CN1726277A - 辅酶结合型葡萄糖脱氢酶 - Google Patents
辅酶结合型葡萄糖脱氢酶 Download PDFInfo
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- CN1726277A CN1726277A CNA2003801056894A CN200380105689A CN1726277A CN 1726277 A CN1726277 A CN 1726277A CN A2003801056894 A CNA2003801056894 A CN A2003801056894A CN 200380105689 A CN200380105689 A CN 200380105689A CN 1726277 A CN1726277 A CN 1726277A
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- glucose dehydrogenase
- coenzyme
- linked glucose
- coenzyme linked
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Abstract
本发明提供一种在电子受体存在下催化葡萄糖氧化反应,对麦芽糖作用率在5%以下,受1,10-菲绕啉阻滞,来源于微生物的可溶性辅酶结合型葡萄糖脱氢酶。而且提供了该辅酶结合型葡萄糖脱氢酶的制造方法及使用该辅酶结合型葡萄糖脱氢酶的测定方法和测定试剂。通过本发明有可能使辅酶结合型葡萄糖脱氢酶应用于产业,也有可能用于包括使用该辅酶结合型葡萄糖脱氢酶的试剂中的葡萄糖测定方法,消除方法,有机化合物的制造方法等的物质生产及分析的用途。另外,也可能提供能够正确测定血糖值的葡萄糖传感器。也就是说可以提供在医药,临床及食品领域中,用于材料的改质加工等利用价值很高的酶。
Description
技术领域
本发明涉及新型可溶性辅酶结合型葡萄糖脱氢酶,该辅酶结合型葡萄糖脱氢酶的制造方法及能够生产该辅酶结合型葡萄糖脱氢酶的微生物。
另外,本发明涉及在测定试剂中的葡萄糖时,使用该辅酶结合型葡萄糖脱氢酶的测定方法,含有该辅酶结合型葡萄糖脱氢酶的试剂及试剂组合物。此外,涉及包括有机化合物的制造方法的制造原料等的物质生产及分析的用途。
而且,本发明涉及迅速并简便地高精度定量试剂中特定成分的生物传感器。具体说,涉及使用该辅酶结合型葡萄糖脱氢酶的葡萄糖传感器。
背景技术
葡萄糖存在于血液中,被用作糖尿病的重要标志。作为葡萄糖的测定方法,历来有化学法和酶法,一般来讲酶法在特异性和安全性方面具有优越性。作为酶法有使用葡萄糖氧化酶,葡萄糖6磷酸脱氢酶或NAD(P)依赖性葡萄糖脱氢酶的测定方法。但是,使用葡萄糖氧化酶或葡萄糖6磷酸脱氢酶的测定方法,由于使用多个酶,反应不够简便。另外,在使用葡萄糖6磷酸脱氢酶及NAD(P)依赖性葡萄糖脱氢酶的测定方法的反应体系中,必须添加辅酶NAD(P),较繁杂。
近年来,提出了针对试样中的特定成分,对试样溶液不进行稀释或搅拌、来进行简易的定量的各种生物传感器。例如,生物传感器是在绝缘性的基板上用丝网印刷等方法形成由作用极,对极及参照极组成的电极系,在该电极系上接上含有亲水性高分子,氧化还原酶和电子受体的酶反应层而形成的。
糖尿病患者每年都在增加,需要不仅在医院,在家中也可以使用的简便的血糖测定方法及血糖值的管理手段。现在,作为血糖测定用途,可以使用简易型葡萄糖传感器,但在这些检测器中,广泛使用葡萄糖氧化酶,由于溶存氧浓度产生测定值误差的可能性很高。另外,使用尼克酰胺类辅酶依赖性葡萄糖脱氢酶的生物传感器背景噪音高,有必要另外添加辅酶或辅助酶等,反应较复杂,而且在用发色系统测定的情况有必须高价的光学系的缺点。
作为不受溶存氧浓度的影响,而且在NAD(P)不存在情况下作用于葡萄糖的酶,已知有以吡咯并喹啉醌为辅酶的葡萄糖脱氢酶,但该吡咯并喹啉醌有易于和酶发生解离的问题。另外,特开2000-350588号公报及特开2001-197888号公报中公开的以吡咯并喹啉醌为辅酶的葡萄糖脱氢酶,具有对葡萄糖选择性低的缺点。由大肠杆菌(Escherichiacoli)来源的吡咯并喹啉醌作为辅酶的葡萄糖脱氢酶(特开平10-243786号公报),Pseudomonas sp.来源的吡咯并喹啉醌作为辅酶葡萄糖脱氢酶(Agric.Biol.Chem.(1980)44:1505-1512),以及G1uconobacter suboxydans来源的吡咯并喹啉醌作为辅酶的葡萄糖脱氢酶(Agric.Biol.Chem(1981)45:851-861)对麦芽糖的作用性分别为3%,3.2%,5%,由于存在菌体的膜成分,有必须通过可溶化进行提取酶的操作的繁杂性。
另外,将葡萄糖3位羟基氧化的辅酶结合型葡萄糖-3-脱氢酶,在J.Biol.Chem.(1967)242:3665-3672,Appl.Microbiol.Biotechnol.(1999)51:58-64,Appl.Biochem.Biotechnol.(1996)56:301-310及Enzyme Microb.Technol.(1998)22:269-274有报道,但对任何葡萄糖的选择性都低。麦芽糖作为输液成分广泛使用,由于给与输液的患者的血中麦芽糖浓度高,所以希望开发出特异性作用于葡萄糖,特别是对麦芽糖作用性低的测定血糖用的酶。
本发明为了适应上述产业上的要求,将提供对葡萄糖有优异的基质识别性,对麦芽糖作用性低的新型葡萄糖脱氢酶,提供其制造方法及具有生产能的微生物作为课题。
另外,本发明将提供利用新型的葡萄糖脱氢酶迅速并简便地高精度定量葡萄糖的优异的测定方法,测定试剂及生物传感器,以及葡萄糖消除试剂作为课题。
发明内容
本发明为了解决前述课题,本发明人从各方面进行了研究,结果着眼于新型的可溶性辅酶结合型葡萄糖脱氢酶。该辅酶结合型葡萄糖脱氢酶,在电子受体存在下催化葡萄糖氧化反应,例如,分类为EC(酶编号)1.1.99的酶。另外,本发明者深入检索了生产该辅酶结合型葡萄糖脱氢酶的各种微生物,结果发现辅酶结合型葡萄糖脱氢酶的生产微生物及辅酶结合型葡萄糖脱氢酶。
本发明提供催化反应时辅酶通常呈结合状态的葡萄糖脱氢酶。该辅酶结合型葡萄糖脱氢酶具有在电子受体存在下,催化葡萄糖,特别是催化葡萄糖的1位羟基氧化反应的理化性质。该辅酶结合型葡萄糖脱氢酶对麦芽糖的作用性在5%以下,优选3%以下,对麦芽糖的作用性低。另外,该辅酶结合型葡萄糖脱氢酶,具有被1,10-菲绕啉阻滞酶反应的性质,终浓度5mM的1,10-菲绕啉可以阻滞酶反应50%以上,优选2mM的1,10-菲绕啉50%以上阻滞,特别优选1mM的1,10-菲绕啉50%以上阻滞。此外,该辅酶结合型葡萄糖脱氢酶在50mM柠檬酸钠缓冲液(pH5.5)存在下,50℃热处理15分钟后,仍残存85%以上酶活性。本发明的与葡萄糖脱氢酶结合的辅酶,例如可以是黄素化合物,可以是黄素腺嘌呤二核苷酸(FAD)等辅酶。另外,本发明中对于该辅酶结合型葡萄糖脱氢酶的性质及/或实质上具有同等性质的蛋白质或它的盐包括,具有编码该蛋白质的氨基酸序列或在该序列中缺失,置换或附加1个或1个以上氨基酸残基而变异的氨基酸序列,而且有稳定生物学活性的蛋白质。进而,本发明的辅酶结合型葡萄糖脱氢酶,为微生物来源的辅酶结合型葡萄糖脱氢酶,优选真核微生物来源的辅酶结合型葡萄糖脱氢酶,特别优选保藏菌株FERM BP-08578来源的辅酶结合型葡萄糖脱氢酶。
现在已经确定米曲霉(Aspergillus oryzae)的细胞质部分及培养液中,存在FAD作为使用辅酶的葡萄糖脱氢酶。(TCHAN-GI BAK(BIOCHEMICA ET BIOPHYSICA ACTA.(1967)139:277-293)。但是,该葡萄糖脱氢酶仅被重金属离子阻滞,具有不被含有1,10-菲绕啉的金属鳌合物所阻滞的特征的理化性质。因此,作为使用该葡萄糖脱氢酶的测定系中的反应停止试剂不能使用重金属以外的试剂,具有处理反应停止后的重金属废液较繁杂的问题。还有,该酶的稳定性较低,实用性差。另外,本发明所发现的辅酶结合型葡萄糖脱氢酶,作为其特征,与已知的米曲霉来源的辅酶结合型葡萄糖脱氢酶相比稳定性高,而且除了重金属离子,微量的1,10-菲绕啉就可以阻滞,具有终止反应的操作简便的性质。
本发明提供新型的可溶性辅酶结合型葡萄糖脱氢酶的制造方法。
本发明提供具有能生产新型的可溶性辅酶结合型葡萄糖脱氢酶的微生物。该微生物优选真核微生物,特别优选曲霉(Aspergillus)属,进一步优选局限曲霉(Aspergillus terreus),最好优选保藏菌株FERM BP-08578。
本发明提供新型的可溶性辅酶结合型葡萄糖脱氢酶的使用方法。优选是在测定试样中的葡萄糖时,使用该辅酶结合型葡萄糖脱氢酶的葡萄糖测定方法。使用辅酶结合型葡萄糖脱氢酶的葡萄糖消除方法及有机化合物的制造方法。
本发明提供含有新型的可溶性辅酶结合型葡萄糖脱氢酶的试剂。该试剂优选含有用于测定试样中葡萄糖的该辅酶结合型葡萄糖脱氢酶的葡萄糖测定试剂及含有辅酶结合型葡萄糖脱氢酶的葡萄糖消除试剂及有机化合物的制造试剂。
本发明提供含有新型可溶性辅酶结合型葡萄糖脱氢酶的试剂组合物。该组合物优选含有用于测定试样中葡萄糖的该辅酶结合型葡萄糖脱氢酶的葡萄糖测定组合物及含有辅酶结合型葡萄糖脱氢酶的葡萄糖消除组合物及有机化合物的制造用原料。
本发明提供使用新型可溶性辅酶结合型葡萄糖脱氢酶的生物传感器,提供可以定量及/或定性试样中的特定成分的生物传感器。该生物传感器,优选使用了该辅酶结合型葡萄糖脱氢酶的葡萄糖传感器。
本发明提供的测定方法,测定试剂,测定化合物,生物传感器中的一种的优选样式,其特征在于在测定反应中使用铁氰化钾(六氰基铁(III)酸钾),使终浓度为2mM-500mM。
本发明中,作为该辅酶结合型葡萄糖脱氢酶的“对麦芽糖的作用性”,“麦芽糖作用性”及与其类似表现的“作用性”的表示值(%),是将该酶对于葡萄糖的酶活性定为100%,其对于麦芽糖或作为作用性对象的物质,以百分率表示的该活性的相对强度数值。
附图的简要说明
图1,是表示实施例3(3.2)的辅酶结合型葡萄糖脱氢酶的相对活性(%)和pH关系的图。记号表示测定值及缓冲液的种类,缓冲液分别为□柠檬酸·磷酸钠缓冲液(pH4.6~6.2),◆磷酸钾缓冲液(pH6.8~7.2),○Tris·盐酸缓冲液(pH7.8~8.6)及▲甘氨酸·氢氧化钠(pH9.2~9.5)。该酶的最适pH为pH7.0~9.0。
图2,是表示实施例3(3.3)的辅酶结合型葡萄糖脱氢酶的残存活性(%)和pH关系的图。记号表示测定值及缓冲液的种类,缓冲液分别为□柠檬酸·磷酸钠缓冲液(pH3.2~6.4),◆磷酸钾缓冲液(pH6.3~6.9),○Tris·盐酸缓冲液(pH7.3~8.6)及▲甘氨酸·氢氧化钠(pH9.1~11.4)。该酶的稳定pH为pH4.5~8.5。
图3,是表示实施例3(3.4)的辅酶结合型葡萄糖脱氢酶的相对活性(%)和温度关系的图。该酶的最适温度是55℃左右。
图4,是表示实施例3(3.5)的辅酶结合型葡萄糖脱氢酶热处理后的残存活性(%)和处理温度关系的图。表示酶在约50℃以下是稳定的。
图5,表示在能够对实施例4的葡萄糖定量的标准曲线,DCIP吸光度变化-葡萄糖浓度的关系。
图6,表示实施例5的辅酶结合型葡萄糖脱氢酶固定化电极的葡萄糖定量的标准曲线。记号分别表示×氩饱和,△氧饱和,○空气饱和的条件。
具体实施方式
本发明的辅酶结合型葡萄糖脱氢酶,是指例如分类为EC1.1.99的酶,优选表示为EC1.1.99.10,EC1.1.99.13或EC1.1.99.17的酶,为辅酶结合型酶,优选可溶性辅酶结合型酶。也就是说,酶的提取及/或精制过程中不使用表面活性剂,在水溶液状态下能取出的酶。这里所说的辅酶可以为黄素化合物,可以列举出FAD,FMN。
本发明的辅酶结合型葡萄糖脱氢酶,在催化反应时,可以是一直结合了该辅酶的萄糖脱氢酶。该辅酶结合型葡萄糖脱氢酶至少具有以下性质。在电子受体存在下,催化葡萄糖的氧化反应,特别是具有催化葡萄糖1位羟基氧化反应的理化性质。另外,该辅酶结合型葡萄糖脱氢酶对麦芽糖的作用性低,例如在5%以下,优选3%以下。而且,该辅酶结合型葡萄糖脱氢酶的酶活性被终浓度5mM 1,10-菲绕啉抑制50%以上,优选2mM 1,10-菲绕啉抑制50%以上,特别优选1mM 1,10-菲绕啉抑制50%以上,更最适为1mM 1,10-菲绕啉抑制60%以上。另外,该辅酶结合型葡萄糖脱氢酶在50mM柠檬酸钠缓冲液(pH5.5)存在下,50℃热处理15分钟后还残存85%以上酶活性率。另外,本发明涉及具有上述理化性质的辅酶结合型葡萄糖脱氢酶,或者涉及对于与该辅酶结合型葡萄糖脱氢酶具有实质同等活性的蛋白质或其盐,具有编码该蛋白质的氨基酸序列或在该序列中缺失,置换或附加1个以上氨基酸残基而引起的变异的氨基酸序列,而且是有稳定生物学活性的蛋白质及其盐,编码其理化性质的氨基酸序列。
本发明的是辅酶结合型葡萄糖脱氢酶的蛋白质及其盐,优选来自于具有上述理化性质的微生物。本发明的辅酶结合型葡萄糖脱氢酶的所来源的微生物,作为具有本发明的辅酶结合型葡萄糖脱氢酶的生产能的微生物,例如属于原核生物的原囊粘菌属(Archangium),古生球菌属(Archaeoglobus),杀雄菌属(Arsenophonus),(Ahrensia)属,金杆菌属(Aureobacterium),气球菌属(Aerococcus),气热菌属(Aeropyrum),气微菌属(Aeromicrobium),气单胞菌属(Aeromonas),产液菌属(Aquifex),水螺菌属(Aquaspirillum),水杆菌属(Aquabacter),Aquabacterium属,污水菌属(Aquamicrobium),异壁放线菌属(Actinoalloteichus),放线动孢菌属(Actinokineospora),珊瑚状放线菌属(Actinocorallia),束丝放线菌属(Actinosynnema),孢囊放线菌属(Actinosporangium),放线杆状菌属(Actinobaculum),放线芽孢杆菌属(Actinobacillus),孢器放线菌属(Actinopycnidium),双孢放线菌属(Actinobispora),游动放线菌属(Actinoplanes),放线多孢菌属(Actinopolyspora),Actinopolymorpha属,放线菌属(Actinomyces),马杜拉放线菌属(Actinomadura),Acrocarpospora属,土壤球菌属(Agrococcus),土壤杆菌属(Agrobacterium),壤霉菌属(Agromyces),无色硫菌属(Achromatium),土壤单胞菌属(Agromonas),无色杆菌属(Achromobacter),无胆甾原体属(Acholeplasma),Asaia属,酸菌属(Acidianus),Acidisphaera属,嗜酸菌属(Acidiphilium),酸微菌属(Acidimicrobium),Acidilobus属,氨基酸球菌属(Acidaminococcus),氨基酸杆菌属(Acidaminobacter),Acidithiobacillus 属,震球菌属(Agitococcus),热酸菌属(Acidothermus),酸胞菌属(Acidocella),酸杆菌属(Acidobacterium),食酸菌属(Acidovorax),酸单胞菌属(Acidomonas),不动杆菌属(Acinetobacter),不粘柄菌属(Asticcacaulis),无甾醇枝原体属(Asteroleplasma),醋香肠菌属(Acetitomaculum),醋弧菌属(Acetivibrio),厌氧醋菌属(Acetoanaerobium),产醋菌属(Acetogenium),醋热菌属(Acetothermus),醋丝菌属(Acetonema),醋杆菌属(Acetobacter),醋酸杆菌属(Acetobacterium),醋盐杆菌属(Acetohalobium),线形醋菌属(Acetofilamentum),醋微菌属(Acetomicrobium),固氮弧菌属(AZoarcus),Azospira,固氮螺菌属(Azospirillum),固氮杆菌属(Azotobacter),Azonexus属,Azovibrio属,氮单胞菌属(Azomonas),固氮丝菌属(Azomonotrichon),固氮根瘤菌属(Azorhizobium),嗜氮根瘤菌属(Azorhizophilus),Atopobacter属,陌生物菌属(Atopobium),无形体属(Anaplasma),解硫胺素杆菌(Aneurinibacillus),Anaeroarcus 属,Anaerococcus属,Anaerosinus属,厌氧杆菌属(Anaerobacter),厌氧小杆菌属(Anaerobaculum),厌氧螺菌属(Anaerobiospirillum),厌氧弧菌属(Anaerovibrio),厌氧细杆菌属(Anaerofilum),厌氧支原体属(Anaeroplasma),厌氧分枝菌属(Anaerobranca),Anaerovorax属,Anaeromusa属,棍状厌氧菌属(Anaerorhabdus),Anoxybacillus属,乏养菌属(Abiotrophia),阿菲波菌属(Afipia),下水道球菌属(Amaricoccus),无枝酸菌属(Amycolata),拟无枝酸菌属(Amycolatopsis),氨基杆菌属(Aminobacter),Aminobacterium属,Aminomonas属,变形杆菌属(Amoebobacter),Ammoniphilus属,制氨菌属(Ammonifex),无定形胞囊菌属(Amorphosporangium),蛛菌属(Arachnia),小链菌属(Alysiella),脂环酸杆菌属(Alicyclobacillus),Alishewanella属,食烷菌属(Alcanivorax),隐稳杆菌属(Arcanobacterium),产碱杆菌属(Alcaligenes),Alkalibacterium属,Alkaliphilus属,弓形菌属(Arcobacter),节杆菌属(Arthrobacter),Alterococcus属,交替单胞菌属(Alteromonas),Albibacter属,差异球菌(Alloiococcus),Allochromatium属,非红单胞菌属(Arhodomonas),异单胞菌属(Allomonas),Allorhizobium属,绿臂菌属(Ancalochloris),臂微菌属(Ancalomicrobium),囊球粘菌属(Angiococcus),角微菌属(Angulomicrobium),弯杆菌属(Ancylobacter),Antarctobacter属,双芽孢杆菌属(Amphibacillus),瓶孢囊菌属(Ampullariella),Ignavigranum属,Idiomarina属,Isochromatium属,等球菌属(Isosphaera),艾德昂菌属(Ideonella),泥杆菌属(Ilyobacter),间孢囊菌属(Intrasporangium),威克斯氏菌属(Weeksella),威格尔斯沃思氏菌属(Wigglesworthia),Williamsia属,沃林氏菌属(Wolinella),沃尔巴克氏体属(Wolbachia),尿枝原体属(Ureaplasma),Ureibacillus属,艾肯氏菌属(Eikenella),埃里希氏体属(Ehrlichia),微小杆菌属(Exiguobacterium),卓孢菌属(Excellospora),外硫红螺菌属(Ectothiorhodospira),埃及小体属(Aegyptianella),Eggerthella属,埃希氏杆菌属(Escherichia),爱德华氏菌属(Edwardsiella),爱文氏菌属(Ewingella),附红血球体(Eperythrozoon),丹毒丝菌属(Erysipelothrix),赤细菌属(Erythrobacter),赤微菌属(Erythromicrobium),红单胞菌属(Erythromonas),鞘孢囊菌属(Elytrosporangium),耶尔森氏菌属(Yersinia),欧文氏杆菌属(Erwinia),Eremococcus属,剑菌属(Ensifer),肠球菌属(Enterococcus),肠杆菌属(Enterobacter),虫原体属(Entomoplasma),水栖菌属(Enhydrobacter),稳杆菌属(Empedobacter),酒球菌属(Oenococcus),厄氏菌属(Oerskovia),Oceanimonas,海洋螺菌属(Oceanospirillum),草酸杆菌属(Oxalobacter),嗜草酸杆菌属(Oxalophagus),产醋杆菌属(Oxobacter),十八杆菌属(Octadecabacter),苍白杆菌属(Ochrobactrum),绿颤细菌属(Oscillochloris),颤螺菌属(Oscillospira),肥杆菌属(Obesumbacterium),东方体属(Orientia),寡原杆菌属(Oligella),寡养菌属(Oligotropha),口腔杆菌属(Oribaculum),Ornithinicoccus属,Ornithinimicrobium属,鸟杆菌属(Ornithobacterium),奥芮氏菌属(Orenia),加德纳氏菌属(Gardnerella),肉胞菌属(Carnimonas),肉杆菌属(Carnobacterium),科氏游动菌属(Couchioplanes),考德里氏体属(Cowdria),柄杆菌属(Caulobacter),乳酪杆菌属(Caseobacter),Catenibacterium属,短链游动菌属(Catenuloplanes),链状球菌属(Catenococcus),链孢菌属(Catellatospora),卡托氏菌属(Catonella),英膜菌属(Capsularis),二氧化碳嗜纤维菌属(Capnocytophaga),报毛菌属(Gallionella),核衣菌属(Caryophanon),Gallicola属,鞘杆菌属(Calymmatobacterium),心杆菌属(Cardiobacterium),热解纤维素菌属(Caldicellulosiruptor),Caldivirga属,热杆菌属(Calderobacterium),Carboxydibrachium属,一氧化碳嗜热菌属(Carboxydothermus),嗜碳菌属(Carbophilus),喜热菌属(Caloramator),黄色杆菌属(Xanthobacter),黄单胞菌属(Xanthomonas),木杆菌属(Xylella),嗜木杆菌属(Xylophilus),致病杆菌属(Xenorhabdus),北里菌属(Kitasatoa),北里孢菌属(Kitasatospora),噬几丁质菌属(Chitinophaga),皮球菌属(Kytococcus),动球菌属(Kineococcus),动孢囊菌属(Kineosporia),奎因氏菌属(Quinella),拟孢囊菌属(Kibdelosporangium),弯曲杆菌属(Campylobacter),金氏菌属(Kingella),库茨涅尔氏菌属(Kutzneria),贪铜菌属(Cupriavidus),脆弱球菌(Craurococcus),Glaciecola,Gracilibacillus,Granulicatella,格拉汉氏体属(Grahamella),棍状杆菌属(Clavibacter),衣原体属(Chlamydia),Chlamydophila,冷杆菌属(Cryobacterium),糖霉菌属(Glycomyces),金矿菌属(Chrysiogenes),脊螺旋体属(Cristispira),金黄杆菌属(chryseobacterium),金色单胞菌属(Chryseomonas),发毛针蓝细菌属(Crinalium),隐孢菌属(Cryptosporangium),Cryptobacterium,克吕沃尔氏菌属(Kluyvera),Kribbella,葡糖酸醋酸杆菌属(Gluconacetobacter),葡糖杆菌属(Gluconobacter),库特氏菌属(Kurthia),短小杆菌属(Curtobacterium),铁细菌属(Crenothrix),克雷白氏杆菌属(Klebsiella),克里夫兰氏菌属(Clevelandina),Crossiella,梭状芽孢杆菌属(Clostridium),格鲁比卡氏菌属(Globicatella),着色菌属(Chromatium),色杆菌属(Chromobacterium),色盐杆菌属(Chromohalobacter),绿丝菌属(Chloronema),绿菌属(Chlorobium),绿屈挠菌属(Chloroflexus),绿滑菌属(Chloroherpeton),杀手杆菌属(Caedibacter),Ketogulonicigenium,出芽菌属(Gemmata),芽殖菌属(Gemmiger),孪生球菌属(Gemella),芽殖杆菌属(Gemmobacter),螯合球菌属(Chelatococcus),螯合杆菌属(Chelatobacter),冰冷杆菌属(Gelidibacter),Coenonia,考克斯氏体属(Coxiella),考克氏菌属(Kocuria),科泽氏菌属(Koserella),粪球菌属(Coprococcus),粪热杆菌属(Coprothermobacter),Coprobacillus,丛毛单胞菌属(Comamonas),科里氏杆菌属(Coriobacterium),棒状杆菌属(Corynebacterium),Collinsella,科尔韦氏菌属(Colwellia),戈登氏菌属(Gordonia),圆团胞菌属(Conglomeromonas),软骨霉状菌属(Chondromyces),Thermaerobacter,栖热菌属(Thermus),Thermacetogenium,Thermanaerovibrio,Thermicanus,Thermithiobacillus,高温放线菌属(Thermoactinomyces),热厌氧杆菌属(Thermoanaerobacter),热厌氧杆菌属(Thermoanaerobacterium),热厌氧菌属(Thermoanaerobium),Thermocladium,热枝菌属(Thermocrispum),Thermocrinis,Thermochromatium,热球菌属(Thermococcus),栖热腔菌属(Thermosipho),热共生菌属(Thermosyntropha),耐热球菌属(Thermosphaera),嗜热丝菌属(Thermothrix),热脱硫杆菌属(Thermodesulfobacterium),Thermodesulfovibrio,热硫还原杆菌属(Thermodesulforhabdus),热土杆菌属(Thermoterrabcterium),栖热孢菌属(Thermotoga),栖热放线菌属(Thermonema),热生水菌属(Thermohydrogenium),栖热拟杆菌属(Thermobacteroides),热杆菌属(Thermobacillus),Thermohalobacter,嗜热双孢菌属(Thermobispora),喜热裂孢菌属(Thermobifida),热丝菌属(Thermofilum),热分歧菌属(Thermobrachium),热原体菌属(Thermoplasma),热变形菌属(Thermoproteus),热微菌属(Thermomicrobium),高温单孢菌属(Thermomonospora),栖热嗜狮菌属(Thermoleophilum),噬纤维菌属(Cytophaga),发酵细菌属(Zymobacter),嗜发酵菌属(Zymophilus),发酵单孢菌属(Zymomonas),箭头菌属(Sagittula),糖球菌属(Saccharococcus),糖丝菌属(Saccharothrix),糖杆菌属(Sacchrobacter),糖多孢菌属(Saccharopolyspora),糖单孢菌属(Saccharomonospora),扎瓦尔金氏菌属(Zavarzinia),Subtercola,腐败螺旋菌属(Saprospira),Samsonia,盐水球菌属(Salinicoccus),盐弧菌属(Salinivibrio),Salibacillus,原生质杆菌属(Sarcobium),八叠球菌属(Sarcina),沙门氏菌属(Salmonella),Salegentibacter,血杆菌属(Sanguibacter),橙色杆菌属(Sandaracinobacter),Dyadobacter,戴阿利斯特杆菌属(Dialister),迪茨氏菌属(Dietzia),希瓦氏菌属(Shewanella),Geothrix,地嗜皮菌属(Geodermatophilus),地袍菌属(Geotoga),土杆菌属(Geobacter),Geobacillus,Geovibrio,网球菌属(Dictyoglomus),解球菌属(Cycloclasticus),冷蛇菌属(Psychroserpens),嗜冷杆菌属(Psychrobacter),圆杆菌属(Cyclobacterium),Psychroflexus,冷单胞菌属(Psychromonas),志贺氏菌属(Shigella),偶蹄形菌属(Dichelobacter),双岐微菌属(Dichotomicrobium),(Dysgonomonas),孢囊杆菌属(Cystobacter),柠檬酸杆菌属(Citrobacter),互养菌属(Synergistes),中华根瘤菌属(Sinorhizobium),双环菌属(Diplocalyx),(Simkania),西蒙斯氏菌属(Simonsiella),两面神菌属(Janibacter),紫色杆菌属(Janthinobacterium),(Pseudaminobacter),类无枝菌酸菌属(Pseudoamycolata),假交替单胞菌属(Pseudoalteromonas),(Pseudoxanthomonas),类杀手杆菌属(Pseudocaedibacter),假诺卡氏菌属(Pseudonocardia),(Pseudobutyrivibrio),假单胞菌属(Pseudomonas),假枝杆菌属(Pseudoramibacter),施瓦茨氏菌属(Schwartzia),琼斯氏菌属(Jonesia),约翰森氏菌属(Johnsonella),(Silicibacter),互养菌属(Syntrophus),互营球菌属(Syntrophococcus),(Syntrophothermus),共养生孢菌属(Syntrophospora),互营杆菌属(Syntrophobacter),共养香肠样杆菌属(Syntrophobotulus),共养单胞菌属(Syntrophomonas),共生小体属(Symbiotes),(Symbiobacterium),动胶菌属(Zoogloea),杜擀氏菌属(Duganella),(Schineria),解琥珀酸菌属(Succiniclasticum),(Succinispira),琥珀酸弧菌属(Succinivibrio),琥珀酸单胞菌属(Succinimonas),斯科曼氏菌属(Skermania),(Skermanella),(Starkeya),(Stappia),葡萄球菌属(Staphylococcus),葡萄嗜热菌属(Staphylothermus),(Staleya),栖冥河菌属(Stygiolobus),锑杆菌属(Stibiobacter),标桩菌属(Stigmatella),斯梯特氏菌属(Stetteria),寡养单胞菌属(Stenotrophomonas),星状菌属(Stella),萨特氏菌属(Sutterella),萨顿氏菌属(Suttonella),口腔球菌属(Stomatococcus),链异壁菌属(Streptoalloteichus),链球菌属(Streptococcus),链孢囊菌属(Streptosporangium),链轮丝菌属(Streptoverticillium),链杆菌属(Streptobacillus),链霉菌属(Streptomyces),(Streptomonospora),螺菌属(Spirillum),游动螺菌属(Spirilliplanes),螺孢菌属(Spirillospora),螺状菌属(Spirosoma),螺原体属(Spiroplasma),螺旋体属(Spirochaeta),鞘氨醇杆菌属(Sphingobacterium),(Sphingobium),(Sphingopyxis),球衣菌属(Sphaerotilus),球杆菌属(Sphaerobacter),鞘氨醇单胞菌属(Sphingomonas),鱼孢菌属(Sporichthya),生孢噬纤维菌属(Sporocytophaga),芽孢八叠球菌属(Sporosarcina),香肠状芽孢菌属(Sporotomaculum),孢杆菌属(Sporobacter),(Sporobacterium),螺旋盐杆菌属(Sporohalobacter),鼠孢菌鼠(Sporomusa),芽孢乳杆菌属(Sporolactobacillus),(Smithella),(Slackia),亚硫酸盐杆菌属(Sulfitobacter),硫化杆菌属(Sulfobacillus),恐硫球菌属(Sulfophobococcus),硫化叶菌属(Sulfolobus),硫代谢球菌属(Sulfurisphaera),硫化球菌属(Sulfurococcus),硫化螺旋菌属(Sulfurospirillum),西地西菌属(Cedecea),鲸杆菌属(Cetobacterium),塞巴鲁德菌属(Sebaldella),沙雷氏菌属(Serratia),塞里伯氏菌属(Seliberia),纤维弧菌属(Cellvibrio),小蛇菌属(Serpula),小蛇菌属(Serpulina),蛇形菌属(Serpens),(Cellulosimicrobium),(Cellulophaga),纤维单胞菌属(Cellulomonas),(Selenihalanaerobacter),月形单胞菌属(Selenomonas),蜈蚣菌属(Centipeda),(Sodalis),(Zobellia),(Solobacterium),索氏菌属(Thauera),指孢囊菌属(Dactylosporangium),塔特姆氏菌属(Tatumella),塔特洛克氏菌属(Tatlockia),Thalassomonas,Thialkalicoccus,(Thialkalivibrio),Thialkalimicrobium,英硫菌属(Thiocapsa),Thiococcus,网硫菌属(Thiodictyon),囊瘤菌属(Thiocystis),硫螺菌属(Thiospira),硫螺旋菌属(Thiospirillum),硫球菌属(Thiosphaera),单岐蓝细菌属(Thiothrix),硫小杆菌属(Thiobacterium),硫杆菌属(Thiobacillus),Thiohalocapsa,Thioflavicoccus,卵硫菌属(Thiovulum),辫硫菌属(Thioploca),板硫菌属(Thiopedia),Thiomargarita,硫微螺菌属(Thiomicrospira),硫单胞菌属(Thiomonas),硫明卵菌属(Thiolamprovum),热红球菌属(Thiorhodococcus),Thiorhodospira,硫红弧菌属(Thiorhodovibrio),组织菌属(Tissierella),钦氏菌属(Chainia),Tindallia,冢村氏菌属(Tsukamurella),苏黎士菌属(Turicella),异常球菌属(Deinococcus),异常杆菌属(Deinobacter),泰勒氏菌属(Taylorella),罩杆菌属(Tectibacter),Dechlorosoma,Dechloromonas,Tessaracoccus,脱硫状菌属(Desulfacinum),脱亚硫酸菌属(Desulfitobacterium),脱硫盒菌属(Desulfocapsa),脱硫球菌属(Desulfococcus),脱硫八叠球菌属(Desulfosarcina),脱硫螺菌属(Desulfospira),脱硫芽孢弯曲菌属(Desulfosporosinus),Desulfocella,Desulfotalea,Desulfotignum,脱硫肠状菌属(Desulfotomaculum),脱硫酸盐碱杆菌属(Desulfonatronum),脱硫酸弯曲杆菌属(Desulfonatronovibrio),Desulfonispora,脱硫线菌属(Desulfonema),Desulfovirga,脱硫菌属(Desulfobacter),脱硫杆菌属(Desulfobacterium),Desulfobacula,Desulfobacca,脱硫叶菌属(Desulfobulbus),脱硫盐菌属(Desulfohalobium),脱硫弧菌属(Desulfovibrio),脱硫棒菌属(Desulfofustis),Desulfofaba,Desulfofrigus,脱硫微菌属(Desulfomicrobium),脱硫单胞菌属(Desulfomonas),脱硫念珠菌属(Desulfomonile),Desulforhabdus,Desulforhopalus,硫还原菌属(Desulfurella),硫还原球菌属(Desulfurococcus),脱硫杆菌属(Desulfurobacterium),硫还原弯形菌属(Desulfuromusa),脱硫单胞菌属(Desulfuromonas),硫还原叶菌属(Desulfurolobus),Desemzia,脱硫代硫酸盐弧菌属(Dethiosulfovibrio),四联球菌属(Tetragenococcus),Tetrasphaera,反硝化杆菌属(Denitropacterium),Denitrovibrio,除卤素杆菌属(Dehalobacter),Tepidimonas,铁还原杆菌属(Deferribacter),Defluvibacter,戴沃斯菌属(Devosia),肥沃菌属(Demetria),土壤球菌属(Terracoccus),地杆菌属(Terrabacter),德克斯氏菌属(Derxia),Delftia,皮生球菌属(Dermacoccus),嗜皮菌属(Dermatophilus),皮杆菌属(Dermabacter),地神菌属(Telluria),德莱氏菌属(Deleya),Dendrosporobacter,曲发菌属(Toxothrix),特拉布斯氏菌属(Trabulsiella),Tricholorobacter,毛球菌属(Trichococcus),甲苯单胞菌属(Tolumonas),密螺旋体属(Treponema),狡诈球菌属(Dolosigranulum),Dolosicoccus,Tropheryma,奈瑟氏球菌属(Neisseria),无色嗜盐菌属(Natrialba),Natrinema,喜碱菌属(Natroniella),Natronincola,嗜盐碱球菌属(Natronococcus),嗜盐碱杆菌属(Natronobacterium),嗜盐碱单胞菌属(Natronomonas),Natronorubrum,侏囊菌属(Nannocystis),硝化球菌属(Nitrococcus),硝化刺菌属(Nitrospina),硝化螺菌属(Nitrospira),亚硝化球菌属(Nitrosococcus),亚硝化螺菌属(Nitrosospira),亚硝化单胞菌属(Nitrosomonas),亚硝化叶菌属(Nitrosolobus),硝化杆菌属(Nitrobacter),(Neochlamydia),新立克次氏体属(Neorickettsia),涅斯捷连科氏菌属(Nesterenkonia),涅瓦河菌属(Nevskia),(Neptunomonas),诺卡氏菌属(Nocardia),类诺卡氏菌属(Nocardioides),拟诺卡氏菌属(Nocardiopsis),(Nonomuraea),(Novosphingobium),枝芽孢杆菌属(virgibacillus),氢杆菌属(Hydrogenobacter),氢弧菌属(Hydrogenovibrio),氢噬胞菌属(Hydrogenophaga),(Hydrogenophilus),栖高温菌属(Hyperthermus),生丝微菌属(Hyphomicrobium),生丝单胞菌属(Hyphomonas),(Paucimonas),丝杆菌属(Bacterionema),(Bacteriovorax),拟杆菌属(Bacteroides),皮肤杆菌属(Bactoderma),漫游球菌属(Vagococcus),巴斯德氏芽菌属(Pasteuria),巴斯德氏菌属(Pasteurella),芽孢杆菌属(Bacillus),(Papillibacter),哈夫尼菌属(Hafnia),(Hahella),副脆弱球菌属(Paracraurococcus),(Parachlamydia),副球菌属(Paracoccus),(Halanaerobacter),(Halanaerobium),(Paralactobacillus),贪噬菌属(Variovorax),束缚杆菌属(Haliscomenobacter),巴尔通氏体属(Bartonella),巴氏丝杆菌属(Balneatrix),(Palaeococcus),霍氏菌属(Hallella),盐盒菌属(Haloarcula),栖盐菌属(Haloincola),(Halochromatium),盐球菌属(Halococcus),盐热发菌属(Halothermothrix),(Halogeometricum),(Halospirulina),嗜盐菌属(Halocella),(Halothiobacillus),(Haloterrigena),(Halonatronum),盐棒杆菌属(Halobaculum),盐杆菌属(Halobacterium),拟盐杆菌属(Halobacteroides),喜盐芽孢杆菌属(Halobacillus),盐弧菌属(Halovibrio),富盐菌属(Haloferax),盐甲烷球菌属(Halomethanococcus),盐单胞菌属(Halomonas),(Ha lorhabdus),盐红菌属(Halorubrum),盐红杆菌属(Halorubrobacterium),需盐红螺菌属(Halorhodos pira),泛菌属(Pantoea),(Pandoraea),吸血弧菌属(Vampirovibrio),嗜酸菌属(Picrophilus),鱼立克次氏体属(Piscirickettsia),(Hippea),透明颤菌属(Vitreoscilla),双岐杆菌属(Bifidobacterium),弧菌属(Vibrio),(Hymenobacter),脂肪杆菌属(Pimelobacter),发仙菌属(Pilimelia),海氏菌属(Hirschia),小梨形菌属(Pirella),小小梨形菌属(Pirellula),热球菌属(Pyrococcus),热网菌属(Pyrodictium),丸状菌属(Pillotina),热棒菌属(Pyrobaculum),嗜胆菌属(Bilophila),(Pyrolobus),干草菌属(Faenia),费克蓝姆氏菌属(Facklamia),考拉杆菌属(Phascolarctobacterium),镰刀弧菌属(Falcivibrio),(Fundibacter),(Finegoldia),丝状杆菌属(Fibrobacter),线状杆菌属(Filibacter),产线菌属(Filifactor),叶杆菌属(Phyllobacterium),(Filobacillus),线状微菌属(Filomicrobium),棕色螺旋菌属(Phaeospirillum),苯基杆菌属(Phenylobacterium),高铁杆菌属(Ferribacterium),铁还原单胞菌属(Ferrimonas),闪烁杆菌属(Fervidobacterium),铁球菌属(Ferroglobus),(Ferroplasma),(Phocoenobacter),发光杆菌属(Photobacterium),光杆状菌属(Photorhabdus),甲酸弧菌属(Formivibrio),梭杆菌属(Fusobacterium),布丘氏菌属(Buttiauxella),丁酸弧菌属(Butyrivibrio),蛭弧菌属(Bdellovibrio),布戴约维采菌属(Budvicia),(Pfennigia),巴克纳氏菌属(Buchnera),(Fusibacter),(Prauserella),布拉格菌属(Pragia),短螺旋菌属(Brachyspira),短状杆菌属(Brachybacterium),短单胞菌属(Brachymonas),绿芽菌属(Blastochloris),芽球菌属(Blastococcus),芽生杆菌属(Blastobacter),芽单胞菌属(Blastomonas),蟑螂杆状体属(Blattabacterium),慢生根瘤菌属(Bradyrhizobium),弗拉特氏菌属(Frateuria),布兰汉氏球菌属(Branhamella),动性球菌属(Planococcus),游动四孢菌属(Planotetraspora),游动双孢菌属(Planobispora),游动多孢菌属(Planopolyspora),(Planomicrobium),游动单胞菌属(Planomonospora),黄色单胞菌属(Flavimonas),黄杆菌属(Flavobacterium),火色杆菌属(Flammeovirga),弗兰克氏菌属(Frankia),浮霉状菌属(Planctomyces),弗朗西斯氏菌属(Francisella),弗莱德门菌属(Friedmanniella),(Frigoribacterium),荧光杆菌属(Fluoribacter),伯克霍尔德氏菌属(Burkholderia),布鲁氏菌属(Brucella),(Bulleidia),(Flexistipes),柔发菌属(Flexithrix),屈挠杆菌属(Flexibacter),弯杆菌属(Flectobacillus),邻单胞菌属(Plesiomonas),(Brenneria),短波单胞菌属(Brevundimonas),短螺旋体属(Brevinema),短杆菌属(Brevibacterium),短小芽孢杆菌属(Brevibacillus),普雷沃氏菌属(Prevotella),(Prochlorococcus),原绿丝蓝细菌属(Prochlorothrix),原绿蓝细菌属(Prochloron),索丝菌属(Brochothrix),突柄绿菌属(Prosthecochloris),突柄杆菌属(Prosthecobacter),突柄微菌属(Prosthecomicrobium),变形菌属(Proteus),原单胞菌属(Protomonas),生丙酸菌属(Propionigenium),丙酸螺菌属(Propionispira),(Propionispora),(Propionibacter),丙酸杆菌属(Propionibacterium),丙酸弧菌属(Propionivibrio),产丙酸菌属(Propioniferax),普罗威登斯菌属(Providencia),原小单胞菌属(Promicromonospora),噬脯氨酸菌属(Prolinoborus),拜叶林克氏菌属(Beijerinckia),韦荣氏菌属(Veillonella),(Beutenbergia),贝日阿托氏菌属(Beggiatoa),梳状菌属(Pectinatus),果胶杆菌属(Pectobacterium),片球菌属(Pediococcus),土地杆菌属(Pedobacter),土微菌属(Pedomicrobium),石袍菌属(Petrotoga),类芽孢杆菌属(Paenibacillus),贝内克氏菌属(Beneckea),消化球菌属(Peptococcus),消化链球菌属(Peptostreptococcus),(Peptoniphilus),血巴通氏体属(Haemobartonella),嗜血菌属(Haemophilus),阳丝菌属(Heliothrix),阳光杆菌属(Heliobacterium),阳光小杆菌属(Heliobacillus),嗜阳菌属(Heliophilum),(Heliorestis),螺杆菌属(Helicobacter),居鸽菌属(Pelistega),佩尔泽氏菌属(Pelczaria),伯杰氏菌属(Bergeyella),创伤球菌属(Helcococcus),(Verrucosispora),疣微菌属(Verrucomicrobium),桃色杆菌属(Persicobacter),草螺菌属(Herbaspirillum),草状孢菌属(Herbidospora),滑柱菌属(Herpetosiphon),暗网菌属(Pelodictyon),(Pelospora),暗杆菌属(Pelobacter),福格斯氏菌属(Vogesella),博戈里亚湖菌属(Bogoriella),包西氏菌属(Bosea),极杆菌属(Polaribacter),极胞菌属(Polaromonas),霍兰德氏菌属(Hollandina),多囊菌属(Polyangium),多核杆菌属(Polynucleobacter),沃氏菌属(Volcaniella),博德特氏菌属(Bordetella),霍尔德曼氏菌属(Holdemania),产卟啉杆菌属(Porphyrobacter),卟啉单胞菌属(Porphyromonas),疏螺旋体属(Borrelia),全孢螺菌属(Holospora),全噬菌属(Holophaga),Hongia,稍热菌属(Meiothermus),分枝杆菌属(Mycobacterium),枝原体属(Mycoplasma),枝动菌属(Mycoplana),Mycetocola,类香味菌属(Myroides),磁螺菌属(Magnetospirillum),巨大球菌属(Macrococcus),大单胞菌属(Macromonas),Massilia,Maricaulis,Marichromatium,海球菌属(Marinococcus),Marinitoga,海滑菌属(Marinilabilia),Marinospirillum,海杆菌属(Marinobacter),海杆菌属(Marinobacterium),海单胞菌属(Marinomonas),Marmoricola,丙二酸单胞菌属(Malonomonas),Mannheimia,云母弧菌属(Micavibrio),粘球菌属(Myxococcus),小英孢囊菌属(Microellobosporia),微球菌属(Micrococcus),微环菌属(Microcyclus),微囊蓝细菌属(Microcystis),微颤蓝细菌属(Microscilla),微球样菌属(Microsphaera),小四孢菌属(Microtetraspora),微枝杆菌属(Microvirgula),微杆菌属(Microbacterium),微泡菌属(Microbulbifer),小双孢菌属(Microbispora),Micropruina,小多孢菌属(Micropolyspora),Micromonas,小单孢菌属(Micromonospora),小月菌属(Microlunatus),光岗菌属(Mitsuokella),巨球形菌属(Megasphaera),巨单胞菌属(Megamonas),中嗜杆菌属(Mesophilobacter),中间原体属(Mesoplasma),中间根瘤菌属(Mesorhizobium),Methanimicrococcus,产甲烷石状菌属(Methanocalculus),甲烷袋状菌属(Methanoculleus),产甲烷菌属(Methanogenium),甲烷粒菌属(Methanocorpusculum),拟甲烷球菌属(Methanococcoides),甲烷球菌属(Methanococcus),甲烷嗜热菌属(Methanothermus),Methanothermobacter,鬃毛甲烷菌属(Methanosaeta),甲烷八叠球菌属(Methanosarcina),甲烷螺菌属(Methanospirillum),甲烷球形菌属(Methanosphaera),甲烷丝菌属(Methanothrix),甲烷杆菌属(Methanobacterium),甲烷盐菌属(Methanohalobium),甲烷嗜盐菌属(Methanohalophilus),甲烷嗜高热菌属(Methanopyrus),Methanofollis,甲烷盘菌属(Methanoplanus),甲烷短杆菌属(Methanobrevibacter),甲烷微菌属(Methanomicrobium),叶形甲烷菌属(Methanolacinia),甲烷叶菌属(Methanolobus),生金球菌属(Metallosphaera),Methylarcula,喜热嗜甲基菌属(Methylocaldum),甲基球菌属(Methylococcus),Methylosarcina,甲基孢囊菌属(Methylocystis),甲基弯曲菌属(Methylosinus),甲基球菌属(Methylosphaera),Methylocella,甲基杆菌属(Methylobacter),甲基杆菌属(Methylobacterium),甲基小杆菌属(Methylobacillus),Methylopila,噬甲基菌属(Methylophaga),嗜甲基菌属(Methylophilus),食甲基菌属(Methylovorus),甲基微菌属(Methylomicrobium),甲基单胞菌属(Methylomonas),耗甲基杆菌属(Methylorhabdus),新月菌属(Meniscus),蜂窝囊菌属(Melittangium),蜜蜂球菌属(Melissococcus),米勒氏菌属(Moellerella),穆尔氏菌属(Moorella),Mogibacterium,Modestobacter,动弯杆菌属(Mobiluncus),莫拉氏菌属(Moraxella),摩根氏菌属(Morganella),Mortiella,桑椹状球菌属(Morococcus),真杆菌属(Eubacterium),紫色小杆菌属(Iodobacter),预研菌属(Yokenella),拉恩氏菌属(Rahnella),Raoultella,乳球菌属(Lactococcus),产乳酸球菌属(Lactosphaera),乳杆菌属(Lactobacillus),毛螺菌属(Lachnospira),拉氏杆菌属(Rathayibacter),杆状色菌属(Rhabdochromatium),双头菌属(Labrys),罗尔斯通氏菌属(Ralstonia),稀有杆菌属(Rarobacter),闪囊菌属(Lamprocystis),闪杆菌属(Lamprobacter),俊片菌属(Lampropedia),立默氏菌属(Riemerella),立克次氏体属(Rickettsia),立克次氏小体属(Rickettsiella),理研菌属(Rikenella),李斯特氏菌属(Listeria),利斯顿氏菌属(Listonella),溶杆菌属(Lysobacter),根瘤杆菌属(Rhizobacter),根瘤菌属(Rhizobium),根单胞菌属(Rhizomonas),溶菌属(Lyticum),Limnobacter,赖文氏菌属(Lewinella),Ruegeria,皱纹单胞菌属(Rugamonas),射光杆菌属(Lucibacterium),Luteimonas,黄球菌属(Luteococcus),古字状菌属(Runella),红长命菌属(Rubrivivax),Rubrimonas,红色杆菌属(Rubrobacter),瘤胃球菌属(Ruminococcus),瘤胃杆菌属(Ruminobacter),Leifsonia,勒克氏菌属(Leclercia),Lechevalieria,军团菌属(Legionella),肾杆菌属(Renibacterium),莱文氏菌属(Levinea),钩端螺旋体属(Leptospira),钩端螺菌属(Leptospirillum),纤发菌属(Leptothrix),纤毛菌属(Leptotrichia),纤线菌属(Leptonema),勒米诺氏菌属(Leminorella),伦茨菌属(Lentzea),亮发菌属(Leucothrix),明串珠菌属(Leuconostoc),无色杆菌属(Leucobacter),劳森氏菌属(Lawsonia),劳特罗普氏菌属(Lautropia),罗卡利马氏体属(Rochalimaea),Roseateles,Roseinatronobacter,Roseibium,Roseivivax,玫瑰球菌属(Roseococcus),蔷薇螺菌属(Roseospira),Roseospirillum,玫瑰杆菌属(Roseobacter),Roseovarius,玫瑰单胞菌属(Roseomonas),罗斯伯里氏菌属(Roseburia),Rhodanobacter,罗斯氏菌属(Rothia),红球菌属(Rhodococcus),红嗜热盐菌属(Rhodothermus),红环菌属(Rhodocyclus),红篓菌属(Rhodocista),红假单胞菌属(Rhodopseudomonas),玫瑰螺旋菌属(Rhodospira),红螺菌属(Rhodospirillum),海玫瑰菌属(Rhodothalassium),Rhodobaca,红细菌属(Rhodobacter),红菌属(Rhodobium),玫瑰弧菌属(Rhodovibrio),红球形菌属(Rhodopila),红育菌属(Rhodoferax),红游动菌属(Rhodoplanes),小红卵菌属(Rhodovulum),红微菌属(Rhodomicrobium),隆派恩菌属(Lonepinella),魏斯氏菌属(Weissella),Waddlia属。
例如,可以举出属于真核微生物的伊萨酵母属(Issatchenkia),假丝酵母属(candida),隐球酵母属(cryptococcus),克鲁维酵母属(Kluyveromyces),克勒克酵母属(Kloeckera),类酵母属(Saccharomycodes),酵母属(Saccharomyces),接合酵母属(Zygosaccharomyces),裂殖酵母属(Schizosaccharomyces),链担尔属(Sirobasidium),梗孢酵母属(Sterigmatomyces),锁掷酵母属(Sporidobolus),掷孢酵母属(Sporobolomyces),德克酵母属(Dekkera),德巴利酵母属(Debaryomyces),丝孢酵母属(Trichosporon),三角酵母属(Trigonopsis),有孢圆酵母属(Torulaspora),银耳属(Tremella),拿逊酵母属(Nadsonia),针属酵母属(Nematospora),有孢汉逊酵母属(Hanseniaspora),毕赤酵母属(Pichia),Fibulobasidium属,Filobasidium属,Filobasidiella属,布勒弹孢酵母属(Bullera),酒香酵母属(Brettanomyces),胶珊瑚酵母属(Holtermannia),鳞斑霉属(Malassezia),梅奇酵母属(Metschnikowia),油脂酵母属(Lipomyces),白冬孢酵母属(Leucosporidium),红冬孢酵母属(Rhodosporidium),红酵母属(Rhodotorula),无柄霉属(Acaulopage),水被孢霉属(Aquamortierella),内孢毛菌属(Asellaria),变形毛菌属(Amoebidium),嗜虫霉属(Amoebophilus),微苇毛菌属(Arundinula),星玉霉属(Utharomyces),刺孢囊霉属(Echinosporangium),肠藓毛菌属(Enterobryus),内囊霉属(Endogone),虫霉属(Entomophthora),梳霉属(Kickxella),Genistellospora属,Choanephora属,下梳霉属(Coemansia),旋体霉属(Cochlonema),耳霉属(Conidiobolus),瓶霉属(Saksenaea),枝霉属(Thamnidium),头枝霉属(Thamnocephalis),双卷霉属(Dispira),双珠霉属(Dimargaris),共头霉属(Syncephalastrum),集珠霉菌属(Syncephalis),捕虫霉菌属(Zoopage),实果内囊霉菌属(Sclerocystis),Smittium属,蛙类霉菌属(Basidiobolus),附丝外毛菌属(Parataeniella),副变毛菌属(Paramoebidium),裂丝毛菌属(Palavascia),钩孢毛菌属(Harpella),夹珠菌属(Piptocephalis),水玉霉菌属(Pilobolus),牙甲囊霉属(Phycomyces),布拉霉属(Blakeslea),赫塞霉属(Hesseltinella),(Helicocephalum),蒲头霉属(Mycotypha),辅枝霉属(Radiomyces),Legeriomyces属,Rhopalomyces属,Acrasis属,无胞丝菌属(Acytostelium),团网菌属(Arcyria),刺丝菌属(Echinostelium),Echinosteliopsis属,Oligonema属,腔丝菌属(Cavostelium),Guttulinopsis属,Clastoderma属,筛菌属(Cribraria),Coenonia属,Copromyxa属,发菌属(Comatricha),Colloderma属,实线菌属(Dianema),网柄菌属(Dictyostellium),钙皮菌属(Didymium),双皮菌属(Diderma),发网菌属(Stemonitis),破囊壶菌属(Thraustochytrium),鹅绒菌属(Ceratiomyxa),Ceratiomyxella属,团毛菌属(Trichia),绒泡菌属(Physarum),根肿菌属(Plasmodiophora),煤绒菌属(Fuligo),Bursulla属,原柱菌属(Protostelium),Protosporangium属,半网菌属(Hemitrichia),盖碗菌属(Perichaena),Polysphondylium属,多粘霉属(Polymyxa),网粘菌属(Labyrinthula),亮皮菌属(Lamproderma),粉瘤菌属(Lycogala),无丝菌属(Licea),Wardmyces属,Actinopelte属),星盘孢属(Asterosporium),节菱孢属(Arthrinium),毛连格孢属(Alternaria),粉孢属(Oidium),Clabosporium属,Cladobotryum属,粘束孢属(Graphium),刺盘孢属(Colletotrichum),小核菌属(Sclerotium),壳多孢属(Stagonospora),Stibella属,瘤座孢属(Tubercularia),Bactridium属,Pycnothytium属,Phaeoisaria属,Pestalozziella属,丝核菌属(Rhizoctonia),Rhinocladiella属,细盾霉属(Leptothyrium),绵壶菌属(Achlyogeton),异壶菌属(Anisolpidium),白锈属(Albugo),外壶菌属(Ectrogella),油壶菌属(Olipidium),拟油壶菌属(Olpidiopsis),链枝菌属(Catenaria),壶菌属(Chytridium),壶歧菌属(Cladochytrium),雕蚀菌属(Coelomomyces),节水霉属(Gonapodya),水霉属(Saprolegnia),离壶菌属(Sirolpidium),集壶菌属(Synchytrium),Haliphthoros属,肋壶菌属(Harpochytrium),腐霉属(Pythium),丝壶菌属(Hyphochytrium),节壶菌属(Physoderma),泡壶菌属(Phlyctidium),芽枝霉属(Blastocladia),霜霉属(Peronospora),Peronophythora属,Micromycopsis属,大壶菌属(Megachytrium),单毛菌属(Monoblepharis),链壶菌属(Lagenidium),根前毛菌属(Rhizidiomyces),根壶菌属(Rhizidium),囊轴霉属(Rhipidium),水节霉属(Leptomitus),小细囊霉属(Leptolegniella),Acremonium属,曲霉属(Aspergillus),梨头霉属(Absidia),蛛网霉属(Arachniotus),节丛孢属(Arthrobotrys),Ulocladium属,棘瓶孢属(Echinobotryum),Exophiala属,附球菌属(Epicoccum),树粉孢属(Oidiodendron),珠头霉属(Oedocephalum),短梗霉属(Aureobasidium),弯孢属(Curvularia),Candelabrella属,小克银汉霉属(Cunninghamella),裸囊菌属(Gymnoascus),枝孢属(Cladosporium),粘束孢属(Graphium),粘帚霉属(Gliocladium),Chrysosporium属,Chromelosporium属,地霉属(Geotrichum),Geomyces属,毛壳菌属(Chaetomium),Geniculifera属,Gonatobotrys属,盾壳霉属(Coniothyrium),卷霉属(Circinella),接霉属(Zygorhynchus),色二孢属(Diplodia),柱孢属(Cylindrocarpon),帚霉属(Scopulariopsis),葡萄穗霉属(Stachybotrys),匍柄霉属(Stemphylium),Sporothrix属,瘤孢属(Sepedonium),Dactylella属,Talaromyces属,Dratomyces属,毛束霉属(Trichurus),Trichocladium属,单端孢属(Trichothecium),木霉属(Trichoderma),发癣菌属(Trichophyton),黑孢属(Nigrospora),Verticicladiella属,轮枝孢属(Verticillium),拟青霉属(Paecilomyces),Pithomyces属,Bipolaris属,棘壳孢属(Pyrenochaeta),Phialocephala属,瓶霉属(Phialophora),茎点霉(Phoma),镰刀菌属(Fusarium),Pestalotiopsis属,青霉属(Penicillium),葡萄孢属(Botrytis),小孢霉属(Microsporum),漆斑菌属(Myrothecium),毛霉属(Mucor),黑乌霉属(Memnoniella),Monacrosporium属,从梗孢属(Monilia),被孢霉属(Mortierella),正青霉属(Eupenicillium),散囊菌属(Eurotium),根霉属(Rhizopus),Leptographium属,三毛孢属(Robillarda),Austroboletus属,木耳属(Auricularia),耳匙菌属(Auriscalpium),蘑菇属(Agaricus),田头菇属(Agrocybe),Asterophona属,硬皮地星属(Astraeus),星头鬼笔属(Aseroe),Anellaria属,乌芝属(Amauroderma),毒伞属(Amanita),蜜环菌属(Armillaria),假蜜环菌属(Armillariella),网孢盘菌属(Aleuria),薄皮孔菌属(Ischnoderma),丝盖伞属(Inocybe),纤孔菌属(Inonotus),Ileodictyon属,从耳属(Wynnea),钟菌属(Verpa),小包脚菇属(Volvariella),脚瓶盘菌属(Urnula),木齿菌属(Echinodontium),黑耳属(Exidia),扁芝属(Elfvingia),小奥德磨属(Oudemansiella),亚脐菇属(Omphalina),翁孔菌属(Onnia),乳头蘑属(Catathelasma),灵芝属(Ganoderma),拱顶菌属(Camarophyllus),Chalciporus属,盔孢伞属(Galerina),胶角耳属(Calocera),丽口孢属(Calostoma),鸡油菌属(Cantharellus),Cantharellula属,黑蛋巢菌属(Cyathus),环褶菌属(Cyclomyces),囊皮伞属(Cystoderma),Cyptotrama属,Cymatoderma属,裸伞属(Gymnopilus),库恩菇属(Kuehneromyces),圆牛肝菌属(Gyrodon),圆孔牛肝菌属(Gyroporus),鹿花菌属(Gyromitra),桂花耳属(Guepinia),Xanthoconium属,炭角菌属(xylaria),绒盖牛肝菌属(Xerocomus),干脐菇属(Xeromphalina),地锤菌属(Cudonia),Clavatia属,珊瑚菌属(Clavaria),棒瑚菌属(Clavariadelphus),冠瑚菌属(Clavicorona),锁瑚菌属(Clavulina),拟锁瑚菌属(Clavulinopsis),喇叭菌属(Craterellus),笼头菌属(Clathrus),杯伞属(Clitocybe),斜盖伞属(Clitopilus),毛皮伞属(Crinipellis),奇果菌属(Grifola),隐皮菌属(Cryptoderma),隐孔菌属(Cryptoporus),白蛋巢菌属(Crucibulum),Creolophus属,靴耳属(Crepidotus),Chroogomphus属,绿盘菌属(Chlorosplenium),地星属(Geastrum),Geolossum属,Cotylidia属,锥盖伞属(Conocybe),Kobayasia属,鬼伞属(Coprinus),铆钉菇属(Gomphidius),钉菇属(Gomphus),革盖菌属(Coriolus),虫草属(Cordyceps),丝膜菌属(Cortinarius),集毛菌属(Coltricia),金钱菌属(Collybia),肉杯菌属(Sarcoscypha),肉齿菌属(Sarcodon),Sarcodontia属,乳牛肝菌属(Suillus),裂褶菌属(Schizophyllum),Squamanita属,盾盘菌属(Scutellinia),硬皮马勃属(Scleroderma),韧革菌属(Stereum),松塔牛肝菌属(Strobilomyces),球盖菇属(Stropharia),地匙菌属(Spathularia),绣球菌属(Sparassis),拟迷孔菌属(Daedaleopsis),花耳属(Dacryomyces),轮层炭壳属(Daldinia),竹荪属(Dictyophora),粉孢牛肝菌属(Tylopilus),干酪菌属(Tyromyces),圆头伞属(Descolea),革菌属(Thelephora),柄灰孢属(Tulostoma),栓菌属(Trametes),毛舌菌属(Trichoglossum),发菌属(Trichocoma),Tricoloma属,Tricholomopsis属,银耳属(Tremella),刺银耳属(Tremellodon),沿丝伞属(Naematoloma),红蛋巢菌属(Nidula),Neobulgaria属,Baeospora属,桩菇属(Paxillus),钉灰孢属(Battarea),斑褶菇属(Panaeolus),革耳属(Panus),扇菇属(Panellus),烟白齿菌属(Bankera),Hygrocybe属,蜡伞属(Hygrophorus),拟蜡伞属(Hygrophoropsis),Bisporella属,豆马勃属(Pisolithus),齿菌属(Hydnum),亚齿菌属(Hydnellum),Hypsizygus属,滴孔菌属(Piptoporus),炭团菌属(Hypoxylon),刺革菌属(Hymenochaete),囊孔菌属(Hirschioporus),Pyrrhoderma属,棱孔菌属(Favolus),暗孔菌属(Phaeolus),褐伞属(Phaeolepiota),鬼笔属(Phallus),牛排菌属(Fistulina),Phyllotopsis属,褶孔菌属(Phylloporus),丝牛肝菌属(Filoboletus),木层孔菌属(Phellinus),拟木层孔菌属(Fomitopsis),层孔菌属(Fomes),鳞伞属(Pholiota),小脆柄菇属(Psathyrella),裸盖菇属(Psilocybe),Pseudocolus属,Pseudohiatula属,羽瑚菌属(Pterula),Flammulina属,粉末牛肝菌属(Pulveroboletus),胶鼓菌属(Bulgaria),光柄菇属(Pluteus),Pleurocybella属,侧耳属(Pleurotus),红盘菌属(Plectania),焰耳属(Phlogiotis),盘菌属(Peziza),Penicilliopsis属,粘滑菇属(Hebeloma),猴头菌属(Hericium),马鞍菌属(Helvella),肉座壳属(Podostroma),Polyozellus属,多孔菌属(Polyporus),拟多孔菌属(Polyporellus),胶珊瑚属(Holtermannia),粪伞属(Bolbitius),红牛肝菌属(Porphyrellus),小牛肝菌属(Boletinus),牛肝菌属(Boletellus),Boletus属,Boletopsis属,Porodisculus属,刺孢多孔菌属(Bondarzewia),Macrocystidia属,大足盘菌属(Macropodia),大环柄菇属(Macrolepiota),小皮伞属(Marasmius),微皮伞属(Marasmiellus),小孔菌属(Microporus),小菇属(Mycena),地杖菌属(Mitrula),蛇头菌属(Mutinus),Melanoleuca属,干朽菌属(Merulius),羊肚菌属(Morchella),(Laetiporus),乳菇属(Lactarius),毛球马勃属(Lasiosphaera),蜡菇属(Laccaria),杖瑚菌属(Ramaria),Lampteromyces属,离褶伞属(Lyophyllum),硬孔菌属(Rigidoporus),马勃属(Lycoperdon),根盘菌属(Rhizina),散尾鬼笔属(Lysurus),粘伞属(Limacella),Lindera属,红菇属(Russula),白鬼伞属(Leucocoprinus),白桩菇属(Leucopaxillus),锤舌菌属(Leotia),伏褶菌属(Resupinatus),疣柄牛肝菌属(Leccinum),环柄菇属(Lepiota),Lepista属,革裥菌属(Lenzites),木瑚菌属(Lentaria),香菇属(Lentinus),Lentinula属,小香菇属(Lentinellus),罗鳞伞属(Rozites),红盖菇属(Rhodocybe),Rhodotus属,赤褶菇属(Rhodophyllus)等。
具有本发明的辅酶结合型葡萄糖脱氢酶生产能的上述微生物,以保藏编号的形式在IFO或ATCC保藏,可以从已知的分支机关,法人处得到。作为该微生物,可以列举出优选属于真核微生物,特别优选属于丝状菌的微生物。另外,可以使用命名为“97508”,在独立行政法人产业技术综合研究所特许生物保藏中心保藏的保藏号为FERMBP-08578的微生物。
作为本发明的辅酶结合型葡萄糖脱氢酶的制造方法的一种方式,可以列举出在营养培养基中培养具有本发明的辅酶结合型葡萄糖脱氢酶生产能的微生物,在培养液中生成并累积辅酶结合型葡萄糖脱氢酶,采集后得到辅酶结合型葡萄糖脱氢酶的蛋白质及其盐。
以下,说明本发明的保藏菌株FERM BP-08578,来源于该菌株的辅酶结合型葡萄糖脱氢酶及该酶的取得方法。
1.FERM BP-08578来源的该酶的理化性质
(1)作用:国际生化联合会(IUB)分类为EC1.1.99.10的酶,在电子受体存在下催化以下所示的氧化葡萄糖1位的羟基、生成葡糖酸-δ-内酯的反应。
葡萄糖+电子受体→葡糖酸-δ-内酯+还原型电子受体
本发明中,作为电子受体,例如可以列举出利用吩嗪硫酸二甲酯,1-甲氧基-5-甲基吩嗪甲硫酸盐(1-methoxy-5-methylphenaziniummethyl sulfate),2,6-二氯苯酚靛酚,氰铁酸化合物等。
(2)基质特异性:按下述活性测定方法1,使用D-葡萄糖及其他的各种基质(除了D-纤维二糖193mM,D-海藻糖及D-棉子糖121mM,其他的终浓度均为333mM)时,该酶的相对反应性(基质特异性),如表1所示。另外,使用终浓度550mM,100mM的D-葡萄糖及麦芽糖时的相对反应性(基质特异性),如表2所示。对D-葡萄糖有强作用,对D-甘露糖,1,5-脱水-D-山梨醇,D-纤维二糖,D-海藻糖,麦芽糖,D-半乳糖,D-葡萄糖-6-磷酸,D-果糖作用较弱。另外,对L-阿拉伯糖,乳糖,D-山梨醇,葡糖酸,蔗糖,D-甘露糖醇,L-山梨糖,D-核糖,L-鼠李糖,D-葡萄糖-1-磷酸,D-棉子糖,乙醇,甘油几乎没有作用。
(3)最适pH:pH7.0至pH9.0
(4)稳定pH:pH4.5至pH8.5
(5)最适温度:55℃左右
(6)温度稳定性:50℃以下稳定
(7)分子量:凝胶过滤法测定的分子量约130kDa,SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胺)电泳法测定约85kDa。
(8)Km值:49.7mM D-葡萄糖
(9)等电点:用等电点电泳法测定的辅酶结合型葡萄糖脱氢酶的等电点(pI)约为4.4
(10)阻滞剂:下面所记载的活性测定方法1中,添加终浓度为1mM的各种添加物,测定活性,结果相对于对照区,由各添加物确定如表3所示的阻滞效果。另外,下面所记载的活性测定方法1中,在添加各终浓度的1,10-菲绕啉(溶于甲醇)时,结果确定如表4所示的阻滞效果。该酶的活性,被重金属离子(Ag+、Cu2+、Hg2+)强烈阻滞,被1,10-菲绕啉,硫酸原黄素,Mn2+阻滞60%以上。
(11)辅酶:FAD
涉及辅酶结合型葡萄糖脱氢酶的氨基酸序列及编码他的基因
的碱基序列也属于本发明。
在制造本发明的辅酶结合型葡萄糖脱氢酶时,用于制造该辅酶结合型葡萄糖脱氢酶的微生物,可以是能够生产本发明的辅酶结合型葡萄糖脱氢酶的生物,例如,微生物,优选真核微生物,特别优选使用属于丝状菌的微生物,可以高效地制造该酶。特别是,可以使用命名为“97508”,在独立行政法人产业技术综合研究所特许生物保藏中心保藏为FERM BP-08578的微生物。该保藏菌株,是本发明者从土壤中分离得到的,其菌学性质,如下所示。另外,本发明中,也可以使用前述菌株的变异株。变异菌株可以通过紫外线,x线等放射线或化学变异剂(NTG等)的处理所得到。
2.FERM BP-08578的菌学性质
(1)形态的性质:土豆·葡萄糖琼脂培养基上生长的该菌株在光学显微镜下的形态特征如下所示。菌丝宽度由2μm到4μm,具有规则的隔壁。菌丝的大半呈直线生长,膨化菌丝基本不可见,具有分支。数根菌丝很好地聚合,形成菌丝束。菌丝宽度大致一定。菌丝表面平滑,壁略厚。有以气中菌丝基部为中心形成的结晶体。未见锔子连接。培养2周后,未见由菌体生出的有性·无性生殖器官,也没有发现分裂子及厚壁孢子的存在。
(2)在各培养基的生长状态:在全部为琼脂的培养基中菌丝为绵毛状。气中菌丝的色调呈白色。在土豆·葡萄糖琼脂培养基,里面着色为淡橙色至橙色。生长至中等程度时,25℃1周培养后的菌落,在土豆·葡萄糖及麦片粥琼脂培养基的直径为30mm至35mm,在麦芽提取物琼脂培养基的直径为37mm到38mm。另外,在土豆·葡萄糖琼脂培养基上可产生淡黄色色素,在麦片粥琼脂培养基可产生浅红色至灰红色的可溶色素。培养2周后,未见由菌体生出的分生孢子柄等生殖器官,也未见产生渗出液。
(3)生理性质:该菌株为好氧性,在土豆·葡萄糖琼脂培养基中生长的最适温度为37℃左右。
3.FERM BP-08578的分类学特征
基于以上性质的保藏菌株“97508”按ホHawksworth,Sutton,Ainsworth编的Ainsworth&Bisby′sDictionary of the Fungi第7版进行检索。该检索显示这个保藏菌株属于曲霉属微生物。进而,进行BLAST同源性检索。以基因组DNA为模板,通过PCR法,进行18s rDNA片断扩增后,解读精制的PCR产物序列。由于从基因库(GenBank/EMBL/DDBJ国际DNA序列数据库)中检索到类似的碱基序列,通过BLAST(Altschul et al.,1997)进行同源性检索,结果判定该保藏菌株“97508”为局限曲霉。
作为本发明中辅酶结合型葡萄糖脱氢酶的制造方法的一种方式,可以列举出培养上述微生物,在微生物菌体内及/或菌体外表达或生产该辅酶结合型葡萄糖脱氢酶的方法。
对于本发明中使用的微生物的培养,可以使用通常的微生物培养用培养基,除碳源,氮源,无机物以外,只要适当含有对使用微生物必需的微量营养素,合成培养基,天然培养基均可使用。作为碳源,可以使用葡萄糖,蔗糖,糊精,淀粉,甘油,糖汁等。作为氮源,
可以使用氯化铵,硝酸铵,硫酸铵,磷酸铵等无机盐类,DL-丙氨酸,L-谷氨酸等氨基酸类,蛋白胨,肉浸膏,酵母浸膏,麦芽浸膏,玉米浆等含有氮的天然物。作为无机物,可以使用磷酸二氢钠,磷酸氢二钠,磷酸二氢钾,磷酸氢二钾,硫酸镁,氯化铁等。
为了得到本发明的辅酶结合型葡萄糖脱氢酶而进行的培养,通常,可以在震荡培养或通气培养等方法的有氧条件下进行,优选25℃至60℃,而且pH5到pH8的范围内进行。培养时间,优选2天到4天的范围。通过这样的方法进行培养,培养物中,特别是培养液中,可以生成并蓄积辅酶结合型葡萄糖脱氢酶。另外,通过这一培养方法,培养微生物内也可以生成并蓄积辅酶结合型葡萄糖脱氢酶。进而,由培养物中获得辅酶结合型葡萄糖脱氢酶的方法,可以使用通常的蛋白质精制方法。作为这个方法,有例如培养微生物后,通过离心分离除去微生物,得到培养上清液的方法,或培养微生物后,离心分离培养液得到培养微生物,用适当方法破碎该培养微生物,从破碎液通过离心分离等方法,得到上清液的方法。这些上清液中含有的辅酶结合型葡萄糖脱氢酶,可以通过盐析,溶剂沉淀,透析,离子交换色谱,疏水吸附色谱,凝胶过滤,亲和层析,电泳等适当的精制操作的组合操作而精制。
另外,对于为了得到本发明的辅酶结合型葡萄糖脱氢酶的培养,可以使用固体培养基。对培养方法没有特别的限制,可以通过静置培养,也可以通过旋转培养或流动层培养等经常混合培养物的方法进行培养,作为设备投资少的培养装置,优选静置培养。进而,由培养物中得到辅酶结合型葡萄糖脱氢酶的方法,可以使用通常的蛋白质精制方法。也就是,可以在培养物中加入水等提取剂并搅拌后,通过离心分离,过滤等分离方法除去麸子等培养基固体成分,得到提取液。另外,对于菌体内蓄积的辅酶结合型葡萄糖脱氢酶的回收,可以通过将得到了上述提取液的培养物残渣与海砂等研磨剂一起研磨后,加入水等,提取由菌体游离出的辅酶结合型葡萄糖脱氢酶而进行。另外,为了得到全辅酶结合型葡萄糖脱氢酶,可以将培养物全部与海砂等研磨剂一起研磨后,加入水等,从菌体游离出的和培养基中分泌的辅酶结合型葡萄糖脱氢酶两部分一起提取。这些上清液中含有的辅酶结合型葡萄糖脱氢酶,可以通过盐析,溶剂沉淀,透析,离子交换色谱,疏水吸附色谱,凝胶过滤,亲和层析,电泳等适当的精制操作的组合操作而精制。
另外,本发明的辅酶结合型葡萄糖脱氢酶,通过合成得到辅酶结合型葡萄糖脱氢酶或通过遗传工程得到的重组型辅酶结合型葡萄糖脱氢酶均可。本领域技术人员,可以基于来源于本发明的辅酶结合型葡萄糖脱氢酶的理化性质的蛋白质及其盐的公开,容易地得到辅酶结合型葡萄糖脱氢酶。例如,对于辅酶结合型葡萄糖脱氢酶,可以从含有丝状菌的微生物或动物等天然物中提取,此外,也可以其氨基酸序列及编码氨基酸序列的基因的碱基序列为基础通过合成方法得到,也可以通过将该辅酶结合型葡萄糖脱氢酶基因的基因片断,插入到市售的表达载体等公知的表达载体中,利用得到的质粒对大肠杆菌等宿主进行转化,培养转化体,得到目的辅酶结合型葡萄糖脱氢酶的通过遗传工程进行工业制造辅酶结合型葡萄糖脱氢酶的方法。
对于该酶的活性测定,优选将该酶的终浓度适当稀释至0.1-1unit/ml使用。另外,该酶的酶活性单位(unit)为1分钟氧化1μmol的葡萄糖的酶活性。本发明的辅酶结合型葡萄糖脱氢酶的酶活性可以用以下方法测定。
(i)酶活性测定法-1
将0.1M磷酸钾缓冲液(pH7.0)1.0ml、1.0M D-葡萄糖1.0ml、3mM 2,6-二氯苯酚靛酚(以下简称DCIP)0.1ml、3mM 1-甲氧基-5-甲基吩嗪甲基硫酸盐0.2ml、水0.65ml,添加到3ml石英杯中(光路长1cm),放入带有恒温箱的分光光度计中,37℃培养5分钟后,添加0.05ml酶溶液,测定DCIP在600nm处的吸光度变化(ΔABS/min)。以DCIP在pH7.0时的摩尔吸光系数为16.3×103cm-1M-1,由于在1分钟1μmol的DCIP被还原的酶活性实质上等价于该酶活性为1unit,可以通过吸光度的变化按下面的公式求得该酶的活性。
(ii)酶活性测定法-2
1.0M磷酸钾缓冲液(pH7.0)3.4μl,1.0M D-葡萄糖0.1ml,20mMDCIP 86.6μl,37℃培养5分钟后,添加0.01ml酶溶液并搅拌,反应5分钟后在100℃培养3分钟、终止反应。然后,加入100mM甘氨酸·钠缓冲液(pH13.0)0.19ml,2.0N氢氧化钾0.01ml,37℃培养10分钟,溶液中的D-葡糖酸变换成D-葡糖酸-δ内酯后,加入100mM的tris·盐酸缓冲液(pH7.5)0.39ml、1.0N盐酸0.01ml,使pH为中性。溶液中的D-葡糖酸用D-葡糖酸/D-葡糖酸-δ-内酯定量试剂盒(Beringa·manhaim公司制)进行定量。由于1分钟生成1μmol的D-葡糖酸-δ-内酯的酶活性实质等价于该酶活性1unit,可以从D-葡糖酸-δ-内酯的生成量推算该酶的活性。
本发明涉及提供使用本发明的辅酶结合型葡萄糖脱氢酶的物质生产及分析用途,涉及在医药用或食品素材的改质加工方面的使用。作为一例,将该辅酶结合型葡萄糖脱氢酶作为试剂,在含有生体物质的样品中的葡萄糖的消除方法,测定方法及它们的试剂,试剂组合物中使用。另外,在使用本发明的辅酶结合型葡萄糖脱氢酶的有机化合物的制造方法及制造原料中使用。
本发明的辅酶结合型葡萄糖脱氢酶,是在电子受体存在下,催化葡萄糖氧化反应的酶。本发明是在前述反应中,使用本发明的辅酶结合型葡萄糖脱氢酶。作为辅酶结合型葡萄糖脱氢酶没有限制,优选来源于生产该辅酶结合型葡萄糖脱氢酶的真核微生物的辅酶结合型葡萄糖脱氢酶,特别是优选来源于丝状菌的辅酶结合型葡萄糖脱氢酶。
然后,对由本发明得到的辅酶结合型葡萄糖脱氢酶的用途进行说明。由于辅酶结合型葡萄糖脱氢酶是在电子受体存在下,催化氧化葡萄糖的反应的酶,只要是能够利用这个反应引起的变化的用途,没有特别的限制。例如,辅酶结合型葡萄糖脱氢酶可以用于含有生体物质的该试样中的葡萄糖的测定用试剂,消除用试剂。另外,也可以在医疗领域,临床领域使用,在使用辅酶结合型葡萄糖脱氢酶的物质生产及分析的用途中也可使用。
本发明的生物传感器可以是用于在含有本发明的辅酶结合型葡萄糖脱氢酶作为酶的反应层中使用的检测器。例如该生物传感器是通过以下方法制成的,在绝缘性的基板上利用丝网印刷等方法形成由作用极,它的配极以及参比极组成的电极系,连接这个电极系,形成含有亲水性高分子,氧化还原酶和电子受体的酶反应层。含有基质的试样液滴到这个生物传感器的酶反应层上,酶反应层溶解,酶与基质发生反应,同时伴随着电子受体被还原。酶反应结束后,被还原的电子受体通过电气化学法氧化,这时,这个生物传感器由得到的氧化电流值就可以测定试样中的基质浓度。另外,除此以外,可以构筑利用发色强度或pH的变化等检测方式的生物传感器。利用这些生物传感器,通过选择以测定对象物质作为基质的酶,可以测定各种各样的物质。例如,对于酶,如果选择本发明的辅酶结合型葡萄糖脱氢酶,就可以制作测定试样液中葡萄糖浓度的葡萄糖传感器。
作为生物传感器的电子受体,可以应用具有优异的电子授受能的化学物质。作为具有优异电子授受能的化学物质,一般是被称为“电子传递体”,“介体”,或“氧化还原媒介剂”的化学物质,作为这一类化学物质,可以利用例如,特表2002-526759中列举的电子传递体或氧化还原媒介剂等。
生物传感器,多使用便宜的铁氰化钾(六氰基铁(Ⅲ)酸钾)作为电子受体,一般在终浓度1mM以下使用。但是,由于本发明的辅酶结合型葡萄糖脱氢酶使用高浓度的铁氰化钾2-500mM,优选30-100mM,可以更感度良好地测定D-葡萄糖。本发明的测定方法,测定试剂,测定化合物,生物传感器等的优选方式,其特征在于所述测定反应系统中使用终浓度2-500mM的铁氰化钾。
实施例
以下,通过实施例具体说明本发明,只要不超过本发明的宗旨,不受以下实施例的限定。以下的实施例中辅酶结合型葡萄糖脱氢酶活性的定量按前述方法进行。
实施例1
保藏菌株97508的培养
将葡萄糖(和光纯药工业社制)1%,脱脂大豆(日本食贩社制)2%,玉米浆(corn steep liquor)(共同商事社制)0.5%及硫酸镁(ナカライテスク社制)0.1%制成的100ml培养基(pH6.0)装入500ml培养烧瓶中,121℃灭菌20分钟,冷却后,在培养烧瓶中接种1白金耳保藏菌株97508,30℃震荡培养88小时,作为该菌株的种培养液。与前述相同组成的培养基中加入消泡剂的4L培养基装入5L的发酵罐中,121℃灭菌30分钟,冷却后,前述的种培养液40ml接种至发酵罐中,28℃培养31小时,通气搅拌条件下培养,作为该菌株的前培养液。然后,与前述相同组成的培养基中加入消泡剂的160L培养基装入200L的发酵罐中,121℃灭菌20分钟,冷却后,接种前述的前培养液1.6L,28℃培养41小时,通气搅拌条件下培养该菌株。培养结束后,离心分离培养液,回收培养上清液。
实施例2
培养上清液中分离辅酶结合型葡萄糖脱氢酶
通过以下步骤2.1~2.5分离辅酶结合型葡萄糖脱氢酶。
2.1.浓缩:
实施例1的培养上清液160L用超滤膜“Pellicon 2Module”(Miripoa社制)浓缩,置换到20mM磷酸钾缓冲液(pH7.5)中,得到粗酶液。
2.2.利用Butyl-TOYOPEARL650M(东曹社制)进行精制(第一次):
前述粗酶液,调整成65%饱和硫酸铵(pH7.5)后,离心分离得到上清液。粗酶液通过用含有65%硫酸铵的20mM磷酸钾缓冲液(pH7.5)预先平衡的Butyl-TOYOPEARL650M柱(直径4.7cm×高7.7cm),使酶吸附。用相同的缓冲液冲洗柱子后,用含有30%硫酸铵的20mM磷酸钾缓冲液(pH7.5)使酶溶出,收集活性部分。然后,以从相同的缓冲液到20mM磷酸钾缓冲液(pH7.5)的梯度溶出法使酶溶出,与前述的活性部分合并。
2.3.利用DEAE-cellulofine A-500(生化学工业社制)进行精制:
用超滤膜“Pellicon 2Module”浓缩前述活性部分,脱盐后,用15mM Tris·盐酸缓冲液(pH8.5)进行平衡。该部分通过用相同缓冲液平衡的DEAE-cellulofine A-500柱子(直径4.7cm×高5.2cm),收集溶出液。
2.4.利用Butyl-TOYOPEARL650M(东曹社制)进行精制(第二次):
前述溶出液调整成65%饱和硫酸铵液(pH7.5)后,离心分离得到上清液。该上清液通过用合有65%硫酸铵的20mM磷酸钾缓冲液(pH7.5)预先平衡的Butyl-TOY0PEARL650M柱(直径4.7cm×高3.6cm),使酶吸附。用相同的缓冲液冲洗柱子后,用含有30%硫酸铵的20mM磷酸钾缓冲液(pH7.5)使酶溶出,收集活性部分。
2.5.TSKgel G3000SW(东曹社制)进行精制:
前述活性部分用苄基(バンジル)型膜浓缩モジエ-ル“ACP-0013”(旭化成工业社制)进行浓缩,脱盐后用含有0.2M氯化钠的50mM磷酸钾缓冲液(pH5.5)进行平衡。该部分通过用前述缓冲液平衡的TSKgel G 3000SW柱子(直径2.15cm×高60cm),用相同的缓冲液溶出酶,分取活性部分。活性部分用centriplus 10(アミコン社制)浓缩,脱盐后置换为50mM柠檬酸·磷酸钠缓冲液(pH5.5)。所得的酶比活性为约1100unit/mg,其精制度相对于粗酶液约为170倍。
实施例3
辅酶结合型葡萄糖脱氢酶的性质试验
对于通过上述实施例2分离的辅酶结合型葡萄糖脱氢酶,调查了其作用性,最适pH,稳定pH,最适温度,温度稳定性,基质特异性,分子量,阻滞剂及辅酶。
3.1.作用性
使辅酶结合型葡萄糖脱氢酶在8.66mM的DCIP存在下与500mM的D-葡萄糖反应,反应产物用D-葡糖酸/D-葡糖酸-δ-内酯定量试剂盒进行定量。其结果是确认生成了D-葡糖酸,由此可知本发明的辅酶结合型葡萄糖脱氢酶是催化D-葡萄糖的1位羟基的氧化反应的酶。
3.2.最适pH:
将酶活性测定法-2中的缓冲液分别适当置换为柠檬酸·磷酸钠缓冲液(pH4.0~5.5),磷酸钾缓冲液(pH6.5~7.5),Tris·盐酸缓冲液(pH8.0~9.0)或甘氨酸·氢氧化钠缓冲液(pH9.5~10.0)(各缓冲液终浓度均为17mM),按与酶活性测定法-2相同的方法在各种pH范围内测定该精制酶的酶活性(图1)。其结果是辅酶结合型葡萄糖脱氢酶的最适pH为7.0~9.0。
3.3.稳定pH:
辅酶结合型葡萄糖脱氢酶分别溶解于50mM的缓冲液,也就是柠檬酸·磷酸钠缓冲液(pH3.2~6.4),磷酸钾缓冲液(pH6.3~6.9),Tris·盐酸缓冲液(pH7.3~8.6)及甘氨酸·氢氧化钠缓冲液(pH9.1~11.4),40℃保温60分钟后,按活性测定方法-1测定酶活性,分析酶活性的残存率(图2)。其结果是辅酶结合型葡萄糖脱氢酶的稳定pH为4.5~8.5。
3.4.最适温度:
辅酶结合型葡萄糖脱氢酶溶解于50mM柠檬酸·磷酸钠缓冲液(pH5.5)中,通过上述活性测定方法-1在30℃~62℃范围测定酶活性(图3)。其结果是辅酶结合型葡萄糖脱氢酶的最适温度在55℃左右。
3.5.温度稳定性:
辅酶结合型葡萄糖脱氢酶溶解于50mM柠檬酸·磷酸钠缓冲液(pH5.5)中,0℃到55℃的几个温度下保持15分钟后,按活性测定方法1测定酶的活性,分析酶活性的残存率(图4)。这里,酶活性的残存率以在0℃保持15分钟时的酶活性值为100%算出。其结果是辅酶结合型葡萄糖脱氢酶,即使在50℃也可以保持89%的酶活性,在大约50℃以下稳定。
3.6.基质特异性及Km值:
在活性测定方法-1中用于活性测定的反应液的基质,使用D-葡萄糖及其他各种基质(全部终浓度333mM,D-纤维二糖193mM,D-海藻糖及D-棉子糖121mM),按酶活性测定方法-1测定该酶的酶活性。将该酶对D-葡萄糖的活性值定设为100%,对各基质的活性值相对于其的相对值如表1所示。
另外,同样地分别设定D-葡萄糖及麦芽糖550mM及100mM 2个终浓度,测定相对反应性(酶活性)。结果以D-葡萄糖的值为基准的相对值如表2所示。
通过以上测定可知辅酶结合型葡萄糖脱氢酶对D-葡萄糖具有很强的作用性,对D-甘露糖,1,5-脱水-D-山梨醇,D-纤维二糖,D-海藻糖,麦芽糖,D-半乳糖,D-葡萄糖-6-磷酸,D-果糖作用较弱。另外,该酶对L-阿拉伯糖,乳糖,D-山梨醇,葡糖酸,蔗糖,D-甘露糖醇,L-山梨糖,D-核糖,L-鼠李糖,D-葡萄糖-1-磷酸,D-棉子糖,乙醇,甘油几乎没有作用。该酶的Km值相对于D-葡萄糖为49.7mM。
表1
基质 | 相对活性(%) |
D-葡萄糖 | 100 |
2-去氧-D-葡萄糖 | 48 |
D-木糖 | 9.1 |
D-甘露糖 | 2.8 |
1,5-脱水-D-山梨醇 | 2 |
D-纤维二糖 | 2 |
D-海藻糖 | 1.7 |
麦芽糖 | 1.4 |
D-半乳糖 | 1.2 |
D-葡萄糖-6-磷酸 | 1.1 |
D-果糖 | 0.86 |
L-阿拉伯糖 | 0.1> |
乳糖 | 0.1> |
D-山梨醇 | 0.1> |
葡糖酸 | 0.1> |
蔗糖 | 0.1> |
D-甘露糖醇 | 0.1> |
L-山梨糖 | 0.1> |
D-核糖 | 0.1> |
L-鼠李糖 | 0.1> |
D-葡萄糖-1-磷酸 | 0.1> |
D-棉子糖 | 0.1> |
乙醇 | 0.1> |
甘油 | 0.1> |
表2
基质 | 终浓度(mM) | 相对活性(%) |
D-葡萄糖 | 550 | 100 |
麦芽糖 | 550 | 2.8 |
D-葡萄糖 | 100 | 100 |
麦芽糖 | 100 | 0.5 |
3.7.分子量及亚单位分子量:
将辅酶结合型葡萄糖脱氢酶溶解于含有0.2M NaCl的50mM磷酸钾缓冲液(pH7.5)中,利用TSKgel-3000SW(直径0.75cm×长60cm,东曹社制)进行分析,使用相同的缓冲液作为流动相。以分子量标记物(オリェンタン酵母工业社制)作为指标,辅酶结合型葡萄糖脱氢酶的分子量为约130kDa。使用12.5%聚酰胺凝胶,按照Laemmli等的方法(Nature(1970)227:680-685),对该发明的辅酶结合型葡萄糖脱氢酶进行SDS-PAGE电泳。电泳后,用考马斯亮蓝染色,其移动距离与分子量标记物(アマシヤムフアルマシアバイオテク社制)进行比较,结果显示本发明的辅酶结合型葡萄糖脱氢酶的亚单位分子量为约85kDa。
3.8.阻滞剂
分别添加表3的各种添加物作为阻滞剂,使活性测定方法-1的反应系统终浓度为1mM,按照活性测定方法-1测定辅酶结合型葡萄糖脱氢酶的活性。对照区除不添加表3的添加物以外,按活性测定方法-1操作。该对照区中,该酶活性值作为100%,分别添加各添加物时的相对活性值与对照区的差作为阻滞效果。其结果确认为表3所示的阻滞效果。
另外,前述活性测定方法-1中,分别添加溶解于甲醇中的1,10-菲绕啉使各终浓度为1mM,5mM,10mM,25mM及50mM,按活性测定方法-1测定本发明的辅酶结合型葡萄糖脱氢酶的活性。另外,对于反应系统的甲醇的终浓度全部为10%(V/V)。对照区按活性测定方法-1添加甲醇使终浓度为10%(V/V)进行测定。结果如表-4所示。结果1,10-菲绕啉的阻滞结果是,1,10-菲绕啉最终浓度1mM时62.0%,5mM时76%,10mM时85%,25mM时91%,50mM时95%(表3)。
对本发明的辅酶结合型葡萄糖脱氢酶的阻滞效果按各添加物的不同而不同,重金属离子(Ag+,Cu2+及Hg2+)效果最强,1,10-菲绕啉,原黄素及Mn2+的阻滞效果在60%以上。
表3
添加物 | 阻滞效果(%) | 添加物 | 阻滞效果(%) |
无 | 0 | 米帕林 | 5.0 |
NaN3 | 0 | 粹通(トリトン)X-100 | 6.2 |
ZnCl2 | 0 | CoCl2 | 7.0 |
AlCl3 | 0 | 苹果酸 | 8.1 |
苯甲酸 | 0 | D-酒石酸 | 8.5 |
EDTA | 0.4 | 碘乙酸 | 9.5 |
CdCl2 | 0.8 | 半胱胺 | 9.8 |
LiCl | 0.9 | 2,2’-联二吡啶 | 10.8 |
氨基胍硫酸盐 | 1.1 | 8-喹啉酚 | 13.9 |
H2O2 | 1.7 | KCN | 14.5 |
N-乙基马来酰亚胺 | 1.8 | NiCl2 | 16.5 |
尿素 | 1.9 | FeCl3 | 25.0 |
NaCl | 2.5 | 马来酸 | 26.2 |
钛试剂 | 2.5 | 利凡诺(アクリノ-ル) | 29.0 |
BaCl2 | 2.6 | 2-硝基苯甲酸 | 44.3 |
PbCl2 | 2.7 | SnCl2 | 45.5 |
MgCl2 | 2.8 | 吖啶黄 | 49.0 |
富马酸 | 3.4 | 1,10-菲绕啉 | 62.0 |
环丝氨酸(シクロセリン) | 3.6 | 原黄素 | 62.0 |
DL-青霉胺 | 4.3 | MnCl2 | 75.5 |
meso-酒石酸 | 5.6 | AgNO3 | 99.4 |
柠檬酸 | 5.6 | CuCl2 | 100 |
CaCl2 | 5.7 | HgCl2 | 100 |
表4
1,10-菲绕啉终浓度(mM) | 阻滞效果(%) |
0 | 0 |
50 | 95 |
25 | 91 |
10 | 85 |
5 | 76 |
3.9.辅酶:
在本发明的辅酶结合型葡萄糖脱氢酶溶液中添加D-葡萄糖,进行吸光分析,结果在385nM及465nM处的最大吸收由于加入该添加物而消失,因此确定辅酶为FAD。另外,这些最大吸收是FAD特有的,在建立仅没有加入FAD的对照反应系统中没有观察到该吸收。
实施例4
葡萄糖的定量
使用前述实施例2精制的保藏菌株97508来源的辅酶结合型葡萄糖脱氢酶,对活性测定方法1中用0.333至33mM范围的D-葡萄糖替换333mM的D-葡萄糖时的吸光度的减少速度进行测定。也就是,测定D-葡萄糖的浓度为0.333mM,0.5mM,1mM,2mM,5mM,6.67mM,10mM,20mM,33mM各浓度时的吸光度变化。测定结果如图5所示。
其结果,做成标准曲线(相关系数r=0.997)。利用这一标准曲线可以定量使用辅酶结合型葡萄糖脱氢酶的D-葡萄糖。
实施例5
用酶固定化电极测定葡萄糖
使用前述实施例2精制的保藏菌株97508来源的辅酶结合型葡萄糖脱氢酶,用酶固定化电极测定D-葡萄糖。使用固定化3.4U该酶的玻璃碳电极(GC),测定相对葡萄糖浓度的应答电流值。电解池中,添加100mM磷酸钠缓冲液(pH7.0)2.7ml及1M六氰基铁(III)酸钾(铁氰化钾)水溶液0.3ml。GC电极接到稳压器BAS100B/W(BAS制)上,在40℃,氩饱和,氧饱和,空气饱和的各种条件下搅拌溶液,相对于银氯化银参比电极施加+500mV。向这些系统中添加1M的D-葡萄糖溶液30μl,测定常规状态的电流值。进而添加同量的1MR D-葡萄糖溶液,进行测定电流值的操作,分别重复3次。该电流值相对于已知的葡萄糖浓度(约10,20,30,40mM)作图,作成标准曲线(图6)。利用这一标准曲线可以用使用本发明的辅酶结合型葡萄糖脱氢酶的酶固定化电极定量葡萄糖。而且,由于不论气体的饱和条件如何,标准曲线均一致,由此表明该辅酶结合型葡萄糖脱氢酶对氧非常惰性,且可以在不受氧的影响下通过使用了该酶的酶固定化电极定量D-葡萄糖。
实施例6
用酶固定化电极测定标准血清中的葡萄糖
基于实施例5的方法,以酶固定化电极,使用对照血清I和光-B(和光纯药制)对血清中的葡萄糖浓度进行测定。电解池中,添加100mM磷酸钠缓冲液(pH7.0)2.4ml及1M六氰基铁(III)酸钾(铁氰化钾)水溶液0.3ml,使电流值达到正常状态,添加血清,测定电流值。同样用已知浓度的D-葡萄糖溶液进行测定,作成标准曲线。该血清中的葡萄糖浓度,用标准曲线法推定为4.5mM,这与用己糖激酶·葡萄糖-6-磷酸脱氢酶法测定的该血清中的葡萄糖浓度一致。由此说明,通过使用了本发明中使用的辅酶结合型葡萄糖脱氢酶的酶固定化电极,可以定量血清中的D-葡萄糖。
通过本发明,可以提供对麦芽糖的作用性在5%以下,受1,10-菲绕啉阻滞的可溶性辅酶结合型葡萄糖脱氢酶。而且提供适于工业生产的该辅酶结合型葡萄糖脱氢酶的制造方法,制造该酶用的生产微生物。因此,辅酶结合型葡萄糖脱氢酶可以应用于产业的用途,详细说,就是可以正确测定将麦芽糖作为成分的输液给与糖尿病患者的血糖值。另外,由于使用本发明的辅酶结合型葡萄糖脱氢酶可以测定葡萄糖传感器中的微量的葡萄糖,该酶有可能得到应用。另外,该辅酶结合型葡萄糖脱氢酶可以用于包括使用该辅酶结合型葡萄糖脱氢酶的试样中的葡萄糖的测定方法,消除方法,有机化合物的制造方法等的物质生产及分析的用途,可以用于医药,临床领域及食品领域的素材改质加工,有可能提供利用价值高的酶。
Claims (21)
1.一种可溶性辅酶结合型葡萄糖脱氢酶,其特征是在电子受体存在下,对氧化葡萄糖的反应进行催化,对麦芽糖的作用性低。
2.权利要求1记载的辅酶结合型葡萄糖脱氢酶,其特征是在电子受体存在下,对氧化葡萄糖的反应进行催化,对麦芽糖的作用性在5%以下,被1,10-菲绕啉阻滞。
3.权利要求1或2记载的辅酶结合型葡萄糖脱氢酶,其特征是被终浓度1mM的1,10-菲绕啉阻滞50%以上。
4.权利要求1至3中的任何一项记载的辅酶结合型葡萄糖脱氢酶,其特征是辅酶为黄素化合物。
5.权利要求1至4中的任何一项记载的辅酶结合型葡萄糖脱氢酶,其特征是氧化葡萄糖的1位的羟基。
6.权利要求1至5中的任何一项记载的辅酶结合型葡萄糖脱氢酶,是来源于微生物。
7.权利要求6中记载的辅酶结合型葡萄糖脱氢酶,是来源于真核微生物。
8.权利要求7中记载的辅酶结合型葡萄糖脱氢酶,是来源于局限曲霉(Aspergillus terreus)。
9.权利要求8中记载的辅酶结合型葡萄糖脱氢酶,是来源于保藏编号FERM BP-08578的局限曲霉。
10.一种辅酶结合型葡萄糖脱氢酶,是具有与权利要求1至5中的任何一项记载的辅酶结合型葡萄糖脱氢酶的性质或实质上相同性质的蛋白质,其特征是具有编码该蛋白质的氨基酸序列或含由该序列中缺失,置换或附加1个以上氨基酸残基而引起的变异的氨基酸序列,而且生物学活性稳定的蛋白质。
11.一种微生物,其特征是具有生产权利要求1至5中的任何一项记载的辅酶结合型葡萄糖脱氢酶的能力。
12.权利要求11中记载的微生物,其是真核微生物。
13.权利要求12中记载的微生物,其是局限曲霉。
14.权利要求13中记载的微生物,其是保藏编号为FERMBP-08578的局限曲霉。
15.一种辅酶结合型葡萄糖脱氢酶的制造方法,是权利要求1至5中的任何一项记载的辅酶结合型葡萄糖脱氢酶的制造方法,其特征是培养权利要求11至14中的任何一项中记载的微生物,通过培养使辅酶结合型葡萄糖脱氢酶生成,进行收集。
16.一种葡萄糖的测定方法,其特征是使用权利要求1至10中的任何一项中记载的辅酶结合型葡萄糖脱氢酶。
17.权利要求16中记载的测定方法,其特征是使用时,使用终浓度为2mM-500mM的铁氰化物。
18.一种葡萄糖测定试剂组合物,其特征是含有权利要求1至10中的任何一项中记载的辅酶结合型葡萄糖脱氢酶。
19.权利要求18中记载的测定试剂组合物,其特征是使用时,使用终浓度为2mM-500mM的铁氰化物。
20.一种测定葡萄糖用的生物传感器,其特征是使用权力要求1至10中的任何一项中记载的辅酶结合型葡萄糖脱氢酶。
21.权利要求20中记载的生物传感器,其特征是使用时,使用终浓度为2mM-500mM的铁氰化物。
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2003
- 2003-12-24 CN CNB2003801056894A patent/CN100415877C/zh not_active Expired - Lifetime
- 2003-12-24 CA CA002511656A patent/CA2511656A1/en not_active Abandoned
- 2003-12-24 WO PCT/JP2003/016603 patent/WO2004058958A1/ja active Application Filing
- 2003-12-24 AU AU2003296186A patent/AU2003296186A1/en not_active Abandoned
- 2003-12-24 US US10/540,025 patent/US7514250B2/en active Active
- 2003-12-24 DE DE60331477T patent/DE60331477D1/de not_active Expired - Lifetime
- 2003-12-24 EP EP03789625A patent/EP1584675B1/en not_active Expired - Lifetime
- 2003-12-24 JP JP2004562920A patent/JP4494978B2/ja not_active Expired - Lifetime
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2009
- 2009-03-03 US US12/396,724 patent/US20090176262A1/en not_active Abandoned
-
2010
- 2010-08-06 US US12/851,668 patent/US20100297743A1/en not_active Abandoned
-
2019
- 2019-02-15 US US16/278,019 patent/US11225645B2/en not_active Expired - Lifetime
- 2019-02-15 US US16/278,008 patent/US10988738B2/en not_active Expired - Lifetime
- 2019-02-15 US US16/278,025 patent/US11345897B2/en not_active Expired - Lifetime
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CN102559846A (zh) * | 2009-04-30 | 2012-07-11 | 池田食研株式会社 | 蛋白质性电子介质 |
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US8716442B2 (en) | 2009-04-30 | 2014-05-06 | Ikeda Food Research Co., Ltd. | Protein electron mediator |
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CN110607248A (zh) * | 2018-06-16 | 2019-12-24 | 华中农业大学 | 修复土壤镉污染的福格斯氏菌菌株a6及用途 |
CN110607248B (zh) * | 2018-06-16 | 2021-01-01 | 华中农业大学 | 修复土壤镉污染的福格斯氏菌菌株a6及用途 |
CN109596750A (zh) * | 2019-01-09 | 2019-04-09 | 新疆医科大学 | 一种基于sec-hplc测定蒜酶的方法 |
WO2020228086A1 (zh) * | 2019-05-10 | 2020-11-19 | 中国农业科学院饲料研究所 | HIF1α蛋白作为生物标志物在筛选安全降肝脂饲用添加物中的应用 |
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US20190177700A1 (en) | 2019-06-13 |
US20060063217A1 (en) | 2006-03-23 |
US20100297743A1 (en) | 2010-11-25 |
EP1584675A4 (en) | 2006-05-24 |
CA2511656A1 (en) | 2004-07-15 |
EP1584675A1 (en) | 2005-10-12 |
JP4494978B2 (ja) | 2010-06-30 |
JPWO2004058958A1 (ja) | 2006-04-27 |
WO2004058958A1 (ja) | 2004-07-15 |
US11155789B2 (en) | 2021-10-26 |
US11345897B2 (en) | 2022-05-31 |
AU2003296186A1 (en) | 2004-07-22 |
US7514250B2 (en) | 2009-04-07 |
US11225645B2 (en) | 2022-01-18 |
US10988738B2 (en) | 2021-04-27 |
CN100415877C (zh) | 2008-09-03 |
US20190177699A1 (en) | 2019-06-13 |
DE60331477D1 (de) | 2010-04-08 |
EP1584675B1 (en) | 2010-02-24 |
US20190177701A1 (en) | 2019-06-13 |
US20190177698A1 (en) | 2019-06-13 |
US20090176262A1 (en) | 2009-07-09 |
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