CN110484520B - 比活提高的植酸酶appa突变体及其基因和应用 - Google Patents

比活提高的植酸酶appa突变体及其基因和应用 Download PDF

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CN110484520B
CN110484520B CN201810454326.XA CN201810454326A CN110484520B CN 110484520 B CN110484520 B CN 110484520B CN 201810454326 A CN201810454326 A CN 201810454326A CN 110484520 B CN110484520 B CN 110484520B
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李阳源
黄江
何小梅
江民华
陈丽芝
高芝
刘金山
唐业
王勇
王平
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Guangdong Vtr Bio Tech Co ltd
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Abstract

本发明涉及生物技术领域，具体涉及比活提高的植酸酶APPA突变体及其基因和应用。本发明采用定点饱和突变的方法对氨基酸序列如SEQ ID NO.1所示的大肠杆菌植酸酶APPA的第53位、第68位、第77位、第96位、第112位、第202位、第233位、第247位和第362位进行分子改造，结合高通量筛选方法获得一系列比活提高的植酸酶突变体，突变体的比活均优于原始酶。

Description

比活提高的植酸酶APPA突变体及其基因和应用
技术领域
本发明涉及生物技术领域，具体涉及比活提高的植酸酶APPA突变体及其基因和应用。
背景技术
植酸(Phytate，Phytic acid，IP6)又称肌醇六磷酸脂，含有6个磷酸基团，带有丰富的磷，是饲料中磷的重要贮存形式。植酸酶(Phytase)能够催化植酸及植酸盐水解成肌醇与磷酸(或磷酸盐)。饲料中添加植酸酶，不仅可以提高动物对饲料中植酸磷的利用率，减少磷对环境的污染，还可以降低植酸磷的抗营养作用。
植酸酶广泛存在于动物、植物和微生物中，目前市场上商品化的植酸酶均来源于微生物。研究表明来源于大肠杆菌的植酸酶(APPA)是目前分解植酸能力最强的植酸酶。目前市场上商品化的植酸酶制剂大多数都是经过优化改良的，优化改良的植酸酶在热稳定上有了较大的提高，但是比活和生产效率的下降也令人堪忧，因此怎样提高植酸酶比活和生产效率是企业提高产品质量、降低成本的关注焦点。
采用分子生物学技术对现有产业化的酶蛋白进行改造，获得能够适应不同产业需求的酶。理性设计是通过研究酶蛋白的三维空间结构与作用机理，找出可能影响酶活性或酶特性的关键氨基酸，并对这些氨基酸进行定点突变，从而得到更优的酶蛋白。与非理性设计相比，理性设计效率更高，往往也更能得到好的结果。
但是相比随机基因突变，理性设计只是提高实验的成功率，序列分析软件不能保证突变的可靠性。理论预测只是提供了一些可能性，这些预测并不能保证绝对准确，尤其是突变的情况比较复杂的时候，对于其性质和功能的研究还需要详细实验验证。并且，一个突变区域有N个突变的位点，一个突变位点有18个突变体。几个突变位点的组合又是千变万化，不可能通过软件得出一样的数据。
发明内容
本发明的目的是通过对来源于大肠杆菌的植酸酶APPA进行分子改造，使改造后的植酸酶具有更高的比活，降低生产成本。
本发明的目的是提供比活提高的植酸酶APPA突变体。
本发明的再一目的是提供比活提高的植酸酶APPA突变体基因。
本发明的再一目的是提供包含上述提供比活提高的植酸酶APPA突变体基因的重组载体。
本发明的再一目的是提供包含上述提供比活提高的植酸酶APPA突变体基因的重组菌株。
本发明的再一目的是提供制备上述比活提高的植酸酶APPA突变体的方法。
本发明的再一目的是提供上述比活提高的植酸酶APPA突变体的应用。
本发明采用定点饱和突变的方法对氨基酸序列如SEQ ID NO.1所示的大肠杆菌植酸酶APPA的第53位、第68位、第77位、第96位、第112位、第202位、第233位、第247位和第362位进行分子改造，结合高通量筛选方法获得一系列比活提高的植酸酶突变体。这些突变体的氨基酸序列如SEQ ID NO.2到SEQ ID NO.11所示。
本发明还提供了包含上述植酸酶突变体基因的重组载体。
本发明还提供了包含上述植酸酶突变体基因的重组菌株，优选重组菌株是毕赤酵母菌株X33。
本发明还提供了表达上述植酸酶突变体的方法，包括以下步骤：
1)用上述的改良的重组表达载体转化宿主细胞，得重组菌株；
2)重组菌株进行发酵，诱导重组植酸酶的表达；
3)发酵结束后，回收并纯化所表达的植酸酶。
本发明通过蛋白理性设计对植酸酶APPA进行分子改造，结合高通量筛选方法筛选出比活提高的植酸酶突变体。与原始植酸酶APPA的比活相比，突变后植酸酶比活的提高幅度为24％-86％。
附图说明
图1显示了原始植酸酶和突变体的pH酶学性质对比。
具体实施方式
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；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实验材料和试剂：
1、菌株与载体
大肠杆菌Topl0、大肠杆菌菌株OrigamiB、毕赤酵母X33、载体pPICZαA、pET-22b(+)，Zeocin购自Invitrogen公司。
2、酶与试剂
超保真2×Master Mix PCR聚合酶、限制性内切酶购自NEB(北京)有限公司；universai DNAPurification Kit、TIANprep Mini PlasmidKit购自天根生化(北京)科技有限公司，其它试剂均为国产分析纯。
3、培养基
大肠杆菌培养基为LB培养基(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LB+Amp培养基为LB培养基加入终浓度为100ug/mL的氨苄青霉素。
酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPDzeo(YPD+100mg/L zeocin)。
酵母诱导培养基BMGY(I％酵母提取物、2％蛋白胨、1.34％YNB、0.00004％Biotin、1％甘油(V/V))和BMMY(除以0.5％甲醇代替甘油，其余成份相与BMGY相同)。
重组酵母发酵培养基本盐培养基：磷酸氢二铵5％、磷酸二氢钾0.5％、七水硫酸镁1.5％、硫酸钾1.95％、硫酸钙0.1％、氢氧化钾0.1％、消泡剂0.03％。高压后每升加4.35毫升PTM1。
PTM1(微量盐溶液)：硫酸铜0.6％、碘化钾0.018％、一水硫酸锰0.3％、二水钼酸钠0.02％、硼酸0.002％、六水氯化钴0.05％、氯化锌2％、七水硫酸铁6.5％、浓硫酸0.5％、生物素0.02％。
实施例1、植酸酶基因的合成及克隆
以大肠杆菌来源的植酸酶APPA的氨基酸序列作为参考(如SEQ ID NO.10所示)，人工合成该酶的基因序列(核酸序列如SEQ ID NO.1所示)。
根据基因序列设计引物，5'端引入Nde I酶切位点，3'端引入EcoR I酶切位点，引物序列如下：
5'端引物Nde I-APPA-F1：
GGATTACCATATGATGTCAGATATGAAAAGCGGAAACA
3'端引物EcoR I-APPA-R1：
CGGAATTCTTATTTGTCTTGATGGAGAGCGCAC
以合成基因APPA为模板，用上述引物进行PCR扩增，获得大小约为1.3kb的DNA条带，回收目的片段，用Nde I和EcoR I进行双酶切，连接到具有相同酶切位点的线性pET-22b(+)载体上，转化至Top10大肠杆菌，LB+Amp平板培养获得pET-22b-APPA阳性克隆子。
实施例2、基因定点饱和突变
最终确定对12个氨基酸进行定点饱和突变，分别为：第47位、第53位、第68位、第77位、第96位、第112位、第193位、第202位、第233位、第245位、第247位和第362位。针对性的设计了12对饱和突变引物，目的基因突变位点统一为NNS，NNS左右各取15个碱基构成正向引物；反向引物与正向引物完全互补，其中，N代表A、T、G、C四种碱基，S代表C、G两种碱基。
以重组载体pET-22b-APPA为模板，加入突变位点的正反向引物，超保真2×Master Mix PCR聚合酶进行PCR扩增，产物经Dpn I酶切处理后电击转化OrigamiB大肠杆菌感受态细胞，在LB+Amp平板筛选阳性突变重组克隆。每个突变位点挑取186个单克隆接种到96孔深孔板。每个平板挑选3个未突变的克隆为对照。每孔含500uL培养基LB+Amp。37℃摇床200rpm培养5h，转移50uL菌液到新的96孔深孔板保藏菌种后，在剩余菌液中添加含有IPTG的50uL LB+Amp培养基，并使每孔的IPTG终浓度为0.5mM，37℃摇床200rpm过夜诱导表达植酸酶。含有过夜培养诱导表达植酸酶的菌液深孔板在75℃水浴锅加热处理5min后细胞裂解，检测粗酶液中植酸酶的活性。植酸酶初步耐热活性检测根据中华人民共和国国家标准《GB/T18634-2002》进行。
根据植酸酶活性检测结果，植酸酶活性高于对照克隆组的突变为阳性克隆。从保种板上挑选阳性克隆集中到96孔深孔板重复上述的培养、诱导表达、酶活测定筛选试验。确定酶活提高的突变体为阳性突变克隆，提取阳性克隆质粒DNA进行基因测序。
实施例3、组合突变
第53位：D53C，D53S，D53A；
第68位：W68N，W68Q；
第77位：I77P，I77A，I77V，I77N，I77Q；
第96位：K96A，K96D，K96P；
第112位：D112W，D112H，D112Y，D112N；
第202位：N202H，N202D；
第233位：V233D，V233H，V233Y，V233Q；
第247位：Q247R，Q247N，Q247W，Q247E；
第362位：R362Y，R362D，R362A，R362W，R362V。
实施例2中酶比活提高的单突变位点为D53C，D53S，D53A；W68N，W68Q；I77P，I77A，I77V，I77N，I77Q；K96A，K96D，K96P；D112W，D112H，D112Y，D112N；N202H，N202D；V233D，V233H，V233Y，V233Q；Q247R，Q247N，Q247W，Q247E；R362Y，R362D，R362A，R362W，R362V。对这些单点突变体分别进行组合，实验过程同实施例2相同。通过实验最终获得10个组合突变，分别命名为APPA-1、APPA-2、APPA-3、APPA-4、APPA-5、APPA-6、APPA-7、APPA-8、APPA-9、APPA-10。
APPA-1包含的突变位点为：D53C，W68N，I77P，K96A，D112W，N202H，V233D，Q247R，R362D。
APPA-2包含的突变位点为：D53A，W68Q，I77A，K96D，D112H，N202H，V233Y，Q247E，R362D。
其中APPA-3包含的突变位点为：D53C，W68N，I77N，K96A，D112W，N202D，V233H，Q247W，R362A。
APPA-4包含的突变位点为：D53C，W68N，I77V，K96P，D112Y，N202D，V233Q，Q247R，R362W。
APPA-5包含的突变位点为：D53C，W68Q，I77Q，K96P，D112W，N202D，V233Q，Q247R，R362V。
APPA-6包含的突变位点为：D53S，W68N，I77P，K96A，D112H，N202H，V233D，Q247N，R362Y。
APPA-7包含的突变位点为：D53A，W68N，I77Q，K96D，D112Y，N202D，V233Y，Q247R，R362A。
其中APPA-8包含的突变位点为：D53S，W68Q，I77V，K96P，D112N，N202H，V233D，Q247E，R362W。
APPA-9包含的突变位点为：D53C，W68Q，I77A，K96P，D112N，N202D，V233Q，Q247W，R362D。
APPA-10包含的突变位点为：D53C，W68N，I77Q，K96A，D112W，N202H，V233H，Q247E，R362W。
实施例4、植酸酶APPA毕赤酵母表达载体的构建
根据植酸酶APPA基因，5'端设计PCR引物含有EcoRI内切酶位点，3'端设计PCR引物含NotI内切酶位点，引物序列如下：5'端引物EcoRI-PHd-F1：GTAGAATTCCAGAGTGAGCCTGAGTTG3'端引物NotI-PHd-R1：ATTGCGGCCGCTTACTACAAGGAACAAGCTGG
以合成基因APPA为模板，用上述引物进行PCR扩增，将扩增得到的片段，用限制性内切酶EcoRI和NotI双酶切，纯化，连接到pPICZαA载体EcoRI和NotI位点，使植酸酶APPA基因插入到上述表达载体的信号肽序列的下游。连接产物转化Top10大肠杆菌，LB-Zeo平板培养获得pPICZαA-APPA阳性菌落，提取pPICZαA-APPA阳性菌落质粒。
实施例5、原始植酸酶及突变植酸酶的比活分析
植酸酶的活性测定按照中华人民共和国国家标准《GB/T 18634-2002》进行。植酸酶活性单位(U)的定义：指样品在植酸钠浓度为5.0mmol/L、温度37℃、pH值5.5的条件下，每min从植酸钠中释放l pmol无机磷，即为一个植酸酶活性单位，以U表示。
U＝FxC/(Vx30)
式中：U-试样中植酸酶的活性，U/mL；C-根据实际样液的吸光值由直线回归方程计算出的酶活性，U；F-试样溶液反应前的总稀释倍数；V-试样体积，mL；30-反应时间，min。
标准曲线的制定如表1：
磷浓度(mmol/L) 0 1.5625 3.125 6.25 12.5 25
OD415 0 0.054 0.109 0.212 0.437 0.942
采用镍亲和层析分别对原始植酸酶APPA和突变植酸酶进行纯化，测定纯化的原始植酸酶和突变植酸酶的酶活和蛋白含量，计算出它们的比活。以突变体提高的比活与原始植酸酶比活之比表示突变体比活的提高幅度。与原始APPA相比，突变后的植酸酶比活提高幅度为14％-66％(具体结果见表2)。
本发明通过理定点饱和突变，预测9个可以使植酸酶比活力提高的氨基酸位点，再通过组合突变筛选到10个植酸酶酶活提高的突变体，大大降低植酸酶生产成本，为酶制剂的进一步应用奠定了基础。
表2原始植酸酶和植酸酶突变体的相对比活
实施例6、原始植酸酶及突变植酸酶的酶学性质测定。
将已知酶活的植酸酶用不同pH缓冲液提取并稀释到一定酶活，并用对应pH底物按照国标法测定并计算相对酶活(％)。每组对三个平行。将比活提高较高的APPA-1，APPA-2，APPA-4，APPA-6，APPA-8进行测定最适pH酶学性质。如图1所示，这5个突变体的最适pH变化较小。
序列表
<110> 广东溢多利生物科技股份有限公司
<120> 比活提高的植酸酶APPA突变体及其基因和应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 1
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
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Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Lys
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Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Asp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asn Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 2
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 2
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Pro Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Ala
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Trp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu His Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Asp Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Arg Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Asp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 3
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 3
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Ala Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Gln Leu Thr Pro Arg Gly Gly Glu Leu Ala Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Asp
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val His
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu His Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Tyr Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Asp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 4
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 4
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Asn Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Ala
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Trp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asp Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala His Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Trp Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Ala Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 5
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 5
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Val Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Pro
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Tyr
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asp Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Gln Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Arg Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Trp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 6
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 6
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Gln Leu Thr Pro Arg Gly Gly Glu Leu Gln Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Pro
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Trp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asp Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Gln Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Arg Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Val Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 7
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 7
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Ser Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Pro Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Ala
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val His
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu His Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Asp Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Asn Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Tyr Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 8
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 8
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Ala Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Gln Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Asp
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Tyr
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asp Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Tyr Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Arg Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Ala Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 9
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 9
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Ser Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Gln Leu Thr Pro Arg Gly Gly Glu Leu Val Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Pro
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Asn
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu His Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Asp Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Trp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 10
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 10
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Gln Leu Thr Pro Arg Gly Gly Glu Leu Ala Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Pro
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Asn
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Asp Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Gln Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Trp Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Asp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435
<210> 11
<211> 436
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 11
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Cys Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Asn Leu Thr Pro Arg Gly Gly Glu Leu Gln Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Ala
85 90 95
Lys Gly Cys Pro Gln Pro Gly Gln Val Ala Ile Ile Ala Asp Val Trp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu His Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala His Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Trp Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Ala Asn Glu Ala Arg Ile Pro Ala Cys Ala Leu
420 425 430
His Gln Asp Lys
435

Claims (7)

1.比活提高的植酸酶APPA突变体，其特征在于，所述突变体为氨基酸序列如SEQ IDNO.1所示的植酸酶APPA发生以下突变而得，
突变位点为：D53S，W68N，I77P，K96A，D112H，N202H，V233D，Q247N，R362Y。
2.比活提高的植酸酶APPA突变体基因，其特征在于，所述基因编码权利要求1所述的比活提高的植酸酶APPA突变体。
3.包含权利要求2所述比活提高的植酸酶APPA突变体基因的重组载体。
4.包含权利要求2所述比活提高的植酸酶APPA突变体基因的重组菌株。
5.根据权利要求4所述重组菌株，其特征在于，所述重组菌株为大肠杆菌或毕赤酵母。
6.权利要求1所述比活提高的植酸酶APPA突变体用于水解植酸的应用。
7.权利要求1所述比活提高的植酸酶APPA突变体作为饲料添加剂的应用。
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