CN114606216A - 一种表达量提高的α-淀粉酶突变体Q441N/N442H及其编码基因和应用 - Google Patents

一种表达量提高的α-淀粉酶突变体Q441N/N442H及其编码基因和应用 Download PDF

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CN114606216A
CN114606216A CN202210337046.7A CN202210337046A CN114606216A CN 114606216 A CN114606216 A CN 114606216A CN 202210337046 A CN202210337046 A CN 202210337046A CN 114606216 A CN114606216 A CN 114606216A
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郭庆文
王兴吉
王克芬
刘文龙
张�杰
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Shandong Lonct Enzymes Co ltd
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Abstract

本发明属于农业生物技术领域，具体涉及一种表达量提高的α‑淀粉酶突变体Q441N/N442H及其编码基因和应用。通过将氨基酸序列如SEQIDNO:1所示的野生型α‑淀粉酶进行Q441N/N442H两点突变，从而获得所述α‑淀粉酶的突变体。本发明的α‑淀粉酶突变体与野生型α‑淀粉酶相比，表达量大幅提升，粗酶液的活性提高了约15倍，野生型与突变体在90℃下处理10、20、30分钟后的剩余酶活相当。

Description

一种表达量提高的α-淀粉酶突变体Q441N/N442H及其编码基 因和应用
技术领域
本发明属于农业生物技术领域，具体涉及一种表达量提高的α-淀粉酶突变体Q441N/N442H及其编码基因和应用。
背景技术
淀粉工业是最大的水解酶市场之一。淀粉作为一种可再生资源广泛存在于自然界的植物中，是生产果葡糖浆、麦芽糖、生物乙醇及酿酒等多种工业的主要原料。淀粉可分为直链淀粉和支链淀粉，淀粉酶是作用于淀粉内部并水解其α-1,4糖苷键的一类工业酶。淀粉酶约占酶市场的25％左右，涵盖了许多工业过程，如制糖、纺织、洗涤剂、造纸、酿造、蒸馏、制药应用及饲料添加剂等。因此，合理开发利用α-淀粉酶对淀粉酶工业至关重要。
在各种α-淀粉酶中，耐热α-淀粉酶因其具有高热稳定性和更长的保质期而被广泛开发用于全球淀粉工业。特别是，这些酶被广泛用于液化过程中，将淀粉转化为葡萄糖或果糖糖浆。而在淀粉加工过程中，首先要将高浓度的淀粉高温处理后糊化，糊化后的淀粉是一种粘度非常大的固体状态，此时必须迅速液化，否则粘度会急剧上升，故耐热α-淀粉酶主要用于淀粉的高温喷射式液化的淀粉加工生产工艺方面。目前来源于嗜热古菌和芽孢杆菌的耐热的α-淀粉酶已经能够满足工业生产需求，利用蛋白质工程的手段对α-淀粉酶进行分子改良以提高其表达量是降低α-淀粉酶生产成本的有效途径。
发明内容
本发明的目的是提供一种以来源于地衣芽孢杆菌Bacillus licheniformis的α-淀粉酶作为母本经点突变获得的突变体。
本发明的再一目的是提供编码上述突变体的基因。
本发明的再一目的是提供包含上述突变体基因的重组载体。
本发明的再一目的是提供包含上述突变体基因的重组菌株。
根据本发明的具体实施方式，对氨基酸序列如SEQ ID NO:1所示的野生型α-淀粉酶进行定点突变。
SEQ ID NO:1
VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKAISQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKAGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWYWYHFDGTDWDESRKLNRIYKFQGKTWDWEVSNEFGNYDYLMYADFDYDHPDVVAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWSNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQRKTTVS。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的野生型α-淀粉酶进行441、442位点的突变，分别由谷氨酰胺、天冬酰胺突变为天冬酰胺、组氨酸，从而获得α-淀粉酶突变体。
根据本发明的具有在大肠杆菌中高表达量的α-淀粉酶突变体具有如SEQ ID NO:2所示的氨基酸序列，由486个氨基酸组成。
SEQ ID NO.2：
VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKAISQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKAGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWYWYHFDGTDWDESRKLNRIYKFQGKTWDWEVSNEFGNYDYLMYADFDYDHPDVVAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWSNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRNHAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQRKTTVS。
根据本发明的具体实施方式，还提供了编码上述具有高表达量的α-淀粉酶突变体的基因，核苷酸序列如SEQ ID NO:3所示，共为1458bp。
SEQ ID NO.3：
GTAAATGGCACGCTCATGCAGTATTTTGAATGGTATACTCCGAACGACGGCCAGCATTGGAAACGGTTGCAGAATGATGCGGAACATTTGTCGGATATCGGTATTACGGCCGTCTGGATTCCCCCGGCTTATAAGGCGATCAGCCAGGCTGATGTGGGCTACGGTGCGTACGACCTTTATGATTTGGGGGAGTTTCATCAAAAAGGGACAGTTCGGACAAAGTACGGCACGAAAGGAGAGCTGCAATCTGCTATCAAAAGTCTTCATTCCCGGGACATTAACGTTTACGGGGATGTCGTCATCAACCACAAAGCCGGCGCAGATGCGACCGAAGATGTAACAGCGGTTGAAGTCGATCCCGCAGACCGCAACCGCGTAATTTCGGGAGAACACCTAATTAAAGCCTGGACCCATTTTCATTTTCCGGGGCGCGGGAGCACATACAGCGATTTTAAATGGTATTGGTACCATTTTGACGGAACGGATTGGGACGAGTCCCGAAAGTTGAACCGCATCTATAAGTTTCAAGGCAAGACTTGGGATTGGGAAGTTTCGAATGAGTTCGGCAACTATGATTATTTAATGTATGCCGACTTTGATTATGATCATCCTGATGTCGTAGCAGAGATCAAGAGATGGGGCACTTGGTATGCAAATGAACTGCAATTGGACGGATTCCGTCTTGATGCTGTGAAACACATTAAATTTTCTTTTCTGCGGGATTGGGTTAATCATGTCAGAGAAAAAACGGGGAAGGAAATGTTTACCGTAGCTGAGTACTGGTCGAATGACTTAGGCGCGCTGGAAAACTATTTAAACAAAACAAATTTTAATCATTCCGTGTTTGACGTGCCGCTTCATTACCAGTTCCATGCTGCATCAACACAGGGAGGCGGCTATGATATGAGAAAATTGCTGAACGGTACGGTCGTTTCAAAGCATCCGTTGAAATCCGTTACATTTGTCGATAACCACGATACACAGCCGGGGCAATCCCTTGAGTCGACTGTCCAAACGTGGTTTAAGCCGCTGGCTTACGCTTTTATTCTGACAAGGGAATCTGGATACCCGCAGGTTTTCTACGGCGATATGTACGGGACGAAAGGTGACTCCCAGCGCGAAATTCCTGCATTGAAACACAAAATTGAACCTATCTTAAAAGCGAGGAAACAGTATGCGTACGGAGCCCAGCATGATTATTTCGACCATCACGACATTGTCGGCTGGACTAGGGAAGGCGACAGCTCGGTTGCCAATTCAGGTTTGGCGGCATTGATAACAGACGGACCCGGGGGTGCAAAGCGAATGTATGTCGGCCGCAATCATGCCGGTGAGACATGGCACGACATTACCGGAAACCGTTCGGAACCGGTTGTCATCAATTCAGAAGGCTGGGGAGAGTTTCACGTCAACGGCGGGTCGGTTTCAATTTATGTTCAAAGAAAAACGACCGTTTCT。
根据本发明的具体实施方式，还提供了包含上述α-淀粉酶突变体基因的重组载体，所述重组表达载体的出发载体具体为pET-28a(+)。
根据本发明的具体实施方式，还提供了包含上述α-淀粉酶突变体基因的重组菌株，所述重组菌的出发菌株具体为E.coli BL21(DE3)。
所述重组表达载体具体为pET-28a(+)；所述重组菌株具体为E.coli BL21(DE3)。
根据本发明的制备具有高表达量的α-淀粉酶的方法，包括以下步骤：
1)制备包含上述突变体基因的重组载体；
2)以所述重组载体转化宿主；
3)发酵培养所述宿主，并分离α-淀粉酶。
本发明的α-淀粉酶突变体与野生型α-淀粉酶相比，α-淀粉酶水解酶的最适作用温度一致，均为85℃，突变体的粗酶液的活性约为野生型的15.6倍，且在90℃处理10、20、30分钟后，野生型和突变体的剩余酶活相当。
本发明提供了上述具有高表达量的α-淀粉酶突变体的应用，具体可以应用于能源、食品和饲料领域中。
本发明克服了现有技术的不足，提供了一种高表达量的适于在能源、食品和饲料等领域中应用的α-淀粉酶突变体。本发明提供的突变酶的最适作用温度与野生酶一致，均为85℃。突变酶在90℃下热处理10、20、30分钟后的剩余酶活与野生酶基本相当。突变酶在大肠杆菌中的表达量较突变酶大幅提高，突变酶的粗酶液的活性是野生酶的15.6倍。因此，本发明提供的α-淀粉酶突变体能很好的满足能源、食品和饲料等领域中应用，对降低α-淀粉酶的生产成本有着非常广阔的应用前景。
附图说明
图1显示α-淀粉酶野生型及突变体在大肠杆菌BL21(DE3)中表达后的SDS-PAGE电泳检测结果；
图2显示α-淀粉酶野生型及突变体粗酶液的活性；
图3显示纯化后的α-淀粉酶野生型及突变体的最适温度；
图4显示纯化后的α-淀粉酶野生型及突变体的90℃的热稳定性。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主E.coli BL21(DE3)，表达质粒载体pET-28a(+)。
2、酶类及其它生化试剂：内切酶购自Thermo Scientific公司，连接酶购自Invitrog公司,底物可溶性淀粉购自Sigma公司。其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、大肠杆菌培养基LB(1％蛋白胨、0.5％酵母提取物、1％NaCl，pH自然)。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1、重组菌株BL21(pET-28a(+)-blamy)的制备
1.构建重组菌株BL21(pET-28a(+)-blamy)
由华大基因公司合成α-淀粉酶野生型BlAMY的基因，使用内切酶EcoRⅠ和NotⅠ将blamy基因和载体pET-28a(+)切开，通过重组试剂盒将两者连接，获得重组质粒pET-28a(+)-blamy，并转化克隆宿主大肠杆菌XL10，获得重组大肠杆菌菌株XL10(pET-28a(+)-blamy)。涂布于LB(含50μg/mL Kan)进行筛选。经核酸凝胶电泳验证正确后，将克隆子接入50mL LB培养基中，摇床中过夜培养(37℃)，使用质粒小提中量试剂盒提取质粒。将质粒转入表达宿主大肠杆菌BL21(DE3)后，获得重组大肠杆菌菌株BL21(pET-28a(+)-blamy)。
实施例2、重组菌株BL21(pET-28a(+)-blamy-Q441N/N442H)的制备
1.重组质粒pET-28a(+)-blamy-Q441N/N442H的构建
经优化突变位点设计为将441、442的谷氨酰胺、天冬酰胺突变为天冬酰胺、组氨酸，通过点突变试剂盒的方法引入突变位点。所用引物如表1所示：
表1.突变体α-淀粉酶特异性引物
Figure BDA0003574755900000051
2.构建重组菌株BL21(pET-28a(+)-blamy-Q441N/N442H)
将测序正确的单克隆接种至50mL LB培养基中，摇床中过夜培养(37℃)，使用质粒小提中量试剂盒提取质粒。将质粒转入表达宿主大肠杆菌BL21(DE3)后，并涂布于LB(含50μg/mL Kan)进行筛选。获得重组大肠杆菌菌株BL21(pET-28a(+)-blamy-Q441N/N442H)。
实施例3、α-淀粉酶蛋白野生型BlAMY及突变体Q441N/N442H的获得
1.蛋白BlAMY及Q441N/N442H的诱导表达
将得到的重组表达菌株BL21(pET-28a(+)-blamy)及BL21(pET-28a(+)-blamy-Q441N/N442H)接种至50ml LB培养基中进行种子培养，200rpm，37℃培养16h后，以1％接种量转接至400mL LB培养基中，200rpm，37℃培养2-4h后，测定菌体浓度，用酶标仪读取在波长600nm下的吸光值，达到0.6-0.8时加入IPTG至终浓度为0.5mM,200rpm，16℃进行诱导表达。
2.蛋白BlAMY及Q441N/N442H的纯化
将诱导表达后的菌液12000rpm,10min离心，收集菌体，再用20mM Tris-HCl溶液(pH 7.6)进行重悬，然后超声破碎，离心收集上清。将上清在60℃水浴锅中保温10分钟使部分杂蛋白沉淀，然后12000rpm离心10min收集上清。用镍亲和层析法纯化蛋白，洗脱液为0.4M咪唑，20mMTris-HCl，0.5M NaCl，收集洗脱液，进行SDS-PAGE，BlAMY及Q441N/N442H的破碎上清液(即粗酶液)、破碎上清热处理及纯化结果见图1。Q441N/N442H显示出显著提高的表达量。
实施例4、α-淀粉酶BlAMY及Q441N/N442H粗酶液活性的检测
诱导表达后经过破碎及初步的热处理去除部分杂蛋白后对BlAMY及Q441N/N442H进行纯化及酶活的测定。
酶活的测定方法(DNS(3,5-二硝基水杨酸)法)：将配制的2％可溶性淀粉用pH7.0的缓冲溶液(0.04M Na2HPO4-NaH2PO4)稀释至1％终浓度淀粉溶液作为底物，测量体系包括900μL的底物和100μL适当稀释的酶液，在90℃水浴锅中反应30min，加入1.5mL的DNS试剂终止反应后，置于沸水浴中处理5min，快速冷却至室温后取250μL混合液用酶标仪读取在波长540nm下的吸光值，每组反应设置1个空白对照及3个平行。结果见图2，BlAMY和Q441N/N442H粗酶液的活性分别为35.80、558.07U/mL，Q441N/N442H约为BlAMY的15.6倍。
酶活单位(U)定义：在最适条件下，每分钟水解可溶性淀粉生成1μmoL葡萄糖所需的酶量为一个酶活单位。
实施例5、α-淀粉酶BlAMY及Q441N/N442H的最适作用温度的检测
诱导表达后对BlAMY及Q441N/N442H进行纯化及酶活的测定。将稀释适当倍数的酶液与底物分别在75、70、80、85、90、95、100℃水浴锅中反应30min，测量体系和方法同实施例4。结果见图3，BlAMY及Q441N/N442H的最适作用温度均为85℃，在高于90℃时仍能保持80％以上的活性。
实施例6、α-淀粉酶BlAMY及Q441N/N442H的热稳定性的检测
取纯化后的酶液100μL在90℃下分别热处理0、10、20、30min后，在冰上冷却，而后分别在其最适作用温度下测定其剩余酶活，测量体系和方法同实例4，结果见图4。BlAMY及Q441N/N442H在90℃处理30分钟后均保持75％左右的活性，显示出良好的热稳定性。
序列表
<110> 山东隆科特酶制剂有限公司
<120> 一种表达量提高的α-淀粉酶突变体Q441N/N442H及其编码基因和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 486
<212> PRT
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<400> 1
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Ala Ile Ser
35 40 45
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu
65 70 75 80
Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr
85 90 95
Gly Asp Val Val Ile Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser
115 120 125
Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg
130 135 140
Gly Ser Thr Tyr Ser Asp Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly
145 150 155 160
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln
165 170 175
Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Tyr Asp
180 185 190
Tyr Leu Met Tyr Ala Asp Phe Asp Tyr Asp His Pro Asp Val Val Ala
195 200 205
Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp
210 215 220
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg
225 230 235 240
Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr
245 250 255
Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu
260 265 270
Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr
275 280 285
Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys
290 295 300
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr
305 310 315 320
Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr
325 330 335
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg
340 345 350
Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys
355 360 365
Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro
370 375 380
Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr
385 390 395 400
Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser
405 410 415
Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly
420 425 430
Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His
435 440 445
Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly
450 455 460
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln
465 470 475 480
Arg Lys Thr Thr Val Ser
485
<210> 2
<211> 486
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Ala Ile Ser
35 40 45
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu
65 70 75 80
Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr
85 90 95
Gly Asp Val Val Ile Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser
115 120 125
Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg
130 135 140
Gly Ser Thr Tyr Ser Asp Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly
145 150 155 160
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln
165 170 175
Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Tyr Asp
180 185 190
Tyr Leu Met Tyr Ala Asp Phe Asp Tyr Asp His Pro Asp Val Val Ala
195 200 205
Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp
210 215 220
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg
225 230 235 240
Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr
245 250 255
Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu
260 265 270
Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr
275 280 285
Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys
290 295 300
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr
305 310 315 320
Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr
325 330 335
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg
340 345 350
Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys
355 360 365
Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro
370 375 380
Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr
385 390 395 400
Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser
405 410 415
Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly
420 425 430
Ala Lys Arg Met Tyr Val Gly Arg Asn His Ala Gly Glu Thr Trp His
435 440 445
Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly
450 455 460
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln
465 470 475 480
Arg Lys Thr Thr Val Ser
485
<210> 3
<211> 1458
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtaaatggca cgctcatgca gtattttgaa tggtatactc cgaacgacgg ccagcattgg 60
aaacggttgc agaatgatgc ggaacatttg tcggatatcg gtattacggc cgtctggatt 120
cccccggctt ataaggcgat cagccaggct gatgtgggct acggtgcgta cgacctttat 180
gatttggggg agtttcatca aaaagggaca gttcggacaa agtacggcac gaaaggagag 240
ctgcaatctg ctatcaaaag tcttcattcc cgggacatta acgtttacgg ggatgtcgtc 300
atcaaccaca aagccggcgc agatgcgacc gaagatgtaa cagcggttga agtcgatccc 360
gcagaccgca accgcgtaat ttcgggagaa cacctaatta aagcctggac ccattttcat 420
tttccggggc gcgggagcac atacagcgat tttaaatggt attggtacca ttttgacgga 480
acggattggg acgagtcccg aaagttgaac cgcatctata agtttcaagg caagacttgg 540
gattgggaag tttcgaatga gttcggcaac tatgattatt taatgtatgc cgactttgat 600
tatgatcatc ctgatgtcgt agcagagatc aagagatggg gcacttggta tgcaaatgaa 660
ctgcaattgg acggattccg tcttgatgct gtgaaacaca ttaaattttc ttttctgcgg 720
gattgggtta atcatgtcag agaaaaaacg gggaaggaaa tgtttaccgt agctgagtac 780
tggtcgaatg acttaggcgc gctggaaaac tatttaaaca aaacaaattt taatcattcc 840
gtgtttgacg tgccgcttca ttaccagttc catgctgcat caacacaggg aggcggctat 900
gatatgagaa aattgctgaa cggtacggtc gtttcaaagc atccgttgaa atccgttaca 960
tttgtcgata accacgatac acagccgggg caatcccttg agtcgactgt ccaaacgtgg 1020
tttaagccgc tggcttacgc ttttattctg acaagggaat ctggataccc gcaggttttc 1080
tacggcgata tgtacgggac gaaaggtgac tcccagcgcg aaattcctgc attgaaacac 1140
aaaattgaac ctatcttaaa agcgaggaaa cagtatgcgt acggagccca gcatgattat 1200
ttcgaccatc acgacattgt cggctggact agggaaggcg acagctcggt tgccaattca 1260
ggtttggcgg cattgataac agacggaccc gggggtgcaa agcgaatgta tgtcggccgc 1320
aatcatgccg gtgagacatg gcacgacatt accggaaacc gttcggaacc ggttgtcatc 1380
aattcagaag gctggggaga gtttcacgtc aacggcgggt cggtttcaat ttatgttcaa 1440
agaaaaacga ccgtttct 1458

Claims (9)

1.一种表达量提高的α-淀粉酶突变体，其特征在于，所述α-淀粉酶突变体的氨基酸序列如SEQ ID NO:2所示。
2.一种α-淀粉酶基因，其特征在于，编码权利要求1所述的表达量提高的α-淀粉酶突变体。
3.根据权利要求2所述的α-淀粉酶基因，其特征在于，所述α-淀粉酶基因的核苷酸序列如SEQ ID NO:3所示。
4.包含权利要求2所述的α-淀粉酶基因的重组载体。
5.包含权利要求2所述的α-淀粉酶基因的重组菌株。
6.一种制备高表达量的α-淀粉酶的方法，其特征在于，所述方法包括以下步骤：
1)制备包含权利要求2所述α-淀粉酶基因的重组载体；
2)以步骤1)所得重组载体转化宿主细胞；
3)发酵培养所述宿主细胞，并分离α-淀粉酶。
7.一种提高α-淀粉酶的表达量的方法，其特征在于，所述方法包括以下步骤：
将氨基酸序列如SEQ ID NO：1所示的野生型α-淀粉酶进行Q441N/N442H两点突变。
8.权利要求1所述表达量提高的α-淀粉酶突变体的应用。
9.权利要求1所述表达量提高的α-淀粉酶突变体在能源、食品和饲料方面的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111601887A (zh) * 2017-12-08 2020-08-28 诺维信公司 α-淀粉酶变体以及对其进行编码的多核苷酸

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111601887A (zh) * 2017-12-08 2020-08-28 诺维信公司 α-淀粉酶变体以及对其进行编码的多核苷酸

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIANGRONG WU等: "Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423" *
罗丹等: "地衣芽胞杆菌 α - 淀粉酶酸性 pH稳定性提升突变体的生化特征" *
顾燕等: "耐高温 α-淀粉酶分子结构与异源表达研究进展" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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