培养板中进行重复筛选。培养耐热性提高的突变酶重组菌，提取其基因组，并用PCR技术获得其相应的突变阿魏酸酯酶A的基因，送上海英骏生物技术公司测序。详细方案见实施例2。
经上述随机突变库构建和筛选，获得了12个由单个氨基酸残基的突变引起耐热性有不同程度提高的阿魏酸酯酶A突变体，分别将其命名为：Z1、Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12，序列信息见SEQ ID NO.3-NO.13，其特征如下：
Z1：该酶的第177位的谷氨酰胺突变为丝氨酸；DNA序列由CAG变为CAT。
Z2：该酶的第63位的苏氨酸突变为异亮氨酸；DNA序列由ACC变为ATC。
Z3：该酶的第140位的丙氨酸突变为苏氨酸；DNA序列由GCA突变为ACA。
Z4：该酶的第235位由半胱氨酸突变为丝氨酸；DNA序列由TGT突变为AGT。
Z5：该酶的第57位由苏氨酸突变为异亮氨酸；DNA序列由ACC突变为ATC。
Z6：该酶的第178位由缬氨酸突变为丙氨酸；DNA序列由GTC突变为GCC。
Z7：该酶的第14位由亮氨酸突变为苯丙氨酸；DNA序列由TTA突变为TTC。
Z8：该酶的第163位由丝氨酸突变为苏氨酸；DNA序列由AGC突变为ACC。
Z9：该酶的第37位由赖氨酸突变为异亮氨酸；DNA序列由AAA突变为ATA。
Z10：该酶的第121位由谷氨酰胺突变为组氨酸；DNA序列由CAG突变为CAC。
Z11：该酶的第185位由谷氨酰胺突变为精氨酸；DNA序列由CAG突变为CGG。
Z12：该酶的第35位由苏氨酸突变为异亮氨酸；DNA序列由ACT突变为ATT。
12个位点的整合：通过全序列基因合成整合以上12个突变位点，将合成的基因片段连入pGAPZαA载体并在毕赤酵母(Pichia pastoris KM71)中表达(组合突变体阿魏酸酯酶A，命名为CV-FAEA，氨基酸序列见SEQ ID NO.14)。母本和CV-FAEA纯化后做耐热性比较发现，与母本相比，整合了12个突变位点的突变体CV-FAEA的热耐受性有了极大提高。如母本在55℃处理15分钟时，其酶活性就丧失了一半，而CV-FAEA在55℃下处理4000分钟后仍保持90％以上的酶活性。需要指出的是，C235S突变位点(Z4)在50-90℃温度下处理一个小时能保持50％以上残余活力，其对组合突变体CV-FAEA耐热性提高有很大贡献。阿魏酸酯酶A突变体耐热性的显著提高显示了该酶在生物能源、饲料、造纸、保健食品、医药等行业潜在的应用价值。
附图说明
图1为母本阿魏酸酯酶A和组合突变体CV-FAEA的耐热性比较。其中，
●为母本阿魏酸酯酶A的酶活变化曲线；
为组合突变型阿魏酸酯酶A的酶活变化曲线。T为温度，Y为残余酶活力。
具体实施方式
以下结合实施实例对本发明做进一步说明，需要指出的是，本实施例仅用于解释本发明，而非对本发明范围的限制。
实施例1易错PCR(error-prone PCR)方法构建阿魏酸酯酶文库
利用
II Random Mutagenesis kit对阿魏酸酯酶基因(见SEQ ID NO.1)进行随机突变。
所用引物为：faeA-F：5’-taggaggt gaattc gcctccacgcaaggcatctc-3’
faeA-R：5’-taggaggt gcggccgc ttaccaagtacaagctccgctcg-3’
反应条件为：94℃预变性5min，94℃变性30s，61℃退火1min和72℃延伸1min，共25个循环，电泳后回收基因片段。
将回收片段用NotI和EcoRI双酶切，与经过相同酶切的pGAPZαA载体(含有博莱霉素抗性基因)进行连接反应。双酶切消化后的pGAPZαA载体和目的基因片段的摩尔量比1∶3混合后，加入600个单位的T4连接酶，16℃连接过夜。然后将连接产物用酚/氯仿抽提两次，离心，浓缩连接产物并用ddH2O溶解后，电击法转入大肠杆菌DH5α，得到约5×104个克隆的一级突变库。收集一级突变库平板上的菌落培养2-3小时后提取质粒，用NotI和EcoRI双酶切回收的目的片段，与经过相同酶切的pPIC9K载体(含有氨苄青霉素抗性基因)进行连接反应，连接产物浓缩、转化等操作均和一级突变库构建方法一致。最后得到约5×104个克隆的二级突变库。
实施例2阿魏酸酯酶突变体库的筛选
将实施例1中二级突变库克隆收集后提取质粒并经PmeI内切酶线性化后，电击转化毕赤酵母(Pichia pastoris KM71)感受态细胞并涂布MD平板(1.34％YNB，4×10-5％生物素，2％葡萄糖)。培养2天后，挑取单克隆于96孔板，每孔含有150μl BMGY培养基(含有100mM、pH值6.0的磷酸钾缓冲液，2％胰蛋白胨，1％酵母提取物，1％甘油，4x 10-5％生物素)，30℃，280rpm，震荡培养2天。用96孔板复制器复制各单克隆于MD固体培养基平板，30℃培养2天后存放于4℃冰箱中。离心96孔板培养物后移出上清液，并加入150μl BMMY(培养基成分中除了用0.5％甲醇代替1％甘油外，其他成分和BMGY相同)，然后于30℃，280rpm摇床中震荡培养36小时，诱导阿魏酸酯酶表达。离心，用排枪轻轻吸出96孔板各孔中的上清液，按相应位置分别加入20μl上清液于两块96孔板中。一块装有20μl上清液的96孔板加入180μl反应液(0.85倍体积的含2.5％TritonX-100的100mM、pH 6.4的磷酸钠缓冲液和0.05倍体积的含有20mM 2-氯对硝基苯酚底物的二甲基亚砜溶液混合而成)，40℃反应15min。另一块装有20μl上清液的96板置于63℃烘箱，30min处理后迅速放置在4℃快速降温。如上述反应以测定突变体残余活性。残余活性比母本残余活性高的菌株挑到新的96孔培养板中进行重复筛选。筛选到27个突变体，残余活性是母本对照的2-5倍不等(见表1)。培养这些突变酶重组菌，提取其基因组，并用PCR技术获得其相应的突变阿魏酸酯酶基因，送上海英骏生物技术公司测序。对27个耐热性提高的阿魏酸酯酶A突变体编码序列测序结果分析后，确定突变酶热稳定的提高是由12个氨基酸突变引起的。12个单点突变体分别命名为：Z1、Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12，其具体特征见本说明书发明内容部分。
实施例3阿魏酸酯酶突变位点的整合
将以上获得的12个突变位点整合到一条核苷酸编码序列上并由上海生工生物技术服务有限公司合成全序列。为了方便后续蛋白纯化，将合成的全序列用NotI和EcoRI双酶切回收目的片段，连入经过相同酶切的pGAPZαA载体(含有博莱霉素抗性基因)中(连接方法如实施例1)。将连接产物转化大肠杆菌DH5α，挑取转化子培养并提取重组质粒。将质粒用BspHI内切酶线性化后，转化毕赤酵母(Pichia pastoris KM71)感受态细胞，涂布含有100μg/ml的博莱霉素的YPDS培养基(含有2％胰蛋白胨，1％酵母提取物，2％葡萄糖，1M山梨醇)中，30℃培养3天。挑取单克隆于YPD培养基(除不含有1M山梨醇外，培养基成分与YPDS培养基相同)中培养2天以表达该组合突变体酶。
实施例4母本和12个单点突变的阿魏酸酯酶A粗酶液的热稳定测定
将表达母本和12个单点突变的阿魏酸酯酶A的单克隆分别挑到25ml的BMGY培养基中培养两天后离心弃上清，用BMMY培养基重悬菌体。0.5％甲醇诱导表达两天后，取培养物上清于55℃温度下分别测定母本和除Z4突变酶外的其他10个单点突变的阿魏酸酯酶A突变体的半失活时间(t1/2)；而Z4突变酶于90℃温度下处理并测定其半失活时间(t1/2)。培养方法和酶活测定方法如实施例2。实验结果见表1。
表1母本阿魏酸酯酶A以及单点突变体t1/2测定
**注解：Z4突变酶的热稳定性于90℃温度下处理10分钟。
实施例5母本和组合突变型阿魏酸酯酶A的纯化以及热稳定性
5.1母本和组合突变体CV-FAEA的纯化
将同样转化到毕赤酵母(Pichia pastoris KM71)的母本和组合突变体CV-FAEA在YPD培养基中培养2天以表达蛋白。然后离心取上清，并用200mM，pH 8.0的Tris-HCl缓冲液调其pH为8.0，然后通过Bio-Scale Mini UNOsphere Q阴离子交换柱(Bio-Rad，Hercules，CA，USA)分离纯化目的蛋白。对纯化蛋白质的酶活测定方法如实施例2所述。
5.2母本和组合突变体CV-FAEA的热稳定性
将纯化后蛋白浓度为118.5μg/ml，pH 6.4的母本以及组合突变体CV-FAEA酶液50μl置于200μl离心管中，每个样品3个重复，在PCR仪中不同温度下处理15min，迅速放置冰上冷却。取20μl酶液在96孔板中测定残余活性(如实施例2所述)。与未经过高温处理的酶液比较，得到残余酶活百分比。结果显示：组合突变体CV-FAEA的热稳定性比母本有显著提高，母本在55℃处理15min后即丧失了50％活力，而组合突变体在同样条件下的酶活基本没有降低；母本在70℃处理15min后基本没有残余活性，而组合突变体在99℃处理15min后仍保持60％以上的活力(结果见说明书附图)。
实施例6组合突变型阿魏酸酯酶A的表征以及应用实例分析
组合突变型阿魏酸酯酶A是在母本阿魏酸酯酶A的基础上由12个氨基酸替换产生的，其氨基酸序列及蛋白质结构未发生重大变化。对母本及组合突变型的阿魏酸酯酶A的底物分析表明它们具有相同的底物谱。根据阿魏酸酯酶A的分类原则(V.F.Crepin，et al.Functional classification of the microbial feruloyl esterases.Appl.Microbiol.Biotechnol.63(2004)647-652.)，组合突变型阿魏酸酯酶A仍属于阿魏酸酯酶A。
用来源嗜热脂肪土芽孢杆菌的木聚糖酶突变体(Zhang Zhi-gang，et al.Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis.Bioresource Technology 101(2010)9272-9278)分别与母本阿魏酸酯酶A和组合突变型阿魏酸酯酶A于55℃条件下水解玉米秸秆1小时，木聚糖酶突变体和组合型阿魏酸酯酶A的反应体系比木聚糖酶突变体与母本阿魏酸酯酶A的反应体系中的阿魏酸的释放量要高10倍以上，其反应体系为0.1mg木聚糖酶突变体粗酶和0.1mg母本阿魏酸酯酶A或组合突变型阿魏酸酯酶A和含有2g处理后的玉米秸秆混合与50ml pH6.4的100mM磷酸钠缓冲液。这可能是由于母本阿魏酸酯酶A在60℃条件下很快失活有关。此结果也说明组合突变型阿魏酸酯酶A的热稳定性提高有利于其在相关领域的应用。
SEQ ID NO.1(野生型阿魏酸酯酶A的编码序列)：
GCCTCCACGCAAGGCATCTCCGAGGACCTCTACAATCGCTTAGTAGAGATGGCCACTATCTCTCAAGCCGCCTACGCCGACCTATGCAATATTCCATCGACTACTATCAAAGGAGAGAAAATTTACAACGCTCAAACTGATATCAACGGATGGATCCTCCGCGACGACACCAGCAAAGAAATTATCACCGTCTTCCGTGGCACTGGCAGTGACACAAACCTACAGCTCGATACTAACTACACGCTCACGCCATTCGAAACTCTACCTCAATGCAGCGGTTGCGAGGTACACGGTGGATACTATATTGGATGGATCTCAATCCAAGACCAAGTCGAGTCGCTTGTCAAACAACAGGCTAGCCAGTATCCGGACTATGCGCTTACCATGACAGGCCATAGTCTGGGAGCGTCGATGGCAGCACTCACTGCCGCCCAGCTGTCCGCGACATACGACAACGTCCGCCTGTACACTTTCGGCGAACCGCGCAGCGGCAACCAGGCCTTCGCGTCGTACATGAACGATGCGTTCCAGGTCTCGAGCCCGGAGACGACCCAGTACTTCCGGGTCACTCATTCCAACGACGGCATCCCAAACTTGCCCCCGGCGGAGCAGGGTTACGCCCATGGCGGTGTAGAGTACTGGAGCGTTGATCCTTACAGCGCCCAGAACACGTTTGTCTGTACTGGGGATGAAGTACAGTGCTGTGAGGCACAGGGCGGACAGGGTGCGAATGATGCGCATACTACTTATTTTGGGATGACGAGCGGAGCTTGTACTTGGTAA
SEQ ID NO.2(母本阿魏酸酯酶A氨基酸序列)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.3：(Z1)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFKTLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCSEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.4：(Z2)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIIIVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.5：(Z3)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTSSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMATLTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.6：(Z4)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCSEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.7：(Z5)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDISKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW.
SEQ ID NO.8：(Z6)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQASSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.9：(Z7)
ASTQGISEDLYNRFVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.10：(Z8)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRTGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.11：(Z9)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIIGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVS SPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.12：(Z10)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASHYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.13：(Z11)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTTIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTRYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.14：(Z12)
ASTQGISEDLYNRLVEMATISQAAYADLCNIPSTIIKGEKIYNAQTDINGWILRDDTSKEIITVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASQYPDYALTMTGHSLGASMAALTAAQLSATYDNVRLYTFGEPRSGNQAFASYMNDAFQVSSPETTQYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQ ID NO.15(组合突变体阿魏酸酯酶A氨基酸序列)
ASTQGISEDLYNRFVEMATISQAAYADLCNIPSTIIIGEKIYNAQTDINGWILRDDISKEIIIVFRGTGSDTNLQLDTNYTLTPFETLPQCSGCEVHGGYYIGWISIQDQVESLVKQQASHYPDYALTMTGHSLGASMATLTAAQLSATYDNVRLYTFGEPRTGNQAFASYMNDAFHASSPETTRYFRVTHSNDGIPNLPPAEQGYAHGGVEYWSVDPYSAQNTFVCTGDEVQCCEAQGGQGANDAHTTYFGMTSGACTW
SEQUENCE LISTING
<110> 中国科学院成都生物研究所
<120> 一种阿魏酸酯酶A突变体及其用途
<130> 说明书
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 783
<212> DNA
<213> Aspergillus niger
<400> 1
gcctccacgc aaggcatctc cgaggacctc tacaatcgct tagtagagat ggccactatc 60
tctcaagccg cctacgccga cctatgcaat attccatcga ctactatcaa aggagagaaa 120
atttacaacg ctcaaactga tatcaacgga tggatcctcc gcgacgacac cagcaaagaa 180
attatcaccg tcttccgtgg cactggcagt gacacaaacc tacagctcga tactaactac 240
acgctcacgc cattcgaaac tctacctcaa tgcagcggtt gcgaggtaca cggtggatac 300
tatattggat ggatctcaat ccaagaccaa gtcgagtcgc ttgtcaaaca acaggctagc 360
cagtatccgg actatgcgct taccatgaca ggccatagtc tgggagcgtc gatggcagca 420
ctcactgccg cccagctgtc cgcgacatac gacaacgtcc gcctgtacac tttcggcgaa 480
ccgcgcagcg gcaaccaggc cttcgcgtcg tacatgaacg atgcgttcca ggtctcgagc 540
ccggagacga cccagtactt ccgggtcact cattccaacg acggcatccc aaacttgccc 600
ccggcggagc agggttacgc ccatggcggt gtagagtact ggagcgttga tccttacagc 660
gcccagaaca cgtttgtctg tactggggat gaagtacagt gctgtgaggc acagggcgga 720
cagggtgcga atgatgcgca tactacttat tttgggatga cgagcggagc ttgtacttgg 780
taa 783
<210> 2
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 2
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 3
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 3
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Lys Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Ser Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 4
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 4
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Ile Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 5
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 5
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Ser Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Thr Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 6
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 6
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Ser Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 7
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 7
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Ile Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 8
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 8
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Ala Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 9
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 9
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Phe Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 10
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 10
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Thr Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 11
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 11
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Ile Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 12
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 12
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser His Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 13
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 13
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Thr Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Arg Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 14
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 14
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Phe Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Ile Ile Ile Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Ile Ser Lys Glu Ile Ile Ile Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser His Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Thr Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Thr Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
His Ala Ser Ser Pro Glu Thr Thr Arg Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
<210> 15
<211> 34
<212> DNA
<213> Aspergillus niger
<400> 15
taggaggtga attcgcctcc acgcaaggca tctc 34
<210> 16
<211> 39
<212> DNA
<213> Aspergillus niger
<400> 16
taggaggtgc ggccgcttac caagtacaag ctccgctcg 39
<210> 17
<211> 260
<212> PRT
<213> Aspergillus niger
<400> 17
Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val Glu
1 5 10 15
Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile Pro
20 25 30
Ser Thr Ile Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp Ile
35 40 45
Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr Val
50 55 60
Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu Asp Thr Asn Tyr
65 70 75 80
Thr Leu Thr Pro Phe Glu Thr Leu Pro Gln Cys Ser Gly Cys Glu Val
85 90 95
His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Ile Gln Asp Gln Val Glu
100 105 110
Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu Thr
115 120 125
Met Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala Ala
130 135 140
Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly Glu
145 150 155 160
Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala Phe
165 170 175
Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His Ser
180 185 190
Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Glu Gln Gly Tyr Ala His
195 200 205
Gly Gly Val Glu Tyr Trp Ser Val Asp Pro Tyr Ser Ala Gln Asn Thr
210 215 220
Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly Gly
225 230 235 240
Gln Gly Ala Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser Gly
245 250 255
Ala Cys Thr Trp
260
Claims (3)
1.一种阿魏酸酯酶A突变体，其特征在于：以SEQ ID NO.2所示的阿魏酸酯酶A氨基酸序列为基础，经突变后具有以下突变位点的氨基酸序列：将其第14位的亮氨酸突变为苯丙氨酸或/和第35位的苏氨酸突变为异亮氨酸或/和第37位的赖氨酸突变为异亮氨酸或/和第57位的苏氨酸突变为异亮氨酸或/和第63位的苏氨酸突变为异亮氨酸或/和第121位的谷氨酰胺突变为组氨酸或/和第140位的丙氨酸突变为苏氨酸或/和第163位的丝氨酸突变为苏氨酸或/和第177位的谷氨酰胺突变为组氨酸或/和第178位的缬氨酸突变为丙氨酸或/和第185位的谷氨酰胺突变为精氨酸或/和第235位的半胱氨酸突变为丝氨酸。
2.将权利要求书1所述的阿魏酸酯酶A突变体的基因连入pPIC9K或pGAPZαA载体，并在宿主菌Pichia pastoris KM71中表达，获得突变体酶蛋白。
3.权利要求1所述的阿魏酸酯酶A突变体在半纤维素降解中的用途。
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| CN104531729A (zh) * | 2014-12-10 | 2015-04-22 | 江南大学 | 一种添加二硫键以提高阿魏酸酯酶a热稳定性的方法 |
| CN104651333A (zh) * | 2015-03-23 | 2015-05-27 | 河南工业大学 | 一种产黄青霉阿魏酸酯酶a在毕赤酵母中的表达及其酶制剂的制备方法 |
| CN108220270A (zh) * | 2018-01-31 | 2018-06-29 | 江南大学 | 一种催化活性提高的酸性脂肪酶突变体 |
| CN109402087A (zh) * | 2018-12-07 | 2019-03-01 | 南京农业大学 | 一种新型阿魏酸酯酶及其制备方法和应用 |
| CN110760489A (zh) * | 2019-11-18 | 2020-02-07 | 西南交通大学 | 一种川芎咖啡酸-o-甲基转移酶的突变体及其用途 |
| CN111094560A (zh) * | 2017-07-17 | 2020-05-01 | 地中海感染基金会 | 新型突变型pte酶 |
| CN111748538A (zh) * | 2020-04-28 | 2020-10-09 | 南京农业大学 | 一种新型阿魏酸酯酶、突变体和应用 |
| CN114958805A (zh) * | 2021-03-16 | 2022-08-30 | 南京农业大学 | 一种阿魏酸酯酶及其突变体n.9-98与应用 |
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| CN111094560B (zh) * | 2017-07-17 | 2023-11-07 | 地中海感染基金会 | 新型突变型pte酶 |
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| CN114958805B (zh) * | 2021-03-16 | 2023-04-11 | 南京农业大学 | 一种阿魏酸酯酶及其突变体n.9-98与应用 |
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