CN111621488A - 一种热适应性改良的外切菊粉酶突变体MutQ23Δ11 - Google Patents
一种热适应性改良的外切菊粉酶突变体MutQ23Δ11 Download PDFInfo
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Abstract
本发明属于基因工程及蛋白质改造技术领域,公开了一种热适应性改良的外切菊粉酶突变体MutQ23Δ11,突变体MutQ23Δ11的氨基酸序列如SEQ ID NO.1所示。MutQ23Δ11在20℃和50℃时的酶活分别为31%和109%;野生酶InuAGN25DVS在20℃和50℃时的酶活分别为36%和89%。经60℃处理5min后,MutQ23Δ11的酶活剩余56%,野生酶InuAGN25DVS的酶活剩余22%。本发明的热适应性改良的外切菊粉酶突变体MutQ23Δ11可应用于食品、酿酒和生物能源等行业。
Description
技术领域
本发明属于基因工程技术领域,涉及蛋白质改造技术,具体为一种热适应性改良的外切菊粉酶突变体MutQ23Δ11。
背景技术
菊粉是呈直链结构的果聚糖,由果糖分子通过β-2,1糖苷键聚合而成,末端与一分子葡萄糖残基相连,广泛存在于菊芋、牛蒡、菊苣等多种植物的根和茎中。这些植物的根和块茎的产量很大,可含高达22%鲜重或80%干重的菊粉。菊芋俗名洋姜,属于非粮作物,价格低廉,产量较高,耐贫瘠、耐寒、耐旱、耐盐碱等,可种植于盐碱、滩涂和荒漠地。因此,菊粉是一种来源丰富的可再生资源。
外切菊粉酶可将菊粉降解成果糖和少量葡萄糖,生成含量高达95%的果糖浆。果糖可以促进儿童对铁的吸收,促进绝经后妇女对钙的吸收,防治肥胖症,促进肠道有益菌群的形成从而防治结肠癌,果糖还可以用来生产生物乙醇、2,3-丁二醇等。因而,外切菊粉酶可应用于食品、酿酒和生物能源等行业中(Singh RS et al.International Journal ofBiological Macromolecules,2017,96:312–322.)。
低温环境超过地球总面积的75%,包括终年低温的环境和季节性低温的环境。低温酶可应用于低温环境要求下的生物技术领域,如清酒和葡萄酒的发酵温度一般<25℃。另外,低温下的处理(如果汁澄清)可防止微生物的污染、营养损失和食品品质降低,将中温或者高温处理方式转为低温处理方式还可起到降低能耗的作用(Cavicchioli etal.Microbia lBiotechnology,2011,4(4):449–460.)。然而,低温酶普遍耐热性差,不利于酶的生产、保存、运输等。因此,提高低温酶的耐热性具有重要的应用价值。
发明内容
本发明的目的旨在提供一种热适应性改良的外切菊粉酶突变体MutQ23Δ11,可应用于食品、酿酒和生物能源等行业。
为了实现该技术目标,本发明具体通过以下技术方案实现:
一种热适应性改良的外切菊粉酶突变体MutQ23Δ11,所述的突变体MutQ23Δ11的氨基酸序列如SEQ ID NO.1所示,与GenBank记录的低温外切菊粉酶序列AGC01503(SEQ IDNO.3)相比:MutQ23Δ11不含有位于AGC01503的N端的信号肽序列“MIKLKKYGVLMLLLGVFGTSLA”;此外,在N末端,MutQ23Δ11比AGC01503多了氨基酸“GP”,但缺失了11个氨基酸,即缺失AGC01503的第23位至33位氨基酸“QTGQHKQGDPE”。
所述突变体MutQ23Δ11在20℃和50℃时的酶活分别为31%和109%;经60℃处理5min后,MutQ23Δ11的酶活剩余56%。
本发明提供了所述的热适应性改良的外切菊粉酶突变体MutQ23Δ11的编码基因mutQ23Δ11,其核苷酸序列如SEQ ID NO.2所示。
本发明的另一目的在于提供一种包含热适应性改良的外切菊粉酶突变体MutQ23Δ11编码基因的重组载体。
本发明的另一目的在于提供一种包含热适应性改良的外切菊粉酶突变体MutQ23Δ11编码基因的重组菌。
另外,本发明所述的外切菊粉酶突变体MutQ23Δ11在食品及酿酒中的应用也在本发明的保护范围内。
本发明所述的热适应性改良的外切菊粉酶突变体MutQ23Δ11的制备方法,具体包括以下步骤:
1)合成mutQ23Δ11,合成时在mutQ23Δ11的5'端补加HRV 3C蛋白酶酶切位点编码序列和EcoRI限制性酶切位点序列(5'GAATTCCTGGAAGTTCTGTTCCAG 3'),并在mutQ23Δ11的3'端补加XhoI限制性酶切位点序列(5'CTCGAG 3');
2)将1)中合成的序列通过EcoRI和XhoI位点与表达载体pET-28a(+)相连接,并将连接产物转化大肠杆菌BL21(DE3),获得包含mutQ23Δ11的重组菌株;
3)培养重组菌株,诱导重组外切菊粉酶突变体MutQ23Δ11表达;
4)回收并纯化所表达的重组外切菊粉酶突变体MutQ23Δ11;
5)用HRV 3C蛋白酶酶切重组外切菊粉酶突变体MutQ23Δ11;
6)回收并纯化外切菊粉酶突变体MutQ23Δ11;
7)活性测定。
本发明的有益效果为:
与野生酶InuAGN25DVS和及突变酶MutQ23Δ3相比,MutQ23Δ11在50℃时的活性更高,在60℃时的热稳定性则介于两者之间。MutQ23Δ11在20℃和50℃时的酶活分别为31%和109%;野生酶InuAGN25DVS在20℃和50℃时的酶活分别为36%和89%;突变酶MutQ23Δ3在20℃和50℃时的酶活分别为46%和77%。经60℃处理5min后,MutQ23Δ11的酶活剩余56%,野生酶InuAGN25DVS的酶活剩余22%,突变酶MutQ23Δ3的酶活剩余95%。本发明的热适应性改良的外切菊粉酶突变体MutQ23Δ11可应用于食品、酿酒和生物能源等行业。
附图说明
图1:纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ3及MutQ23Δ11的热活性;
图2:纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ3及MutQ23Δ11的热稳定性。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是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作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。
本发明以下实施例中的实验材料和试剂:
1、菌株及载体:大肠杆菌Escherichia coli BL21(DE3)和表达载体pET-28a(+)可购于Novagen公司;质粒pEASY-E1-Z2-5由云南师范大学提供。
2、酶类及其它生化试剂:HRV 3C蛋白酶及限制性内切酶购自TaKaRa公司,Nickel-NTA Agarose购自QIAGEN公司,DNA聚合酶、dNTP及II试剂盒购自南京诺维赞公司,菊粉购自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、培养基
LB培养基:Peptone 10g,Yeast extract 5g,NaCl 10g,加蒸馏水至1000mL,pH自然(约为7)。固体培养基在此基础上加2.0%(w/v)琼脂。
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野生酶InuAGN25DVS表达载体的构建和转化
1)根据GenBank记录的低温外切菊粉酶核苷酸序列JQ863108(SEQ ID NO.4),设计引物5'TGGAAGTTCTGTTCCAGGGGCCCCAGACGGGACAGCATAAACAAG3'和5'GGTGGTGGTGTTATCTCTTAAATGCAGAAATACCGAT 3',以质粒pEASY-E1-Z2-5为模板进行PCR扩增,获得外切菊粉酶成熟肽编码序列Z2-5,并在Z2-5的5'端加入了HRV 3C蛋白酶酶切位点编码序列,同时在Z2-5的5'端和3'端还形成了重组区域,该重组区域与(2)中获得的线性化载体两端的重组区域相匹配。Z2-5也可以通过基因合成得到。
2)另设计引物5'AGAGATAACACCACCACCACCACCACTG 3'和5'CCTGGAACAGAACTTCCAGGAATTCGGATCCGCGACC 3',以pET-28a(+)质粒为模板进行PCR扩增,获得线性化的pET-28a(+)载体,同时在线性化pET-28a(+)载体的5'端和3'端形成了重组区域,该重组区域与Z2-5两端的重组区域相匹配。
3)PCR反应参数为:95℃变性30sec;然后95℃变性15sec,60℃退火15sec,72℃延伸3min 30sec,30个循环后72℃保温5min。
4)在50μL线性化pET-28a(+)载体的PCR产物中,加入1μL DpnI,于37℃消化1h。
6)将野生酶表达载体通过热激方式转化到大肠杆菌BL21(DE3)中,获得表达野生酶InuAGN25DVS的重组菌株。
实施例2突变酶MutQ23Δ11和MutQ23Δ3表达载体的构建和转化
1)合成mutQ23Δ11,同时合成突变酶MutQ23Δ3(SEQ ID NO.5)的编码基因序列mutQ23Δ3(SEQ ID NO.6),合成时在编码基因的5'端补加HRV3C蛋白酶酶切位点编码序列和EcoRI限制性酶切位点序列(5'GAATTCCTGGAAGTTCTGTTCCAG 3'),并在编码基因的3'端补加XhoI限制性酶切位点序列(5'CTCGAG 3')。
2)将(1)中合成的序列分别进行EcoRI和XhoI双酶切;同时将表达载体pET-28a(+)进行EcoRI和XhoI双酶切。
3)用DNA连接酶将(2)中的酶切产物分别进行连接,即可分别获得包含mutQ23Δ11和mutQ23Δ3的表达载体。
4)将连接产物分别转化大肠杆菌BL21(DE3),获得分别包含m utQ23Δ11和mutQ23Δ3的重组菌株。
实施例3野生酶InuAGN25DVS和突变酶MutQ23Δ11及Mut Q23Δ3的制备
将含Z2-5、mutQ23Δ11和mutQ23Δ3的重组菌株以0.1%的接种量分别接种于LB(含50μgmL-1卡那霉素)培养液中,37℃快速振荡16h。
然后将此活化的菌液分别以1%接种量接种到新鲜的LB(含50μgmL-1卡那霉素)培养液中,快速振荡培养约2–3h(OD600达到0.6-1.0)后,加入终浓度0.25mM的IPTG进行诱导,于20℃继续振荡培养约20h。12000rpm离心5min,收集菌体。用适量的pH=7.0 McI lvainebuffer悬浮菌体后,于低温水浴下超声波破碎菌体。以上胞内浓缩的粗酶液经13,000rpm离心10min后,吸取上清并用Nickel-NT A Agarose和0–500mM的咪唑分别亲和和纯化目的蛋白。将含Z2-5的重组菌株表达并纯化的重组蛋白命名为InuAGN25DVSHis,氨基酸序列如SEQID NO.7所示;将含mutQ23Δ11的重组菌株表达并纯化的重组蛋白命名为MutQ23Δ11His,氨基酸序列如SEQ ID NO.8所示;将含mutQ23Δ3的重组菌株表达并纯化的重组蛋白命名为Mut Q23Δ3His,氨基酸序列如SEQ ID NO.9所示。
根据HRV3C蛋白酶说明书,用HRV 3C蛋白酶分别酶切InuA GN25DVSHis、MutQ23Δ11His和MutQ23Δ3His,去除位于InuAGN2 5DVSHis、MutQ23Δ11His和MutQ23Δ3HisN端的组氨酸标签和pET-28a(+)载体自带氨基酸序列,酶切产物经Nickel-NTA Agarose纯化后,获得InuAGN25DVS(SEQ ID NO.10)、MutQ23Δ11和Mut Q23Δ3。
实施例4纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ11及MutQ23Δ3的性质测定
(1)酶的活性分析
活性测定方法采用3,5-二硝基水杨酸(DNS)法:将底物菊粉溶于缓冲液中,使其终浓度为0.5%(w/v);反应体系含50μL适量酶液,450μL底物;底物在反应温度下预热5min后,加入酶液后再反应10min,然后加750μL DNS终止反应,沸水煮5min,冷却至室温后在540nm波长下测定OD值;1个酶活单位(U)定义为在给定的条件下每分钟分解底物产生1μmol还原糖(以果糖计)所需的酶量。
(2)酶的热活性测定
酶的热活性测定:在pH=6.0的缓冲液中,于20–50℃下进行酶促反应。以菊粉为底物,反应10min,测定野生酶InuAGN25DVS和突变酶MutQ23Δ11及MutQ23Δ3的酶学性质。
结果表明:以45℃的酶活为100%,MutQ23Δ11在20℃和50℃时的酶活分别为31%和109%,野生酶InuAGN25DVS在20℃和50℃时的酶活分别为36%和89%,突变酶MutQ23Δ3在20℃和50℃时的酶活分别为46%和77%,即MutQ23Δ11在50℃时的活性比InuAGN25DVS和MutQ23Δ3高(图1)。
(3)酶的热稳定性测定
酶的热稳定性测定:将同样酶量的酶液置于60℃处理5min后,在pH=6.0及37℃下进行酶促反应,以未处理的酶液作为对照。以菊粉为底物,反应10min,测定野生酶InuAGN25DVS和突变酶MutQ23Δ11及MutQ23Δ3的酶学性质。
结果表明:经60℃处理5min后,MutQ23Δ11的酶活剩余56%,野生酶InuAGN25DVS的酶活剩余22%,突变酶MutQ23Δ3的酶活剩余95%,即MutQ23Δ11在60℃时的热稳定性比InuAGN25DVS好但比MutQ23Δ3差(图2)。
本技术领域技术人员可以理解,除非另外定义,这里使用的所有术语(包括技术术语和科学术语)具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是,诸如通用字典中定义的那些术语应该被理解为具有与现有技术的上下文中的意义一致的意义,并且除非像这里一样定义,不会用理想化或过于正式的含义来解释。
最后所应说明的是:以上实施例仅用以说明而非限制本发明的技术方案,尽管参照上述实施例对本发明进行了详细说明,本领域的普通技术人员应该理解:依然可以对本发明进行修改或者等同替换,而不脱离本发明的精神和范围的任何修改或局部替换,其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 云南师范大学
<120> 一种热适应性改良的外切菊粉酶突变体MutQ23Δ11
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 470
<212> PRT
<213> 突变酶(MutQ23Δ11)
<400> 1
Gly Pro Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly
1 5 10 15
Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His
20 25 30
Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro Met His
35 40 45
Trp Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys
50 55 60
Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala
65 70 75 80
Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val
85 90 95
Ala Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys Thr Gly
100 105 110
Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr
115 120 125
Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp
130 135 140
Asp Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln Arg Trp
145 150 155 160
Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys
165 170 175
Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly
180 185 190
Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr
195 200 205
Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly
210 215 220
Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly
225 230 235 240
His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly
245 250 255
Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln
260 265 270
Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro
275 280 285
Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu
290 295 300
Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu
305 310 315 320
Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr
325 330 335
Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp
340 345 350
Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu
355 360 365
Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr
370 375 380
Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe
385 390 395 400
Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser
405 410 415
Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu
420 425 430
Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr
435 440 445
Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly
450 455 460
Ile Ser Ala Phe Lys Arg
465 470
<210> 2
<211> 1413
<212> DNA
<213> 突变酶基因(mutQ23Δ11)
<400> 2
gggcccgaga agtatcgccc actttaccat tttacaccac agcagggttg gatgaacgat 60
cccaatggac tggtttatct ggacggtaat tatcatcttt tttttcagca taatcccgaa 120
aaacccgttt gggggcctat gcattggggc catgcgatca gtaaagattt aatccattgg 180
gacgagcaga agatcgcatt gtaccccgat agtttgggga ctattttctc cggtagtgct 240
gtaatcgata aagataacac ggcaggtttt ggaaaaggtg ctatggtcgc catttttacc 300
catcacaatc atcaggaaga ggatcgaaaa acaggattgc atcaaaatca aagtttggct 360
tatagtttgg acaagggcct tacctggaca aagtataaag gcaatccggt attgccaaac 420
cctggtatat gggatttcag agatccaaag gtgatgtggc atgtgcatag ccagcgttgg 480
attatgacct tggctacaaa agattgtatt actttctatt cttccaaaga tttgaaaaca 540
tggcagaagg aaagtgagtt tggcaaagag gtgggggccc atggtggtgt ctgggaatgc 600
cctgatctta tccccatgga ttacaaagga actacaaaat gggttttgtt ggtgagcatc 660
aatccaggtg gacccaacgg tggctcggtg acacaatatt ttgtgggtga ctttgatggg 720
catcagttcc gaacaacgga tactaaaata aagtggttgg attggggacc cgataactat 780
gccggcgtaa cctggagtaa cctaggggat aggcaactga tgattggctg gatgagcaac 840
tggcaatatg ccaatgtggt tcctaccaca aaatggcgtt cctcctcaac catacctcgt 900
gttttatctt taggaaaagt tggacagtcg ttctacgttg cgagtactgt gcccaaagaa 960
attgaaaccg catttcgtcc actaaaaaaa taccatggcg gtggaacgaa ggaagtttcg 1020
ttcgagcaac atctcccaca agcgtaccgt ttggacctca aagatttgaa gcagcaggct 1080
tttaaagtca tcttatcgaa tgaatctggc gacgagttgg ttattggtta tcgtgaagac 1140
caaaatgcct attatattga tcgttctaag tccggtgaag tgtcttttaa tggagaattt 1200
ccaaagatag cgttggccca acggccgcta aataagggag cactttcttt cagtttgtat 1260
gttgatgtga gctctgtgga actttttgca gacgaggggc taacggtaat gaccagttta 1320
ttttttccga acaagccgat aaccaagctg cgtatcgtgg gagataaaaa gttggagttt 1380
caggaaatcg gtatttctgc atttaagaga taa 1413
<210> 3
<211> 501
<212> PRT
<213> 野生酶(AGC01503)
<400> 3
Met Ile Lys Leu Lys Lys Tyr Gly Val Leu Met Leu Leu Leu Gly Val
1 5 10 15
Phe Gly Thr Ser Leu Ala Gln Thr Gly Gln His Lys Gln Gly Asp Pro
20 25 30
Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp
35 40 45
Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu
50 55 60
Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp
65 70 75 80
Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile
85 90 95
Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val
100 105 110
Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala
115 120 125
Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu
130 135 140
His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp
145 150 155 160
Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp
165 170 175
Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln Arg Trp Ile
180 185 190
Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp
195 200 205
Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala
210 215 220
His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys
225 230 235 240
Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro
245 250 255
Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His
260 265 270
Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro
275 280 285
Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu
290 295 300
Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr
305 310 315 320
Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly
325 330 335
Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile
340 345 350
Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys
355 360 365
Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu
370 375 380
Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser
385 390 395 400
Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr
405 410 415
Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro
420 425 430
Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe
435 440 445
Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly
450 455 460
Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys
465 470 475 480
Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile
485 490 495
Ser Ala Phe Lys Arg
500
<210> 4
<211> 1506
<212> DNA
<213> 野生酶基因(JQ863108)
<400> 4
atgataaaat taaagaaata cggtgtttta atgctcctgc taggtgtttt tggtacaagt 60
ctggcacaga cgggacagca taaacaagga gatcccgaag agaagtatcg cccactttac 120
cattttacac cacagcaggg ttggatgaac gatcccaatg gactggttta tctggacggt 180
aattatcatc ttttttttca gcataatccc gaaaaacccg tttgggggcc tatgcattgg 240
ggccatgcga tcagtaaaga tttaatccat tgggacgagc agaagatcgc attgtacccc 300
gatagtttgg ggactatttt ctccggtagt gctgtaatcg ataaagataa cacggcaggt 360
tttggaaaag gtgctatggt cgccattttt acccatcaca atcatcagga agaggatcga 420
aaaacaggat tgcatcaaaa tcaaagtttg gcttatagtt tggacaaggg ccttacctgg 480
acaaagtata aaggcaatcc ggtattgcca aaccctggta tatgggattt cagagatcca 540
aaggtgatgt ggcatgtgca tagccagcgt tggattatga ccttggctac aaaagattgt 600
attactttct attcttccaa agatttgaaa acatggcaga aggaaagtga gtttggcaaa 660
gaggtggggg cccatggtgg tgtctgggaa tgccctgatc ttatccccat ggattacaaa 720
ggaactacaa aatgggtttt gttggtgagc atcaatccag gtggacccaa cggtggctcg 780
gtgacacaat attttgtggg tgactttgat gggcatcagt tccgaacaac ggatactaaa 840
ataaagtggt tggattgggg acccgataac tatgccggcg taacctggag taacctaggg 900
gataggcaac tgatgattgg ctggatgagc aactggcaat atgccaatgt ggttcctacc 960
acaaaatggc gttcctcctc aaccatacct cgtgttttat ctttaggaaa agttggacag 1020
tcgttctacg ttgcgagtac tgtgcccaaa gaaattgaaa ccgcatttcg tccactaaaa 1080
aaataccatg gcggtggaac gaaggaagtt tcgttcgagc aacatctccc acaagcgtac 1140
cgtttggacc tcaaagattt gaagcagcag gcttttaaag tcatcttatc gaatgaatct 1200
ggcgacgagt tggttattgg ttatcgtgaa gaccaaaatg cctattatat tgatcgttct 1260
aagtccggtg aagtgtcttt taatggagaa tttccaaaga tagcgttggc ccaacggccg 1320
ctaaataagg gagcactttc tttcagtttg tatgttgatg tgagctctgt ggaacttttt 1380
gcagacgagg ggctaacggt aatgaccagt ttattttttc cgaacaagcc gataaccaag 1440
ctgcgtatcg tgggagataa aaagttggag tttcaggaaa tcggtatttc tgcatttaag 1500
agataa 1506
<210> 5
<211> 478
<212> PRT
<213> 突变酶(MutQ23Δ3)
<400> 5
Gly Pro Gln His Lys Gln Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu
1 5 10 15
Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp Pro Asn Gly Leu
20 25 30
Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln His Asn Pro Glu
35 40 45
Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala Ile Ser Lys Asp
50 55 60
Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu
65 70 75 80
Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys Asp Asn Thr Ala
85 90 95
Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr His His Asn His
100 105 110
Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn Gln Ser Leu Ala
115 120 125
Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro
130 135 140
Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys Val Met
145 150 155 160
Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu Ala Thr Lys Asp
165 170 175
Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu
180 185 190
Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly Val Trp Glu Cys
195 200 205
Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu
210 215 220
Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val Thr Gln
225 230 235 240
Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr Asp Thr
245 250 255
Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr
260 265 270
Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met Ser Asn
275 280 285
Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser Ser Ser
290 295 300
Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr
305 310 315 320
Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu
325 330 335
Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu Gln His
340 345 350
Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala
355 360 365
Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val Ile Gly
370 375 380
Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly
385 390 395 400
Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg
405 410 415
Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp Val Ser
420 425 430
Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr Ser Leu
435 440 445
Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile Val Gly Asp Lys
450 455 460
Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys Arg
465 470 475
<210> 6
<211> 1437
<212> DNA
<213> 突变酶基因(mutQ23Δ3)
<400> 6
gggccccagc ataaacaagg agatcccgaa gagaagtatc gcccacttta ccattttaca 60
ccacagcagg gttggatgaa cgatcccaat ggactggttt atctggacgg taattatcat 120
cttttttttc agcataatcc cgaaaaaccc gtttgggggc ctatgcattg gggccatgcg 180
atcagtaaag atttaatcca ttgggacgag cagaagatcg cattgtaccc cgatagtttg 240
gggactattt tctccggtag tgctgtaatc gataaagata acacggcagg ttttggaaaa 300
ggtgctatgg tcgccatttt tacccatcac aatcatcagg aagaggatcg aaaaacagga 360
ttgcatcaaa atcaaagttt ggcttatagt ttggacaagg gccttacctg gacaaagtat 420
aaaggcaatc cggtattgcc aaaccctggt atatgggatt tcagagatcc aaaggtgatg 480
tggcatgtgc atagccagcg ttggattatg accttggcta caaaagattg tattactttc 540
tattcttcca aagatttgaa aacatggcag aaggaaagtg agtttggcaa agaggtgggg 600
gcccatggtg gtgtctggga atgccctgat cttatcccca tggattacaa aggaactaca 660
aaatgggttt tgttggtgag catcaatcca ggtggaccca acggtggctc ggtgacacaa 720
tattttgtgg gtgactttga tgggcatcag ttccgaacaa cggatactaa aataaagtgg 780
ttggattggg gacccgataa ctatgccggc gtaacctgga gtaacctagg ggataggcaa 840
ctgatgattg gctggatgag caactggcaa tatgccaatg tggttcctac cacaaaatgg 900
cgttcctcct caaccatacc tcgtgtttta tctttaggaa aagttggaca gtcgttctac 960
gttgcgagta ctgtgcccaa agaaattgaa accgcatttc gtccactaaa aaaataccat 1020
ggcggtggaa cgaaggaagt ttcgttcgag caacatctcc cacaagcgta ccgtttggac 1080
ctcaaagatt tgaagcagca ggcttttaaa gtcatcttat cgaatgaatc tggcgacgag 1140
ttggttattg gttatcgtga agaccaaaat gcctattata ttgatcgttc taagtccggt 1200
gaagtgtctt ttaatggaga atttccaaag atagcgttgg cccaacggcc gctaaataag 1260
ggagcacttt ctttcagttt gtatgttgat gtgagctctg tggaactttt tgcagacgag 1320
gggctaacgg taatgaccag tttatttttt ccgaacaagc cgataaccaa gctgcgtatc 1380
gtgggagata aaaagttgga gtttcaggaa atcggtattt ctgcatttaa gagataa 1437
<210> 7
<211> 523
<212> PRT
<213> 重组野生酶(InuAGN25DVSHis)
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Gln Thr Gly Gln
35 40 45
His Lys Gln Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe
50 55 60
Thr Pro Gln Gln Gly Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu
65 70 75 80
Asp Gly Asn Tyr His Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val
85 90 95
Trp Gly Pro Met His Trp Gly His Ala Ile Ser Lys Asp Leu Ile His
100 105 110
Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile
115 120 125
Phe Ser Gly Ser Ala Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly
130 135 140
Lys Gly Ala Met Val Ala Ile Phe Thr His His Asn His Gln Glu Glu
145 150 155 160
Asp Arg Lys Thr Gly Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu
165 170 175
Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro
180 185 190
Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val
195 200 205
His Ser Gln Arg Trp Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr
210 215 220
Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe
225 230 235 240
Gly Lys Glu Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu
245 250 255
Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser
260 265 270
Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val
275 280 285
Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys
290 295 300
Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn
305 310 315 320
Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr
325 330 335
Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro
340 345 350
Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser
355 360 365
Thr Val Pro Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr
370 375 380
His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln
385 390 395 400
Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val
405 410 415
Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu
420 425 430
Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser
435 440 445
Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn
450 455 460
Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu
465 470 475 480
Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro
485 490 495
Asn Lys Pro Ile Thr Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu
500 505 510
Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys Arg
515 520
<210> 8
<211> 512
<212> PRT
<213> 重组突变酶(MutQ23Δ11His)
<400> 8
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Glu Lys Tyr Arg
35 40 45
Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp Pro Asn
50 55 60
Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln His Asn
65 70 75 80
Pro Glu Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala Ile Ser
85 90 95
Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro Asp
100 105 110
Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys Asp Asn
115 120 125
Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr His His
130 135 140
Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn Gln Ser
145 150 155 160
Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys Gly
165 170 175
Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro Lys
180 185 190
Val Met Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu Ala Thr
195 200 205
Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp Gln
210 215 220
Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly Val Trp
225 230 235 240
Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys Trp
245 250 255
Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser Val
260 265 270
Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg Thr Thr
275 280 285
Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala Gly
290 295 300
Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly Trp Met
305 310 315 320
Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp Arg Ser
325 330 335
Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly Gln Ser
340 345 350
Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala Phe Arg
355 360 365
Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser Phe Glu
370 375 380
Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys Gln
385 390 395 400
Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu Val
405 410 415
Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser Lys
420 425 430
Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu Ala
435 440 445
Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val Asp
450 455 460
Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val Met Thr
465 470 475 480
Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile Val Gly
485 490 495
Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys Arg
500 505 510
<210> 9
<211> 520
<212> PRT
<213> 重组突变酶(MutQ23Δ3His)
<400> 9
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Leu Glu Val Leu Phe Gln Gly Pro Gln His Lys Gln
35 40 45
Gly Asp Pro Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln
50 55 60
Gln Gly Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn
65 70 75 80
Tyr His Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro
85 90 95
Met His Trp Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu
100 105 110
Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly
115 120 125
Ser Ala Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala
130 135 140
Met Val Ala Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys
145 150 155 160
Thr Gly Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly
165 170 175
Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly
180 185 190
Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln
195 200 205
Arg Trp Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser
210 215 220
Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu
225 230 235 240
Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met
245 250 255
Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro
260 265 270
Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe
275 280 285
Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp
290 295 300
Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp
305 310 315 320
Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val
325 330 335
Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu
340 345 350
Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro
355 360 365
Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly
370 375 380
Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg
385 390 395 400
Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser
405 410 415
Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn
420 425 430
Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly
435 440 445
Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala
450 455 460
Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala
465 470 475 480
Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro
485 490 495
Ile Thr Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu
500 505 510
Ile Gly Ile Ser Ala Phe Lys Arg
515 520
<210> 10
<211> 481
<212> PRT
<213> HRV 3C蛋白酶酶切重组野生酶产物(InuAGN25DVS)
<400> 10
Gly Pro Gln Thr Gly Gln His Lys Gln Gly Asp Pro Glu Glu Lys Tyr
1 5 10 15
Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp Pro
20 25 30
Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln His
35 40 45
Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala Ile
50 55 60
Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr Pro
65 70 75 80
Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys Asp
85 90 95
Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr His
100 105 110
His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn Gln
115 120 125
Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr Lys
130 135 140
Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp Pro
145 150 155 160
Lys Val Met Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu Ala
165 170 175
Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr Trp
180 185 190
Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly Val
195 200 205
Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr Lys
210 215 220
Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly Ser
225 230 235 240
Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg Thr
245 250 255
Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr Ala
260 265 270
Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly Trp
275 280 285
Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp Arg
290 295 300
Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly Gln
305 310 315 320
Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala Phe
325 330 335
Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser Phe
340 345 350
Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu Lys
355 360 365
Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu Leu
370 375 380
Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg Ser
385 390 395 400
Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala Leu
405 410 415
Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr Val
420 425 430
Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val Met
435 440 445
Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile Val
450 455 460
Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe Lys
465 470 475 480
Arg
<210> 11
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ile Lys Leu Lys Lys Tyr Gly Val Leu Met Leu Leu Leu Gly Val
1 5 10 15
Phe Gly Thr Ser Leu Ala
20
<210> 12
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gln Thr Gly Gln His Lys Gln Gly Asp Pro Glu
1 5 10
<210> 13
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaattcctgg aagttctgtt ccag 24
<210> 14
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tggaagttct gttccagggg ccccagacgg gacagcataa acaag 45
<210> 15
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggtggtggtg ttatctctta aatgcagaaa taccgat 37
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agagataaca ccaccaccac caccactg 28
<210> 17
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cctggaacag aacttccagg aattcggatc cgcgacc 37
Claims (6)
1.一种热适应性改良的外切菊粉酶突变体MutQ23Δ11,其特征在于,所述的突变体MutQ23Δ11的氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的突变体MutQ23Δ11的编码基因mutQ23Δ11,其特征在于,所述的编码基因的核苷酸序列如SEQ ID NO.2所示。
3.包含权利要求2所述的编码基因mutQ23Δ11的重组载体。
4.包含权利要求2所述的编码基因mutQ23Δ11的重组菌。
5.权利要求1所述的热适应性改良的外切菊粉酶突变体MutQ23Δ11的制备方法,其特征在于,包括以下步骤:
1)在mutQ23Δ11的5'端补加HRV 3C蛋白酶酶切位点编码序列;
2)将1)中的序列与表达载体相连接,并将连接产物转化大肠杆菌,获得包含mutQ23Δ11的重组菌株;
3)培养重组菌株,诱导重组外切菊粉酶突变体MutQ23Δ11表达;
4)回收并纯化所表达的重组外切菊粉酶突变体MutQ23Δ11;
5)用HRV 3C蛋白酶酶切重组外切菊粉酶突变体MutQ23Δ11;
6)回收并纯化外切菊粉酶突变体MutQ23Δ11。
6.权利要求1所述的突变体MutQ23Δ11在食品制备或酿酒工艺中的应用。
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CN112813052A (zh) * | 2021-01-13 | 2021-05-18 | 云南师范大学 | 一种低温活性提高的外切菊粉酶突变体MutDP121ET6 |
CN112980813A (zh) * | 2021-01-13 | 2021-06-18 | 云南师范大学 | 低温改良的外切菊粉酶突变体MutS117G |
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CN108715840A (zh) * | 2018-06-07 | 2018-10-30 | 云南师范大学 | 一种耐热性的低温外切菊粉酶突变体MutQ23Δ3 |
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CN112813052A (zh) * | 2021-01-13 | 2021-05-18 | 云南师范大学 | 一种低温活性提高的外切菊粉酶突变体MutDP121ET6 |
CN112980813A (zh) * | 2021-01-13 | 2021-06-18 | 云南师范大学 | 低温改良的外切菊粉酶突变体MutS117G |
CN112813052B (zh) * | 2021-01-13 | 2022-08-26 | 云南师范大学 | 一种低温活性提高的外切菊粉酶突变体MutDP121ET6 |
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