CN111647579B

CN111647579B - 一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用

Info

Publication number: CN111647579B
Application number: CN202010536907.5A
Authority: CN
Inventors: 周峻沛; 张蕊; 黄遵锡; 何丽梅; 韩楠玉; 唐湘华; 吴倩; 慕跃林
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2022-06-24
Anticipated expiration: 2040-06-12
Also published as: CN111647579A

Abstract

本发明公开了一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用，该突变体MutQ23Δ9具有如SEQ ID NO.1所示的氨基酸序列。与野生酶InuAGN25DVS和突变酶MutQ23Δ3相比，MutQ23Δ9在50℃时活性低，而且在55℃下热稳定性差，其对较高温度更加敏感。在经55℃处理5‑30min后，MutQ23Δ9的酶活从52％下降到5％，野生酶InuAGN25DVS的酶活从71％下降到20％，突变酶MutQ23Δ3的酶活从90％下降到22％。因此，本发明的不耐热的外切菊粉酶突变体MutQ23Δ9更适用于食品、酿酒和生物能源等行业。

Description

一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用

技术领域

本发明涉及一种外切菊粉酶突变体，具体涉及一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用。

背景技术

菊粉广泛存在于菊芋、牛蒡、菊苣等多种植物中。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。

菊粉由果糖分子通过β-2,1糖苷键聚合而成，末端与一分子葡萄糖残基相连。外切菊粉酶逐个水解菊粉中的β-2,1糖苷键，最终将菊粉全部降解成果糖和少量葡萄糖，生成含量高达95％的果糖浆，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.)。

不耐热的酶容易热变性，热变性后酶容易被降解，该特性使酶的催化反应得以简易控制，同时使酶的使用更具安全性，在低温生物技术特别是食品领域具有应用的价值。低温酶常常具有不耐热的特点，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 et al.Microbial Biotechnology,2011,4(4):449–460.)。因此，需要开发低温或不耐热的外切菊粉酶。

发明内容

本发明的目的是提供一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用，对温度敏感，能够应用于食品、酿酒和生物能源等行业。

为了达到上述目的，本发明提供了一种不耐热的外切菊粉酶突变体MutQ23Δ9，该突变体MutQ23Δ9具有如SEQ ID NO.1所示的氨基酸序列。该突变体与GenBank记录的低温外切菊粉酶序列AGC01503(SEQ ID NO.3)相比：MutQ23Δ9不含有位于AGC01503的N端的信号肽序列“MIKLKKYGVLMLLLGVFGTSLA”；此外，在N末端，MutQ23Δ9比AGC01503多了氨基酸“GP”，但缺失了9个氨基酸，即缺失AGC01503的第23位至31位氨基酸“QTGQHKQGD”。

本发明的另一目的是提供一种所述的突变体MutQ23Δ9的编码基因mutQ23Δ9。

优选地，该编码基因mutQ23Δ9具有如SEQ ID NO.2所示的核苷酸序列。

本发明的另一目的是提供一种包含所述的编码基因mutQ23Δ9的重组载体。

本发明的另一目的是提供一种包含所述的编码基因mutQ23Δ9的重组菌。

本发明的另一目的是提供一种所述的突变体MutQ23Δ9的制备方法，该方法包含：

在所述的mutQ23Δ9的5'端补加HRV3C蛋白酶酶切位点编码序列；

将所述的mutQ23Δ9的核苷酸序列与表达载体pET-28a(+)相连接，并将连接产物转化大肠杆菌BL21(DE3)，获得包含mutQ23Δ9的重组菌株；

培养重组菌株，诱导重组外切菊粉酶突变体MutQ23Δ9表达，将得到的重组外切菊粉酶突变体MutQ23Δ9用HRV3C蛋白酶酶切，以获得外切菊粉酶突变体MutQ23Δ9。

优选地，所述培养重组菌株，采用含50μg·mL^-1卡那霉素的LB培养液。

优选地，所述培养重组菌株，在37℃在含50μg·mL^-1卡那霉素的LB培养液中振荡培养。

优选地，所述诱导，当OD₆₀₀达到0.6～1.0时，加入IPTG进行诱导。

本发明的另一目的是提供所述的突变体MutQ23Δ9在食品或酿酒中的应用。

本发明的不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用，具有以下优点：

与野生酶InuAGN25DVS和突变酶MutQ23Δ3相比，MutQ23Δ9在50℃时活性低，而且在55℃下热稳定性差，其对较高温度更加敏感。MutQ23Δ9在20℃和50℃时的酶活分别为45％和11％，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％。在经55℃处理5min–30min后，MutQ23Δ9的酶活从52％下降到5％，野生酶InuAGN25DVS的酶活从71％下降到20％，突变酶MutQ23Δ3的酶活从90％下降到22％。因此，本发明的不耐热的外切菊粉酶突变体MutQ23Δ9更适用于食品、酿酒和生物能源等行业。

附图说明

图1为纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ3及MutQ23Δ9的热活性。

图2为纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ3及MutQ23Δ9的热稳定性。

具体实施方式

下面将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。

本发明以下实验例中使用的实验材料和试剂：

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

HRV3C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Clon

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。

实验例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'-TGGAAGTTCTGTTCCAGGGGCCCCAGACGGGACAGCATAAACAAG-3'(SEQ ID NO.11)和5'-GGTGGTGGTGTTATCTCTTAAATGCAGAAATACCGAT-3'(SEQ ID NO.12)，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，该重组区域与下述(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)设计引物5'-AGAGATAACACCACCACCACCACCACTG-3'(SEQ ID NO.13)和5'-CCTGGAACAGAACTTCCAGGAATTCGGATCCGCGACC-3'(SEQ ID NO.14)，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3min30sec，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。

5)根据Clon

II试剂盒的说明书，将(4)中的消化产物和Z2-5在37℃下重组连接30min，即可获得野生酶InuAGN25DVS的表达载体。

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。

实验例2突变酶MutQ23Δ9和MutQ23Δ3表达载体的构建和转化

(1)合成mutQ23Δ9，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mutQ23Δ9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mutQ23Δ9和mutQ23Δ3的重组菌株。

实验例3野生酶InuAGN25DVS和突变酶MutQ23Δ9及MutQ23Δ3的制备

将含Z2-5、mutQ23Δ9和mutQ23Δ3的重组菌株以0.1％的接种量分别接种于LB(含50μg mL^-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50μg mL^-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＝7.0McIlvainebuffer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-NTAAgarose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SEQ ID NO.7所示；将含mutQ23Δ9的重组菌株表达并纯化的重组蛋白命名为MutQ23Δ9His，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MutQ23Δ3His，氨基酸序列如SEQ IDNO.9所示。

根据HRV 3C蛋白酶说明书，用HRV 3C蛋白酶分别酶切InuAGN25DVSHis、MutQ23Δ9His和MutQ23Δ3His，去除位于InuAGN25DVSHis、MutQ23Δ9His和MutQ23Δ3His N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-NTAAgarose纯化后，获得InuAGN25DVS(SEQ ID NO.10)、MutQ23Δ9和MutQ23Δ3。

实验例4纯化的野生酶InuAGN25DVS和突变酶MutQ23Δ9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750μLDNS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MutQ23Δ9及MutQ23Δ3的酶学性质。

结果表明：以45℃的酶活为100％，MutQ23Δ9在20℃和50℃时的酶活(相对酶活)分别为45％和11％，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MutQ23Δ9在50℃时的活性比InuAGN25DVS和MutQ23Δ3低(参见图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55℃处理5min–30min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MutQ23Δ9及MutQ23Δ3的酶学性质。

结果表明：经55℃处理5min–30min后，MutQ23Δ9的酶活从52％下降到5％，野生酶InuAGN25DVS的酶活从71％下降到20％，突变酶MutQ23Δ3的酶活从90％下降到22％，即MutQ23Δ9在55℃时的热稳定性比InuAGN25DVS和MutQ23Δ3差(参见图2)。

尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。

序列表

<110> 云南师范大学

<120> 一种不耐热的外切菊粉酶突变体MutQ23Δ9及其制备和应用

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 472

<212> PRT

<213> 突变酶(MutQ23Δ9)

<400> 1

Gly Pro Pro Glu Glu Lys Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln

1 5 10 15

Gln Gly Trp Met Asn Asp Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn

20 25 30

Tyr His Leu Phe Phe Gln His Asn Pro Glu Lys Pro Val Trp Gly Pro

35 40 45

Met His Trp Gly His Ala Ile Ser Lys Asp Leu Ile His Trp Asp Glu

50 55 60

Gln Lys Ile Ala Leu Tyr Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly

65 70 75 80

Ser Ala Val Ile Asp Lys Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala

85 90 95

Met Val Ala Ile Phe Thr His His Asn His Gln Glu Glu Asp Arg Lys

100 105 110

Thr Gly Leu His Gln Asn Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly

115 120 125

Leu Thr Trp Thr Lys Tyr Lys Gly Asn Pro Val Leu Pro Asn Pro Gly

130 135 140

Ile Trp Asp Phe Arg Asp Pro Lys Val Met Trp His Val His Ser Gln

145 150 155 160

Arg Trp Ile Met Thr Leu Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser

165 170 175

Ser Lys Asp Leu Lys Thr Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu

180 185 190

Val Gly Ala His Gly Gly Val Trp Glu Cys Pro Asp Leu Ile Pro Met

195 200 205

Asp Tyr Lys Gly Thr Thr Lys Trp Val Leu Leu Val Ser Ile Asn Pro

210 215 220

Gly Gly Pro Asn Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp Phe

225 230 235 240

Asp Gly His Gln Phe Arg Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp

245 250 255

Trp Gly Pro Asp Asn Tyr Ala Gly Val Thr Trp Ser Asn Leu Gly Asp

260 265 270

Arg Gln Leu Met Ile Gly Trp Met Ser Asn Trp Gln Tyr Ala Asn Val

275 280 285

Val Pro Thr Thr Lys Trp Arg Ser Ser Ser Thr Ile Pro Arg Val Leu

290 295 300

Ser Leu Gly Lys Val Gly Gln Ser Phe Tyr Val Ala Ser Thr Val Pro

305 310 315 320

Lys Glu Ile Glu Thr Ala Phe Arg Pro Leu Lys Lys Tyr His Gly Gly

325 330 335

Gly Thr Lys Glu Val Ser Phe Glu Gln His Leu Pro Gln Ala Tyr Arg

340 345 350

Leu Asp Leu Lys Asp Leu Lys Gln Gln Ala Phe Lys Val Ile Leu Ser

355 360 365

Asn Glu Ser Gly Asp Glu Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn

370 375 380

Ala Tyr Tyr Ile Asp Arg Ser Lys Ser Gly Glu Val Ser Phe Asn Gly

385 390 395 400

Glu Phe Pro Lys Ile Ala Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala

405 410 415

Leu Ser Phe Ser Leu Tyr Val Asp Val Ser Ser Val Glu Leu Phe Ala

420 425 430

Asp Glu Gly Leu Thr Val Met Thr Ser Leu Phe Phe Pro Asn Lys Pro

435 440 445

Ile Thr Lys Leu Arg Ile Val Gly Asp Lys Lys Leu Glu Phe Gln Glu

450 455 460

Ile Gly Ile Ser Ala Phe Lys Arg

465 470

<210> 2

<211> 1419

<212> DNA

<213> 突变基因(mutQ23Δ9)

<400> 2

gggccccccg aagagaagta tcgcccactt taccatttta caccacagca gggttggatg 60

aacgatccca atggactggt ttatctggac ggtaattatc atcttttttt tcagcataat 120

cccgaaaaac ccgtttgggg gcctatgcat tggggccatg cgatcagtaa agatttaatc 180

cattgggacg agcagaagat cgcattgtac cccgatagtt tggggactat tttctccggt 240

agtgctgtaa tcgataaaga taacacggca ggttttggaa aaggtgctat ggtcgccatt 300

tttacccatc acaatcatca ggaagaggat cgaaaaacag gattgcatca aaatcaaagt 360

ttggcttata gtttggacaa gggccttacc tggacaaagt ataaaggcaa tccggtattg 420

ccaaaccctg gtatatggga tttcagagat ccaaaggtga tgtggcatgt gcatagccag 480

cgttggatta tgaccttggc tacaaaagat tgtattactt tctattcttc caaagatttg 540

aaaacatggc agaaggaaag tgagtttggc aaagaggtgg gggcccatgg tggtgtctgg 600

gaatgccctg atcttatccc catggattac aaaggaacta caaaatgggt tttgttggtg 660

agcatcaatc caggtggacc caacggtggc tcggtgacac aatattttgt gggtgacttt 720

gatgggcatc agttccgaac aacggatact aaaataaagt ggttggattg gggacccgat 780

aactatgccg gcgtaacctg gagtaaccta ggggataggc aactgatgat tggctggatg 840

agcaactggc aatatgccaa tgtggttcct accacaaaat ggcgttcctc ctcaaccata 900

cctcgtgttt tatctttagg aaaagttgga cagtcgttct acgttgcgag tactgtgccc 960

aaagaaattg aaaccgcatt tcgtccacta aaaaaatacc atggcggtgg aacgaaggaa 1020

gtttcgttcg agcaacatct cccacaagcg taccgtttgg acctcaaaga tttgaagcag 1080

caggctttta aagtcatctt atcgaatgaa tctggcgacg agttggttat tggttatcgt 1140

gaagaccaaa atgcctatta tattgatcgt tctaagtccg gtgaagtgtc ttttaatgga 1200

gaatttccaa agatagcgtt ggcccaacgg ccgctaaata agggagcact ttctttcagt 1260

ttgtatgttg atgtgagctc tgtggaactt tttgcagacg aggggctaac ggtaatgacc 1320

agtttatttt ttccgaacaa gccgataacc aagctgcgta tcgtgggaga taaaaagttg 1380

gagtttcagg aaatcggtat ttctgcattt aagagataa 1419

<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> 514

<212> PRT

<213> 重组突变酶(MutQ23Δ9His)

<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 Pro Glu Glu Lys

35 40 45

Tyr Arg Pro Leu Tyr His Phe Thr Pro Gln Gln Gly Trp Met Asn Asp

50 55 60

Pro Asn Gly Leu Val Tyr Leu Asp Gly Asn Tyr His Leu Phe Phe Gln

65 70 75 80

His Asn Pro Glu Lys Pro Val Trp Gly Pro Met His Trp Gly His Ala

85 90 95

Ile Ser Lys Asp Leu Ile His Trp Asp Glu Gln Lys Ile Ala Leu Tyr

100 105 110

Pro Asp Ser Leu Gly Thr Ile Phe Ser Gly Ser Ala Val Ile Asp Lys

115 120 125

Asp Asn Thr Ala Gly Phe Gly Lys Gly Ala Met Val Ala Ile Phe Thr

130 135 140

His His Asn His Gln Glu Glu Asp Arg Lys Thr Gly Leu His Gln Asn

145 150 155 160

Gln Ser Leu Ala Tyr Ser Leu Asp Lys Gly Leu Thr Trp Thr Lys Tyr

165 170 175

Lys Gly Asn Pro Val Leu Pro Asn Pro Gly Ile Trp Asp Phe Arg Asp

180 185 190

Pro Lys Val Met Trp His Val His Ser Gln Arg Trp Ile Met Thr Leu

195 200 205

Ala Thr Lys Asp Cys Ile Thr Phe Tyr Ser Ser Lys Asp Leu Lys Thr

210 215 220

Trp Gln Lys Glu Ser Glu Phe Gly Lys Glu Val Gly Ala His Gly Gly

225 230 235 240

Val Trp Glu Cys Pro Asp Leu Ile Pro Met Asp Tyr Lys Gly Thr Thr

245 250 255

Lys Trp Val Leu Leu Val Ser Ile Asn Pro Gly Gly Pro Asn Gly Gly

260 265 270

Ser Val Thr Gln Tyr Phe Val Gly Asp Phe Asp Gly His Gln Phe Arg

275 280 285

Thr Thr Asp Thr Lys Ile Lys Trp Leu Asp Trp Gly Pro Asp Asn Tyr

290 295 300

Ala Gly Val Thr Trp Ser Asn Leu Gly Asp Arg Gln Leu Met Ile Gly

305 310 315 320

Trp Met Ser Asn Trp Gln Tyr Ala Asn Val Val Pro Thr Thr Lys Trp

325 330 335

Arg Ser Ser Ser Thr Ile Pro Arg Val Leu Ser Leu Gly Lys Val Gly

340 345 350

Gln Ser Phe Tyr Val Ala Ser Thr Val Pro Lys Glu Ile Glu Thr Ala

355 360 365

Phe Arg Pro Leu Lys Lys Tyr His Gly Gly Gly Thr Lys Glu Val Ser

370 375 380

Phe Glu Gln His Leu Pro Gln Ala Tyr Arg Leu Asp Leu Lys Asp Leu

385 390 395 400

Lys Gln Gln Ala Phe Lys Val Ile Leu Ser Asn Glu Ser Gly Asp Glu

405 410 415

Leu Val Ile Gly Tyr Arg Glu Asp Gln Asn Ala Tyr Tyr Ile Asp Arg

420 425 430

Ser Lys Ser Gly Glu Val Ser Phe Asn Gly Glu Phe Pro Lys Ile Ala

435 440 445

Leu Ala Gln Arg Pro Leu Asn Lys Gly Ala Leu Ser Phe Ser Leu Tyr

450 455 460

Val Asp Val Ser Ser Val Glu Leu Phe Ala Asp Glu Gly Leu Thr Val

465 470 475 480

Met Thr Ser Leu Phe Phe Pro Asn Lys Pro Ile Thr Lys Leu Arg Ile

485 490 495

Val Gly Asp Lys Lys Leu Glu Phe Gln Glu Ile Gly Ile Ser Ala Phe

500 505 510

Lys Arg

<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> 45

<212> DNA

<213> Artificial Sequence

<400> 11

tggaagttct gttccagggg ccccagacgg gacagcataa acaag 45

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 12

ggtggtggtg ttatctctta aatgcagaaa taccgat 37

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 13

agagataaca ccaccaccac caccactg 28

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 14

cctggaacag aacttccagg aattcggatc cgcgacc 37

Claims

1.一种不耐热的外切菊粉酶突变体MutQ23Δ9，其特征在于，该突变体MutQ23Δ9的氨基酸序列如SEQ ID NO. 1所示。

2.一种如权利要求1所述的突变体MutQ23Δ9的编码基因mutQ23Δ9，其特征在于，该编码基因mutQ23Δ9的核苷酸序列如SEQ ID NO. 2所示。

3.一种包含权利要求2所述的编码基因mutQ23Δ9的重组载体。

4.一种包含权利要求2所述的编码基因mutQ23Δ9的重组菌。

5.一种如权利要求1所述的突变体MutQ23Δ9的制备方法，其特征在于，该方法包含：

在如权利要求2所述的mutQ23Δ9的5'端补加HRV 3C蛋白酶酶切位点编码序列；

将所述的mutQ23Δ9的核苷酸序列与表达载体pET-28a（+）相连接，并将连接产物转化大肠杆菌BL21(DE3)，获得包含mutQ23Δ9的重组菌株；

培养重组菌株，诱导重组外切菊粉酶突变体MutQ23Δ9表达，将得到的重组外切菊粉酶突变体MutQ23Δ9用HRV 3C蛋白酶酶切，以获得外切菊粉酶突变体MutQ23Δ9。

6.根据权利要求5所述的制备方法，其特征在于，所述培养重组菌株，采用含50µg·mL⁻¹卡那霉素的LB培养液。

7.根据权利要求6所述的制备方法，其特征在于，所述培养重组菌株，在37℃在含50µg·mL⁻¹卡那霉素的LB培养液中振荡培养。

8.根据权利要求5所述的制备方法，其特征在于，所述诱导，当OD₆₀₀达到0.6~1.0时，加入IPTG进行诱导。

9.如权利要求1所述的突变体MutQ23Δ9在食品中的应用。

10.根据权利要求9所述的应用，其特征在于，所述食品包括：酿酒。