CN106318957B - 土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用 - Google Patents
土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用 Download PDFInfo
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Abstract
土曲霉CCF 3059 α‑L‑鼠李糖苷酶突变体及其应用，包括如SEQ ID NO:2所示的D594Q基因、SEQ ID NO:3所示的D594R基因、SEQ ID NO:4所示的D594C基因、SEQ ID NO:5所示的G827K基因、SEQ ID NO:6所示的G827M基因和SEQ ID NO:7所示的G828A基因。突变酶MRha‑D594Q最适温度和原酶MRha均为65℃；但与原酶MRha相比，突变酶MRha‑D594Q在70℃和75℃时仍保持较高的酶活性；添加山梨醇能够进一步提高突变酶MRha‑D594Q的热稳定性，在70℃的半衰期提高了7.8倍。
Description
技术领域
本发明属于分子生物学及酶工程领域，具体涉及土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用。
背景技术
α-L-鼠李糖苷酶(EC 3.2.1.40)是一种重要的糖苷水解酶，它能够特异性水解许多末端含有非还原性α-L-鼠李糖的天然的糖苷类化合物，如芦丁，柚皮苷，橘皮苷，甜菊苷，杨梅苷等。α-L-鼠李糖苷酶的来源比较广，广泛分布在自然界的植物、细菌、真菌、动物肝脏中。CAZy(http://www.cazy.org/)数据库中糖苷水解酶的分类依据是氨基酸序列的相似性，α-L-鼠李糖苷酶包含在三个糖苷水解酶家族(glycoside hydrolase family，GH)，分别是GH13、GH78和GH106。
α-L-鼠李糖苷酶在食品工业中具有广泛的应用价值。众所周知，很多水果都含有柚皮苷、橘皮苷等苦味物质。α-L-鼠李糖苷酶能够水解芸香科水果中的柚皮苷和柠檬苦素等苦味物质，从而很大程度上降低苦味，改善果汁的口感；α-L-鼠李糖苷酶还能够水解葡萄酒中大量的键合态芳香物质，释放出糖苷配基，产生游离的芳香物质，使葡萄酒增香并提高风味。同时，α-L-鼠李糖苷酶能定向水解含有鼠李糖苷的天然活性物质或天然药物，提高原有物质的生物活性和生物利用度。此外，α-L-鼠李糖苷酶在化合物的结构鉴定、改进抗癌药物的靶向性方面也有广泛的应用。然而，α-L-鼠李糖苷酶在工业中进行规模化推广应用还存在障碍和技术瓶颈：(1)现有菌株产α-L-鼠李糖苷酶的活力不够高，直接导致了使用成本较高。(2)大多数报道的α-L-鼠李糖苷酶最适反应温度在40～60℃之间，在高温条件下热稳定性较差，这不利于底物的溶解及产物的纯化。
本发明申请人在研究中克隆表达了土曲霉CCF 3059来源的α-L-鼠李糖苷酶基因Rha，通过优化密码子的偏好性，得到了优化后的α-L-鼠李糖苷酶基因MRha，并构建重组质粒pPICZαA-MRha，使其表达水平大幅提高，摇瓶最高酶活力达到1000U/mL，远远高于到目前为止国内外报导的最好水平。该酶具有优良的最适作用温度、最适作用pH及较好的pH稳定性，但在温度高于70℃条件下该酶不稳定。提高该酶的热稳定性将有望大幅提高该酶实际使用效果。
目前，提高酶的热稳定性的方法主要包括：(1)定向进化：通过随机突变，定点突变，饱和突变等技术，筛选得到热稳定性较好的突变株；(2)从嗜热微生物中筛选热稳定性较好的酶；(3)酶的固定化；(4)酶的化学修饰；(5)添加稳定剂，如多羟基化合物，金属离子等。虽然通过以上技术可以提高酶的热稳定性，但还未见利用这些技术手段，特别是利用两种或两种以上手段提高α-L-鼠李糖苷酶稳定性的报道。
本研究将对已经实现超量表达的土曲霉CCF 3059 α-L-鼠李糖苷酶基因MRha为研究对象，从定点突变和添加稳定剂这两个方面着手来提高其耐热性能。
发明内容
解决的技术问题：本发明提供了一种土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用。本发明分为两步：(1)获得土曲霉CCF 3059 α-L-鼠李糖苷酶的突变基因，其编码对应的突变酶在70℃下的热稳定性有明显提高；(2)通过大量筛选，获得一类多羟基化合物，能进一步提高突变酶在70℃下的热稳定性。从而在工业生产过程中可以采用较高的操作温度，以提高反应速率，增加底物溶解度，降低酶的失活率，从而有利于该酶的生物催化转化。
技术方案：土曲霉CCF 3059 α-L-鼠李糖苷酶突变体基因，包括如SEQ ID NO:2所示的D594Q基因、SEQ ID NO:3所示的D594R基因、SEQ ID NO:4所示的D594C基因、SEQ IDNO:5所示的G827K基因、SEQ ID NO:6所示的G827M基因和SEQ ID NO:7所示的G828A基因。
上述突变体基因翻译对应的土曲霉CCF 3059 α-L-鼠李糖苷酶。
土曲霉CCF 3059 α-L-鼠李糖苷酶，氨基酸序列如SEQ ID NO:1所示。
一种催化转化天然活性物质或天然药物中鼠李糖苷的组合物，包括酶稳定剂和上述土曲霉CCF 3059 α-L-鼠李糖苷酶。
上述酶稳定剂为山梨醇、Ca2+、甘氨酸、甘油或PEG1000，浓度为0.2-2mol/L。
上述组合物在催化转化芦丁制备异槲皮素中的应用。
有益效果：1.突变酶MRha-D594Q最适温度和原酶MRha均为65℃；但与原酶MRha相比，突变酶MRha-D594Q在70℃和75℃时仍保持较高的酶活性；2.突变酶MRha-D594Q的温度稳定性相对于原酶MRha有大幅提高，在70℃的半衰期提高了190％。3.添加山梨醇能够进一步提高突变酶MRha-D594Q的热稳定性，在70℃的半衰期提高了7.8倍。4.相比较于原酶，重组酶MRha-D594Q能够显著提高在70℃下芦丁的转化效率，在重组酶MRha-D594Q中添加一定浓度的山梨醇能够进一步提高在70℃下芦丁的转化效率，摩尔转化率达到98％。
附图说明
图1为原酶及突变α-L-鼠李糖苷酶热稳定性比较示意图；
图2为稳定剂对突变酶D594Q热稳定性的影响示意图；
图3为原酶，突变酶D594Q的最适温度示意图；
图4为原酶，突变酶D594Q及突变酶D594Q添加山梨醇的热稳定性比较示意图；
图5为原酶，突变酶D594Q及突变酶D594Q添加山梨醇对芦丁的转化效率比较示意图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅为本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为了进一步理解本发明，下面结合实施例对本发明进行详细阐述，其中，如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。
实施例1
1.重组质粒pPICZαA-MRha的获得及突变位点的确定
全基因合成并通过酵母密码子偏好性优化土曲霉CCF 3059 α-L-鼠李糖苷酶基因，其核苷酸序列如SEQ ID NO:8，将其连接到pPICZαA质粒上，获得的重组质粒命名为pPICZαA-MRha，转化Pichia pastoris KM71H。
研究表明酶蛋白Loop区对酶的热稳定性密切相关，此外氨基酸B-Factor值越大越不稳定。通过同源建模，获得土曲霉CCF 3059 α-L-鼠李糖苷酶的三维结构，基于上述理论寻找突变位点，利用生物信息学软件Discovery Studio对该突变点的突变能进行分析，筛选突变势能较低的突变体作为研究对象，最终确定的突变体为D594Q，D594R，D594C，G827K，G827M，G828A。
突变引物设计：利用生物信息学分析及同源建模比对，改变α-L-鼠李糖苷酶MRha分子部分氨基酸，设计突变点。利用反向PCR技术获得突变序列，每个突变位点设计正反两条寡核苷酸序列。突变体选用酵母偏爱密码子，突变引物如下表。
表1定点突变引物表
2.携带突变基因的系列重组质粒的构建
2.1重组质粒pPICZαA-MRha-D594Q的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将594位的天冬氨酸突变成谷氨酰胺，获得携带有突变基因SEQ ID NO：2的重组质粒pPICZαA-MRha-D594Q
表2反向PCR反应液的配制(共50μL)
表3反向PCR反应条件
PCR片段5’末端磷酸化反应配制如下表，于37℃下反应1h。
表4磷酸化反应液的配制
37℃磷酸化后，加入1μL T4 ligase，16℃连接3h。质粒DNA转化，挑选转化子，通过测序鉴定。
2.2重组质粒pPICZαA-MRha-D594R的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将594位的天冬氨酸突变成精氨酸，获得携带有突变基因SEQ ID NO：3的重组质粒pPICZαA-MRha-D594R，所用引物为F2和R1，其他过程同2.1。
2.3重组质粒pPICZαA-MRha-D594C的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将594位的天冬氨酸突变成谷氨酰胺，获得携带有突变基因SEQ ID NO：4的重组质粒pPICZαA-MRha-D594C，所用引物为F3和R1，其他过程同2.1。
2.4重组质粒pPICZαA-MRha-G827K的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将827位的甘氨酸突变成赖氨酸，获得携带有突变基因SEQ ID NO：5的重组质粒pPICZαA-MRha-G827K，所用引物为F4和R2，其他过程同2.1。
2.5重组质粒pPICZαA-MRha-G827M的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将827位的甘氨酸突变成甲硫氨酸，获得携带有突变基因SEQ ID NO：6的重组质粒pPICZαA-MRha-G827M，所用引物为F5和R2，其他过程同2.1。
2.6重组质粒pPICZαA-MRha-G828A的构建
以重组质粒pPICZαA-MRha为模板，利用反向PCR技术将828位的甘氨酸突变成丙氨酸，获得携带有突变基因SEQ ID NO：7的重组质粒pPICZαA-MRha-G828A，所用引物为F6和R3，其他过程同2.1。
3.重组酶的表达及纯化
提取测序正确的重组质粒pPICZαA-MRha-D594Q，经SacⅠ线性化后，采用电转化法将其导入毕赤酵母KM71H中，筛选阳性克隆。接种在YPD培养基中进行活化后，转入BMGY培养基中继续活化，收集OD600为2.0-3.0的菌体，转入到BMMY培养基中，置于30℃，180rpm摇床进行α-L-鼠李糖苷酶的诱导表达。每隔24h按照体积比0.6％向诱导的菌液中补加无菌的甲醇，培养15天后离心取上清得粗酶液。重组蛋白的纯化：(1)向粗酶液中加终浓度为80％的硫酸铵沉淀蛋白，离心弃上清，用pH 7.5 50mM Tris-HCl缓冲液溶解沉淀的蛋白；(2)用pH7.5 50mM Tris-HCl缓冲液于4℃下透析四次，每次8h，以去除盐溶液；(3)将透析后的酶液加到装好的DEAE SFF柱子中，用浓度20-300mM的NaCl梯度洗脱；(4)取合适浓度的NaCl洗脱下的酶液，用pH 6.5 10mM PB缓冲液于4℃下透析四次，每次8h，以去除盐溶液得纯酶。
4.α-L-鼠李糖苷酶酶活测定
以对硝基苯酚-α-鼠李糖苷(pNP-R)为底物，水解得到的对硝基苯酚与碳酸钠发生显色反应，在405nm的波长下测定产物的吸光度。100μL反应体系包括75μL 100mM pH 6.5缓冲液，20μL 5mM底物，混匀预热后加入5μL稀释酶液，65℃反应10min，然后加入0.3mL 1MNaCO3终止反应，混匀后在405nm条件下酶标仪测定。同时做有酶液无底物的对照和有底物无酶液的对照。
一个酶活单位(U)定义为：在65℃、pH 6.5的条件下，水解释放1μmol对硝基酚所需的酶量。
对照标准曲线，计算酶活：
α-L-鼠李糖苷酶酶活(U/mL)＝c×V1/(t×V2)×N
c：由对硝基苯酚标准方程计算出的酶反应后的对硝基苯酚含量(μmol/mL)；
V1：反应体系总体积(mL)；
t：酶与底物反应时间(min)；
V2：酶反应时酶液的体积(mL)；
N：酶液稀释倍数。
5.突变酶耐热性能的比较
将原酶及六种突变酶在pH 6.5，70℃条件下保温60min后，按照标准方法在65℃、pH 6.5下测定其残余酶活性，以不进行热处理的酶的酶活性为相对100％。结果表明：突变酶D594Q的热稳定性最好(图1)。
6.稳定剂的筛选
将终浓度为25mM Ca2+，1M甘氨酸，1M山梨醇，10％甘油，10％PEG1000分别添加到突变酶D594Q中，在pH 6.5，70℃条件下保温120min后，按照标准方法在65℃、pH 6.5下测定其残余酶活性，以不进行热处理的酶的酶活性为相对100％。结果表明：添加山梨醇能够明显提高突变酶D594Q的热稳定性(图2中a)。
将终浓度为0.2至2.0M山梨醇分别添加到突变酶D594Q中，在pH 6.5，70℃条件下保温120min后，按照标准方法在65℃、pH 6.5下测定其残余酶活性，以不进行热处理的酶的酶活性为相对100％。结果表明：各浓度的山梨醇都能提高突变酶D594Q的热稳定性，而1.5M的山梨醇效果最佳(图2中b)。
7.酶学性质的测定
7.1最适反应温度
重组酶最适温度的测定：将原酶，突变酶D594Q在pH6.5下，分别于40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃和80℃条件下测定酶活力，根据各温度下酶活力的大小确定酶的最适反应温度，以测得的最高酶活力为相对100％。结果表明：突变酶MRha-D594Q最适温度和原酶MRha均为65℃；但与原酶MRha相比，突变酶MRha-D594Q在70℃和75℃时仍保持较高的酶活性(图3)。
7.2温度稳定性
将原酶，突变酶D594Q及突变酶D594Q添加山梨醇在pH 6.5，70℃条件下保温60min后，按照标准方法在65℃、pH 6.5下测定其残余酶活性，以不进行热处理的酶的酶活性为相对100％。结果表明：突变酶MRha-D594Q的温度稳定性相对于原酶MRha有大幅提高，在70℃的半衰期提高了1.9倍；添加山梨醇能够进一步提高突变酶MRha-D594Q的热稳定性，在70℃的半衰期提高了7.8倍(图4)。
8.酶转化芦丁为异槲皮素
芦丁的浓度为16mmol/L，转化条件为70℃，pH 6.5 100mmol/L柠檬酸-磷酸氢二钠缓冲液，加入的0.2U/mL原酶，0.2U/mL突变酶D594Q，0.2U/mL突变酶D594Q加终浓度为1.5M山梨醇，不同的时间点(0，0.5，1.0，2.0，4.0，6.0，8.0，10.0min)分别取样，通过HPLC进行检测。结果表明：反应10h后，原酶，突变酶D594Q及添加山梨醇突变酶D594Q的芦丁转化率分别为72.0％，85.3％，98％(图5)。
SEQUENCE LISTING
<110> 南京林业大学
<120> 土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用
<130>
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 871
<212> PRT
<213> 人工序列
<400> 1
Met Ala Leu Ser Ile Ser Gln Val Ala Phe Glu His His Arg Thr Ala
1 5 10 15
Leu Gly Ile Gly Glu Thr Gln Pro Arg Val Ser Trp Arg Phe Asp Gly
20 25 30
Asn Val Ser Asp Trp Glu Gln Arg Ala Tyr Glu Ile Glu Val Lys Arg
35 40 45
Ala Gly His Asp Ala Asp Val Phe Arg Ser Glu Ser Ser Asp Ser Val
50 55 60
Leu Val Pro Trp Pro Ser Ser Pro Leu Gln Ser Gly Glu Glu Ala Thr
65 70 75 80
Val Arg Val Arg Ser Phe Gly Ser Asp Gly Gln His Asp Thr Pro Trp
85 90 95
Ser Asp Ala Val Thr Val Glu Pro Gly Leu Leu Thr Pro Asp Asp Trp
100 105 110
His Asp Ala Val Val Ile Ala Ser Asp Arg Pro Thr Glu Val Asp Ala
115 120 125
Thr His Arg Pro Ile Gln Phe Arg Lys Glu Phe Ser Val Asp Asp Ser
130 135 140
Tyr Val Ser Ala Arg Leu Tyr Ile Thr Ala Leu Gly Leu Tyr Glu Ala
145 150 155 160
Arg Ile Asn Asp Gln Arg Val Gly Asp His Val Met Ala Pro Gly Trp
165 170 175
Gln Ser Tyr Gln Tyr Arg His Glu Tyr Asn Thr Tyr Asp Val Thr Asp
180 185 190
Leu Leu Lys Gln Gly Pro Asn Ala Ile Gly Val Thr Val Gly Glu Gly
195 200 205
Trp Tyr Ser Gly Arg Ile Gly Tyr Asp Gly Gly Lys Arg Asn Ile Tyr
210 215 220
Gly Asp Thr Leu Gly Leu Leu Ser Leu Leu Val Val Thr Lys Ser Asp
225 230 235 240
Gly Ser Lys Leu Tyr Ile Pro Ser Asp Ser Ser Trp Lys Ser Ser Thr
245 250 255
Gly Pro Ile Ile Ser Ser Glu Ile Tyr Asp Gly Glu Glu Tyr Asp Ser
260 265 270
Arg Leu Glu Gln Lys Gly Trp Ser Gln Val Gly Phe Asn Ser Thr Gly
275 280 285
Trp Leu Gly Thr His Glu Leu Ser Phe Pro Lys Glu Arg Leu Ala Ser
290 295 300
Pro Asp Gly Pro Pro Val Arg Arg Val Ala Glu His Lys Leu Ala Asn
305 310 315 320
Val Phe Ser Ser Ala Ser Gly Lys Thr Val Leu Asp Phe Gly Gln Asn
325 330 335
Leu Val Gly Trp Leu Arg Ile Arg Val Lys Gly Pro Lys Gly Gln Thr
340 345 350
Ile Arg Phe Val His Thr Glu Val Met Glu Asn Gly Glu Val Ala Thr
355 360 365
Arg Pro Leu Arg Gln Ala Lys Ala Thr Asp His Phe Thr Leu Ser Gly
370 375 380
Glu Gly Val Gln Glu Trp Glu Pro Ser Phe Thr Tyr His Gly Phe Arg
385 390 395 400
Tyr Val Gln Val Asp Gly Trp Pro Ala Asp Thr Pro Leu Asp Glu Asn
405 410 415
Ser Val Thr Ala Ile Val Val His Ser Asp Met Glu Arg Thr Gly Tyr
420 425 430
Phe Glu Cys Ser Asn Pro Leu Ile Ser Lys Leu His Glu Asn Ile Leu
435 440 445
Trp Ser Met Arg Gly Asn Phe Phe Ser Ile Pro Thr Asp Cys Pro Gln
450 455 460
Arg Asp Glu Arg Leu Gly Trp Thr Gly Asp Ile His Ala Phe Ser Arg
465 470 475 480
Thr Ala Asn Phe Ile Tyr Asp Thr Ala Gly Phe Leu Arg Ala Trp Leu
485 490 495
Lys Asp Ala Arg Ser Glu Gln Leu Asn His Ser Tyr Ser Leu Pro Tyr
500 505 510
Val Ile Pro Asn Ile His Gly Asn Gly Glu Thr Pro Thr Ser Ile Trp
515 520 525
Gly Asp Ala Ile Val Gly Val Pro Trp Gln Leu Tyr Glu Ser Phe Gly
530 535 540
Asp Lys Val Met Leu Glu Glu Gln Tyr Gly Gly Ala Lys Asp Trp Val
545 550 555 560
Asp Lys Gly Ile Val Arg Asn Asp Val Gly Leu Trp Asp Arg Ser Thr
565 570 575
Phe Gln Trp Ala Asp Trp Leu Asp Pro Lys Ala Pro Ala Asp Asp Pro
580 585 590
Gly Gln Ala Thr Thr Asn Lys Tyr Leu Val Ser Asp Ala Tyr Leu Leu
595 600 605
His Ser Thr Asp Met Leu Ala Asn Ile Ser Thr Ser Leu Ser Lys Gly
610 615 620
Glu Glu Ala Ser Asn Tyr Thr Glu Trp His Ala Lys Leu Thr Lys Glu
625 630 635 640
Phe Gln Lys Ala Trp Ile Thr Ser Asn Gly Thr Met Ala Asn Glu Thr
645 650 655
Gln Thr Gly Leu Ala Leu Pro Leu Tyr Phe Asp Leu Phe Pro Ser Ala
660 665 670
Glu Gln Ala Gln Ser Ala Ala Lys Arg Leu Val Asn Ile Ile Lys Gln
675 680 685
Asn Asp Tyr Lys Val Gly Thr Gly Phe Ala Gly Thr His Leu Leu Gly
690 695 700
His Thr Leu Ser Lys Tyr Gly Glu Ser Asp Ala Phe Tyr Ser Met Leu
705 710 715 720
Arg Gln Thr Glu Val Pro Ser Trp Leu Tyr Gln Val Val Met Asn Gly
725 730 735
Thr Thr Thr Trp Glu Arg Trp Asp Ser Met Leu Pro Asn Gly Ser Ile
740 745 750
Asn Pro Gly Gln Met Thr Ser Phe Asn His Tyr Ala Val Gly Ser Val
755 760 765
Gly Ser Trp Leu His Glu Val Ile Gly Gly Leu Ser Pro Ala Glu Pro
770 775 780
Gly Trp Arg Arg Ile Asn Ile Glu Val Val Pro Gly Gly Asp Leu Gln
785 790 795 800
Gln Ala Ser Thr Lys Phe Leu Thr Pro Tyr Gly Met Ala Ser Thr Lys
805 810 815
Trp Trp Leu Asp Gly Gln Asp Gln Ser Cys Gly Gly Phe Asp Phe His
820 825 830
Leu Val Ala Glu Val Pro Pro Asn Thr Arg Ala Thr Val Val Leu Pro
835 840 845
Gly Lys Gly Gly Glu Lys Val Asp Val Gly Ser Gly Val His Glu Tyr
850 855 860
His Val Arg Cys Val Lys Leu
865 870
<210> 2
<211> 2616
<212> DNA
<213> 人工序列
<400> 2
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtc aagcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcgg aggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 3
<211> 2616
<212> DNA
<213> 人工序列
<400> 3
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggta gagcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcgg aggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 4
<211> 2616
<212> DNA
<213> 人工序列
<400> 4
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtt gtgcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcgg aggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 5
<211> 2616
<212> DNA
<213> 人工序列
<400> 5
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtg acgcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcaa aggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 6
<211> 2616
<212> DNA
<213> 人工序列
<400> 6
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtc aagcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcat gggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 7
<211> 2616
<212> DNA
<213> 人工序列
<400> 7
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtc aagcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcgg agcttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 8
<211> 2616
<212> DNA
<213> 人工序列
<400> 8
atggccttgt ctatttctca agtcgcattt gagcatcaca gaacagcatt gggtattggt 60
gaaacccagc caagagtttc ttggagattt gatggtaacg tctctgactg ggagcagaga 120
gcttacgaaa tcgaagtcaa gagagccggt cacgacgctg acgtttttag gtctgaatct 180
tctgactctg ttttggttcc ttggccatct tctccattgc agtctggtga agaagctact 240
gttagagtca ggtctttcgg ttctgatgga caacatgata caccttggtc tgatgccgtt 300
actgttgagc caggattgtt gacacctgac gactggcatg acgcagttgt tattgcctct 360
gacagaccta ccgaggtcga tgcaacacat agacctattc agttcagaaa ggagttctct 420
gttgatgact cttatgtctc tgccagattg tatattacag ctttgggatt gtacgaagcc 480
agaattaatg atcaaagagt cggagatcat gtcatggctc caggatggca atcttatcaa 540
tacagacatg agtacaacac ttatgatgtt acagatttgt tgaagcaagg accaaacgct 600
atcggagtta cagtcggtga aggatggtat tctggtagaa tcggatacga tggtggaaaa 660
agaaatatct atggagatac tttgggtttg ttgtctttgt tggtcgtcac aaaatctgac 720
ggttctaagt tgtacattcc atctgattct tcttggaagt cttctacagg tcctatcatt 780
tcttctgaaa tctatgacgg agaggagtat gattctaggt tggagcaaaa aggttggtct 840
caagttggat tcaattctac tggatggttg ggtacacacg aattgtcttt tcctaaagaa 900
agattggctt ctcctgatgg tcctccagtt agaagagttg ctgagcacaa attggctaat 960
gtcttctctt ctgcatctgg taaaaccgtt ttggacttcg gacaaaattt ggtcggttgg 1020
ttgagaatta gagttaaggg acctaagggt cagactatta gattcgttca tactgaagtc 1080
atggaaaacg gagaagttgc tactagacct ttgagacagg ctaaggccac agatcacttt 1140
actttgtctg gagagggtgt tcaggaatgg gagccttctt tcacctatca cggtttcaga 1200
tacgttcagg ttgatggatg gcctgctgac acccctttgg acgaaaattc tgttaccgct 1260
attgttgttc actctgatat ggaaagaact ggttacttcg aatgctctaa cccattgatt 1320
tctaaattgc atgaaaacat cttgtggtct atgagaggaa acttcttttc tatcccaact 1380
gactgtcctc agagagatga gagattggga tggaccggag acatccatgc attctctagg 1440
actgctaatt tcatctacga tactgctgga tttttgagag catggttgaa ggacgcaagg 1500
tctgaacaat tgaaccattc ttattctttg ccttatgtta ttcctaatat ccacggtaac 1560
ggtgagacac ctacttctat ctggggagac gccattgtcg gtgtcccttg gcaattgtat 1620
gagtcttttg gtgacaaggt tatgttggaa gaacagtacg gaggtgctaa ggattgggtc 1680
gataagggaa ttgttagaaa cgatgtcggt ttgtgggaca ggtctacttt ccaatgggcc 1740
gactggttgg accctaaagc ccctgccgat gaccctggtc aagcaactac aaataagtat 1800
ttggtttctg acgcttactt gttgcactct actgacatgt tggcaaacat ttctacctct 1860
ttgtctaaag gtgaggaagc atctaattac actgagtggc atgcaaagtt gactaaagag 1920
ttccaaaagg cttggattac ctctaacggt actatggcaa atgagactca gaccggattg 1980
gcattgcctt tgtactttga cttgttccca tctgctgaac aggcacagtc tgctgctaag 2040
agattggtta acattatcaa acaaaacgat tataaggtcg gaactggatt cgctggaaca 2100
cacttgttgg gacatacatt gtctaagtac ggtgaatctg atgctttcta ttctatgttg 2160
agacaaactg aggttccatc ttggttgtat caggtcgtta tgaatggtac tactacctgg 2220
gaaagatggg actctatgtt gccaaacgga tctattaatc caggtcagat gacatctttt 2280
aatcactacg cagtcggatc tgttggttct tggttgcacg aggttatcgg tggattgtct 2340
ccagctgaac caggttggag aagaatcaat atcgaggttg ttcctggtgg tgacttgcag 2400
caggcttcta ctaagttttt gactccatac ggaatggcat ctacaaaatg gtggttggat 2460
ggacaggatc agtcttgcgg aggttttgat tttcacttgg tcgccgaagt tcctccaaac 2520
actagagcaa ccgttgtttt gccaggaaag ggaggtgaga aggttgacgt tggatctggt 2580
gtccatgaat atcacgttag atgtgttaag ttgtaa 2616
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
caagcaacta caaataagta tttgg 25
<210> 10
<211> 18
<212> DNA
<213> 人工序列
<400> 10
accagggtca tcggcagg 18
<210> 11
<211> 25
<212> DNA
<213> 人工序列
<400> 11
agagcaacta caaataagta tttgg 25
<210> 12
<211> 18
<212> DNA
<213> 人工序列
<400> 12
accagggtca tcggcagg 18
<210> 13
<211> 25
<212> DNA
<213> 人工序列
<400> 13
tgtgcaacta caaataagta tttgg 25
<210> 14
<211> 18
<212> DNA
<213> 人工序列
<400> 14
accagggtca tcggcagg 18
<210> 15
<211> 28
<212> DNA
<213> 人工序列
<400> 15
aaaggttttg attttcactt ggtcgccg 28
<210> 16
<211> 28
<212> DNA
<213> 人工序列
<400> 16
gcaagactga tcctgtccat ccaaccac 28
<210> 17
<211> 28
<212> DNA
<213> 人工序列
<400> 17
atgggttttg attttcactt ggtcgccg 28
<210> 18
<211> 28
<212> DNA
<213> 人工序列
<400> 18
gcaagactga tcctgtccat ccaaccac 28
<210> 19
<211> 29
<212> DNA
<213> 人工序列
<400> 19
gcttttgatt ttcacttggt cgccgaagt 29
<210> 20
<211> 25
<212> DNA
<213> 人工序列
<400> 20
tccgcaagac tgatcctgtc catcc 25
Claims (6)
1.土曲霉CCF 3059 α-L-鼠李糖苷酶突变体基因，其特征在于包括如SEQ ID NO:2所示的D594Q基因、SEQ ID NO: 3所示的D594R基因、SEQ ID NO: 4所示的D594C基因、SEQ IDNO: 5所示的G827K基因、SEQ ID NO: 6所示的G827M基因和SEQ ID NO: 7所示的G828A基因。
2.权利要求1所述突变体基因翻译对应的土曲霉CCF 3059 α-L-鼠李糖苷酶。
3.根据权利要求2所述土曲霉CCF 3059 α-L-鼠李糖苷酶，其特征在于氨基酸序列如SEQ ID NO: 1所示。
4.一种催化转化天然活性物质或天然药物中鼠李糖苷的组合物，其特征在于包括酶稳定剂和权利要求1或2所述的土曲霉CCF 3059 α-L-鼠李糖苷酶。
5.根据权利要求4所述催化转化天然活性物质或天然药物中鼠李糖苷的组合物，其特征在于所述酶稳定剂为山梨醇、Ca2+、甘氨酸、甘油或PEG1000，浓度为0.2-2 mol/L。
6.权利要求5所述组合物在催化转化芦丁制备异槲皮素中的应用。
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| CN107641621B (zh) * | 2017-06-14 | 2021-07-23 | 江苏康缘药业股份有限公司 | 一种糖苷酶组合物及酶法制备淫羊藿苷元的方法 |
| CN109312375B (zh) * | 2018-04-25 | 2022-06-17 | 邦泰生物工程(深圳)有限公司 | 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶 |
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