CN108660121A - 一种热稳定性提高的淀粉分支酶突变体 - Google Patents

一种热稳定性提高的淀粉分支酶突变体 Download PDF

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CN108660121A
CN108660121A CN201810531948.8A CN201810531948A CN108660121A CN 108660121 A CN108660121 A CN 108660121A CN 201810531948 A CN201810531948 A CN 201810531948A CN 108660121 A CN108660121 A CN 108660121A
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李兆丰
班宵逢
顾正彪
李才明
程力
洪雁
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Abstract

本发明公开了一种热稳定性提高的淀粉分支酶突变体，属于酶工程技术领域。本发明通过将淀粉分支酶中的氨基酸残基突变为与内源性氨基酸残基带相反电荷的氨基酸残基，与附近内源性、带相反电荷的氨基酸残基形成静电相互作用，得到热稳定提高的淀粉分支酶突变体，与野生型淀粉分支酶相比，本发明所得的淀粉分支酶突变体Q231R、Q231K、T339E、T339D、V37E、V37D和I571D的热稳定性在60C下的半衰期t1/2(min,60℃)分别延长40％、38％、21％、26％、16％和21％，在65C下的半衰期t1/2(min,65℃)分别延长1.5‑2.0倍。

Description

一种热稳定性提高的淀粉分支酶突变体
技术领域
本发明涉及一种热稳定性提高的淀粉分支酶突变体，属于酶工程技术领域。
背景技术
淀粉分支酶(1,4-α-glucan branching enzyme；EC 2.4.1.18)是属于糖苷水解酶家族13(GH 13)的一类糖基转移酶，能够催化淀粉分子α-1,4-糖苷键的断裂形成游离短链，并通过转糖苷作用将切割下的短链以α-1,6-糖苷键的形式连接于受体链上，在淀粉分子原主链上形成新的α-1,6-分支点。
通过该种转糖基反应，淀粉分支酶能够增加淀粉的分支度，提高淀粉的抗消化性和慢消化性，延缓淀粉的回生过程，增强淀粉的稳定性并改善淀粉的使用性能，可用于生产具有良好应用价值的淀粉衍生物。
在淀粉的改性过程中，淀粉分支酶的热稳定性是一个关键因素，淀粉分支酶的热稳定性强不仅有利于延长酶的贮藏时间、减少酶在保存、运输过程中活力的损失，而且可以使酶在较高的温度下保持较高的活力，从而提高反应效率，缩短生产周期，进而降低生产成本。
目前，已被报道的用来提高淀粉分支酶热稳定性的方法有物理法、化学法、生物法等。在这些方法中，物理法存在产量低和稳定性差等问题，化学法在食品领域中的应用受到一定限制。
相比于物理法和化学法，生物法主要通过蛋白质工程、基因工程等技术手段，从根本上改变酶蛋白分子的空间构象，改善酶蛋白的使用性能，具有环境危害小、可良性遗传等优点，成为了改善酶蛋白分子热稳定性的重要技术手段。
发明内容
本发明的技术目的是通过将淀粉分支酶突变体为将淀粉分支酶中的氨基酸残基突变为与该氨基酸残基附近的内源性氨基酸残基带相反电荷的氨基酸残基的淀粉分支酶突变体内源性氨基酸残基带相反电荷的氨基酸残基，与附近内源性、带相反电荷的氨基酸残基形成静电相互作用，得到热稳定性提高的淀粉分支酶突变体。
本发明的技术方案如下：
本发明提供了一种热稳定性提高的淀粉分支酶突变体，所述淀粉分支酶突变体是通过将淀粉分支酶中的氨基酸残基突变为与该氨基酸残基附近的内源性氨基酸残基带相反电荷的氨基酸残基得到的。
在本发明的一种实施方式中，所述淀粉分支酶突变体的突变位点为第231位、第339位、第37位或第571位。
在本发明的一种实施方式中，所述淀粉分支酶突变体是将淀粉分支酶第231位的谷氨酰胺突变为精氨酸或赖氨酸，或将淀粉分支酶第339位的苏氨酸突变为谷氨酸或天冬氨酸，或将淀粉分支酶第37位的缬氨酸突变为谷氨酸或天冬氨酸，或将淀粉分支酶第571位的异亮氨酸突变为天冬氨酸的淀粉分支酶突变体。
在本发明的一种实施方式中，所述淀粉分支酶为来源于Geobacillusthermoglucosidans STB02的淀粉分支酶。
在本发明的一种实施方式中，所述淀粉分支酶的氨基酸序列为SEQ ID NO.1。
在本发明的一种实施方式中，所述淀粉分支酶突变体的氨基酸序列分别为SEQ IDNO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ IDNO.8。
本发明提供了上述热稳定性提高的淀粉分支酶突变体在水解淀粉方面的应用。
本发明提供了编码上述热稳定性提高的淀粉分支酶突变体的基因。
本发明提供了携带上述基因的基因工程菌。
在本发明的一种实施方式中，所述基因工程菌以pET-20b(+)为表达载体，以Escherichiacoli BL 21(DE3)为表达宿主。
本发明提供了上述基因工程菌在水解淀粉方面的应用。
有益效果：
与野生型淀粉分支酶相比，本发明所得的淀粉分支酶突变体Q231R、Q231K、T339E、T339D、V37E、V37D和I571D的热稳定性在60℃下的半衰期t1/2(min,60℃)分别延长40％、38％、21％、26％、16％和21％，在65℃下的半衰期t1/2(min,65℃)分别延长1.5-2.0倍。
附图说明
图1为本发明实施例中各淀粉分支酶突变体及野生型淀粉分支酶的热失活速率曲线；
图2为本发明实施例中各淀粉分支酶突变体及野生型淀粉分支酶的热失活速率曲线；
图3为本发明实施例中各淀粉分支酶突变体及野生型淀粉分支酶的热失活速率曲线。
具体实施方式
本发明的实施例仅作为本发明内容的进一步说明，不能作为本发明的限定内容或范围。
本发明涉及的检测方法如下：
淀粉分支酶热稳定性的分析方法：
将淀粉分支酶在一定温度下保温，不同时间点取样，迅速冷却至0℃，测定酶的残余活力，以未保温酶液的活力为100％。绘制相对酶活-时间曲线。
淀粉分支酶活力的测定方法：
用10mM磷酸缓冲液(pH7.5)配制0.25％(w/v)的马铃薯支链淀粉溶液，加入0.01g/mL淀粉分支酶后混合均匀并置于50℃水浴条件下反应15min。反应结束后沸水浴灭酶。取175L反应混合液加入2.5mL显色液(0.05％(w/v)KI，0.005％(w/v)I2，pH7.5)置于室温下静置15min以充分显色。显色15min后在530nm处测定吸光值。一个酶活力单位定义为：在530nm处，吸光值每分钟降低1％所需加入酶的量为一个酶活力单位。
本发明所用的培养基如下：
LB培养基：酵母粉5g/L，胰蛋白胨10g/L，NaCl 10g/L，pH 7.0。
TB培养基：酵母粉24g/L，胰蛋白胨12g/L，甘油5g/L，KH2PO4 17mM，K2HPO4 72mM，pH7.0。
实施例1：表达本发明淀粉分支酶突变体基因序列的制备
以表达载体gbe/pET-20(+)为模板，设计实验所需的互补引物链(见表1)，引物由金唯智生物科技有限公司合成，参照TaKaRa公司STAR Primer GXL试剂盒说明书所示方法进行定点突变。PCR反应体系依照STAR Primer试剂盒说明书中所设定条件：5×PrimeSTARBuffer(Mg2+Plus)10μL，模板DNA1μL，正向和反向引物(10μM)均为1μL，PrimeSTAR HS DNAPolymerase(2.5U/μL)0.5μL，dNTPs(各2.5mM)4μL，最后加入超纯水32.5μL。PCR扩增条件为：98℃条件下预变性5min；随后以98℃ 10s，55℃ 10s，72℃ 7min为一个循环，在以上条件下进行35个循环；最后72℃下保温15min。
表1淀粉分支酶突变位点的引入
引物 引物序列(5'-3')1
Q231R-For SEQ ID NO.9：ATCGCTTCCATCGAGCGGGCATTGG
Q231R-Rev SEQ ID NO.10：CCAATGCCCGCTCGATGGAAGCGAT
Q231K-For SEQ ID NO.11：ATCGCTTCCATAAAGCGGGCATTGG
Q231K-Rev SEQ ID NO.12：CCAATGCCCGCTTTATGGAAGCGAT
Q231A-For SEQ ID NO.13：ATCGCTTCCATGCAGCGGGCATTGG
Q231A-Rev SEQ ID NO.14：CCAATGCCCGCTGCATGGAAGCGAT
T339E-For SEQ ID NO.15：ATTAAATGAGGAGGTATTTGCGC
T339E-Rev SEQ ID NO.16：GCGCAAATACCTCCTCATTTAAT
T339D-For SEQ ID NO.17：ATTAAATGAGGACGTATTTGCGC
T339D-Rev SEQ ID NO.18：GCGCAAATACGTCCTCATTTAAT
T339A-For SEQ ID NO.19：ATTAAATGAGGCAGTATTTGCGC
T339A-Rev SEQ ID NO.20：GCGCAAATACTGCCTCATTTAAT
V37E-For SEQ ID NO.21：CAAAACGACGTTGAAGGAACCCGGT
V37E-Rev SEQ ID NO.22：ACCGGGTTCCTTCAACGTCGTTTTG
V37D-For SEQ ID NO.23：CAAAACGACGTTGACGGAACCCGGT
V37D-Rev SEQ ID NO.24：ACCGGGTTCCGTCAACGTCGTTTTG
V37A-For SEQ ID NO.25：CAAAACGACGTTGCAGGAACCCGGT
V37A-Rev SEQ ID NO.26：ACCGGGTTCCTGCAACGTCGTTTTG
I571D-For SEQ ID NO.27：AATACCGGGAAGACATCAGCAGCG
I571D-Rev SEQ ID NO.28：CGCTGCTGATGTCTTCCCGGTATT
I571A-For SEQ ID NO.29：AATACCGGGAAGCAATCAGCAGCG
I571A-Rev SEQ ID NO.30：CGCTGCTGATTGCTTCCCGGTATT
1下划线碱基对应于相应突变氨基酸。
实施例2：含有表达本发明淀粉分支酶突变体基因的基因工程菌的构建
含有淀粉分支酶突变体基因的工程菌的构建按照如下方法进行：
(1)将PCR产物按照E.coli DH5α感受态转化方法转入到E.coli DH5α中，并将受体菌涂布到含有100μg/mL氨苄青霉素LB固体培养基上；
(2)将涂布后的LB固体培养基倒置于37℃恒温培养箱中培养12h；
(3)挑取阳性单克隆接种于含有100μg/mL氨苄青霉素的LB液体培养基并于37℃下培养10～12h；
(4)将收集到的菌体提取质粒并采用双酶切电泳和测序鉴定；
(5)在37℃下，用DpnI处理PCR产物2h，随后将处理好的PCR产物转化到E.coliJM109中，将转化的E.coli JM 109涂布到含有100μg/mL氨苄霉素的LB琼脂培养基中，在37℃恒温箱中过夜培养12h，从中挑选出单菌落接种到含有100μg/mL氨苄霉素的LB液体培养基中，在37℃下、200r/min培养过夜并按照质粒提取试剂盒说明书所示方法提取质粒鉴定测序。将构建好的目的质粒通过化学转化法转入表达宿主E.coli BL21(DE 3)感受态中。最终得到基因工程菌E.coli BL21(DE 3)(pET-22b(+)/gbe)。
实施例3：本发明淀粉分支酶突变体的表达
宿主菌活化培养：将含有表达载体质粒pET-20b(+)/gbe的E.coli BL21(DE 3)在LB固体培养基上进行划线分离，置于37℃恒温培养箱中过夜培养，挑取阳性单菌落接种于含有15mLLB液体培养基的灭菌50mL离心管中。将该离心管置于200r/min的回转式摇床中，在37℃下培养12h。
发酵培养：将活化好的菌种培养液200μL接种于含有50mL TB液体培养基的250mL三角瓶中，并置于回转式摇床中以200r/min的速率，在37℃下培养至OD600达到1.0-1.5之间。加入终浓度为0.01mM/L的IPTG并降低培养温度至25℃，在此条件下继续诱导16h。
各培养基在使用前添加终浓度为100μmg/mL的氨苄青霉素。
实施例4：本发明淀粉分支酶突变体的热稳定性检测
将酶浓度为1μmol/L的淀粉分支酶突变体Q231R、Q231K、Q231A、T339E、T339D、T339A、V37E、V37D、V37A、I571D和I571A分别在60℃和65℃下保温一段时间，间隔一定时间取样，在50℃下按照淀粉分支酶活力测定方法测定该种酶溶液的活力。
与野生型淀粉分支酶相比，Q231R、Q231K、T339E、T339D、V37E、V37D和I571D突变体在60℃下的半衰期t1/2(min，60℃)分别延长40％、38％、21％、26％、16％和21％，在65℃下的半衰期t1/2(min，65℃)的延长1.5-2.0倍；Q231A、T339A、V37A和I571A突变体在60℃和65℃下的半衰期与野生型淀粉分支酶相似。(热稳定性检测结果如图1和表2)
表2野生型和突变体GBE的半衰期
1每个值为3次平行实验的平均值。
实施例5：本发明淀粉分支酶突变体的酶活检测
以10mM磷酸缓冲液(pH 7.5)配制浓度为0-5mg/mL的马铃薯支链淀粉溶液，加入一定量的纯化后淀粉分支酶突变体Q231R、Q231K、Q231A、T339E、T339D、T339A、V37E、V37D、V37A、I571D和I571A分别在50℃下进行反应并按照上述所示方法测定淀粉分支酶突变体的活力。将所得数据导入到SigmaPlot程序(Systat Software Inc.,version 10.0forWindows)中得到米氏(Michaelis-Menten)方程。
其中：V为不同底物浓度下淀粉分支酶突变体的比活；S为底物浓度；Vmax为最大反应速率；KM为米氏常数。(酶活检测结果如表3)
表3野生型和突变体GBE的酶活力和KM值
1每个值为3次平行实验的平均值。
实施例6：本发明淀粉分支酶突变体的失活动力学分析
以10mM磷酸缓冲液(pH 7.5)配制浓度为0-5mg/mL的马铃薯支链淀粉溶液，加入一定量的纯化后淀粉分支酶在50℃下进行反应并按照以上方法测定活力。计算出野生型和突变体淀粉分支酶在热失活过程的吉布斯能量ΔG(kJ·mol-1)、焓变ΔH(kJ·mol-1)、熵变ΔS(J·K-1·mol-1)，这些参数可由以下公式获得：
ΔH＝Ea-RT
其中，KB表示玻尔兹曼常数(1.381×10-23J·K-1)，h表示普朗克常数(1.104×10- 35J·min)，R表示气体常数(8.314J·K-1·mol-1)，T的单位为绝对温度(K)。(失活动力学分析结果如表4)
表4野生型和突变体GBE失活动力学分析
1每个值为3次平行实验的平均值。
实施例7：本发明淀粉分支酶突变体的加酶量分析
配制10％的马铃薯淀粉溶液为底物，调节pH 7.5，按25U/g加酶量加入淀粉分支酶，在60℃下反应改性24h，反应结束后沸水浴灭酶。在与野生型淀粉分支酶对淀粉有相同改性效果时，具有较高热稳定性的淀粉分支酶突变体可以使用较少的量。(加酶量分析结果如表5)
表5野生型和突变体GBE的应用
1每个值为3次平行实验的平均值。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 江南大学
<120> 一种热稳定性提高的淀粉分支酶突变体
<160> 30
<170> PatentIn version 3.3
<210> 1
<211> 643
<212> PRT
<213> 人工序列
<400> 1
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 2
<211> 643
<212> PRT
<213> 人工序列
<400> 2
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Arg Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 3
<211> 643
<212> PRT
<213> 人工序列
<400> 3
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Lys Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 4
<211> 643
<212> PRT
<213> 人工序列
<400> 4
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Glu Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 5
<211> 643
<212> PRT
<213> 人工序列
<400> 5
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Asp Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 6
<211> 643
<212> PRT
<213> 人工序列
<400> 6
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Glu Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 7
<211> 643
<212> PRT
<213> 人工序列
<400> 7
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Asp Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 8
<211> 643
<212> PRT
<213> 人工序列
<400> 8
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Asp Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
atcgcttcca tcgagcgggc attgg 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
ccaatgcccg ctcgatggaa gcgat 25
<210> 11
<211> 25
<212> DNA
<213> 人工序列
<400> 11
atcgcttcca taaagcgggc attgg 25
<210> 12
<211> 25
<212> DNA
<213> 人工序列
<400> 12
ccaatgcccg ctttatggaa gcgat 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列
<400> 13
atcgcttcca tgcagcgggc attgg 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列
<400> 14
ccaatgcccg ctgcatggaa gcgat 25
<210> 15
<211> 23
<212> DNA
<213> 人工序列
<400> 15
attaaatgag gaggtatttg cgc 23
<210> 16
<211> 23
<212> DNA
<213> 人工序列
<400> 16
gcgcaaatac ctcctcattt aat 23
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<400> 17
attaaatgag gacgtatttg cgc 23
<210> 18
<211> 23
<212> DNA
<213> 人工序列
<400> 18
gcgcaaatac gtcctcattt aat 23
<210> 19
<211> 23
<212> DNA
<213> 人工序列
<400> 19
attaaatgag gcagtatttg cgc 23
<210> 20
<211> 23
<212> DNA
<213> 人工序列
<400> 20
gcgcaaatac tgcctcattt aat 23
<210> 21
<211> 25
<212> DNA
<213> 人工序列
<400> 21
caaaacgacg ttgaaggaac ccggt 25
<210> 22
<211> 25
<212> DNA
<213> 人工序列
<400> 22
accgggttcc ttcaacgtcg ttttg 25
<210> 23
<211> 25
<212> DNA
<213> 人工序列
<400> 23
caaaacgacg ttgacggaac ccggt 25
<210> 24
<211> 25
<212> DNA
<213> 人工序列
<400> 24
accgggttcc gtcaacgtcg ttttg 25
<210> 25
<211> 25
<212> DNA
<213> 人工序列
<400> 25
caaaacgacg ttgcaggaac ccggt 25
<210> 26
<211> 25
<212> DNA
<213> 人工序列
<400> 26
accgggttcc tgcaacgtcg ttttg 25
<210> 27
<211> 24
<212> DNA
<213> 人工序列
<400> 27
aataccggga agacatcagc agcg 24
<210> 28
<211> 24
<212> DNA
<213> 人工序列
<400> 28
cgctgctgat gtcttcccgg tatt 24
<210> 29
<211> 24
<212> DNA
<213> 人工序列
<400> 29
aataccggga agcaatcagc agcg 24
<210> 30
<211> 24
<212> DNA
<213> 人工序列
<400> 30
cgctgctgat tgcttcccgg tatt 24

Claims (10)

1.一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶突变体是通过将淀粉分支酶中的氨基酸残基突变为与该氨基酸残基附近的内源性氨基酸残基带相反电荷的氨基酸残基得到的。
2.如权利要求1所述的一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶突变体的突变位点为第231位、第339位、第37位或第571位。
3.如权利要求1或2所述的一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶突变体是将淀粉分支酶第231位的谷氨酰胺突变为精氨酸或赖氨酸，或将淀粉分支酶第339位的苏氨酸突变为谷氨酸或天冬氨酸，或将淀粉分支酶第37位的缬氨酸突变为谷氨酸或天冬氨酸，或将淀粉分支酶第571位的异亮氨酸突变为天冬氨酸的淀粉分支酶突变体。
4.如权利要求1-3任一所述的一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶为来源于Geobacillus thermoglucosidans STB02的淀粉分支酶。
5.如权利要求1-4任一所述的一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶的氨基酸序列为SEQ ID NO.1。
6.如权利要求1-5任一所述的一种热稳定性提高的淀粉分支酶突变体，其特征在于，所述淀粉分支酶突变体的氨基酸序列为SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8。
7.权利要求1-6任一所述的一种热稳定性提高的淀粉分支酶突变体在水解淀粉方面的应用。
8.编码权利要求1-6任一所述的一种热稳定性提高的淀粉分支酶突变体的基因。
9.携带权利要求8所述基因的基因工程菌。
10.权利要求9所述的宿主细胞在水解淀粉方面的应用。
CN201810531948.8A 2018-05-29 2018-05-29 一种热稳定性提高的淀粉分支酶突变体 Pending CN108660121A (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110283881A (zh) * 2019-06-24 2019-09-27 江南大学 一种提高酶稳定性的方法及其应用
CN110684751A (zh) * 2019-10-23 2020-01-14 江南大学 一种催化能力提高的淀粉分支酶突变体
CN114181919A (zh) * 2022-01-10 2022-03-15 江南大学 一种提高淀粉分支酶热稳定性的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106190998A (zh) * 2016-08-25 2016-12-07 江南大学 一种提高淀粉分支酶活力的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106190998A (zh) * 2016-08-25 2016-12-07 江南大学 一种提高淀粉分支酶活力的方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110283881A (zh) * 2019-06-24 2019-09-27 江南大学 一种提高酶稳定性的方法及其应用
CN110283881B (zh) * 2019-06-24 2020-11-06 江南大学 一种提高酶稳定性的方法及其应用
CN110684751A (zh) * 2019-10-23 2020-01-14 江南大学 一种催化能力提高的淀粉分支酶突变体
CN110684751B (zh) * 2019-10-23 2021-06-25 江南大学 一种催化能力提高的淀粉分支酶突变体
CN114181919A (zh) * 2022-01-10 2022-03-15 江南大学 一种提高淀粉分支酶热稳定性的方法
CN114181919B (zh) * 2022-01-10 2023-07-18 江南大学 一种提高淀粉分支酶热稳定性的方法

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