CN107058249A - 变异葡萄糖脱氢酶及其使用 - Google Patents

变异葡萄糖脱氢酶及其使用 Download PDF

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CN107058249A
CN107058249A CN201610973614.7A CN201610973614A CN107058249A CN 107058249 A CN107058249 A CN 107058249A CN 201610973614 A CN201610973614 A CN 201610973614A CN 107058249 A CN107058249 A CN 107058249A
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CN107058249B (zh
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小岛胜博
森茂
森一茂
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Arkray Inc
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Abstract

本发明涉及变异葡萄糖脱氢酶及其使用。本发明提供一种对木糖的反应性降低的变异葡萄糖脱氢酶(GDH)，其包含具有在依次包含下面的肽序列(i)～(v)的520～550个氨基酸的GDHα亚单位的氨基酸序列中、以其他氨基酸取代了肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺中的一个以上的氨基酸序列的变异GDHα亚单位，所述变异葡萄糖脱氢酶具有GDH活性，对木糖的反应性降低。(i)Val/Ile Val/Ile Val/Ile Gly Ser Gly Val Ala Gly(ii)Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys(iii)Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly(iv)Lys Lys Ile/Leu His Leu Ser Asn(v)Phe Ala Pro/Asn Asn Asn His Ile。

Description

变异葡萄糖脱氢酶及其使用
技术领域
本发明涉及底物特异性提高的变异葡萄糖脱氢酶(GDH)。详细地讲，本发明涉及包含变异型α亚单位的GDH。本发明的GDH可以适当地用于葡萄糖传感器及葡萄糖检测试剂盒等，在生物化学领域、临床领域等中有用。
背景技术
目前，将野生型的含细胞色素C的GDH(CyGDH)及以吡咯并喹啉苯醌作为辅酶的PQQGDH用于血糖自测传感器。然而，野生型的CyGDH及PQQGDH有以下缺点：由于其不仅与葡萄糖反应而且还与麦芽糖反应，因此不能测定准确的血糖值。
专利文献1中虽然公开了对二糖类的反应性低、且在α亚单位的326位、365位以及472位具有变异的变异GDH，但显示反应性低的仅仅是针对麦芽糖，并没有公开对木糖以及半乳糖的反应性低的变异GDH。
在先技术文献
专利文献
专利文献1：日本特开2012-090563号公报。
发明内容
发明要解决的技术问题
本发明的课题在于，提供一种对木糖、优选木糖以及半乳糖的反应性低、且对葡萄糖的底物特异性提高的GDH。
用于解决技术问题的手段
本发明人为了解决如上所述的课题而进行了潜心研究，结果发现，通过在依次包含下面叙述的肽序列(i)～(v)的520～550个氨基酸的GDHα亚单位的氨基酸序列中、以其他氨基酸取代肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺中的一个以上，会降低对木糖以及半乳糖的反应性，并提高对葡萄糖的底物特异性，而且，根据该发现，成功地得到葡萄糖传感器等中有用的变异GDH，从而完成了本发明。
即，本发明为以下的内容。
(1)一种变异GDH，其包含变异葡萄糖脱氢酶α亚单位，所述变异葡萄糖脱氢酶α亚单位具有在依次包含下面的肽序列(i)～(v)的520～550氨基酸的葡萄糖脱氢酶α亚单位的氨基酸序列中、以其他氨基酸取代肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺的一个以上的氨基酸序列，其中，所述变异葡萄糖脱氢酶具有葡萄糖脱氢酶活性，对木糖的反应性降低。
(i)Val/Ile Val/Ile Val/Ile Gly Ser Gly Val Ala Gly(序列号21)
(ii)Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys(序列号22)
(iii)Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly(序列号23)
(iv)Lys Lys Ile/Leu His Leu SerAsn(序列号24)
(v)Phe Ala Pro/Asn Asn Asn His Ile(序列号25)
(2)根据(1)所述的变异GDH，其中，与从肽序列(ii)的8位的Met、肽序列(iii)的12位的Gly、肽序列(iv)的6位的Ser、肽序列(v)的2位的Ala、肽序列(v)的3位的Pro/Asn、肽序列(v)的5位的Asn以及肽序列(v)的7位的Ile中选择的至少一个氨基酸残基相当的氨基酸残基还被其他氨基酸残基取代。
(3)根据(1)或(2)所述的变异GDH，其特征在于，还对半乳糖的反应性降低。
(4)根据(1)至(3)中任一者所述的变异GDH，其中，所述变异GDHα亚单位包含和序列号3至少有60％的同一性的氨基酸序列。
(5)根据(1)至(3)中任一者所述的变异GDH，其中，所述变异GDHα亚单位包含和序列号3(SM4)、序列号4(YP_002234347)、序列号5(ZP_02370914)、序列号6(ZP_02007109)、序列号7(YP_002260434)或者序列号8(YP_001890482)至少有80％的同一性的氨基酸序列。
(6)根据(1)至(4)中任一者所述的变异GDH，其中，所述变异GDHα亚单位具有序列号3(SM4)、序列号4(YP_002234347)、序列号5(ZP_02370914)、序列号6(ZP_02007109)、序列号7(YP_002260434)或者序列号8(YP_001890482)中取代、缺失、插入或者添加了的一个或者多个氨基酸的氨基酸序列。
(7)一种变异GDH，其包含具有和序列号3至少有60％的同一性的氨基酸序列、且具有葡萄糖脱氢酶活性的蛋白质，其特征在于，在所述氨基酸序列中，与序列号3的474位的天冬酰胺相当的氨基酸残基被丝氨酸取代，与序列号3的322位的甘氨酸相当的氨基酸残基被谷氨酰胺取代，与序列号3的363位的组氨酸相当的氨基酸残基被谷氨酰胺或者丝氨酸取代，并且，与序列号3的475位的天冬酰胺相当的氨基酸残基被丝氨酸取代，并且，所述变异葡萄糖脱氢酶对木糖的反应性降低。
(8)根据(1)至(3)中任一者所述的变异GDH，其中，所述变异葡萄糖脱氢酶还包含电子传递亚单位。
(9)根据(8)所述的变异GDH，其特征在于，电子传递亚单位是细胞色素C。
(10)一种DNA，其编码(1)至(9)中任一者所述的变异GDH。
(11)一种重组载体，其包含(10)所述的DNA。
(12)一种微生物，其由(11)所述的重组载体转化。
(13)一种葡萄糖检测试剂盒，其包含(1)至(9)中任一者所述的变异GDH。
(14)一种葡萄糖传感器，其包含(1)至(9)中任一者所述的变异GDH。
发明效果
根据本发明，提供一种对葡萄糖的特异性提高了的GDH，并适当地用于葡萄糖传感器等的用途中。
附图说明
图1-1是示出GDH的序列比对的图～之一；
图1-2是示出GDH的序列比对的图～之一(续)；
图1-3是示出GDH的序列比对的图～之一(续)；
图1-4是示出GDH的序列比对的图～之一(续)；
图1-5是示出GDH的序列比对的图～之一(续)；
图1-6是示出GDH的序列比对的图～之一(续)；
图1-7是示出GDH的序列比对的图～之一(续)；
图1-8是示出GDH的序列比对的图～之一(续)；
图1-9是示出GDH的序列比对的图～之一(续)；
图1-10是示出GDH的序列比对的图～之一(续)；
图2-1是示出GDH的序列比对的图～之二；
图2-2是示出GDH的序列比对的图～之二(续)；
图2-3是示出GDH的序列比对的图～之二(续)；
图2-4是示出GDH的序列比对的图～之二(续)；
图2-5是示出GDH的序列比对的图～之二(续)；
图2-6是示出GDH的序列比对的图～之二(续)；
图2-7是示出GDH的序列比对的图～之二(续)；
图2-8是示出GDH的序列比对的图～之二(续)；
图2-9是示出GDH的序列比对的图～之二(续)；
图2-10是示出GDH的序列比对的图～之二(续)；
图3-1是示出GDH的序列比对的图～之三；
图3-2是示出GDH的序列比对的图～之三(续)；
图3-3是示出GDH的序列比对的图～之三(续)；
图3-4是示出GDH的序列比对的图～之三(续)；
图3-5是示出GDH的序列比对的图～之三(续)；
图3-6是示出GDH的序列比对的图～之三(续)；
图3-7是示出GDH的序列比对的图～之三(续)；
图3-8是示出GDH的序列比对的图～之三(续)；
图3-9是示出GDH的序列比对的图～之三(续)；
图3-10是示出GDH的序列比对的图～之三(续)。
具体实施方式
下面，详细地说明本发明。
本发明的变异GDH的特征在于，其包含变异GDHα亚单位，上述变异GDHα亚单位具有在依次包含下面的肽序列(i)～(v)的520～550氨基酸的GDHα亚单位的氨基酸序列中、以其他氨基酸取代肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺中的一个以上的氨基酸序列，其中，上述变异GDH具有GDH活性，对木糖的反应性降低。
(i)Val/Ile Val/Ile Val/Ile Gly Ser Gly Val Ala Gly序列号21
(ii)Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys序列号22
(iii)Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly序列号23
(iv)Lys Lys Ile/Leu His Leu Ser Asn序列号24
(v)Phe Ala Pro/Asn Asn Asn His Ile序列号25
如图1～3所示，上面所述的肽序列(i)～(v)是被保存在各种GDHα亚单位蛋白质之间、且对GDH活性重要的区域。
图1的各序列为如下。
Sequence 3序列号3(CyGDH)
WP_006396898.1 choline dehydrogenase[Burkholderia multivorans]
WP_035974223.1 MULTISPECIES：choline dehydrogenase[Burkholderiacepacia complex]
EGD03130.1 glucose-methanol-choline oxidoreductase[Burkholderiasp.TJI49]
WP_040131619.1 choline dehydrogenase[Burkholderia cenocepacia]
WP_040140036.1 choline dehydrogenase[Burkholderia cenocepacia]
WP_034204694.1 choline dehydrogenase[Burkholderia cepacia]
WP_044843287.1 choline dehydrogenase[Burkholderia sp.USM B20]
WP_023476482.1 glucose dehydrogenase[Burkholderia cenocepacia]
WP_027780833.1 choline dehydrogenase[Burkholderia cepacia]
WP_011658979.1 choline dehydrogenase[Burkholderia ambifaria]
WP_050012791.1 choline dehydrogenase[Burkholderia cenocepacia]
WP_011547562.1 choline dehydrogenase[Burkholderia cenocepacia]
WP_034202233.1 choline dehydrogenase[Burkholderia cepacia]
WP_011349244.1 choline dehydrogenase[Burkholderia lata]
WP_034180249.1 choline dehydrogenase[Burkholderia pyrrocinia]
WP_006752014.1 choline dehydrogenase[Burkholderia ambifaria]
WP_047903069.1 choline dehydrogenase[Burkholderia pyrrocinia]
YP_002234347putative oxidoreductase[Burkholderia cenocepacia J2315](序列号7)
WP_039320756.1 choline dehydrogenase[Burkholderia sp.A9]
WP_011882360.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_021162033.1 MULTISPECIES：glucose dehydrogenase[Burkholderia]
WP_014724779.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_012366205.1 choline dehydrogenase[Burkholderia ambifaria]
WP_046548034.1 choline dehydrogenase[Burkholderia contaminans]
WP_039351161.1 choline dehydrogenase[Burkholderia contaminans]
WP_027791851.1 MULTISPECIES：choline dehydrogenase[Burkholderiacepacia complex]
WP_047849808.1 choline dehydrogenase[Burkholderia contaminans]
WP_043184375.1 choline dehydrogenase[Burkholderia cepacia]
WP_014899066.1 choline dehydrogenase[Burkholderia cepacia]
WP_048252127.1 choline dehydrogenase[Burkholderia cepacia]
WP_031400524.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_010089726.1 choline dehydrogenase[Burkholderia ubonensis]
WP_011354898.1 choline dehydrogenase[Burkholderia lata]
WP_034187894.1 choline dehydrogenase[Burkholderia cenocepacia]
WP_017329146.1 choline dehydrogenase[Burkholderia pyrrocinia]
WP_045565070.1 choline dehydrogenase[Burkholderia ubonensis]
WP_010804580.1 glucose-methanol-choline oxidoreductase[Pandoraeasp.SD6-2]
WP_042588018.1 choline dehydrogenase[Burkholderia sp.MSHR3999]
WP_038746878.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_010118596.1 choline dehydrogenase[Burkholderia oklahomensis]
WP_010108853.1 choline dehydrogenase[Burkholderia oklahomensis]
ZP_02370914hypothetical protein BthaT_07876[Burkholderiathailandensis TXDOH](序列号8)
WP_045602806.1 choline dehydrogenase[Burkholderia thailandensis]
WP_004528231.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_009897186.1 choline dehydrogenase[Burkholderia thailandensis]
WP_038763142.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_038781432.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_038778573.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_038779482.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_004198666.1 MULTISPECIES：choline dehydrogenase [pseudomalleigroup]
WP_041195444.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_044490678.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_041189202.1 choline dehydrogenase[Burkholderia pseudomallei]
WP_038789867.1 MULTISPECIES：choline dehydrogenase[pseudomallei group]
WP_041198446.1 choline dehydrogenase[Burkholderia pseudomallei]
KGT02773.1 FAD dependent oxidoreductase family protein[Burkholderiapseudomallei]
WP_015602981.1 FAD-binding protein[Burkholderia thailandensis]
WP_006027349.1 MULrISPECIES：choline dehydrogenase[Burkholderia]
WP_027778581.1 choline dehydrogenase[Burkholderia caledonica]
图2的各序列为如下：
Sequence 3序列号3(CyGDH)
WP_038789867.1 MULTISPECIES：choline dehydrogenase[pseudomallei group]
WP_041198446.1 choline dehydrogenase[Burkholderia pseudomallei]
KGT02773.1 FAD dependent oxidoreductase family protein[Burkholderiapseudomallei]
WP_015602981.1 FAD-binding protein[Burkholderia thailandensis]
WP_006027349.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_027778581.1 choline dehydrogenase[Burkholderia caledonica]
WP_020067867.1 choline dehydrogenase[Burkholderia bryophila]
WP_027798558.1 choline dehydrogenase[Burkholderia dilworthii]
WP_017774216.1 choline dehydrogenase[Burkholderia kururiensis]
WP_035557405.1 choline dehydrogenase[Burkholderia sp.9120]
WP_028194543.1 MULTISPECIES：choline dehydrogenase[Burkholderia]
WP_048931828.1 choline dehydrogenase[Ralstonia sp.MD27]
WP_039597686.1 choline dehydrogenase[Ralstonia sp.A12]
WP_021195199.1 MULTISPECIES：choline dehydrogenase[Ralstonia]
WP_027677929.1 choline dehydrogenase[Ralstonia sp.UNC404CL21Col]
WP_004629448.1 MULTISPECIES：choline dehydrogenase[Ralstonia]
WP_024976326.1 choline dehydrogenase[Ralstonia pickettii]
WP_045204558.1 choline dehydrogenase[Burkholderiaceae bacterium 26]
ZP_02007109FAD dependent oxidoreductase[Ralstonia pickettii 12D](序列号9)
WP_045786289.1 choline dehydrogenase[Ralstonia mannitolilytica]
WP_009238767.1 MULTISPECIES：choline dehydrogenase[Bacteria]
CBJ51936.1 putative transmembrane dehydrogenase(Large subunit)oxidoreductase
[Ralstonia solanacearum PSI07]
WP_020749404.1 dehydrogenase(Large subunit)oxidoreductase[Ralstoniasolanacearum]
WP_011000725.1 choline dehydrogenase[Ralstonia solanacearum]
WP_028852718.1 choline dehydrogenase[Ralstonia solanacearum]
WP_016727135.1 choline dehydrogenase[Ralstonia solanacearum]
WP_019717688.1 choline dehydrogenase[Ralstonia solanacearum]
WP_020831435.1 2-Keto-D-gluconate dehydrogenase[Ralstoniasolanacearum]
YP_002260434transmembrane dehydrogenase(1arge subunit)protein[Ralstonia solanaceamm IPO1609](序列号10)
WP_003279244.1 choline dehydrogenase[Ralstonia solanacearum]
WP_039568928.1 choline dehydrogenase[Ralstonia solanacearum]
WP_050138572.1 choline dehydrogenase[Yersinia enterocolitica]
WP_049608172.1 choline dehydrogenase[Yersinia pekkanenii]
WP_050140675.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050101384.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050151802.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050107996.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050146890.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050135114.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050122940.1 choline dehydrogenase[Yersinia frederiksenii]
WP_050113306.1 choline dehydrogenase[Yersinia kristensenii]
WP_019081810.1 choline dehydrogenase[Yersinia enterocolitica]
CQH40496.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_050076365.1 MULTISPECIES：choline dehydrogenase[Yersinia]
CFQ84255.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
CFB68626.1 2-keto-D-gluconate dehydrogenase membrane-boundnavoprotein[Yersinia enterocolitica]
WP_019083704.1 choline dehydrogenase[Yersinia enterocolitica]
WP_004391242.1 choline dehydrogenase[Yersinia kristensenii]
WP_050130822.1 choline dehydrogenase[Yersinia frederiksenii]
WP_048616917.1 MULTISPECIES：choline dehydrogenase[Yersinia]
WP_050157528.1 choline dehydrogenase[Yersinia enterocolitica]
CNB97617.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
CRE83670.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_038635282.1 MULTISPECIES：choline dehydrogenase[Yersinia]
CQQ92408.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_049679386.1 choline dehydrogenase[Yersinia mollaretii]
WP_049648565.1 choline dehydrogenase[Yersinia mollaretii]
WP_050159872.1 choline dehydrogenase[Yersinia enterocolitica]
WP_050144494.1 choline dehydrogenase[Yersinia enterocolitica]
CFR10819.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia frederiksenii]
WP_050299034.1 choline dehydrogenase[Yersinia frederiksenii]
WP_049610421.1 choline dehydrogenase[Yersinia mollaretii]
WP_004877956.1 choline dehydrogenase[Yersinia mollaretii]
WP_046050437.1 choline dehydrogenase[Yersinia enterocolitica]
CFR23261.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia kristensenii]
图3的各序列为如下。
Sequence 3序列号3(CyGDH)
WP_013650283.1 choline dehydrogenase[Yersinia enterocolitica]
CQJ52766.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_050539080.1 choline dehydrogenase[Yersinia mollaretii]
WP_049562931.1 choline dehydrogenase[Yersinia kristensenii]
CCV62586.1 hypothetical protein YE3094_31131[Yersinia enterocolitica(type O：2)str.YE3094/96]
WP_005172530.1 choline dehydrogenase[Yersinia enterocolitica]
WP_011815792.1 choline dehydrogenase[Yersinia enterocolitica]
WP_050290584.1 choline dehydrogenase[Yersinia kristensenii]
WP_004707861.1 choline dehydrogenase[Yersinia frederiksenii]
CQJ18911.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_049600682.1 choline dehydrogenase[Yersinia bercovieri]
WP_049603209.1 choline dehydrogenase[Yersinia aldovae]
CFQ30984.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_004702484.1 choline dehydrogenase[Yersinia aldovae]
WP_049688140.1 choline dehydrogenase[Yersinia aldovae]
WP_042548059.1 choline dehydrogenase[Yersinia aldovae]
CRX73404.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_014609437.1 choline dehydrogenase[Yersinia enterocolitica]
CFQ15314.1 2-keto-D-gluconate dehydrogenase membrane-boundflavoprotein[Yersinia enterocolitica]
WP_050128783.1 choline dehydrogenase[Yersinia enterocolitica]
WP_018433926.1 choline dehydrogenase[Burkholderia sp.JPY251]
WP_018424152.1 choline dehydrogenase[Burkholderia sp.WSM4176]
WP_008921043.1 choline dehydrogenase[Burkholderia sp.H160]
WP_013092964.1 choline dehydrogenase[Burkholderia sp.CCGE1002]
WP_027195331.1 choline dehydrogenase[Burkholderia sprentiae]
WP_028223852.1 choline dehydrogenase[Burkholderia ferrariae]
WP_027796285.1 choline dehydrogenase[Burkholderia acidipaludis]
WP_028196142.1 choline dehydrogenase[Burkholderia fungorum]
YP_001890482glucose-methanol-choline oxidoreductase[Burkholderiaphytofirmans
因此，在本发明中，关于在引入使对木糖的反应性降低的变异之前的GDHα亚单位的序列，只要具有这些肽序列(i)～(v)的序列、全长氨基酸为520～550(优选525～544)且GDH活性被维持，就没有特别限制。
在本发明中，在引入使对木糖的反应性降低的变异之前的GDHα亚单位的序列优选和序列号3有60％以上的同一性。
序列号3是洋葱伯克霍尔德菌(Burkholderia cepacia)KS1株保持的GDHα亚单位的氨基酸序列。KS1株在平成12年9月25日以保藏号码为第FERM BP-7306保藏在独立行政法人产业技术综合研究所专利生物保藏中心(305-8566日本茨城县筑波市东1丁目1番地1中央第6)。肽序列(i)～(v)分别对应于序列号3中的氨基酸编号12-20、212-222、313-324、360-366、471-477的区域。
作为和序列号3有60％以上同一性的GDHα亚单位的氨基酸序列，例示洋葱伯克霍尔德菌J2315株的putative oxidoreductase(序列号7)、泰国伯克菌(Burkholderiathailandensis)TXDOH株的hypothetical protein BthaT#07876(序列号8)、皮氏罗尔斯顿氏菌(Ralstonia pickettii)12D株的FAD dependent oxidoreductase(序列号9)、茄科雷尔氏菌(Ralstonia solanacearum)IPO1609株的transmembrane dehydrogenase(序列号10)以及伯克霍尔德氏菌(Burkholderia phytofirmans)PsJN株的glucose-methanol-choline oxidoreductase(序列号11)的各自的α亚单位。
此外，关于序列号7～11所示的氨基酸序列，均在美国国家生物技术信息中心(National Center for Biotechnology Information，NCBI)的数据库中登录。各自的登录号码分别为：序列号7为YP_002234347、序列号8为ZP_02370914、序列号9为YP_002980762、序列号10为YP_002260434、序列号11为YP_001890482。
另外，洋葱伯克霍尔德菌J2315株以LMG16656、ATCCBAA-245、CCM4899、CCUG48434、NCTC13227保藏。伯克霍尔德氏菌PsJN株以LMG22487、CCUG49060保藏。
另外，作为和序列号3有60％以上同一性的GDHα亚单位，也可以举出以下：作为和洋葱伯克霍尔德菌KS1株同属的洋葱伯克霍尔德菌株，来自例如JCM2800、JCM2801、JCM5506、JCM5507、IFO14595的各GDH的α亚单位(序列号12～16)。此外，JCM2800、JCM2801、JCM5506及JCM5507保存于日本独立行政法人理化学研究所微生物菌种保藏中心(JapanCollection of Microorganisms，JCM)。IFO14595保存于日本财团法人发酵研究所(IFO)。
此外，序列号3示出的氨基酸序列相对于序列号7～11分别有96％、93％、82％、82％、63％的氨基酸序列同一性。
在图1～3中，关于含有这些的149个GDHα亚单位的序列，与序列号3进行序列比对。图1～3所示的各种GDHα亚单位的序列中的、具有和序列号3有最低同一性的是YP_001890482，YP_001890482和序列号3有63％同一性。如上所述，已知有多个GDHα亚单位，均和序列号3有60％以上的同一性，并被保存在如上所述的区域。因此，通过在依次包含肽序列(i)～(v)的520～550个氨基酸的GDHα亚单位的氨基酸序列、优选依次包含肽序列(i)～(v)、且和序列号3有60％以上(优选63％以上)的同一性的520～550个氨基酸的氨基酸序列中、以其他氨基酸取代肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺中的一个以上，能够获得包含本发明的变异GDHα亚单位的变异GDH。
取代肽序列(iii)的10位的甘氨酸的氨基酸可以是甘氨酸以外的任何氨基酸，优选谷氨酰胺、组氨酸、蛋氨酸、天冬酰胺、天冬酰胺酸、半胱氨酸、缬氨酸、亮氨酸、丙氨酸、异亮氨酸、苯丙氨酸、谷氨酰胺酸、丝氨酸、苏氨酸、精氨酸，特别优选谷氨酰胺。
取代肽序列(iv)的4位的组氨酸的氨基酸可以是组氨酸以外的任何氨基酸，优选谷氨酰胺、天冬酰胺、精氨酸、丝氨酸，特别优选谷氨酰胺。
取代肽序列(v)的4位的天冬酰胺的氨基酸可以是天冬酰胺以外的任何氨基酸，优选丝氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、谷氨酰胺、蛋氨酸、酪氨酸、半胱氨酸、苏氨酸、谷氨酰胺酸、天冬酰胺酸，特别优选丝氨酸。
本发明的变异GDHα亚单位只要具有这些三个变异中的任何一个即可，但优选具有两个以上，特别优选三个。
优选肽序列(iii)的10位的甘氨酸的取代和肽序列(v)的4位的天冬酰胺的取代的组合，更优选肽序列(iii)的10位的甘氨酸的取代、肽序列(v)的4位的天冬酰胺的取代、以及肽序列(iv)的4位的组氨酸的取代的组合。
肽序列(iii)的10位的甘氨酸在序列号3中对应于322位的甘氨酸，在序列号7中对应于322位的甘氨酸，在序列号8中对应于320位的甘氨酸，在序列号9中对应于323位的甘氨酸，在序列号10中对应于323位的甘氨酸，在序列号11中对对应于318位的甘氨酸。
肽序列(iv)的4位的组氨酸在序列号3中对应于363位的组氨酸，在序列号7中对应于363位的组氨酸，在序列号8中对应于361位的组氨酸，在序列号9中对应于364位的组氨酸，在序列号10中对应于364位的组氨酸，在序列号11中对应于359位的组氨酸。
肽序列(v)的4位的天冬酰胺在序列号3中对应于474位的天冬酰胺，在序列号7中对应于474位的天冬酰胺，在序列号8中对应于472位的天冬酰胺，在序列号9中对应于475位的天冬酰胺，在序列号10中对应于475位的天冬酰胺，在序列号11中对应于468位的天冬酰胺。
此外，如上所述的氨基酸取代变异的位置是序列号3、7、8、9、10、11中的位置，但是在序列号3、7、8、9、10、11的氨基酸序列中除上述特定的变异以外还具有取代、缺失、插入或添加了一个或多个氨基酸残基的氨基酸序列的GDHα亚单位的同源物或变体中，在和原氨基酸序列的比对中，表示与上述氨基酸取代的位置相当的位置。例如，在1～321位的区域中具有1个氨基酸残基缺失的GDHα亚单位的保守变体中，322位表示所述变体的321位。
作为本发明的变异型α亚单位的例子，除了所述特定的变异以外，可以举出具有序列号3、7～11所示的氨基酸序列的蛋白质。另外，对于变异型α亚单位而言，只要具有GDH活性、且相对于木糖的反应性降低，也可以是具有如下氨基酸序列的蛋白质：所述氨基酸序列为序列号3、7～11的氨基酸序列中，除了具有如上所述的特定的变异之外，还取代、缺失、插入或添加了一个或多个氨基酸残基。所述“一个或多个”例如为1～20个、优选为1～10个、更优选为1～5个、特别优选为1～3个。另外，如上所述的取代优选为保守的取代，“保存的取代”是指酸性氨基酸之间的取代、中性氨基酸之间的取代、碱基性氨基酸之间的取代等性质相似的氨基酸之间的取代。
此外，本发明的变异型α亚单位优选为相对于序列号3、7～11中的任一个氨基酸序列而言，具有至少80％、优选85％、更优选90％的氨基酸同一性的蛋白质。
本发明的变异型α亚单位除了如上所述的变异之外，还可以以其他氨基酸残基取代相当于选自肽序列(ii)的8位的蛋氨酸、肽序列(iii)的12位的甘氨酸、肽序列(iv)的6位的丝氨酸、肽序列(v)的2位的丙氨酸、肽序列(v)的3位的脯氨酸或者天冬酰胺、肽序列(v)的5位的天冬酰胺以及肽序列(v)的7位的异亮氨酸中的至少一个氨基酸残基的氨基酸残基。
这些氨基酸对应于序列号3中的下面的位置。
肽序列(ii)的8位的蛋氨酸…序列号3的219位的蛋氨酸肽序列(iii)的12位的甘氨酸…序列号3的324位的甘氨酸肽序列(iv)的6位的丝氨酸…序列号3的365位的丝氨酸肽序列(v)的2位的丙氨酸…序列号3的472位的丙氨酸肽序列(v)的3位的脯氨酸或者天冬酰胺…序列号3的473位的脯氨酸肽序列(v)的5位的天冬酰胺…序列号3的475位的天冬酰胺肽序列(v)的7位的异亮氨酸…序列号3的477位的异亮氨酸
除此之外，可以取代相当于序列号3的59位的蛋氨酸、序列号3的61位的苯丙氨酸、序列号3的326位的丝氨酸、序列号3的341位的谷氨酰胺酸、序列号3的367位的亮氨酸、序列号3的369位的精氨酸、序列号3的400位的酪氨酸、序列号3的402位的谷氨酰胺、序列号3的404位的天冬酰胺酸的氨基酸的一个以上。
下面，示出优选的变异体的例子。
G322Q-H363Q-N474S-N475S
G322Q-H363Q-S365A-N474S-N475S
G322Q-H363Q-S365G-N474S-N475S
G322Q-H363S-S365N-N474S-N475S
G322Q-H324Q-N474S-N475S
G322Q-H324S-N474S-N475S
G322Q-D404E-N474S-N475S
G322Q-A472M-N474S-N475S
这些变异点表示序列号3中的位置，例如，G322Q-H363Q-N474S-N475S是序列号3中的、322位的甘氨酸被谷氨酰胺取代、363位的组氨酸被谷氨酰胺取代、474位的天冬酰胺被丝氨酸取代、475位的天冬酰胺被丝氨酸取代的变异体，也可以在序列号3中的除了这些以外的氨基酸中引入变异，当然，在序列号7～11等的其他GDH的序列中的相同位置引入变异的也被包含在本发明的变异GDH。
例如，下面所示的变异体被包含在本发明的变异体。
一种变异GDH，其包含具有和序列号3至少有60％的同一性的氨基酸序列、且具有葡萄糖脱氢酶活性的蛋白质，其特征在于，相当于序列号3的474位的天冬酰胺的氨基酸残基被丝氨酸取代，相当于序列号3的322位的甘氨酸的氨基酸残基被谷氨酰胺取代，并且相当于序列号3的475位的天冬酰胺的氨基酸残基被丝氨酸取代，并且，对木糖的反应性降低。
一种变异GDH，其是包含具有和序列号3至少有60％的同一性的氨基酸序列、且具有葡萄糖脱氢酶活性的蛋白质，其特征在于，与序列号3的474位的天冬酰胺相当的氨基酸残基被丝氨酸取代，与序列号3的322位的甘氨酸相当的氨基酸残基被谷氨酰胺取代，与序列号3的363位的组氨酸相当的氨基酸残基被谷氨酰胺或者丝氨酸取代，并且与序列号3的475位的天冬酰胺相当的氨基酸残基被丝氨酸取代，并且，对木糖的反应性降低。
本发明的变异GDH通过具有如上所述的特定的变异，来降低对木糖的反应性。例如，对木糖的反应性是野生型GDH的50％以下、优选为20％以下、更优选为10％以下。
此外，在与对葡萄糖的反应性相比，对木糖的反应性相对低的情况也被包含在“对木糖的反应性降低”中。例如，如下所述的底物特异性(Xyl/Glc)对应于比野生型GDH低的情况。例如，底物特异性(Xyl/Glc)是野生型GDH的50％以下、优选为20％以下、更优选为10％以下。底物特异性＝(对木糖的比活性/对葡萄糖的比活性)×100。
本发明的变异GDH由于具有如上所述的特定的变异，不仅对木糖的反应性比野生型GDH低，对半乳糖的反应性也比野生型GDH低。例如，对半乳糖的反应性与野生型GDH相比，例如为50％以下、优选为20％以下、更优选为10％以下。
此外，在与对葡萄糖的反应性相比，对半乳糖的反应性相对低的情况也包含在“对半乳糖的反应性降低”中。例如，如下所述的底物特异性(Gal/Glc)对应于比野生型GDH低的情况。例如，底物特异性(Gal/Glc)是野生型GDH的50％以下、优选为20％以下、更优选为10％以下。
底物特异性＝(对半乳糖的比活性/对葡萄糖的比活性)×100)
本发明的变异GDH可以仅仅是α亚单位，可以是包括α亚单位及β亚单位的复合体、α亚单位及γ亚单位的复合体、或者α亚单位、β亚单位及γ亚单位的复合体。
序列号1示出包含洋葱伯克霍尔德菌KS1株的GDHα亚单位基因、以及β亚单位基因的一部分的染色体DNA片段的碱基序列(美国专利申请公开第2004/0023330号)。该碱基序列中存在3个开放阅读框(ORF)，从5′末端侧开始第2个及第3个ORF分别编码α亚单位(序列号3)及β亚单位(序列号4)。另外，第1个ORF编码γ亚单位(序列号2)。另外，序列号5示出含有β亚单位基因全长的片段的碱基序列。而且，序列号6示出β亚单位的氨基酸序列(欧洲专利申请公开第1498484号)。序列号6中的氨基酸编号1～22为信号肽。
在本发明的变异GDH包含β亚单位的情况下，关于β亚单位，只要作为β亚单位而起作用即可，没有特别的限制，可以使用包括公知的在内的、来自各种生物的β亚单位，具体而言，例如，可以使用具有来自洋葱伯克霍尔德菌KS1株的序列号6的氨基酸编号23～425的氨基酸序列的蛋白质。但是，只要可以作为CyGDH的β亚单位起作用，也可以是具有序列号6的氨基酸号码23～425的氨基酸序列中取代、缺失、插入或添加了一个或多个氨基酸残基的氨基酸序列的蛋白质。另外，只要可以作为CyGDH的β亚单位起作用，也可以是具有KS1株以外的β亚单位及该β亚单位的氨基酸序列中取代、缺失、插入或添加了一个或多个氨基酸残基的氨基酸序列的蛋白质。所述“一个或多个”优选为1～20个、更优选为1～10个、特别优选为1～5个。此外，作为CyGDH的β亚单位起作用是指，当和α亚单位一起形成复合体时，不损害该复合体的GDH活性而作为电子传递亚单位、即细胞色素C而起作用。
在本发明的变异GDH包含γ亚单位的情况下，关于γ亚单位，只要作为γ亚单位而起作用即可，没有特别的限制，可以使用包含公知的在内、来自各种生物的γ亚单位，具体而言，例如，可以使用来自洋葱伯克霍尔德菌KS1株的序列号2的氨基酸序列的蛋白质。但是，只要可以作为γ亚单位起作用，也可以是具有由序列号2构成的氨基酸序列中取代、缺失、插入或添加了一个或多个氨基酸残基的氨基酸序列的蛋白质。另外，只要可以作为γ亚单位起作用，也可以是具有KS1株以外的γ亚单位的氨基酸序列中取代、缺失、插入或添加了一个或多个氨基酸残基的氨基酸序列的蛋白质。所述“一个或多个”优选为1～20个、更优选为1～10个、特别优选为1～5个。此外，作为γ亚单位起作用是指，当和α亚单位一起形成复合体时，提高该复合体的GDH活性的作用。
作为编码本发明的变异GDH的α亚单位的基因，只要具有与变异GDHα亚单位的氨基酸序列对应的碱基序列即可，但作为具体例，可以举出包含在由序列号1的碱基号764～2380构成的碱基序列中具有与如上所述的氨基酸取代对应的密码子取代的碱基序列的DNA。另外，α亚单位基因可以是具有包含序列号1的碱基序列的碱基号764～2380的碱基序列的DNA，或者可以是在严谨条件下与可由该序列制备的探针杂交、并且编码具有GDH活性的蛋白质的DNA。
作为β亚单位基因的具体例，可以举出包含包括序列号5的碱基号187～1398的碱基序列的DNA。另外，β亚单位基因可以是具有包括序列号5的碱基号187～1398的碱基序列的DNA，或者可以是在严谨条件下与可由该序列制备的探针杂交、并且编码可作为β亚单位而起作用的蛋白质的DNA。
作为γ亚单位基因的具体例，可以举出包含包括序列号1的碱基号258～761的碱基序列的DNA。另外，γ亚单位基因可以是具有包括序列号1的碱基号258～761的碱基序列的DNA、或者可以是在严谨条件下与可由该序列制备的探针杂交、并且编码可作为γ亚单位而起作用的蛋白质的DNA。
作为所述严谨条件，可以举出优选具有80％、更优选具有90％以上、特别优选具有95％以上的同一性的DNA之间杂交的条件，具体而言，在0.1×SSC、0.1％SDS、60℃下洗涤的条件。
α亚单位基因、β亚单位基因及γ亚单位基因例如可以通过以洋葱伯克霍尔德菌KS1株的染色体DNA为模板的PCR来获取。PCR用引物可以通过基于上述的碱基序列而进行的化学合成来制备。另外，还可以通过将基于所述序列而制作的寡核苷酸作为探针的杂交，能够从洋葱伯克霍尔德菌KS1株的染色体DNA获取。此外，也可以使用KS1株以外的新洋葱伯克霍尔德菌J2315株、泰国伯克菌TXDOH株、皮氏罗尔斯顿氏菌12D株、茄科雷尔氏菌IPO1609株及伯克霍尔德氏菌PsJN株。
具有期望的变异的GDHα亚单位可通过如下方法来获取，利用位点特异性变异法，在编码GDHα亚单位的DNA(α亚单位基因)上，引入对应于期望的氨基酸变异的核苷酸变异，将获得的变异DNA用适当的表达体系来表达。另外，通过将编码变异GDHα亚单位的DNA、与编码β亚单位的DNA(β亚单位基因)或β亚单位基因和编码γ亚单位的DNA(γ亚单位基因)一起表达，可以获得变异CyGDH复合体。此外，关于向编码GDHα亚单位的DNA引入变异，也可以使用依次编码γ亚单位、α亚单位及β亚单位的多顺反子DNA片段。
关于依次编码γ亚单位、α亚单位及β亚单位的多顺反子DNA片段，例如能够通过以洋葱伯克霍尔德菌KS1株的染色体DNA为模板、且将具有序列号19、20的碱基序列的寡核苷酸作为引物的PCR来获取。
作为GDH的各亚单位的基因的获取、变异的引入、基因的表达等的载体，可以举出例如通过Escherichia属细菌起作用的载体，具体地pTrc99A、pBR322、pUC18、pUC118、pUC19、pUC119、pACYC184、pBBR122等。作为用于基因表达的启动子，例如可以举出lac、trp、tac、trc、PL、tet、PhoA等。另外，通过在包含启动子的表达载体的适当位点插入α亚单位基因或其他亚单位基因，能够在相同步骤中进行向这些基因的载体的插入和启动子的连接。作为这样的表达载体，可以举出pTrc99A、pBlucscript、pKK223-3等。
另外，α亚单位基因或其他亚单位基因可以以能够表达的方式整合进宿主微生物的染色体DNA中。
在使用重组载体转化微生物时，例如可以举出利用钙处理的感受态细胞法、原生质粒法或电穿孔法等。
作为宿主微生物，可以举出枯草芽孢杆菌等芽孢杆菌属细菌、酿酒酵母等酵母、黑曲霉等丝状真菌，但并不限于这些，只要是适合生产异源蛋白质的宿主微生物就可以使用。
例如通过实施例中所记载的方法来调查对各种糖类的反应性，并与包含野生型α亚单位的GDH的反应性进行比较，由此能够决定对包含变异型α亚单位的GDH的糖类的底物特异性。
包含本发明的变异α亚单位的GDH或者对其进行表达的微生物，可以作为葡萄糖传感器的酶电极的构成要素来使用。作为葡萄糖传感器，可以举出将酶电极作为工作电极而使用的葡萄糖传感器，其中，在酶电极中，包含本发明的变异α亚单位的GDH被固定在金电极、铂电极、碳电极等的电极表面上。传感器是指电化学测定作为目标的待测物质的浓度的测定系统，通常包含工作电极(酶电极)、对极(铂等)、以及参照极(Ag/AgCl等)的三个电极。或者，也可以是如在惯用的简易血糖值系统中使用的、由工作电极以及对极构成的两个电极系统。传感器还优选包括放入缓冲液以及待测试样的恒温小室、向工作电极施加电压的电源、电流表、记录仪等。传感器可以是间歇式，也可以是流式。尤其作为流式的传感器，可以是能够连续性测量血糖值的传感器。即，可以是在被连续性供给的血液试样或该透析试样、或者血液中或细胞间液中插入固定了本发明的酶的两个电极系统或者三个电极系统并进行测量的传感器。这样的酶传感器的结构在该技术领域中已被所知，例如记载在Biosensors-Fundamental and Applications-Anthony P.F.Turner，Isao Karube andGeroge S.Wilson，Oxford University Press 1987。
使用本发明的葡萄糖传感器的葡萄糖的浓度的测定能够通过下面的方式进行。将缓冲液添加到传感器的恒温小室，并维持一定温度。作为作用电极，使用固定了本发明的变异GDH的酶电极，作为对极，例如使用铂电极，作为参照电极，例如使用Ag/AgCl电极。向工作电极施加一定的电压，在电流变得稳定之后，将含有葡萄糖的试样添加到恒温小室并测定电流的增加。按照由标准浓度的葡萄糖溶液制作的校正曲线，可以计算试样中的葡萄糖浓度。
包含本发明的变异α亚单位的GDH能够作为葡萄糖检测试剂盒的构成要素而使用。葡萄糖检测试剂盒中除了包含本发明的变异α亚单位的GDH以外，还可以包括发色或者发光试剂、稀释用缓冲液、标准物质、使用说明书等。
例如，使用了洋葱伯克霍尔德菌的野生型GDH的葡萄糖传感器以及葡萄糖检测试剂盒被记载在美国专利公开第2004/0023330A1中。本发明的变异GDH也可以以同样的方式使用。
实施例
接着，举出实施例而更具体地说明本发明，但本发明并不局限于这些实施例。
〔实施例1〕作为表达用于表达洋葱伯克霍尔德菌的GDH的质粒的、构建洋葱伯克霍尔德菌的GDH，准备表达GDH的α亚单位及γ亚单位的质粒。
作为表达α亚单位及γ亚单位的质粒，使用了WO02/036779(对应于EP1331272A1)中记载的质粒pTrc99A/γ+α。该质粒是通过以下的方式形成的质粒：将连续包含洋葱伯克霍尔德菌KS1株的GDH下亚单位结构基因和α亚单位结构基因的DNA片段插入到作为载体pTrc99A的克隆位点的NcoI/HindIII。本质粒中的GDHγα基因通过trc启动子来控制。pTrc99A/γ+α保持有氨苄青霉素抗性基因。
通过以所述质粒pTrc99A/γ+α作为模板、且以具有下面的序列的寡核苷酸作为引物的PCR，来扩增包含DNA片段的整个质粒，其中，该DNA片段通过在GDH的α亚单位C末端附加了6个组氨酸残基而形成。
〔正向引物〕
5’-ACCACCACTGATAAGGAGGTCTGACCGTGCGGAAATCTAC-3’(序列号17)
〔反向引物〕
5’-AGCCTGTGCGACTTCTTCCTTCAGCGATCGGTGGTGGTGG-3’(序列号18)
将扩增得到的片段两端进行平末端化后，将5’末端磷酸化，利用连接反应进行环状化。通过获得的重组载体来转化大肠杆菌DH5α，并采集在包含氨苄青霉素50μg/mL的LB琼脂培养基中产生的菌落。在液体LB培养基中培养获得的转化体并提取质粒，并且分析该插入DNA片段，结果确认了约2.1kb的插入片段。本质粒中GDH的各结构基因利用trc启动子来控制。本质粒保持有氨苄青霉素抗性基因。
〔实施例2〕向GDHα亚单位基因引入变异
使用实施例1中获得的pTrc99A/γ+α中包含的GDHα亚单位基因，并进行位点特异性变异引入，以使得在该基因引起编码的α亚单位的各氨基酸中引入变异。具体而言，使用市售的位点特异性变异引入试剂盒(Stratagene公司，QuikChangeII Site-DirectedMutagenesis Kit)而进行密码子改变，使得实施例1中记载的质粒pTrc99A/γ+α中含有的GDHα亚单位基因的、322位的甘氨酸、363位的组氨酸、以及474位的天冬酰胺等的氨基酸被其他氨基酸取代。
表1示出构建的各变异体。此外，在表示变异的标记中，数字表示氨基酸序列中的位置，数字之前的字母表示氨基酸取代前的氨基酸残基，数字之后的字母表示氨基酸取代之后的氨基酸残基。例如，N474G表示从474位的天冬酰胺取代为甘氨酸。
〔实施例3〕变异GDH的底物特异性的分析
使用实施例2中获得的变异酶表达质粒而制造变异酶，并研究底物特异性。
(1)培养
关于引入了变异GDH表达质粒的大肠杆菌BL21株，分别使用3ml的LB培养基(含有羧苄青霉素100μg/ml)，并使用L形管而在37℃下培养4个小时之后，在温度25℃下进一步培养了20个小时。
(2)粗制酶样品的调制
从以如上所述的方式培养的培养液中收集菌体，将它们悬浮在400μl的BugBuster(默克密理博制造)并在室温下进行20分钟的高速振荡，由此粉碎。对该悬浮液进行离心分离(15000×g，20分，4℃)并去除残渣，将获得的上清液(水溶性级分)作为粗制酶样品。
(3)GDH活性的测定
通过600nm时的吸光度的随时间变化来对由脱氢酶和底物的反应还原的DCIP(2，6-二氯靛酚)的褪色进行定量，由此进行酶活性测定。除非另有规定，反应条件在下面的条件下进行。通过在包含酶溶液的反应溶液(10mM磷酸钾(pH6.5)+6.0mM PMS(甲基吩嗪硫酸盐)+0.12mM DCIP浓度全为最终浓度)中添加底物(葡萄糖、木糖或者半乳糖)，开始进行反应，并测定了600nm的吸光度变化。表1示出底物的最终浓度为40mM时的结果。此外，作为野生型GDH，使用了SM4γα。
表1-1
表1-2
表1-3
表1-4
表1-5
表1-6
表1-7
表1-8
表1-9
其结果为，可以确认当取代了322位的甘氨酸、363位的组氨酸、474位的天冬酰胺的一个以上时，维持对葡萄糖的反应性，并降低与木糖之间的反应性，并且很多这些变异体还同时降低对半乳糖的反应性。
产业上的可应用性
本发明的变异GDH对葡萄糖的底物特异性提高，并且可以适当地用于使用了葡萄糖传感器等的葡萄糖的测定中。
序列表
<110> 爱科来株式会社；究极酵素国际股份有限公司
<120> 变异葡萄糖脱氢酶及其使用
<130> P-150607
<160> 25
<170> PatentIn version 3.3
<210> 1
<211> 2467
<212> DNA
<213> Burkhorderia cepacia
<220>
<221> CDS
<222> (258)..(761)
<220>
<221> CDS
<222> (764)..(2380)
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<222> (2386)..(2466)
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aagctttctg tttgattgca cgcgattcta accgagcgtc tgtgaggcgg aacgcgacat 60
gcttcgtgtc gcacacgtgt cgcgccgacg acacaaaaat gcagcgaaat ggctgatcgt 120
tacgaatggc tgacacattg aatggactat aaaaccattg tccgttccgg aatgtgcgcg 180
tacatttcag gtccgcgccg atttttgaga aatatcaagc gtggttttcc cgaatccggt 240
gttcgagaga aggaaac atg cac aac gac aac act ccc cac tcg cgt cgc 290
Met His Asn Asp Asn Thr Pro His Ser Arg Arg
1 5 10
cac ggc gac gca gcc gca tca ggc atc acg cgg cgt caa tgg ttg caa 338
His Gly Asp Ala Ala Ala Ser Gly Ile Thr Arg Arg Gln Trp Leu Gln
15 20 25
ggc gcg ctg gcg ctg acc gca gcg ggc ctc acg ggt tcg ctg aca ttg 386
Gly Ala Leu Ala Leu Thr Ala Ala Gly Leu Thr Gly Ser Leu Thr Leu
30 35 40
cgg gcg ctt gca gac aac ccc ggc act gcg ccg ctc gat acg ttc atg 434
Arg Ala Leu Ala Asp Asn Pro Gly Thr Ala Pro Leu Asp Thr Phe Met
45 50 55
acg ctt tcc gaa tcg ctg acc ggc aag aaa ggg ctc agc cgc gtg atc 482
Thr Leu Ser Glu Ser Leu Thr Gly Lys Lys Gly Leu Ser Arg Val Ile
60 65 70 75
ggc gag cgc ctg ctg cag gcg ctg cag aag ggc tcg ttc aag acg gcc 530
Gly Glu Arg Leu Leu Gln Ala Leu Gln Lys Gly Ser Phe Lys Thr Ala
80 85 90
gac agc ctg ccg cag ctc gcc ggc gcg ctc gcg tcc ggt tcg ctg acg 578
Asp Ser Leu Pro Gln Leu Ala Gly Ala Leu Ala Ser Gly Ser Leu Thr
95 100 105
cct gaa cag gaa tcg ctc gca ctg acg atc ctc gag gcc tgg tat ctc 626
Pro Glu Gln Glu Ser Leu Ala Leu Thr Ile Leu Glu Ala Trp Tyr Leu
110 115 120
ggc atc gtc gac aac gtc gtg att acg tac gag gaa gca tta atg ttc 674
Gly Ile Val Asp Asn Val Val Ile Thr Tyr Glu Glu Ala Leu Met Phe
125 130 135
ggc gtc gtg tcc gat acg ctc gtg atc cgt tcg tat tgc ccc aac aaa 722
Gly Val Val Ser Asp Thr Leu Val Ile Arg Ser Tyr Cys Pro Asn Lys
140 145 150 155
ccc ggc ttc tgg gcc gac aaa ccg atc gag agg caa gcc tg atg gcc 769
Pro Gly Phe Trp Ala Asp Lys Pro Ile Glu Arg Gln Ala Met Ala
160 165 170
gat acc gat acg caa aag gcc gac gtc gtc gtc gtt gga tcg ggt gtc 817
Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser Gly Val
175 180 185
gcg ggc gcg atc gtc gcg cat cag ctc gcg atg gcg ggc aag gcg gtg 865
Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys Ala Val
190 195 200
atc ctg ctc gaa gcg ggc ccg cgc atg ccg cgc tgg gaa atc gtc gag 913
Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile Val Glu
205 210 215
cgc ttc cgc aat cag ccc gac aag atg gac ttc atg gcg ccg tac ccg 961
Arg Phe Arg Asn Gln Pro Asp Lys Met Asp Phe Met Ala Pro Tyr Pro
220 225 230
tcg agc ccc tgg gcg ccg cat ccc gag tac ggc ccg ccg aac gac tac 1009
Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn Asp Tyr
235 240 245 250
ctg atc ctg aag ggc gag cac aag ttc aac tcg cag tac atc cgc gcg 1057
Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile Arg Ala
255 260 265
gtg ggc ggc acg acg tgg cac tgg gcc gcg tcg gcg tgg cgc ttc att 1105
Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg Phe Ile
270 275 280
ccg aac gac ttc aag atg aag agc gtg tac ggc gtc ggc cgc gac tgg 1153
Pro Asn Asp Phe Lys Met Lys Ser Val Tyr Gly Val Gly Arg Asp Trp
285 290 295
ccg atc cag tac gac gat ctc gag ccg tac tat cag cgc gcg gag gaa 1201
Pro Ile Gln Tyr Asp Asp Leu Glu Pro Tyr Tyr Gln Arg Ala Glu Glu
300 305 310
gag ctc ggc gtg tgg ggc ccg ggc ccc gag gaa gat ctg tac tcg ccg 1249
Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr Ser Pro
315 320 325 330
cgc aag cag ccg tat ccg atg ccg ccg ctg ccg ttg tcg ttc aac gag 1297
Arg Lys Gln Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe Asn Glu
335 340 345
cag acc atc aag acg gcg ctg aac aac tac gat ccg aag ttc cat gtc 1345
Gln Thr Ile Lys Thr Ala Leu Asn Asn Tyr Asp Pro Lys Phe His Val
350 355 360
gtg acc gag ccg gtc gcg cgc aac agc cgc ccg tac gac ggc cgc ccg 1393
Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly Arg Pro
365 370 375
act tgt tgc ggc aac aac aac tgc atg ccg atc tgc ccg atc ggc gcg 1441
Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile Gly Ala
380 385 390
atg tac aac ggc atc gtg cac gtc gag aag gcc gaa cgc gcc ggc gcg 1489
Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Arg Ala Gly Ala
395 400 405 410
aag ctg atc gag aac gcg gtc gtc tac aag ctc gag acg ggc ccg gac 1537
Lys Leu Ile Glu Asn Ala Val Val Tyr Lys Leu Glu Thr Gly Pro Asp
415 420 425
aag cgc atc gtc gcg gcg ctc tac aag gac aag acg ggc gcc gag cat 1585
Lys Arg Ile Val Ala Ala Leu Tyr Lys Asp Lys Thr Gly Ala Glu His
430 435 440
cgc gtc gaa ggc aag tat ttc gtg ctc gcc gcg aac ggc atc gag acg 1633
Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile Glu Thr
445 450 455
ccg aag atc ctg ctg atg tcc gcg aac cgc gat ttc ccg aac ggt gtc 1681
Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn Gly Val
460 465 470
gcg aac agc tcg gac atg gtc ggc cgc aac ctg atg gac cat ccg ggc 1729
Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His Pro Gly
475 480 485 490
acc ggc gtg tcg ttc tat gcg agc gag aag ctg tgg ccg ggc cgc ggc 1777
Thr Gly Val Ser Phe Tyr Ala Ser Glu Lys Leu Trp Pro Gly Arg Gly
495 500 505
ccg cag gag atg acg tcg ctg atc ggt ttc cgc gac ggt ccg ttc cgc 1825
Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro Phe Arg
510 515 520
gcg acc gaa gcg gcg aag aag atc cac ctg tcg aac ctg tcg cgc atc 1873
Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Leu Ser Arg Ile
525 530 535
gac cag gag acg cag aag atc ttc aag gcc ggc aag ctg atg aag ccc 1921
Asp Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met Lys Pro
540 545 550
gac gag ctc gac gcg cag atc cgc gac cgt tcc gca cgc tac gtg cag 1969
Asp Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr Val Gln
555 560 565 570
ttc gac tgc ttc cac gaa atc ctg ccg caa ccc gag aac cgc atc gtg 2017
Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg Ile Val
575 580 585
ccg agc aag acg gcg acc gat gcg atc ggc att ccg cgc ccc gag atc 2065
Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro Glu Ile
590 595 600
acg tat gcg atc gac gac tac gtg aag cgc ggc gcc gcg cat acg cgc 2113
Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Ala His Thr Arg
605 610 615
gag gtc tac gcg acc gcc gcg aag gtg ctc ggc ggc acg gac gtc gtg 2161
Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp Val Val
620 625 630
ttc aac gac gaa ttc gcg ccg aac aat cac atc acg ggc tcg acg atc 2209
Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ser Thr Ile
635 640 645 650
atg ggc gcc gat gcg cgc gac tcc gtc gtc gac aag gac tgc cgc acg 2257
Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys Arg Thr
655 660 665
ttc gac cat ccg aac ctg ttc att tcg agc agc gcg acg atg ccg acc 2305
Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ala Thr Met Pro Thr
670 675 680
gtc ggt acc gta aac gtg acg ctg acg atc gcc gcg ctc gcg ctg cgg 2353
Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala Leu Arg
685 690 695
atg tcg gac acg ctg aag aag gaa gtc tgacc gtg cgg aaa tct act ctc 2403
Met Ser Asp Thr Leu Lys Lys Glu Val Val Arg Lys Ser Thr Leu
700 705 710
act ttc ctc atc gcc ggc tgc ctc gcg ttg ccg ggc ttc gcg cgc gcg 2451
Thr Phe Leu Ile Ala Gly Cys Leu Ala Leu Pro Gly Phe Ala Arg Ala
715 720 725
gcc gat gcg gcc gat c 2467
Ala Asp Ala Ala Asp
730
<210> 2
<211> 168
<212> PRT
<213> Burkhorderia cepacia
<400> 2
Met His Asn Asp Asn Thr Pro His Ser Arg Arg His Gly Asp Ala Ala
1 5 10 15
Ala Ser Gly Ile Thr Arg Arg Gln Trp Leu Gln Gly Ala Leu Ala Leu
20 25 30
Thr Ala Ala Gly Leu Thr Gly Ser Leu Thr Leu Arg Ala Leu Ala Asp
35 40 45
Asn Pro Gly Thr Ala Pro Leu Asp Thr Phe Met Thr Leu Ser Glu Ser
50 55 60
Leu Thr Gly Lys Lys Gly Leu Ser Arg Val Ile Gly Glu Arg Leu Leu
65 70 75 80
Gln Ala Leu Gln Lys Gly Ser Phe Lys Thr Ala Asp Ser Leu Pro Gln
85 90 95
Leu Ala Gly Ala Leu Ala Ser Gly Ser Leu Thr Pro Glu Gln Glu Ser
100 105 110
Leu Ala Leu Thr Ile Leu Glu Ala Trp Tyr Leu Gly Ile Val Asp Asn
115 120 125
Val Val Ile Thr Tyr Glu Glu Ala Leu Met Phe Gly Val Val Ser Asp
130 135 140
Thr Leu Val Ile Arg Ser Tyr Cys Pro Asn Lys Pro Gly Phe Trp Ala
145 150 155 160
Asp Lys Pro Ile Glu Arg Gln Ala
165
<210> 3
<211> 539
<212> PRT
<213> Burkhorderia cepacia
<400> 3
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ala Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Pro Asp Lys Met Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Ser Val Tyr Gly Val Gly Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Leu Glu Pro Tyr Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Gln Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Thr Ala Leu Asn Asn Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Arg Ala
225 230 235 240
Gly Ala Lys Leu Ile Glu Asn Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Val Ala Ala Leu Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Glu His Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Ser Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Leu Ser
355 360 365
Arg Ile Asp Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met
370 375 380
Lys Pro Asp Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Ala His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ser
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ala Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 4
<211> 27
<212> PRT
<213> Burkhorderia cepacia
<400> 4
Val Arg Lys Ser Thr Leu Thr Phe Leu Ile Ala Gly Cys Leu Ala Leu
1 5 10 15
Pro Gly Phe Ala Arg Ala Ala Asp Ala Ala Asp
20 25
<210> 5
<211> 1441
<212> DNA
<213> Burkholderia cepacia
<220>
<221> CDS
<222> (121)..(1398)
<400> 5
tccgaacctg ttcatttcga gcagcgcgac gatgccgacc gtcggtaccg taaacgtgac 60
gctgacgatc gccgcgctcg cgctgcggat gtcggacacg ctgaagaagg aagtctgacc 120
gtg cgg aaa tct act ctc act ttc ctc atc gcc ggc tgc ctc gcg ttg 168
Val Arg Lys Ser Thr Leu Thr Phe Leu Ile Ala Gly Cys Leu Ala Leu
1 5 10 15
ccg ggc ttc gcg cgc gcg gcc gat gcg gcc gat ccg gcg ctg gtc aag 216
Pro Gly Phe Ala Arg Ala Ala Asp Ala Ala Asp Pro Ala Leu Val Lys
20 25 30
cgc ggc gaa tac ctc gcg acc gcc ggc gac tgc atg gcc tgc cac acc 264
Arg Gly Glu Tyr Leu Ala Thr Ala Gly Asp Cys Met Ala Cys His Thr
35 40 45
gtg aag ggc ggc aag ccg tac gcg ggc ggc ctt ggc atg ccg gta ccg 312
Val Lys Gly Gly Lys Pro Tyr Ala Gly Gly Leu Gly Met Pro Val Pro
50 55 60
atg ctc ggc aag atc tac acg agc aac atc acg ccc gat ccc gat acg 360
Met Leu Gly Lys Ile Tyr Thr Ser Asn Ile Thr Pro Asp Pro Asp Thr
65 70 75 80
ggc atc ggc aaa tgg acg ttc gag gac ttc gag cgc gcg gtg cgg cac 408
Gly Ile Gly Lys Trp Thr Phe Glu Asp Phe Glu Arg Ala Val Arg His
85 90 95
ggc gtg tcg aag aac ggc gac aac ctg tat ccg gcg atg ccg tac gtg 456
Gly Val Ser Lys Asn Gly Asp Asn Leu Tyr Pro Ala Met Pro Tyr Val
100 105 110
tcg tac gcg aag atc acg gac gac gac gta cgc gcg ctg tac gcc tac 504
Ser Tyr Ala Lys Ile Thr Asp Asp Asp Val Arg Ala Leu Tyr Ala Tyr
115 120 125
ttc atg cac ggc gtc gag ccg gtc aag cag gcg ccg ccg aag aac gag 552
Phe Met His Gly Val Glu Pro Val Lys Gln Ala Pro Pro Lys Asn Glu
130 135 140
att ccc gcg ctg ctc agc atg cgc tgg ccg ctg aag atc tgg aac tgg 600
Ile Pro Ala Leu Leu Ser Met Arg Trp Pro Leu Lys Ile Trp Asn Trp
145 150 155 160
ctg ttc ctg aag gac ggc ccg tac cag ccg aag ccg tcg cag agc gcc 648
Leu Phe Leu Lys Asp Gly Pro Tyr Gln Pro Lys Pro Ser Gln Ser Ala
165 170 175
gaa tgg aat cgc ggc gcg tat ctg gtg cag ggt ctc gcg cac tgc agc 696
Glu Trp Asn Arg Gly Ala Tyr Leu Val Gln Gly Leu Ala His Cys Ser
180 185 190
acg tgc cac acg ccg cgc ggc atc gcg atg cag gag aag tcg ctc gac 744
Thr Cys His Thr Pro Arg Gly Ile Ala Met Gln Glu Lys Ser Leu Asp
195 200 205
gaa acc ggc ggc agc ttc ctc gcg ggg tcg gtg ctc gcc ggc tgg gac 792
Glu Thr Gly Gly Ser Phe Leu Ala Gly Ser Val Leu Ala Gly Trp Asp
210 215 220
ggc tac aac atc acg tcg gac ccg aat gcg ggg atc ggc agc tgg acg 840
Gly Tyr Asn Ile Thr Ser Asp Pro Asn Ala Gly Ile Gly Ser Trp Thr
225 230 235 240
cag cag cag ctc gtg cag tat ttg cgc acc ggc agc gtg ccg ggc gtc 888
Gln Gln Gln Leu Val Gln Tyr Leu Arg Thr Gly Ser Val Pro Gly Val
245 250 255
gcg cag gcg gcc ggg ccg atg gcc gag gcg gtc gag cac agc ttc tcg 936
Ala Gln Ala Ala Gly Pro Met Ala Glu Ala Val Glu His Ser Phe Ser
260 265 270
aag atg acc gaa gcg gac atc ggt gcg atc gcc acg tac gtc cgc acg 984
Lys Met Thr Glu Ala Asp Ile Gly Ala Ile Ala Thr Tyr Val Arg Thr
275 280 285
gtg ccg gcc gtt gcc gac agc aac gcg aag cag ccg cgg tcg tcg tgg 1032
Val Pro Ala Val Ala Asp Ser Asn Ala Lys Gln Pro Arg Ser Ser Trp
290 295 300
ggc aag ccg gcc gag gac ggg ctg aag ctg cgc ggt gtc gcg ctc gcg 1080
Gly Lys Pro Ala Glu Asp Gly Leu Lys Leu Arg Gly Val Ala Leu Ala
305 310 315 320
tcg tcg ggc atc gat ccg gcg cgg ctg tat ctc ggc aac tgc gcg acg 1128
Ser Ser Gly Ile Asp Pro Ala Arg Leu Tyr Leu Gly Asn Cys Ala Thr
325 330 335
tgc cac cag atg cag ggc aag ggc acg ccg gac ggc tat tac ccg tcg 1176
Cys His Gln Met Gln Gly Lys Gly Thr Pro Asp Gly Tyr Tyr Pro Ser
340 345 350
ctg ttc cac aac tcc acc gtc ggc gcg tcg aat ccg tcg aac ctc gtg 1224
Leu Phe His Asn Ser Thr Val Gly Ala Ser Asn Pro Ser Asn Leu Val
355 360 365
cag gtg atc ctg aac ggc gtg cag cgc aag atc ggc agc gag gat atc 1272
Gln Val Ile Leu Asn Gly Val Gln Arg Lys Ile Gly Ser Glu Asp Ile
370 375 380
ggg atg ccc gct ttc cgc tac gat ctg aac gac gcg cag atc gcc gcg 1320
Gly Met Pro Ala Phe Arg Tyr Asp Leu Asn Asp Ala Gln Ile Ala Ala
385 390 395 400
ctg acg aac tac gtg acc gcg cag ttc ggc aat ccg gcg gcg aag gtg 1368
Leu Thr Asn Tyr Val Thr Ala Gln Phe Gly Asn Pro Ala Ala Lys Val
405 410 415
acg gag cag gac gtc gcg aag ctg cgc tga catagtcggg cgcgccgaca 1418
Thr Glu Gln Asp Val Ala Lys Leu Arg
420 425
cggcgcaacc gataggacag gag 1441
<210> 6
<211> 425
<212> PRT
<213> Burkholderia cepacia
<400> 6
Val Arg Lys Ser Thr Leu Thr Phe Leu Ile Ala Gly Cys Leu Ala Leu
1 5 10 15
Pro Gly Phe Ala Arg Ala Ala Asp Ala Ala Asp Pro Ala Leu Val Lys
20 25 30
Arg Gly Glu Tyr Leu Ala Thr Ala Gly Asp Cys Met Ala Cys His Thr
35 40 45
Val Lys Gly Gly Lys Pro Tyr Ala Gly Gly Leu Gly Met Pro Val Pro
50 55 60
Met Leu Gly Lys Ile Tyr Thr Ser Asn Ile Thr Pro Asp Pro Asp Thr
65 70 75 80
Gly Ile Gly Lys Trp Thr Phe Glu Asp Phe Glu Arg Ala Val Arg His
85 90 95
Gly Val Ser Lys Asn Gly Asp Asn Leu Tyr Pro Ala Met Pro Tyr Val
100 105 110
Ser Tyr Ala Lys Ile Thr Asp Asp Asp Val Arg Ala Leu Tyr Ala Tyr
115 120 125
Phe Met His Gly Val Glu Pro Val Lys Gln Ala Pro Pro Lys Asn Glu
130 135 140
Ile Pro Ala Leu Leu Ser Met Arg Trp Pro Leu Lys Ile Trp Asn Trp
145 150 155 160
Leu Phe Leu Lys Asp Gly Pro Tyr Gln Pro Lys Pro Ser Gln Ser Ala
165 170 175
Glu Trp Asn Arg Gly Ala Tyr Leu Val Gln Gly Leu Ala His Cys Ser
180 185 190
Thr Cys His Thr Pro Arg Gly Ile Ala Met Gln Glu Lys Ser Leu Asp
195 200 205
Glu Thr Gly Gly Ser Phe Leu Ala Gly Ser Val Leu Ala Gly Trp Asp
210 215 220
Gly Tyr Asn Ile Thr Ser Asp Pro Asn Ala Gly Ile Gly Ser Trp Thr
225 230 235 240
Gln Gln Gln Leu Val Gln Tyr Leu Arg Thr Gly Ser Val Pro Gly Val
245 250 255
Ala Gln Ala Ala Gly Pro Met Ala Glu Ala Val Glu His Ser Phe Ser
260 265 270
Lys Met Thr Glu Ala Asp Ile Gly Ala Ile Ala Thr Tyr Val Arg Thr
275 280 285
Val Pro Ala Val Ala Asp Ser Asn Ala Lys Gln Pro Arg Ser Ser Trp
290 295 300
Gly Lys Pro Ala Glu Asp Gly Leu Lys Leu Arg Gly Val Ala Leu Ala
305 310 315 320
Ser Ser Gly Ile Asp Pro Ala Arg Leu Tyr Leu Gly Asn Cys Ala Thr
325 330 335
Cys His Gln Met Gln Gly Lys Gly Thr Pro Asp Gly Tyr Tyr Pro Ser
340 345 350
Leu Phe His Asn Ser Thr Val Gly Ala Ser Asn Pro Ser Asn Leu Val
355 360 365
Gln Val Ile Leu Asn Gly Val Gln Arg Lys Ile Gly Ser Glu Asp Ile
370 375 380
Gly Met Pro Ala Phe Arg Tyr Asp Leu Asn Asp Ala Gln Ile Ala Ala
385 390 395 400
Leu Thr Asn Tyr Val Thr Ala Gln Phe Gly Asn Pro Ala Ala Lys Val
405 410 415
Thr Glu Gln Asp Val Ala Lys Leu Arg
420 425
<210> 7
<211> 539
<212> PRT
<213> Burkholderia cenocepacia
<400> 7
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Pro Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Ala Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Leu Glu His Trp Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Gln Ala Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Asp Ser Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Val Ala Ala Ile Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met
370 375 380
Lys His Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 8
<211> 537
<212> PRT
<213> Burkholderia thailandensis
<400> 8
Met Ala Glu Thr Gln Gln Ala Asp Val Val Val Val Gly Ser Gly Val
1 5 10 15
Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys Ser Val
20 25 30
Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile Val Glu
35 40 45
Arg Phe Arg Asn Gln Pro Asp Lys Met Asp Phe Met Ala Pro Tyr Pro
50 55 60
Ser Ser Ala Trp Ala Pro His Pro Glu Tyr Ala Pro Pro Asn Asp Tyr
65 70 75 80
Leu Val Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile Arg Ala
85 90 95
Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg Phe Ile
100 105 110
Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Gly Arg Asp Trp
115 120 125
Pro Ile Gln Tyr Asp Asp Leu Glu His Phe Tyr Gln Arg Ala Glu Glu
130 135 140
Glu Leu Gly Val Trp Gly Pro Gly Ala Glu Glu Asp Leu Leu Ser Pro
145 150 155 160
Arg Lys Ala Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Tyr Asn Glu
165 170 175
Arg Thr Ile Lys Thr Ala Leu Asn Asn His Asp Pro Lys Tyr His Val
180 185 190
Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly Arg Pro
195 200 205
Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile Gly Ala
210 215 220
Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala Gly Ala
225 230 235 240
Lys Leu Ile Glu Asn Ala Val Val His Lys Leu Glu Val Gly Pro Gln
245 250 255
Lys Lys Ile Val Ala Ala Leu Tyr Lys Asp Pro Lys Gly Ala Glu His
260 265 270
Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile Glu Thr
275 280 285
Pro Lys Leu Met Leu Met Ser Thr Ser His Asp Phe Pro Asn Gly Val
290 295 300
Gly Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His Pro Gly
305 310 315 320
Thr Gly Val Ser Phe Tyr Ala Ser Glu Lys Leu Trp Pro Gly Arg Gly
325 330 335
Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro Phe Arg
340 345 350
Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Leu Ser Arg Ile
355 360 365
Asp Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Leu Lys Pro
370 375 380
Ala Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr Val Gln
385 390 395 400
Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg Ile Val
405 410 415
Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro Glu Ile
420 425 430
Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Ala His Thr Arg
435 440 445
Glu Val Tyr Ala Ser Ala Ala Gln Val Leu Gly Gly Thr Asp Val Val
450 455 460
Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala Thr Ile
465 470 475 480
Met Gly Ala Asp Pro Arg Asp Ser Val Val Asp Lys Asp Cys Arg Thr
485 490 495
Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ala Thr Met Pro Thr
500 505 510
Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala Leu Arg
515 520 525
Ile Ser Asp Gln Leu Lys Lys Glu Ile
530 535
<210> 9
<211> 540
<212> PRT
<213> Ralstonia pickettii
<400> 9
Met Ala Gln Ser Glu Gln Thr Arg Gln Gln Ala Asp Ile Val Val Val
1 5 10 15
Gly Ser Gly Val Ala Gly Ala Leu Val Ala Tyr Glu Leu Ala Arg Ala
20 25 30
Gly Lys Ser Val Leu Met Leu Glu Ala Gly Pro Arg Leu Pro Arg Trp
35 40 45
Glu Ile Val Glu Arg Phe Arg Asn Gln Ala Asp Lys Met Asp Phe Met
50 55 60
Ala Pro Tyr Pro Ser Thr Ala Trp Ala Pro His Pro Glu Tyr Gly Pro
65 70 75 80
Pro Asn Asn Tyr Leu Val Leu Lys Gly Glu His Gln Phe Asn Ser Gln
85 90 95
Tyr Ile Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Thr
100 105 110
Trp Arg Phe Leu Pro Asn Asp Phe Lys Leu Arg Ser Val Tyr Gly Ile
115 120 125
Ala Arg Asp Trp Pro Ile Gln Tyr Gln Asp Leu Glu Arg Tyr Tyr Gly
130 135 140
Leu Ala Glu Glu Ala Leu Gly Val Trp Gly Pro Asn Asp Glu Asp Leu
145 150 155 160
Gly Ser Pro Arg Ser Gln Pro Tyr Pro Met Thr Pro Leu Pro Leu Ser
165 170 175
Phe Asn Glu Arg Thr Ile Lys Glu Ala Leu Asn Ala His Asp Ala Ser
180 185 190
Phe His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp
195 200 205
Gly Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro
210 215 220
Ile Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln
225 230 235 240
Ala Gly Ala Arg Leu Ile Glu Asn Ala Val Val Phe Lys Leu Glu Val
245 250 255
Gly Pro Asn Lys Arg Ile Val Ala Ala Arg Tyr Lys Asp Ser Lys Gly
260 265 270
Ala Glu His Arg Val Glu Gly Lys Trp Phe Val Leu Ala Ala Asn Gly
275 280 285
Ile Glu Thr Pro Lys Leu Met Leu Met Ser Thr Ser Gln Asp Phe Pro
290 295 300
Lys Gly Val Gly Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp
305 310 315 320
His Pro Gly Thr Gly Val Ser Phe Tyr Ala Asp Arg Lys Leu Trp Pro
325 330 335
Gly Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly
340 345 350
Pro Phe Arg Ala Thr Gln Ala Gly Lys Lys Leu His Leu Ser Asn Ile
355 360 365
Ser Arg Ile Glu Gln Glu Thr Gln Arg Ile Phe Lys Glu Gly Lys Leu
370 375 380
Ile Lys Pro Ala Asp Leu Asp Ala Arg Ile Arg Asp Gln Ala Ala Arg
385 390 395 400
Tyr Val Gln Phe Asp Ser Phe His Glu Ile Leu Pro Leu Pro Glu Asn
405 410 415
Arg Ile Val Pro Ser Ala Thr Glu Val Asp Ala Ile Gly Ile Pro Arg
420 425 430
Pro Glu Ile Thr Tyr His Ile Asp Asp Tyr Val Lys Arg Ser Ala Val
435 440 445
His Thr Arg Glu Val Tyr Ala Thr Ala Ala Gln Val Met Gly Gly Thr
450 455 460
Asn Val Glu Phe His Asp Asp Phe Ala Pro Asn Asn His Ile Thr Gly
465 470 475 480
Ala Thr Ile Met Gly Ala Asp Pro Lys Asp Ser Val Val Asp Lys Asp
485 490 495
Cys Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr
500 505 510
Met Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu
515 520 525
Ala Leu Arg Ile Ala Asp Gln Leu Lys Gln Glu Ala
530 535 540
<210> 10
<211> 540
<212> PRT
<213> Ralstonia solanacearum
<400> 10
Met Ala Asp Thr Arg Arg Ala Asp Gln Ala Asp Ile Val Val Val Gly
1 5 10 15
Ser Gly Val Ala Gly Ala Leu Val Ala Tyr Glu Leu Ala Arg Ala Gly
20 25 30
Lys Ser Val Leu Met Leu Glu Ala Gly Pro Arg Leu Pro Arg Trp Glu
35 40 45
Ile Val Glu Arg Phe Arg Asn Gln Ala Asp Lys Met Asp Phe Met Ala
50 55 60
Pro Tyr Pro Ser Thr Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Ser
65 70 75 80
Pro Asn Asp Tyr Leu Val Leu Lys Gly Glu His Lys Phe Asp Ser Gln
85 90 95
Tyr Ile Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Thr
100 105 110
Trp Arg Phe Leu Pro Asn Asp Phe Lys Leu Arg Ser Val Tyr Gly Ile
115 120 125
Ala Arg Asp Trp Pro Leu Gln Tyr Asp Asp Leu Glu Arg Asp Tyr Gly
130 135 140
Arg Ala Glu Ala Ala Leu Gly Val Trp Gly Pro Asn Asp Glu Asp Leu
145 150 155 160
Gly Ser Pro Arg Ser Gln Pro Tyr Pro Met Ala Pro Leu Pro Leu Ser
165 170 175
Phe Asn Glu Arg Thr Ile Lys Glu Ala Leu Asn Ala His Asp Pro Ala
180 185 190
Phe His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp
195 200 205
Gly Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro
210 215 220
Ile Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln
225 230 235 240
Ala Gly Ala Arg Leu Ile Glu Asn Ala Val Val Tyr Lys Leu Glu Val
245 250 255
Gly Ala Gly Arg Arg Ile Val Ala Ala His Tyr Lys Asp Pro Lys Gly
260 265 270
Val Asp His Arg Val Glu Gly Lys Trp Phe Val Leu Ala Ala Asn Gly
275 280 285
Ile Glu Thr Pro Lys Leu Met Leu Met Ser Thr Ser Glu Ala Phe Pro
290 295 300
Arg Gly Val Gly Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp
305 310 315 320
His Pro Gly Thr Gly Val Ser Phe Tyr Ala Asp Arg Lys Leu Trp Pro
325 330 335
Gly Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly
340 345 350
Pro Phe Arg Ala Met Gln Ala Gly Lys Lys Leu His Leu Ser Asn Ile
355 360 365
Ser Arg Ile Glu Gln Glu Thr Ala Arg Ile Phe Lys Ala Gly Lys Leu
370 375 380
Leu Lys Pro Ala Glu Leu Asp Ala Arg Ile Arg Asp Gln Ala Ala Arg
385 390 395 400
Tyr Val Gln Phe Asp Ser Phe His Glu Ile Leu Pro Leu Pro Glu Asn
405 410 415
Arg Ile Val Pro Ser Ala Thr Glu Thr Asp Ala Leu Gly Ile Pro Arg
420 425 430
Pro Glu Ile Thr Tyr Arg Ile Asp Asp Tyr Val Lys Arg Ser Ala Val
435 440 445
His Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Ala Thr
450 455 460
Asp Val Gln Phe His Asp Asp Phe Ala Pro Asn Asn His Ile Thr Gly
465 470 475 480
Ala Thr Ser Met Gly Ala Asp Pro Lys Asp Ser Val Val Asp Lys Asp
485 490 495
Cys Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ala Thr
500 505 510
Met Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu
515 520 525
Ala Leu Arg Ile Ala Asp Arg Leu Lys Lys Glu Ala
530 535 540
<210> 11
<211> 533
<212> PRT
<213> Burkholderia phytofirmans
<400> 11
Met Ala Asn Lys Asn Ser Ala Asp Ile Val Val Val Gly Ser Gly Val
1 5 10 15
Ala Gly Gly Leu Val Ala His Gln Met Ala Leu Ala Gly Ala Ser Val
20 25 30
Ile Leu Leu Glu Ala Gly Pro Arg Ile Pro Arg Trp Gln Ile Val Glu
35 40 45
Asn Phe Arg Asn Ser Pro Val Lys Ser Asp Phe Ala Thr Pro Tyr Pro
50 55 60
Ser Thr Pro Tyr Ala Pro His Pro Glu Tyr Ala Pro Ala Asn Asn Tyr
65 70 75 80
Leu Ile Gln Lys Gly Asp Tyr Pro Tyr Ser Ser Gln Tyr Leu Arg Leu
85 90 95
Val Gly Gly Thr Thr Trp His Trp Ala Ala Ala Ala Trp Arg Leu Leu
100 105 110
Pro Ser Asp Phe Gln Leu His Lys Leu Tyr Gly Val Gly Arg Asp Trp
115 120 125
Pro Tyr Pro Tyr Glu Thr Leu Glu Pro Trp Tyr Ser Ala Ala Glu Val
130 135 140
Gln Leu Gly Val Ser Gly Pro Gly Asn Ser Ile Asp Leu Gly Ser Pro
145 150 155 160
Arg Ser Lys Pro Tyr Pro Met Asn Pro Leu Pro Leu Ser Tyr Met Asp
165 170 175
Gln Arg Phe Ser Asp Val Leu Asn Ala Gln Gly Phe Lys Val Val Pro
180 185 190
Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Ala Arg Pro Thr Cys
195 200 205
Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile Ala Ala Met Tyr
210 215 220
Asn Gly Val Val His Ala Glu Lys Ala Glu Gln Ala Gly Ala Lys Leu
225 230 235 240
Ile Pro Glu Ala Val Val Tyr Arg Val Glu Ala Asp Asn Lys Gly Leu
245 250 255
Ile Thr Ala Val His Tyr Lys Asp Pro Asn Gly Asn Ser Thr Arg Val
260 265 270
Thr Gly Lys Leu Phe Val Leu Ala Ala Asn Gly Ile Glu Thr Pro Lys
275 280 285
Leu Met Leu Met Ser Thr Ser Asp Lys Phe Pro His Gly Val Gly Asn
290 295 300
Ser Ser Asp Gln Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly
305 310 315 320
Val Thr Phe Leu Ala Asn Glu Ala Leu Trp Pro Gly Arg Gly Pro Met
325 330 335
Glu Met Thr Ser Ile Val Asn Phe Arg Asp Gly Ala Phe Arg Ser Asp
340 345 350
Tyr Ala Ala Lys Lys Leu His Leu Ser Asn Gly Val Pro Thr Met Ser
355 360 365
Val Thr Ala Asp Leu Leu Lys Lys Gly Leu Thr Gly Ala Glu Leu Asp
370 375 380
Arg Gln Ile Arg Asp Arg Ala Ala Arg Thr Leu Asn Ile Asn Ser Phe
385 390 395 400
His Glu His Leu Ala Glu Pro Gln Asn Arg Val Val Pro Ser Ala Asp
405 410 415
His Lys Asp Ser Leu Gly Ile Pro Gln Pro Glu Ile Tyr Tyr Ser Ile
420 425 430
Asn Asp Tyr Val Lys Lys Ser Ala Ala Asn Thr His Glu Leu Tyr Ala
435 440 445
Gln Ile Ala Ala Leu Phe Gly Gly Ala Glu Val Thr Phe Asp Asp Thr
450 455 460
Phe Ala Pro Asn Asn His Ile Met Gly Thr Thr Ile Met Gly Ser Asp
465 470 475 480
Pro Ala Asp Ser Val Val Asp Ala Asp Cys Arg Thr His Asp His Ser
485 490 495
Asn Leu Phe Ile Ala Ser Ser Gly Val Met Pro Thr Ala Ala Ser Val
500 505 510
Asn Cys Thr Leu Thr Ile Ala Ala Leu Ser Leu Lys Leu Ala Asp Lys
515 520 525
Leu Lys Arg Glu Ile
530
<210> 12
<211> 539
<212> PRT
<213> Burkholderia cepacia
<400> 12
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Val Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Ala Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Gly Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Ile Glu His Tyr Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Glu Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Asp Ser Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Thr Ala Ala Val Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Ile Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met
370 375 380
Lys Pro Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Glu
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 13
<211> 539
<212> PRT
<213> Burkholderia cepacia
<400> 13
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Val Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Ala Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Gly Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Ile Glu His Tyr Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Glu Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Asp Ser Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Thr Ala Ala Val Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Ile Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Asn Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Gly Gly Lys Leu Met
370 375 380
Lys Pro Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Phe
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Val Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Glu
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 14
<211> 539
<212> PRT
<213> Burkholderia cepacia
<400> 14
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Thr Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Val Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Gly Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Leu Glu His Tyr Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Arg Gln Pro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Glu Asn Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asn Lys Arg Ile Val Ala Ala Ile Tyr Lys Asp Lys Ser Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Val Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Gly Gly Lys Leu Met
370 375 380
Lys Pro Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Cys Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Glu
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 15
<211> 539
<212> PRT
<213> Burkholderia cepacia
<400> 15
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Ile Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Pro Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Ala Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Leu Glu His Trp Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Gln Ala Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Asp Ser Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Val Ala Ala Ile Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met
370 375 380
Lys His Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 16
<211> 539
<212> PRT
<213> Burkholderia cepacia
<400> 16
Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Ile Val Val Val Gly Ser
1 5 10 15
Gly Val Ala Gly Ala Ile Val Ala His Gln Leu Ala Met Ala Gly Lys
20 25 30
Ser Val Ile Leu Leu Glu Ala Gly Pro Arg Met Pro Arg Trp Glu Ile
35 40 45
Val Glu Arg Phe Arg Asn Gln Pro Asp Lys Thr Asp Phe Met Ala Pro
50 55 60
Tyr Pro Ser Ser Pro Trp Ala Pro His Pro Glu Tyr Gly Pro Pro Asn
65 70 75 80
Asp Tyr Leu Ile Leu Lys Gly Glu His Lys Phe Asn Ser Gln Tyr Ile
85 90 95
Arg Ala Val Gly Gly Thr Thr Trp His Trp Ala Ala Ser Ala Trp Arg
100 105 110
Phe Ile Pro Asn Asp Phe Lys Met Lys Thr Val Tyr Gly Val Ala Arg
115 120 125
Asp Trp Pro Ile Gln Tyr Asp Asp Leu Glu His Trp Tyr Gln Arg Ala
130 135 140
Glu Glu Glu Leu Gly Val Trp Gly Pro Gly Pro Glu Glu Asp Leu Tyr
145 150 155 160
Ser Pro Arg Lys Gln Ala Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe
165 170 175
Asn Glu Gln Thr Ile Lys Ser Ala Leu Asn Gly Tyr Asp Pro Lys Phe
180 185 190
His Val Val Thr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly
195 200 205
Arg Pro Thr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile
210 215 220
Gly Ala Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Gln Ala
225 230 235 240
Gly Ala Lys Leu Ile Asp Ser Ala Val Val Tyr Lys Leu Glu Thr Gly
245 250 255
Pro Asp Lys Arg Ile Val Ala Ala Ile Tyr Lys Asp Lys Thr Gly Ala
260 265 270
Asp His Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn Gly Ile
275 280 285
Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp Phe Pro Asn
290 295 300
Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn Leu Met Asp His
305 310 315 320
Pro Gly Thr Gly Val Ser Phe Tyr Ala Asn Glu Lys Leu Trp Pro Gly
325 330 335
Arg Gly Pro Gln Glu Met Thr Ser Leu Ile Gly Phe Arg Asp Gly Pro
340 345 350
Phe Arg Ala Thr Glu Ala Ala Lys Lys Ile His Leu Ser Asn Met Ser
355 360 365
Arg Ile Asn Gln Glu Thr Gln Lys Ile Phe Lys Ala Gly Lys Leu Met
370 375 380
Lys His Glu Glu Leu Asp Ala Gln Ile Arg Asp Arg Ser Ala Arg Tyr
385 390 395 400
Val Gln Phe Asp Cys Phe His Glu Ile Leu Pro Gln Pro Glu Asn Arg
405 410 415
Ile Val Pro Ser Lys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro
420 425 430
Glu Ile Thr Tyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Val His
435 440 445
Thr Arg Glu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp
450 455 460
Val Val Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ala
465 470 475 480
Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys
485 490 495
Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ser Thr Met
500 505 510
Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala Leu Ala
515 520 525
Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val
530 535
<210> 17
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 17
accaccactg ataaggaggt ctgaccgtgc ggaaatctac 40
<210> 18
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 18
agcctgtgcg acttcttcct tcagcgatcg gtggtggtgg 40
<210> 19
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 19
catgccatgg cacacaacga caacac 26
<210> 20
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 20
gtcgacgatc ttcttccagc cgaacatcac 30
<210> 21
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 区域 (i)
<220>
<221> MISC_FEATURE
<222> (1)..(3)
<223> Xaa 是 Val 或 Ile
<400> 21
Xaa Xaa Xaa Gly Ser Gly Val Ala Gly
1 5
<210> 22
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 区域 (ii)
<400> 22
Cys Cys Gly Asn Asn Cys Met Pro Ile Cys
1 5 10
<210> 23
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 区域 (iii)
<400> 23
Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly
1 5 10
<210> 24
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 区域 (iv)
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa 是 Ile 或 Leu
<400> 24
Lys Lys Xaa His Leu Ser Asn
1 5
<210> 25
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 区域 (v)
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa 是 Pro 或 Asn
<400> 25
Phe Ala Xaa Asn Asn His Ile
1 5

Claims (14)

1.一种变异葡萄糖脱氢酶，其包含变异葡萄糖脱氢酶α亚单位，所述变异葡萄糖脱氢酶α亚单位具有在依次包含下面的肽序列(i)～(v)的520～550个氨基酸的葡萄糖脱氢酶α亚单位的氨基酸序列中、以其他氨基酸取代肽序列(iii)的10位的甘氨酸、肽序列(iv)的4位的组氨酸、以及肽序列(v)的4位的天冬酰胺中的一个以上的氨基酸序列，其中，所述变异葡萄糖脱氢酶具有葡萄糖脱氢酶活性，对木糖的反应性降低。
(i)Val/Ile Val/Ile Val/Ile Gly Ser Gly Val Ala Gly(序列号21)
(ii)Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys(序列号22)
(iii)Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly(序列号23)
(iv)Lys Lys Ile/Leu His Leu Ser Asn(序列号24)
(v)Phe Ala Pro/Asn Asn Asn His Ile(序列号25)。
2.如权利要求1所述的变异葡萄糖脱氢酶，其中，
与从肽序列(ii)的8位的Met、肽序列(iii)的12位的Gly、肽序列(iv)的6位的Ser、肽序列(v)的2位的Ala、肽序列(v)的3位的Pro/Asn、肽序列(v)的5位的Asn以及肽序列(v)的7位的Ile中选择的至少一个氨基酸残基相当的氨基酸残基还被其他氨基酸残基取代。
3.如权利要求1所述的变异葡萄糖脱氢酶，其中，
所述变异葡萄糖脱氢酶还对半乳糖的反应性降低。
4.如权利要求1至3中任一项所述的变异葡萄糖脱氢酶，其中，
所述变异葡萄糖脱氢酶α亚单位包含和序列号3至少有60％的同一性的氨基酸序列。
5.如权利要求1至3中任一项所述的变异葡萄糖脱氢酶，其中，
所述变异葡萄糖脱氢酶d亚单位包含和序列号3、序列号4、序列号5、序列号6、序列号7或者序列号8至少有80％的同一性的氨基酸序列。
6.如权利要求1至3中任一项所述的变异葡萄糖脱氢酶，其中，
所述变异葡萄糖脱氢酶α亚单位具有序列号3、序列号4、序列号5、序列号6、序列号7或者序列号8中取代、缺失、插入或者添加了一个或者多个氨基酸的氨基酸序列。
7.如权利要求1至3中任一项所述的变异葡萄糖脱氢酶，其中，所述变异葡萄糖脱氢酶还包含电子传递亚单位。
8.如权利要求7所述的变异葡萄糖脱氢酶，其特征在于，电子传递亚单位是细胞色素C。
9.一种变异葡萄糖脱氢酶，其是包含具有和序列号3至少有60％的同一性的氨基酸序列、且具有葡萄糖脱氢酶活性的蛋白质，其特征在于，
在所述氨基酸序列中，与序列号3的474位的天冬酰胺相当的氨基酸残基被丝氨酸取代，与序列号3的322位的甘氨酸相当的氨基酸残基被谷氨酰胺取代，与序列号3的363位的组氨酸相当的氨基酸残基被谷氨酰胺或者丝氨酸取代，并且，与序列号3的475位的天冬酰胺相当的氨基酸残基被丝氨酸取代，并且，所述变异葡萄糖脱氢酶对木糖的反应性降低。
10.一种DNA，其编码权利要求1至9中任一项所述的变异葡萄糖脱氢酶。
11.一种重组载体，其包含权利要求10所述的DNA。
12.一种微生物，其由权利要求11所述的重组载体转化。
13.一种葡萄糖检测试剂盒，其包含权利要求1至9中任一项所述的变异葡萄糖脱氢酶。
14.一种葡萄糖传感器，其包含权利要求1至9中任一项所述的变异葡萄糖脱氢酶。
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