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Timestamp: 2019-07-18 20:30:42
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CN105026558A - Novel glucose oxidases derived from aspergillus niger - Google Patents
Novel glucose oxidases derived from aspergillus niger Download PDF
CN105026558A
CN105026558A CN201480005873.XA CN201480005873A CN105026558A CN 105026558 A CN105026558 A CN 105026558A CN 201480005873 A CN201480005873 A CN 201480005873A CN 105026558 A CN105026558 A CN 105026558A
CN201480005873.XA
M.博科拉
H.迪费尔
E.U.古蒂雷茨
D.海因德尔
T.迈尔
H.芒德哈达
U.施瓦内贝格
M.塔克
2013-01-28 Priority to EP13152935 priority Critical
2014-01-28 Application filed by 霍夫曼-拉罗奇有限公司 filed Critical 霍夫曼-拉罗奇有限公司
2014-01-28 Priority to PCT/EP2014/051602 priority patent/WO2014114810A1/en
2015-11-04 Publication of CN105026558A publication Critical patent/CN105026558A/en
Disclosed herein are novel glucose oxidase variants having besides the two substitutions T30V and I94V according to SEQ ID NO: 1, additionally at least one further amino acid substitution in the polypeptide sequence in any of the positions S53; A137; A173; A332; F414; and V560. The thus optimized glucose oxidase variants exhibit specificity for glucose and significantly reduced oxygen-consumption rates. In another aspect, the present invention provides for glucose oxidase variants having significantly reduced oxygen-consumption rates and/or increased enzyme activity for electron mediators other than oxygen. The invention also provides for an assay device bearing at least one of the herein disclosed GOx variants for improved blood glucose measurements.
衍生自黑曲霉的新型葡萄糖氧化酶发明领域 Derived from Aspergillus niger glucose oxidase novel Field of the invention
[0001] 本发明设及新型葡萄糖氧化酶变体。 [0001] The present invention is provided and new variant glucose oxidase. 本文提供的葡萄糖氧化酶对于底物葡萄糖特异性，并且从而显示出显著降低的耗氧率。 Provided herein glucose oxidase to the substrate specificity of glucose, and thus show a significant decrease in oxygen consumption rate. 在另一个方面，本发明提供了葡萄糖氧化酶，其对于底物葡萄糖特异性，并且从而显示出显著降低的耗氧率和/或对于除氧外的电子介质增加的酶促活性。 In another aspect, the present invention provides a glucose oxidase, to which glucose substrate specificity, and thus show a significant decrease in oxygen consumption rate and / or enzymatic activity of the electron mediator other than oxygen for the increased. 进一步地，本发明设及用于测量葡萄糖的试剂盒和传感器中的所述葡萄糖氧化酶。 Further, the present invention is provided, and a kit and a glucose sensor for measuring the glucose oxidase.
[0002] 特别地，本发明设及衍生自黑曲霉（心公6巧77扣5 如r)的葡萄糖氧化酶，除根据沈QIDNO: 1的两个置换T30V和I94V之外，另外具有在选自S53 ;A137 ;A173 ;A332 ;F414 ; 和V560的六个位置中的至少一个氨基酸置换。 [0002] In particular, the present invention is provided and derived from Aspergillus niger (well 6 clever heart 77 as fastener 5 r), glucose oxidase, except under heavy QIDNO: two outside and T30V substitution of I94V 1, further having a selected since S53; A137; A173; A332; at least one amino acid substitution six locations in and V560; F414.
[0003] 科学背景糖尿病反映可W在全世界广泛发现的代谢疾病。 [0003] reflects the scientific background of diabetes and metabolic diseases can be found in a wide range of W in the world. 糖尿病患者具有受损或缺失的激素膜岛素产生，所述膜岛素控制血糖水平，并且因此在不足够的膜岛素应用的情况下，运些患者具有高血糖症W及低血糖症的危险你e打心。 Diabetic patients with damaged or missing hormone film islands prime generation, the membrane Insulin control blood sugar levels, and thus without sufficient film Insulin applications, some patients having a transport hyperglycemia and hypoglycemia W your e dangerous heart beat. 〇/7ant/化3抑asis〇/化sAe沁S曲加？ Square / 7ant / asis〇 inhibition of 3 / S-curve of sAe Qin plus? and intermediate hyperglycaemia. WHO,and IDF (2006);肥0 Document Production service ISBN 9241594934]。 . And intermediate hyperglycaemia WHO, and IDF (2006); fat 0 Document Production service ISBN 9241594934].
[0004] 为了确保膜岛素的正确应用，高度特异性、准确和容易处理的葡萄糖测量系统是自测量系统和临床规模上的高流通量测量系统两者所需的。 [0004] In order to ensure the correct application of membrane Insulin, highly specific, precise and easy to handle glucose measurement system is self-measurement system on both high-throughput measurement systems and clinical scale required.
[0005] 为了允许血液中的葡萄糖浓度的准确测定并且使得测量高度特异性，设及酶促反应。 [0005] In order to allow the blood glucose concentration measurement and that the measurement accuracy is highly specific enzymatic reaction and provided. 目前，在糖尿病分析中使用的两类酶由葡萄糖脱氨酶(下文GDH)和葡萄糖氧化酶(下义G&k)反巧k [册nes, J.，伽ller,和Surridge, N. (2008).The T'echnology Behind Glucose Meters: Test Strips. DIABETES TECHNOLOGY & T皿RAPEUTICS 10,10-26]。 Currently, two types of diabetes analysis of glucose by the enzyme deaminase (hereinafter GDH) and glucose oxidase (hyponymy G & k) anti-coincidence K [volumes nes, J., gamma Ller, and Surridge, N. (2008) .The T'echnology Behind Glucose Meters:. Test Strips DIABETES TECHNOLOGY & T boat RAPEUTICS 10,10-26].
[0006] GDH的主要优点在于它的氧不依赖性葡萄糖氧化，但该酶显示对某些其他临床相关糖的轻微副活性，并且因此GDH是非特异性的仍_7別如0177,乂乂游化i打6,乂乂做;. On the mechanism and specificity of soluble,quinoprotein glucose dehydrogenase in the oxidation of aldose sugars. Biochemistry 37，13854-13861]。 [0006] The main advantage of GDH is that it is not oxygen-dependent oxidation of glucose, the enzyme showed a slight sub-clinically relevant activity against certain other sugars, and thus non-specific GDH _7 still not such as 0177, qe qe tour i hit of 6, qe qe do;. On the mechanism and specificity of soluble, quinoprotein glucose dehydrogenase in the oxidation of aldose sugars Biochemistry 37,13854-13861].. 相之下，GOx 对于葡萄糖高度特异性，但它的氧化是强氧依赖性的/化M左a。 In contrast, the GOx highly specific for glucose, but it is the oxidation / M of the strong oxygen-dependent left a. 5：么，化/知，瓜K，57端'AgA RS和Ananthanarayan，L. (2009). Glucose oxidase-an overview. Biotechnology Advances 27,489-501; Bentley, R.和化uberger, A. (1949).The mechanism of the action of notation. Biochem J 45，584-590]。 5: Why, of / known, melon K, 57 end 'AgA RS and Ananthanarayan, L (2009) Glucose oxidase-an overview Biotechnology Advances 27,489-501; Bentley, R., and of uberger, A. (1949)... .The mechanism of the action of notation. Biochem J 45,584-590].
[0007] 更详细地，G化作为黄素蛋白属于氧化还原酶（即PD-葡萄糖：氧1-氧化还原酶）家族。 [0007] In more detail, G belonging of a flavoprotein oxidoreductase (i.e. PD- glucose: oxygen oxidoreductase 1-) family. 通过采用分子氧作为电子受体，天然或野生型（下文WT)G化催化PD-葡萄糖氧化为D-葡糖酸-5 -内醋和&〇2 /参屈嫁妨，化乂度游必邸公6,足."化斯.巧e Oxidation of Glucose and Related Compounds by Glucose Oxidase from Aspergillus 化〇〇/?6化/5的^义旅$*-5'况?7。所述反应由下式描述： D-葡萄糖+ 〇2 -葡糖酸内醋+ &〇2 G化的底物可W分成两组：i)氧化半反应的电子受体，和ii)还原半反应的电子供体， 参嘴獻舰[Leskovac，V.，Trivic，S.，Wohlfalrrt，G.，Kandrac，J•和Pericin，D. (2005) •GlucoseoxidasefromAspergillusniger:themechanismofactionwithmolecular oxygen，quinones，andone-electronacceptors,IntJBiochemCellBiol37， 7J7-7沉?7。本领域技术人员应认识到，除了D-葡萄糖之外，多种D-葡萄糖衍生物是GOx的还原半反应的潜在底物。 By using molecular oxygen as an electron acceptor, native or wild type (hereafter WT) G PD- catalytic oxidation of glucose to gluconic acid, D- -5-- & 〇2 vinegar and / flexor marry hinder reference, the degree of time must qe Di public 6, foot "of Si Qiao e Oxidation of Glucose and Related Compounds by Glucose Oxidase from Aspergillus of thousand and / of 6/5-defined trip $ ^ * -.?? 5 'the reaction is represented by the following condition 7 description of formula: D- glucose + 〇2 - the vinegar gluconic acid + G & 〇2 the substrates W can be divided into two groups: i) oxidation half reaction of the electron acceptor, and ii) a reduction half reaction of the electron donor , the Senate offered mouth-ship [Leskovac, V., Trivic, S., Wohlfalrrt, G., Kandrac, J • and Pericin, D (2005) • GlucoseoxidasefromAspergillusniger:. themechanismofactionwithmolecular oxygen, quinones, andone-electronacceptors, IntJBiochemCellBiol37, 7J7-7 Shen? 7. skilled in the art will recognize that, in addition to the D- glucose, D- glucose derivative is more reduction half reaction of GOx potential substrates.
[000引迄今为止已描述了来自不同起源的GOx。 [000 cited so far have been described GOx from different origins. 例如，来自海藻皱波角叉莱瓜W化AS。 For example, from alginic wrinkled wave of carrageenan, guar W AS. 打5'所/5)的6(^在服7,544,795和服6,924,366中得到描述；来自丝状真菌枝抱属物种(C知施邱ori拙7邱ec.)的GOx在W0 95/29996 ;W0 1998/020136中得到描述；并且来自黄蓝状菌（沾知巧TC6W 的GOx在US6, 054, 318中得到描述D 5 hit 'by / 5) 6 (^ obtained clothing kimono 6,924,366 7,544,795 described;. From a filamentous fungus species hold branches (C-known application Zhuo Herman ori 7 Qiu EC) of GOx in W0 95/29996; W0 1998 / 020136 describe obtained; from the yellow-blue and GOx-like bacteria (Qiao TC6W dip known in US6, the 054, 318 are described D
[0009] 在文献中最佳描述的GOx来自黑曲霉/觀cAf，原乂，况命孤Ayr梦，公.，侣，原和Schmid，民.D. (1993) •The3DstructureofglucoseoxidasefromAspergillus niger.ImplicationsfortheuseofGODasabiosensorenzyme.Biosensors &Bioelectronics8，197-203;Wohlfahrt，G.，Witt，S.，Henclle，J.，Schomburg，D.， Kalisz，HM•和Hecht，HJ (1999) • 1. 8and1. 9Aresolutionstructuresofthe Penici11iumamagasakienseandAspergillusnigerglucoseoxidasesasabasis formodellingsubstratecomplexes.ActaCrystallographicaSectionDBiological Ciystallography55,969-977]。 [0009] GOx best described in the literature from Aspergillus niger / CaF concept, the original qe, Ayr dream solitary life conditions, public., Companion, primary and Schmid, China .D. (1993) • The3DstructureofglucoseoxidasefromAspergillus niger.ImplicationsfortheuseofGODasabiosensorenzyme.Biosensors & Bioelectronics8,197-203;. Wohlfahrt, G., Witt, S., Henclle, J., Schomburg, D, Kalisz, HM • and Hecht, HJ (1999) • 1. 8and1 9Aresolutionstructuresofthe Penici11iumamagasakienseandAspergillusnigerglucoseoxidasesasabasis formodellingsubstratecomplexes.ActaCrystallographicaSectionDBiological Ciystallography55,969. -977].
[0010] W089/126675描述了在重组系统中来自黑曲霉的GOx的生产，并且wo 2008/079227A1涉及在赋予改善的胆存稳定性的组合物中配制的得自黑曲霉的GOx。 [0010] W089 / 126675 describes the production of GOx from Aspergillus niger in a recombinant system, and wo 2008 / 079227A1 relates GOx from Aspergillus niger formulated in the composition of bile impart improved storage stability in.
[0011] 它是充分表征的蛋白质，形成大小160kDa的二聚体，并且其晶体结构已得到解决[Hecht，比J.等人，Crystal-StructureOfGlucose-OxidaseFromAspergillus-Niger RefinedAt2. 3AngstromResolution.JournalOfMolecularBiology，1993. 229(1): 第153-172页]。 [0011] It is a well characterized protein, the size of 160kDa dimer formation, and its crystal structure has been solved [Hecht, J. et al. Ratio, Crystal-StructureOfGlucose-OxidaseFromAspergillus-Niger RefinedAt2. 3AngstromResolution.JournalOfMolecularBiology, 1993. 229 (1): pp. 153-172].
[001引进一步已知，特别地，黑曲霉的野生型GOx(下文GOx-WT)显示出显著的温度稳定性和对于底物葡萄糖的特异性。 [001 cited is further known, in particular, the GOx wild type Aspergillus niger (hereinafter GOx-WT) showed significant temperature stability and substrate specificity for glucose. GOx是具有10-16%的高甘露糖型碳水化合物含量的糖蛋皆化ayashi，S.和Nakamura，S. (1981) •Multipleformsofglucoseoxidasewith differentcarbohydratecompositions.BiochimBiophysActa657,40-51;Pazur，J, H.，Kleppe，K.和Cepure，A. (1965).Aglycoproteinstructureforglucoseoxidase fromAspergillusniger.ArchBiochemBiophys111，351-357]。 GOx is a high mannose type glycoprotein having a carbohydrate content of 10-16% of all ayashi, S and Nakamura, S (1981) • Multipleformsofglucoseoxidasewith differentcarbohydratecompositions.BiochimBiophysActa657,40-51;.. Pazur, J, H., Kleppe , K. and Cepure, A. (1965) .Aglycoproteinstructureforglucoseoxidase fromAspergillusniger.ArchBiochemBiophys111,351-357].
[0013] 目前，GOx通常用于检测工业溶液或受试者体液(例如血液和尿）中的葡萄糖的生物传感器中。 [0013] Currently, GOx solution commonly used to detect or industrial fluid of the subject (e.g. blood and urine) glucose biosensor.
[0014] 目前可用的自测量装置中的大多数是原则上由下述组成的电化学传感器： a) 生物组分，即具有葡萄糖作为底物的各种酶， b) 指示器（电子元件)，和C)信号转换器。 [0014] Most currently available from the measuring device is an electrochemical sensor principle consisting of: a) a biological component, i.e. enzymes having glucose as a substrate, b) an indicator (electronic components) and C) the signal converter.
[0015] 在测量装置中，来自葡萄糖的电子经由介质通过生物组分（a)转移到电极（b)。 [0015] In the measuring device, the transfer of electrons from glucose via the medium through biological components (a) to an electrode (b). 信号转换器（C)随后将电子信号转换成实时葡萄糖浓度，其与转移的电子量成比例。 Signal converter (C) and then convert the electrical signal into glucose concentration in real time, an amount of electron transfer which is proportional to
[0016] 除了上述电化学传感器之外，还存在可用的光测传感器。 [0016] In addition to the electrochemical sensor, there is also available a light detecting sensor. 此处的差异是来自葡萄糖的电子转移到氧化还原指示剂染料(充当指示剂)。 The difference here is of electron transfer from glucose to the oxidation reduction indicator dye (serving as an indicator). 光度测量所得到的还原染料的变色。 Photometric measurements of the resulting dye color reduction.
[0017] 另一种主要应用可能变成(X)x在植入和小型化的生物燃料电池的阳极区室中的用途，所述生物燃料电池燃烧来自血流的葡萄糖，并且从而给微型诊断装置或累提供动力。 [0017] Another major application may become (X) x implant use in the anode compartment and miniaturization of the bio-fuel cell, the combustion of the biofuel cell glucose from the bloodstream, and thereby to the diagnosis of miniature powered means or tired.
[0018] 此外，G化在食品工业中的应用是众多的，因为它生成过氧化氨的能力，所述过氧化氨具有抗微生物效应，可W用于改善某些食物产品的胆存稳定性，所述食物产品包括例如奶酪、黄油和果汁。 [0018] Further, G of the application in the food industry are numerous, as it had the ability to generate oxidation of ammonia, the ammonia peroxide having an antimicrobial effect, W can be used to improve the storage stability of certain bile food product the products include food such as cheese, butter and juice.
[0019] G化在化妆品组合物中的应用同样可W利用抗微生物特性。 [0019] Application of G in cosmetic compositions may likewise W using antimicrobial properties. 己糖氧化酶在药物和化妆品组合物中的潜在用途例如在S6, 924, 366;US6, 251，626和W0 2007/045251A2中提出。 Hexose oxidase potential use in pharmaceutical and cosmetic compositions, for example, in S6, 924, 366; US6, 251,626 and in W0 2007 / 045251A2 proposes.
[0020] 此外，G化的用途在转基因植物及其他生物的生产中提出，所述转基因植物及其他生物对于害虫或疾病具有降低的敏感性或增加的抗性(参见例如W0 1995/021924)。 [0020] Further, G made use of in the production of transgenic plants and other organisms, and the transgenic plant pest or disease to other organisms having an increased sensitivity or resistance to reduction (see, e.g., W0 1995/021924).
[0021] 然而，依照本发明，G化在葡萄糖生物传感器中的用途具有显著兴趣。 [0021] However, according to the present invention, G in the use of a glucose biosensor has a significant interest. 在运点上， W0 2009/104836A1描述了葡萄糖传感器，其包含就其附着至金属表面而改善的遗传改造的(X)x变体。 At the point of operation, W0 2009 / 104836A1 describes a glucose sensor, comprising (X) x engineered variants thereof attached to the metal surface to improve genetic.
[0022] Zhu等人在2006和2007年描述了在T30V和/或I94V中突变的衍生自黑曲霉的(X)x突变体。 [0022] Zhu et al., 2006 and 2007 are described in the T30V mutation and / or I94V derived from Aspergillus niger (X) x mutants. 因此，相应的双重突变体T30V;I94V也已得到描述如/，么，必(畑細，C， Zakhartsev，M.和Schwaneberg，U. (2006).Makingglucoseoxidasefitforbiofuel cellapplicationsbydirectedproteinevolution.BiosensorsandBioelectronics 21,2046-2051;化u,Z. ,Wang,M. ,Gau化m,A.,化zor,J. ,Momeu,C. ,R,P. ,andU,S. (2007).DirectedevolutionofglucoseoxidasefromAspergillusnigerfor ferrocenemethanol-mediatedelectrontransfer.BiotechnologyJournal2,241-248] (下文Zhu等人（2006/2007)。 Thus, the corresponding double mutant T30V; I94V has also been described as /, it shall (Hata thin, C, Zakhartsev, M and Schwaneberg, U (2006) .Makingglucoseoxidasefitforbiofuel cellapplicationsbydirectedproteinevolution.BiosensorsandBioelectronics 21,2046-2051;.. Of u, Z., Wang, m., Gau of m, A., of zor, J., Momeu, C., R, P., andU, S. (2007) .DirectedevolutionofglucoseoxidasefromAspergillusnigerfor ferrocenemethanol-mediatedelectrontransfer.BiotechnologyJournal2,241- 248] (hereinafter, Zhu et al (2006/2007).
[002引具体地，具有置换T30V和I94V的上述双重突变体在酶活性中轻微增加（keat从69. 5/s到137. 7/s)，与(X)x-WT相比较，显示出在58°C至62°C范围内增加的热稳定性，化及在8至11范围内改善的抑稳定性。 [002 cited Specifically, this double mutant having substitution of I94V T30V and a slight increase in the enzyme activity (Keat from 69. 5 / s to 137. 7 / s), compared with (X) x-WT, shows the increase in the 58 ° C to 62 ° C range of thermal stability, and improvement of in the range of 8-11 suppression stability. 然而，与GOx-WT相比较，所述衍生自黑曲霉的双重突变体显不出相等的耗氧率。 However, as compared with GOx-WT, the double mutants derived from Aspergillus niger not show the oxygen consumption rate is equal.
[0024] EP2 415 863A1描述了核酸分子及其多肤，其具有(X)x活性，但在下述氨基酸位置2、13、30、94和152中的至少S个中突变。 [0024] EP2 415 863A1 describes nucleic acid molecules and multiple skin, having (X) x activity, but at least the S mutation in the following amino acid positions 2,13,30,94 and 152. 特别地，除在酿酒酵母巧rces cereWsiae)中增加的表达水平之外，还具有置换N2Y、K13E、T30V、I94V和K152R的EP2 415 863A1的M12变体显示出对于作为电子受体的氧增加两倍的活性。 In particular, in addition to the increase in S. cerevisiae clever rces cereWsiae) the expression level further transposition N2Y, M12 variant K13E, T30V, I94V and K152R of EP2 415 863A1 it shows two for increasing oxygen as an electron acceptor times of activity.
[0025]Hora邑uchi等K阳oraguchi,Y.，Saito,S.，Ferri,S.，Mori,K.，Kojima, K. ,Tsugawa,W.和Sode,K. (2012).TurningGlucoseOxidaseintoEssentially Dehydrogenase.Meet.Abstr. ,]hhmeMA2012-02,Is洲e18,Pages2057]化年鉴定了设及本文描述的(X)x变体的氧化半反应的一个氨基酸残基位置，它是尼崎青霉(化曲洗S3心ease)的GOx和黑曲霉的GOx变体。 [0025] Hora et Yap uchi K male oraguchi, Y., Saito, S., Ferri, S., Mori, K., Kojima, K., Tsugawa, W. And Sode, K. (2012) .TurningGlucoseOxidaseintoEssentially Dehydrogenase. amino acid residue positions a half-reaction oxidation Meet.Abstr.,] hhmeMA2012-02, is Island e18, Pages2057] of the set of identified and described herein (X) x variant, which is Penicillium Kawasaki Nigeria (curl wash S3 heart EASE) of Aspergillus niger and GOx GOx variants. 所述一个位置是尼崎青霉GOx 变体的S114和黑曲霉变体的相应T110。 The position of a corresponding T110 S114 and mildew Aspergillus Penicillium GOx Amagasaki body variant. 两个位置均替换为氨基酸丙氨酸，导致对于作为电子受体的氧活性中的减少。 Two positions were replaced with the amino acid alanine, resulting in reduced activity of the oxygen as an electron acceptor in respect. 例如，黑曲霉的(X)x变体显示出降低6. 6倍的耗氧量，并且因此除363%的介质活性之外，还具有30. 4%的残留氧活性。 For example, Aspergillus niger (X) x variants showed 6.6 fold decrease oxygen consumption, and thus in addition to 363% of the active medium, further comprising 30.4% residual oxygen activity.
[002引本发明的根本问题对于葡萄糖非特异性的GM1和氧依赖性的(X)x代表目前葡萄糖测量系统的关键酶。 [002 underlying problem of the present invention for the primer (X) x GM1 representatives and non-specific glucose-dependent oxygen current key enzyme glucose measurement system.
[0027] 在本公开内容的上下文中，表达"对于葡萄糖非特异性"、"氧依赖性的"和"氧依赖性"具有如定义部分中所述的含义。 [0027] In the context of the present disclosure, the expression "non-specific for glucose", "oxygen-dependent" and "oxygen-dependent" have the meanings as described in the definitions section.
[0028] 本发明的根本问题的解决方案本发明的根本问题的解决方案是提供衍生自真菌黑曲霉的特别修饰且因此优化的GOx变体。 [0028] The solution to the fundamental problem solution to the fundamental problem of the present invention, the present invention is derived from the fungus Aspergillus niger and therefore particularly optimized GOx modified variants.
[0029] 令人惊讶和出乎意料地，本发明人已发现衍生自黑曲霉的新型(X)x变体对于葡萄糖特异性，但不依赖于氧用于葡萄糖氧化且因此对于葡萄糖测量更准确。 [0029] Surprisingly and unexpectedly, the present inventors have found that novel derived from Aspergillus niger (X) x variant specificity for glucose, but not for the oxygen-dependent oxidation of glucose and thus for a more accurate glucose measurement .
[0030] 此外，令人惊讶和出乎意料地，本发明提供了新型(X)x变体，其对于葡萄糖特异性，并且从而显示出显著降低的耗氧率和/或对于除氧外的电子介质显著增加的介质活性。 [0030] Furthermore, surprisingly and unexpectedly, the present invention provides novel (X) x variant thereof, specific for glucose, and thus show a significant decrease in oxygen consumption rate and / or oxygen in the outer electronic media media significantly increased activity.
[003。 [003. 本发明的主题是新型6化变体，除根据SEQIDNO: 1的两个置换T30V和I94V之夕b其另外具有在选自S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个氨基酸置换。 The subject of the invention is a novel variants 6, except under SEQIDNO: 1 is replaced with two Eve b T30V and I94V in which additionally has selected S53; A137; A173; A332; F414; and six position V560 in at least one amino acid substitution.
[0032] 在本公开内容的上下文中，表达"对于葡萄糖的酶特异性"或"对于葡萄糖特异性" 指示：当通过如条目ff)下的材料与方法中概述的介质测定进行测定时，本文提供的(X)x对于底物葡萄糖〉99. 5%或〉99. 9%，特别是100%的活性。 [0032] In the context of the present disclosure, the expression "for the enzyme glucose-specific" or "specific for glucose" indicator: When the medium measurement Materials and Methods) by the entry ff as measured outlined herein provided (X) x for the substrate glucose> 99.5% or> 99.9%, in particular 100% activity. 言下之意，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，本文提供的(X)x对于除葡萄糖外的糖例如半乳糖、麦芽糖、木糖和麦芽=糖的残留活性< 6%。 This implies that when the medium measurement Materials and Methods section) in the entry ff as outlined by the time of measurement, provided herein (X) x glucose in addition to sugar such as galactose, maltose, xylose and malt = sugars residual activity <6%.
[0033] 在本公开内容的上下文中，表达"显著降低的耗氧率"指示：当如根据条目gg)下的材料与方法部分中概述的ABTS测定，通过生色底物ABTS的氧化间接测定氧依赖性时，本文提供的(Xk变体经过3分钟的时间段〉95%的残留氧含量。 [0033] In the context of the present disclosure, the expression "significantly reduced the oxygen consumption rate" indicates: When, as measured according to the Materials and Methods section ABTS entry under gg) outlined by the oxidation of a chromogenic substrate ABTS was determined indirectly when the oxygen-dependent, (Xk variant period after 3 minutes> 95% of the residual oxygen content provided herein.
[0034] 在本公开内容的上下文中，表达"显著降低的氧活性"或"对于作为电子受体的氧显著降低的活性"指示：当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，特征在于残留氧活性《30%、或< 25%、或< 20%，特别是< 15%、或《10%的(X)x活性。 [0034] In the context of the present disclosure, the expression "significantly reduced oxygen activity" or "for oxygen as an electron acceptor significantly reduced activity" indicator: When the Materials and Methods section) lower by as entry gg outlined when the ABTS assay measurement, characterized in that the residual oxygen activity "is 30%, or <25% or <20%, in particular <15% or <10% of (X) x activity.
[0035] 在本公开内容的上下文中，表达"显著增加的介质活性"或"对于除氧外的电子介质显著增加的活性"指示：当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，特征在于根据SEQIDNO: 1的葡萄糖氧化酶对于除氧外的电子介质增加至少1.5 倍的活性的(X)x活性。 [0035] In the context of the present disclosure, the expression "a significant increase in dielectric activity" or "the outer oxygen electronic medium significantly increased activity" indicator: When the Materials and Methods section through such entry ff) under outlined measured as the medium measured, wherein according SEQIDNO: (X) x activity of the glucose oxidase is an electron mediator other than oxygen for at least 1.5-fold increase in activity.
[0036] 因此，本发明的优化(X)x变体适合于植入改善的血糖测量系统中。 [0036] Accordingly, the present invention is to optimize the (X) x improved variants suitable for implantation in a blood glucose measurement system.
[0037] 发明概述令人惊讶和出乎意料地，本发明人发现如Zhu等人（2006 ;2007)中描述的(X)x双重突变体T30V;I94V(下文缩写为G0X-T30V;I94V)，适合作为进一步的特异性氨基酸置换的基础，W获得氧不依赖性(X)x变体，其具有显著降低的氧化酶活性和伴随的显著增加的脱氨酶活性，同时保留对于底物葡萄糖的特异性。 [0037] Summary of the invention Surprisingly and unexpectedly, the present inventors have found that such Zhu et al. (2006; 2007) as described in (X) x double mutant T30V; I94V (hereinafter abbreviated as G0X-T30V; I94V) suitable as a basis for further specific amino acid substitutions, W to obtain an oxygen-independent (X) x variants which have significantly reduced oxidative activity and concomitant significant increase deaminase, while retaining for the substrate glucose specificity. 进一步地，本发明人发现依照本发明的(X)x变体另外或单独对于除氧外的电子介质显示出显著增加的介质活性。 Further, the present inventors have found that further electronic medium or for separate oxygen from outside the media showed a significant increase in activity in accordance with (X) x variant of the present invention. 在运点上，本发明人还发现依照本发明的(X)x变体接受除氧外的某些电子介质用于电子转移。 At the point of operation, in accordance with the present invention also found that (X) x variant of the present invention to accept some electron mediator other than oxygen for the electron transfer. 因此，依照本发明的GOx变体适合于改善的葡萄糖测量，特别是改善的血糖测量。 Thus, in accordance with the present invention GOx variants suitable for improving glucose measurement, especially to improve blood glucose measurement.
[0038] 在本公开内容的上下文中，表达"接受除氧外的某些电子介质用于电子转移"指示：本文提供的(X)x变体与选自下述的介质相互作用且因此接受所述介质用于介导电子转移的能力：亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、紫精、四硫富瓦締及其衍生物和献菁。 [0038] In the context of the present disclosure, the expression "to accept some electron mediator other than oxygen for the electron transfer" indicates: (X) x variant provided herein is selected from the interaction of the medium and thus acceptable the medium used to mediate electron transfer capability: nitrosoaniline and derivatives thereof, azo compounds, and derivatives thereof winded thiophene, thiophene cried winded and derivatives thereof, thiophene and derivatives thereof winded ah, ferrocenium iron and its derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof, casein noise do I, viologen, tetrathiafulvalene and its derivatives offer associative phthalocyanine.
[0039] 本发明设及衍生自黑曲霉的(X)x变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其另外具有在选自S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个氨基酸置换。 [0039] The present invention is provided and derived from Aspergillus niger (X) x variants, except under SEQIDNO: T30V substitution and two outside I94V 1, which additionally has a selected S53; A137; A173; A332; F414; V560 and six position of at least one amino acid substitution.
[0040] 在另一个方面，本发明设及衍生自黑曲霉的(X)x变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其另外具有在选自A173 ;A332 ;F414 ;和V560的四个位置中的至少一个氨基酸置换。 [0040] In another aspect, the present invention is provided and derived from (X) x variant of Aspergillus niger, except under SEQIDNO: T30V substitution and two outside I94V 1, which additionally has a selected A173; A332; F414 at least one amino acid substitution in four positions and V560; and.
[0041] 在进一步方面，本发明设及(X)x变体，其具有在选自根据SEQIDNO: 1的(X)x的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少两个、或S个、或四个、或五个或甚至六个协同且因此不同的氨基酸置换，导致耗氧率中的显著减少。 [0041] In a further aspect, the present invention is provided, and (X) x variants selected in accordance with SEQIDNO: S53 (X) x 1; A137; A173; A332; F414; and six position of V560 at least two, one, or S, or four, five or six or even synergistic, and thus a different amino acid substitution, resulting in a significant reduction in oxygen consumption rate.
[0042] 进一步地，本发明设及(X)x变体，其具有在选自根据SEQIDNO: 1的(X)x的S53 ; A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少两个、或S个、或四个、或五个或甚至六个协同且因此不同的氨基酸置换，导致显著减少的耗氧率和/或对于除氧外的某些电子介质显著增加的介质活性。 [0042] Further, the present invention is provided, and (X) x variants selected in accordance with SEQIDNO: (X) x 1 S53; A137; A173; A332; F414; and six position V560 in at least two, one, or S, or four, five or six or even synergistic, and thus a different amino acid substitution, resulting in a significant reduction in oxygen consumption rate and / or oxygen to the outer medium of certain electronic medium significantly increased active.
[004引因此，本发明的发明人已能够提供(X)x变体，与GOx-WT和根据SEQIDNO: 1的G化-T30V;I94V相比较，其氧化酶活性显著降低，同时与(X)x-WT和GOx-T30V;I94V相比较，该酶的脱氨酶活性显著增加。 [004 cited Accordingly, the present inventors have been able to provide (X) X variants, and GOx-WT and according SEQIDNO: G of -T30V 1 a; I94V compared to the oxidation activity decreased significantly, while the (X ) x-WT and GOx-T30V; I94V compared deaminase enzyme is significantly increased.
[0044] 在运点上，进一步出乎意料和令人惊讶的发现是：当在如条目gg)下的材料与方法部分中概述的ABTS测定中，与GOx-WT和G0X-T30V;I94V相比较时，在位置F414和V560 中的特异性氨基酸置换显著降低本文提供的(X)x变体的耗氧率。 [0044] In the operation point, and further unexpected discovery is surprising: ABTS when measured as outlined in Materials and Methods section in such entry gg) in the, and GOx-WT and G0X-T30V; I94V phase comparison, specific amino acid substitutions at positions F414 and V560 are significantly reduced oxygen consumption rate (X) x variants provided herein. 用于所述置换的合适氨基酸是所有剩余19种蛋白源氨基酸。 Suitable amino acid substitutions for the remaining 19 kinds are all proteinogenic amino acids. 特别地，合适的氨基酸是用于位置F414的Arg、Asn、 Asp、切S、Gly、His、Met、lie、Leu、Ser、1"虹、Tyr和Val，W及用于位置V560 的Ala、lie、 Leu、Met、Pro、Tyr、T虹和Val。 Specifically, amino acids are suitable for the location of the Arg F414, Asn, Asp, cut S, Gly, His, Met, lie, Leu, Ser, 1 "rainbow, Tyr and Val, W, and the location of Ala V560, lie, Leu, Met, Pro, Tyr, T rainbow and Val.
[0045] 作为另一个出乎意料和令人惊讶的发现：当与(X)x-T30V;I94V相比较时，除本文提供的(X)x变体的两个置换T30V和I94V之外，对组合的两个位置F414和V560的特异性氨基酸置换显著增加对于除氧外的电子介质的介质活性。 [0045] As a further unexpected and surprising findings: When the (X) x-T30V; I94V when compared, and in addition two substitutions T30v I94V (X) x variants provided herein, two specific amino acid substitutions to positions V560 and F414 combinations significantly increased the electronic medium for the active oxygen of the outer medium. 用于所述置换的合适氨基酸是所有剩余19种蛋白源氨基酸。 Suitable amino acid substitutions for the remaining 19 kinds are all proteinogenic amino acids. 特别地，合适的氨基酸是用于位置F414的Arg、Asn、AspXys、 Gly、His、lie、Leu、Met、Ser、T虹、Tyr和Val，W及用于位置V560 的F414 和Ala、lie、Leu、 Met、Pro、T虹、Tyr和Val。 Specifically, amino acids are suitable for the location of the Arg F414, Asn, AspXys, Gly, His, lie, Leu, Met, Ser, T rainbow, Tyr and Val, W, and the location of the F414 V560 and Ala, lie, Leu, Met, Pro, T rainbow, Tyr and Val.
[0046] 作为进一步发现，本发明掲示当与(X)x-WT和G0X-T30V;I94V相比较时，除本文提供的(X)x变体的两个置换T30V和I94V之外，位置A173和A332各自一般导致(X)x活性中的增加。 [0046] As a further found that, when the present invention is shown and kei (X) x-WT and G0X-T30V; I94V when compared to two other substitutions T30v and I94V (X) x variants provided herein other than the position A173 and A332 are each generally leads to an increase (X) x the activity. 用于所述置换的合适氨基酸是所有剩余19种蛋白源氨基酸。 Suitable amino acid substitutions for the remaining 19 kinds are all proteinogenic amino acids. 特别地，合适的氨基酸是用于位置A173的Ile、T虹和Val，W及用于位置A332的Ser和Asn。 Specifically, amino acids are suitable for the position A173 of Ile, T rainbow and Val, W, and a position for A332 and Ser Asn.
[0047] 因此，本发明允许本领域技术人员获得具有显著降低的耗氧率的(X)x变体。 [0047] Accordingly, the present invention allows the skilled artisan to obtain (X) x variant has significantly reduced the rate of oxygen consumption.
[0048] 另外，本发明允许本领域技术人员获得(X)x变体，其经由特异性氨基酸置换个别地或对于组合的两个特点，具有显著降低的耗氧率和/或对于除氧外的某些电子介质增加的介质活性。 [0048] Further, the present invention allows the skilled artisan to obtain (X) x variant thereof via a specific amino acid substitutions individually or in combination to the two characteristics of having significantly reduced rates of oxygen consumption and / or for addition to the oxygen some electronic media increased media activity.
[004引发明详述糖尿病患者需要在其日常生活期间的准确的血糖测量。 [004 initiated out detailed diabetics require accurate measurement of blood glucose during their daily lives. 如科学背景部分中概述的，现有酶促测量系统基于氧依赖性(X)X和对于葡萄糖非特异性的GDH。 As outlined in the background section SCIENCES, existing measurement systems based on oxygen-dependent enzymatic (X) X and non-specific for glucose GDH.
[0050] 在来自黑曲霉的(X)x-WT中，氧化酶活性比其脱氨酶活性高约S至四倍。 [0050] In the (X) x-WT from Aspergillus niger, oxidase activity higher than the activity of the ammonia removal from about S to four times. 相应地， 当溶解氧存在于血糖测定系统中时，在底物葡萄糖氧化期间生成的电子也将转移至氧。 Accordingly, when the dissolved oxygen present in a blood glucose measuring system, during oxidation of the substrate glucose electrons will be transferred to the oxygen. 因此，在电子介质的存在下测量的酶活性可W受溶解氧浓度影响。 Thus, the enzymatic activity measured in the presence of the electronic media W may be affected by the concentration of dissolved oxygen. 然而，来自黑曲霉的GOx-WT 对于葡萄糖特异性且显示出足够的溫度稳定性。 However, GOx-WT from Aspergillus niger glucose to exhibit sufficient specificity and temperature stability.
[0051] 相比之下，GDH对血样中的溶解氧抗性，但对于葡萄糖不够特异性，并且因此也可W受血样中存在的其他糖类型影响，所述其他糖类型例如麦芽糖、半乳糖、木糖和麦芽= 糖。 [0051] In contrast, the GDH of the dissolved oxygen in the blood sample resistance, but not specific for glucose, and thus also affect other types of sugar may be present in the blood sample by W, the types of other sugars such as maltose, galactose , = xylose and malt sugar.
[0052] 本发明的目的是消除在基于GOx的血糖测量期间的溶解氧的效应，同时保存且进一步增加其有利特性。 [0052] The object of the present invention is to eliminate the effect of dissolved oxygen based on blood glucose measurement during the GOx, and further increases while preserving its advantageous properties.
[0053] 因此，本发明的发明人已能够提供(X)x变体，与(X)x-WT和根据SEQIDNO: 1的G化-T30V;I94V相比较，其氧化酶活性显著降低，同时与(X)x-WT和GOx-T30V;I94V相比较，该酶的脱氨酶活性显著增加。 [0053] Accordingly, the present invention is capable of providing (X) x variant, and (X) x-WT and according SEQIDNO: G of -T30V 1 a; I94V compared to the oxidation activity decreased significantly, while and (X) x-WT and GOx-T30V; I94V compared deaminase enzyme is significantly increased.
[0054] 如本文使用的，"氧化酶活性"是本文提供的(X)x变体催化葡萄糖氧化W生成葡糖酸内醋的酶促活性，其中利用氧作为电子受体。 [0054] As used herein, "oxidase activity" as provided herein (X) x W variants generated in the catalytic oxidation of glucose to gluconic acid, vinegar, enzymatic activity, where the use of oxygen as an electron acceptor. "氧化酶活性"可W通过本领域已知的任何方法通过测量生成的&〇2量进行评价。 Any method "oxidase activity" W may be known in the art & 〇2 evaluated by measuring the amount of generated. 例如氧化酶活性"可W通过用于H202检测的试剂例如4AA/T0DB/P0D(4-氨基安替比林（N，N-双（4-横下基）-3-甲基丙氨酸二钢盐/辣根过氧化物酶）或销电极进行测定。 E.g. oxidase activity "by W may be, for example, reagents for detecting H202 4AA / T0DB / P0D (4- amino-antipyrine (N, N- bis (the cross-4- yl) -3-methyl-alanine diacetic steel salt / horseradish peroxidase) or the pin electrodes was measured.
[0055] 如本文在相对或定量活性的上下文中使用的，氧化酶活性特别限定为通过在lOmM PPB，抑7. 0、1.5mlT0DB、2U/血辣根过氧化物酶（POD)、和1. 5ml4-氨基安替比林（4AA) 中，在25°C下生成的&〇2量测量的氧化底物(葡萄糖)的摩尔量/单位时间。 [0055] As used herein, the relative activity or quantitative context, oxidase activity is defined by the particular lOmM PPB, suppression 7. 0,1.5mlT0DB, 2U / blood horseradish peroxidase (the POD), and 1 molar amount. 5ml4- aminoantipyrine than Lin (4AA), at 25 ° C for measuring the amount of generated & 〇2 oxide substrate (glucose) / unit of time. 酿亚胺染料的形成可W在546nm处进行分光光度计测量。 Stuffed imine dyes formed can be measured spectrophotometrically at W at 546nm.
[0056] 如本文使用的，"脱氨酶活性"是通过利用除氧外的电子介质作为电子受体，本文提供的(X)x变体催化葡萄糖氧化W生成葡糖酸内醋的酶促活性。 [0056] As used herein, "deaminase activity" as an electron acceptor, provided herein (X) x W variants generated enzymatically catalyzed oxidation of glucose within gluconic acid vinegar by using the electronic medium out of oxygen active. "脱氨酶活性"可W通过测量转移至所使用的除氧外的介质的电子量进行测定。 "Deaminase activity" W may be transferred by measuring the amount of the medium to the electronic-oxygen used for measurement.
[0057] 如本文在相对或定量活性的上下文中使用的，"脱氨酶活性"特别限定为通过在10 mlPPB(pH7. 0)，0. 6mlmetho巧PMS(mPMS)中，在25°C下转移至除氧外的介质的电子量测量的氧化底物(葡萄糖）的摩尔量/单位时间。 [0057] As used herein, the relative or quantitative activity context, "deaminase" particularly limited to by 10 mlPPB (pH7. 0), 0. In 6mlmetho Qiao PMS (mPMS), at 25 ° C for the molar amount of electrons transferred to the measuring medium outside of the oxygen oxide substrate (glucose) / unit of time.
[005引在本公开内容的上下文中，表达"一种或多种电子介质"和"除氧外的一种或多种介质"指示能够W氧化和还原形式两者存在，并且快速反应W贡献或接受电子的小有机或无机化学制品。 [005 incorporated in the context of the present disclosure, the expression "one or more electronic media" and "one or more oxygen outside medium" can be indicative of both the oxidized and reduced form of W, and W contribution to the rapid reaction or accept electrons small organic or inorganic chemicals. 特别地，表达"一种或多种电子介质"和"除氧外的一种或多种介质"指示其为葡萄糖的电子受体且从而从氧化形式转换成还原形式的小有机或无机化学制品。 In particular, the expression "one or more electronic media" and "one or more oxygen outside medium" indicates that small organic or inorganic electron acceptor chemicals glucose and thereby converted from an oxidized form to the reduced form of . 根据运点，所述介质将还原形式的电子递送至工作电极用于电化学葡萄糖测量，或递送至指示剂分子用于血糖测定系统中的比色测量。 The point of operation, the reduced form of the electronic medium is delivered to the working electrode for an electrochemical glucose measurement, or delivered to the indicator molecule for measuring blood glucose measuring system color ratio. 某些电子受体自身充当指示剂分子，并且可W直接用于葡萄糖的比色测量(参见例如EP8 313 27)。 Some indicator molecule itself serves as an electron acceptor, and W can be used directly in color than the measurement of glucose (see, e.g. EP8 313 27).
[0059] 在本发明的特定方面，电子介质或除氧外的介质选自亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、紫精、四硫富瓦締及其衍生物和献菁。 [0059] In a particular aspect of the present invention, the medium is selected from ethylene-nitroaniline and derivatives thereof, azo compounds, and thiophene derivatives winded outer electron mediator or oxygen, cried winded thiophene and derivatives thereof, thiophene ah winded and its derivatives, ferrocene and derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof, casein noise do I, viologen, tetrathiafulvalene derivatives and their associated matter and offer Ching.
[0060]在本发明的另一个更特定的方面，电子介质或除氧外的介质选自酿类，例如菲酿、 1，4二氨基蔥酿和菲酿的金属络合物，W及亚硝基苯胺，例如N，N-双（2-径乙基）-4-亚硝基苯胺或N，N-双（2-径乙基）-2-甲氧基-4-亚硝基苯胺。 [0060] more particular aspect, an electronic medium, or the external medium is selected from oxygen brewed In another class of the present invention, e.g. stuffed phenanthrene, 1,4-amino-metal complexes of onions stuffed and stuffed phenanthrene, W, and ethylene nitroaniline, for example N, N- bis (2-diameter ethyl) -4-nitroaniline or N, N- bis (2-ethyl-diameter) -2-methoxy-4-nitroaniline . 后面两种亚硝基苯胺中的任一种在下文将被称为"亚硝基苯胺介质"。 Either of the latter two in the nitrosoaniline it will be hereinafter referred to as "nitrosoaniline media."
[006。 [006. 如上所述的此类电子介质已例如在US5, 393, 615;US5, 498, 542;US5,520, 786 ；W0 2009/129108；US2009/0095642 ；W0 93/25898JP57-128678；EP0 441 222A孜Prevoteau,A.等人，ElectrochemistryCommunications(2010), 12(2),213-2巧; Berchimns,S.等人，Materials化emistryand曲ysics(2003)，77 (2)，390-396啼京描述。 Such electronic medium as described above has, for example, in US5, 393, 615; US5, 498, 542; US5,520, 786; W0 2009/129108; US2009 / 0095642; W0 93 / 25898JP57-128678; EP0 441 222A Zi Prevoteau , A et al., ElectrochemistryCommunications (2010), 12 (2), 213-2 clever;.. Berchimns, S et al., Materials of the song emistryand ysics (2003), 77 (2), 390-396 describes sings Beijing.
[0062] 尤其适合于本公开内容的主题的是通过FA畑2快速还原但通过抗坏血酸缓慢还原或不还原的电子介质；此类介质是例如2-[4-(二甲基氨基）苯基]-二氮締甲酯胺的衍生物。 [0062] particularly suitable for the subject matter of the present disclosure it is by Hata quickly restore FA slowly reduced by ascorbic acid but not reduced electron mediator or 2; Such media are, for example 2- [4- (dimethylamino) phenyl] - association nitrous ester amine derivatives.
[0063] 本文描述的方法的另一种用途是生成定制的"氧依赖性(X)x变体"，W使各自的G化变体适应固定的介质。 [0063] Another use of the method described herein is to produce customized "oxygen-dependent (X) x variant", W G so that the respective variants of fixed media adaptation.
[0064] 在运点上，术语"定制的"意指本发明的各自0化变体的多肤序列允许(X)x与某些固定的介质相互作用，并且因此接受来自某些固定的介质的电子。 Multi skin sequence [0064] at the point of operation, the term "custom" means that the present invention each of the 0 variant allows (X) x interact with certain fixed media, and thus receiving from certain fixed media e.
[0065] 表达"某些固定的介质"指示选自下述的除氧外的一种或多种电子介质：亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、紫精、四硫富瓦締及其衍生物和献菁。 [0065] The expression "certain fixed medium" indicates one or more electron mediator other than oxygen is selected from the following: Sub-nitroaniline and derivatives thereof, azo compounds, and derivatives thereof winded thiophene, thiophene cried winded and derivatives thereof, thiophene and derivatives thereof winded ah, ferrocene and derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof, casein noise do I, viologen , tetrathiafulvalene and its derivatives offer associative phthalocyanine.
[0066] 在本公开内容的另一个非常特定的方面，来自亚硝基苯胺特别是对亚硝基苯胺或其衍生物的介质，且特别是亚硝基苯胺介质或其衍生物优选作为依照本发明的电子介质。 [0066] In another embodiment of the disclosure a very specific aspect, especially nitrosoaniline from p-nitroaniline or a derivative thereof media, and in particular an alkylene or a nitroaniline derivative is preferably used as medium in accordance with the present electronic media invention. 一般而言，来自亚硝基苯胺的介质例如在测试条上与葡萄糖和(X)x原位反应，W形成充当电子介质的种类。 In general, the media, for example, from nitrosoaniline on glucose test strips and (X) x situ reaction with, W-forming species act as electron mediator. 在葡萄糖测量和介质活性上下文中的亚硝基苯胺在化bes，乂，做7心6。 Nitrosoaniline glucose measurements and in the active medium in the context of BES, qe, doing 6 7 heart. P.和Surridge,N. (2008).TheTechnologyBehindGlucoseMeters:TestStrips. DiabetesTechnology&^erapeutics10,10-26;和EP0 354 441啤'常城据卷。 . P. and Surridge, N (2008) .TheTechnologyBehindGlucoseMeters: TestStrips & ^ erapeutics10,10-26 DiabetesTechnology; EP0 354 441 and beer 'Chang Cheng data volume.
[0067] 上文列表不应限于依照本发明除氧外的介质，因为本领域技术人员已知的另外其他商购可得的电子介质完全由本公开内容包含。 [0067] In accordance with the above list should not be limited to the outer medium of the present invention, the oxygen, as known to those skilled in yet other commercially available electronic medium is completely encompassed by the present disclosure.
[0068] 在本公开内容的另一个非常特定的方面，当通过如条目ff)下的材料与方法中概述的介质测定进行测定时，与根据SEQIDNO: 1的G0x-T30V;I94V的各自介质活性相比较，根据序列沈QIDNO: 2至沈QIDNO: 9的(X)x变体显示出对于介质N，N-双（2-径乙基）-4-亚硝基苯胺至少150%的介质活性。 When [0068] In another very specific disclosure of the present aspect, when the medium measurement method according to the item material ff) under outlined was measured in accordance with SEQIDNO: G0x-T30V 1; and each of the medium for the active I94V comparing their sequences Shen QIDNO: 2 to Shen QIDNO: (X) x 9 shows a variant to the medium N, N- bis (2-diameter ethyl) -4-nitroaniline at least 150% of the active medium .
[0069] 在本公开内容的另一个非常特定的方面，当通过如条目ff)下的材料与方法中概述的介质测定进行测定时，与根据SEQIDNO: 1的G0x-T30V;I94V的各自介质活性相比较，除根据SEQIDNO: 1的两个置换T30V和I94V之外，还具有下述另外的氨基酸置换的G化变体显示出对于介质N，N-双（2-径乙基)-2-甲氧基-4-亚硝基苯胺至少150%的介质活性： • A173V;A332S;F414Y;和V560A或• A173V;A332S;和F414Y。 When [0069] In another very specific disclosure of the present aspect, when the medium measurement method according to the item material ff) under outlined was measured in accordance with SEQIDNO: G0x-T30V 1; and each of the medium for the active I94V comparison, except under SEQIDNO: T30V substitution than two and I94V 1, further having the following additional amino acid substitutions of the variants showed G to the medium N, N- bis (2-ethyl-diameter) -2- methoxy-4-nitroaniline at least 150% of the active medium: • A173V; A332S; F414Y; and V560A or • A173V; A332S; and F414Y.
[0070] 除根据沈QIDNO: 1的两个置换T30V和I94V之外，上述置换• A173V;A332S;F414Y;和V560A或• A173V;A332S;和F414Y。 [0070] The addition Shen QIDNO: I94V substitution T30V and two outside 1, said replacement • A173V; A332S; F414Y; and V560A or • A173V; A332S; and F414Y.
[0071] 反映本文提供的(X)x变体的另一个特征，即接受除氧外的某些电子介质（在运种情况下，N，N-双（2-径乙基）-2-甲氧基-4-亚硝基苯胺介质）用于电子转移的能力。 [0071] Another feature of the reflected (X) x variants provided herein, i.e. accept some electron mediator other than oxygen (in the transport case, N, N- bis (2-ethyl-diameter) -2- methoxy-4-nitroaniline medium) for electron transfer capability.
[0072] 在该上下文中，表达"除氧外的某些电子介质"指示选自下述的除氧外的一种或多种电子介质：亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、 紫精、四硫富瓦締及其衍生物和献菁。 [0072] In this context, the expression of one or more of the outer electron mediator "Some electron mediator other than oxygen" indicates an oxygen selected from: alkylene nitroaniline and derivatives thereof, azo compounds, winded thiophene and derivatives thereof, thiophene and derivatives thereof cried winded, repetitious ah thiophene and its derivatives, ferrocene and derivatives, ferric chloride, bell, and Os- Ru- complexes, and derivatives thereof stuffed , I do noise casein, viologen, tetrathiafulvalene and its derivatives offer associative phthalocyanine.
[0073] 在第一个方面，本发明设及选自下述的根据SEQIDNO: 1的葡萄糖氧化酶的主题： a) 根据SEQIDNO: 1的葡萄糖氧化酶，除两个氨基酸置换T30V和I94V之外，还具有在选自所述沈QIDNO: 1中的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个另外的氨基酸置换；或b) 显示出与根据a)的葡萄糖氧化酶至少70%，特别是至少80%或〉90%的氨基酸序列同一性的葡萄糖氧化酶，条件是除两个氨基酸置换T30V和I94V之外，还具有在选自根据沈QIDNO: 1的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个另外的氨基酸置换， 并且条件是根据b)的葡萄糖氧化酶显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80%或〉90%的酶活性，并且显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80% 或〉90%的对于葡萄糖的酶特异性， 并且条件是根据b)的葡萄糖氧化酶显示出根据SEQIDNO: 1的葡萄糖氧 [0073] In a first aspect, the present invention is selected from and disposed according SEQIDNO: glucose oxidase Topic 1: a) According SEQIDNO: 1 glucose oxidase, in addition to two amino acid substitutions outside T30V and I94V further having a selected said counterbore QIDNO: in 1 S53; at least one additional six amino acid substitutions in position and V560;; A137; A173; A332; F414 or b) exhibits glucose according to a), oxidase least 70%, especially at least 80% or> 90% amino acid sequence identity glucose oxidase, with the proviso that two amino acid substitutions in addition to and T30V I94V, but also has QIDNO selected according Shen: S53 1 of ; at least one additional amino acid substitution in the six position and V560, and shows that according to b) glucose oxidase according to a) glucose oxidase least 70%, especially at least; A137; A173; A332; F414 80% glucose oxidation or> 90% of the enzyme activity, and shows according to a) glucose oxidase at least 70%, especially at least 80% or> 90% for the glucose-specific enzyme, and that according to b) of the enzyme exhibits SEQIDNO: 1 oxygen glucose 酶对于作为电子受体的氧降低至少5倍的活性，或显示出根据SEQIDNO: 1的葡萄糖氧化酶对于除氧外的电子介质增加至少1. 5倍的活性，或两者；或C)根据a)或b)的葡萄糖氧化酶的活性片段，条件是当与根据a)或b)的葡萄糖氧化酶相比较时，根据C)的活性片段具有如在a)或b)下概述的氨基酸置换， 并且条件是根据C)的葡萄糖氧化酶显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80%或〉90%的酶活性，并且显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80% 或〉90%的对于葡萄糖的酶特异性， 并且条件是根据C)的葡萄糖氧化酶显示出根据SEQIDNO: 1的葡萄糖氧化酶对于作为电子受体的氧降低至少5倍的活性，或显示出根据SEQIDNO: 1的葡萄糖氧化酶对于除氧外的电子介质增加至少1. 5倍的活性，或两者。 For the enzyme activity of at least 5 times as electron acceptor oxygen, or display according SEQIDNO: 1 glucose oxidase for the outer electron mediator is at least 1.5-fold increase in oxygen activity, or both; or C) according to active fragment of a) or b) is glucose oxidase, with the proviso that when when according to a) or b) is glucose oxidase compared according to C) is an active fragment has as amino acid outlined under a) or b) replacing , and the condition is based on C), glucose oxidase exhibits according to a) glucose oxidase at least 70%, especially at least 80% or> 90% of the enzyme activity, and shows according to a) glucose oxidase at least 70% , especially at least 80% or> 90% for the glucose-specific enzyme, and that in accordance with C) exhibits glucose oxidase according SEQIDNO: 1 glucose oxidase to reduce oxygen as an electron acceptor is at least 5 times activity, or exhibit according SEQIDNO: 1 glucose oxidase to increase the electron mediator other than oxygen activity at least 1.5 times, or both.
[0074] 在本说明书的上下文中，表达"在氨基酸序列中的一种或多种另外的修饰"和"一种或多种另外的氨基酸置换"指示：在根据SEQIDNO: 1的位置S53 ;A137 ;A173 ;A332 ; F414 ;和V560的，至少一种氨基酸由剩余19种蛋白源氨基酸中任一种的置换。 [0074] In the context of this specification, the expression "one or more further modifications in the amino acid sequence" and "one or more additional amino acid substitution" indicates: In SEQIDNO: S53 at position 1; A137 ; A173; A332; F414; and V560 at least one amino acid according to any of the remaining 19 kinds of amino acid of one protein source replacement. 进一步地， 上述表达还包含由剩余19种蛋白源氨基酸中任一种或其化学等价物的协同氨基酸置换， 条件是在选自根据沈QIDNO: 1的位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560中置换至少两个、或=个、或四个、或五个或甚至六个位置。 Further, the expression of a further synergistic amino acid substitutions comprises the remaining 19 kinds of amino acids in the protein source any one or a chemical equivalent thereof, is selected according to the conditions Shen QIDNO: 1 position S53; A137; A173; A332; F414; and V560 in the replacement of at least two, or = a, or four, or even five or six positions.
[0075] 与本发明的(X)x变体有关的术语"酶活性"指定蛋白质，其催化PD-葡萄糖氧化成D-葡糖酸-1,5-内醋（D-葡萄糖+ 〇2 -葡糖酸内醋+ &〇2)，其随后可W水解为葡糖酸。 [0075] The term "activity" of a given protein (X) x variant of the invention relating, which catalyzes the oxidation of glucose PD- D- glucono-1,5 vinegar (D- glucose + 〇2 - the gluconic acid + & 〇2 vinegar), which may then be hydrolyzed to gluconic acid W.
[0076] 在第二个方面，本发明设及下述实施方案的主题，其指定本发明的第一个方面： 在一个方面，本发明的主题是根据本发明的第一个方面的(X)x变体，其中•通过ABTS测定来测定对于作为电子受体的氧的所述活性，其包括下述步骤： a) 将75化样品酶溶液转移至含有100化憐酸盐缓冲液（pH7)的96孔平底微孔板b) 将20化反应混合物加入每个孔，导致下述浓度：0.91U/mLHRP(辣根过氧化物酶)； 2. 3mlABTS(2, 2' -联氮-双（3-乙基苯并嚷挫嘟-6-横酸)） C)通过添加25化葡萄糖底物溶液，且随后将板W1000巧m振荡共30秒来起始反应d)使用微孔板阅读器在414nm处动态测定ABTS的氧化•通过介质测定来测定对于除氧外的电子介质的所述活性，其包括下述步骤： a) 将75化酶溶液样品转移至96孔平底微孔板b) 加入100化介质溶液a9.05mMN，N-双（2-径乙基）-4-亚硝基苯胺）；5%(w/w) 聚乙締化咯烧 [0076] In a second aspect, embodiments of the subject matter provided and the present invention is described below, which specifies a first aspect of the present invention: In one aspect, the subject matter of the present invention is the first aspect of the present invention (X ) x variant, wherein • determined by the ABTS assay for reactive oxygen as the electron acceptor, comprising the steps of: a) 75 of the enzyme solution was transferred to a sample of 100 Rei formate buffer (pH 7 ) in 96 well flat bottom microplate b) the 20 reaction mixture was added to each well, resulting in the following concentrations: 0.91U / mLHRP (horseradish peroxidase); 2. 3mlABTS (2, 2 '- azino - bis (3-ethyl-benzo cried setback beep -6- cross acid)) C) 25 glucose by addition of a substrate solution, and then the plates were shaken W1000 Qiao m for 30 seconds starting reaction d) using a microplate a) the enzyme solution was 75 samples were transferred to 96 well flat bottom microplates: ABTS oxidation is determined • the active oxygen of the outer electron mediator by the measuring medium, comprising the steps of measuring in a dynamic reader at 414nm b) medium was added a solution of 100 a9.05mMN, N- bis (2-diameter ethyl) -4-nitroaniline); 5% (w / w) of polyglycolide associative slightly burned 酬，pH7和20化25ml憐钢酸C)通过添加25化葡萄糖底物溶液，且随后将板W1000rpm振荡共1分钟来起始反应d)使用微孔板阅读器在700nm处监控憐钢酸还原的动力学。 Pay, pH7 and 20 of steel 25ml Rei acid C) by the addition of 25 glucose substrate solution, and then the plates were shaken for 1 minute W1000rpm starting reaction d) monitored using a microplate reader at 700nm pity steel reducing acid the kinetics.
[0077] 在设及上述方面的另一个方面，除氧外的电子介质选自亚硝基苯胺及其衍生物、 偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、 铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、紫精、四硫富瓦締及其衍生物和献菁。 [0077] and further provided in the above aspect, the electronic medium is selected from ethylene-nitroaniline and derivatives thereof, azo compounds, thiophene and derivatives thereof excluding oxygen winded, repetitious cried thiophene and derivatives thereof, thiophene ah winded and its derivatives, ferrocene and derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof, casein noise do I, viologen, tetrathiafulvalene and its associated and phthalocyanine derivatives offer.
[0078] 在设及上述方面的另一个方面，本发明的主题是(X)x变体，除根据SEQIDNO: 1 的两个置换T30V和I94V之外，其还具有在选自根据SEQIDNO: 1的S53;A137;A173; A332 ;F414 ;和V560的六个位置中的另外两个、或S个、或四个、或五个或六个氨基酸置换。 [0078] In another aspect of the above aspects and provided, subject of the invention is (X) x variants, except under SEQIDNO: two T30V substitution than 1 and I94V, further having a selected according SEQIDNO: 1 the S53; A137; A173; A332; F414; V560 and the other two positions in the six or more S, or four, or five, or six amino acid substitutions.
[0079] 在设及上述方面的另一个方面，本发明提供了0化变体，其中关于所述一个或多个另外置换的氨基酸选自Ala、Arg、Asn、Asp、切S、Gin、Glu、Gly、His、lie、Leu、Lys、Met、 ?116、？ [0079] In another aspect of the above aspects, and is provided, the present invention provides variants 0, wherein on the one or more additional amino acid substitutions selected from Ala, Arg, Asn, Asp, cut S, Gin, Glu , Gly, His, lie, Leu, Lys, Met,? 116 ,? '〇、56'、1'虹、1'巧、了7'和¥日1，条件是取代氨基酸是如根据569 10^:1的各自位置中存在的其他氨基酸。 'Square, 56', 1 'rainbow, 1' clever, 7 'and ¥ Day 1, with the proviso that such substitution is in accordance with the amino acid ^ 56,910: 1, the respective positions of other amino acids present.
[0080] 在设及上述方面中任一个的另一个方面，本发明提供了0化变体，其中关于一个或多个另外置换的氨基酸选自Ala、lie、1"虹、Tyr、Val、Ser、Asn、Arg、Asp、切S、Gly、His、 Met、Leu、Phe、Met和Pro,条件是取代氨基酸是如根据SEQIDNO: 1的各自位置中存在的其他氨基酸。 [0080] In another aspect is provided, and any one of the above aspects, the present invention provides variants 0, wherein on the one or more additional amino acid substitutions selected from Ala, lie, 1 "rainbow, Tyr, Val, Ser , Asn, Arg, Asp, cut S, Gly, His, Met, Leu, Phe, Met and Pro, with the proviso that such substitution is in accordance with the amino acid SEQIDNO: 1, the respective positions of other amino acids present.
[0081] 在设及上述方面中任一个的另一个方面，本发明提供了6化变体，其中关于一个或多个另外置换的氨基酸选自： 用于位置S53的化e ;和/或用于位置A137的Ser、Leu ;和/或用于位置A173的lie、虹、Val ;和/或用于位置A332的Ser、Asn、Val ;和/或用于位置F414 的Arg、Asn、Asp、切S、Gly、His、lie、Met、Ser、1"虹、Tyr、Val;和/ 或用于位置V560 的Ala、116、1^611、]\161、？'〇、1'虹、17'和¥日1。 [0081] In another aspect is provided, and any one of the above aspects, the present invention provides 6 variants, of which about one or more additional amino acid substitutions selected from: a position S53 of the e; and / or with the position of Ser A137, Leu; and / or location of A173 lie, rainbow, Val; and / or a position A332 of Ser, Asn, Val; and / or Arg in position F414, Asn, Asp, cut S, Gly, His, lie, Met, Ser, 1 "rainbow, Tyr, Val; Ala, and / or a position of V560, 116,1 ^ 611,] \ 161, 'square, a' rainbow, 17? 'day and ¥ 1.
[0082] 在设及上述方面中任一个的另一个方面，本发明提供了0化变体，其中除根据SEQ IDNO: 1的置换T30V和I94V之外，位置F414和/或V560中的至少一个另外置换的氨基酸置换与位置A137和/或A173和/或A332中的至少一个氨基酸置换组合。 [0082] In another aspect is provided, and any one of the above aspects, the present invention provides variants 0, according to which, inter SEQ IDNO: T30V substitution and addition I94V 1, the position of F414 and / or at least one of V560 Further substituted amino acid substitutions position A137 and / or A173 and / or A332 of at least one amino acid substitution in combination.
[0083] 在设及上述方面中任一个的另一个方面，本发明提供了根据SEQIDNO: 4的GOx 变体，除根据SEQIDNO: 1的置换T30V和I94V之外，还具有另外的氨基酸置换A173I; A332S;和F414L。 [0083] In another aspect is provided and any one of the above aspects, the present invention provides an SEQIDNO: GOx variant 4, except under SEQIDNO: T30V and substitution of I94V 1, but also have additional amino acid substitution A173I; A332S; and F414L.
[0084] 在设及上述方面中任一个的另一个方面，本发明提供了根据SEQIDNO: 3的GOx 变体，除根据SEQIDNO: 1的置换T30V和I94V之外，还具有另外的氨基酸置换A173V; A332S;F414I;和V560T。 [0084] In another aspect is provided and any one of the above aspects, the present invention provides an SEQIDNO: GOx 3 variants, except under SEQIDNO: T30V substitution and addition I94V 1, further having an additional amino acid substitution A173V; A332S; F414I; and V560T.
[0085] 在设及上述方面中任一个的另一个方面，本发明提供了根据SEQIDNO: 6的GOx 变体，除根据SEQIDNO: 1的置换T30V和I94V之外，还具有另外的氨基酸置换A173V; A332N;F414V;和V560L。 [0085] In another aspect is provided and any one of the above aspects, the present invention provides an SEQIDNO: GOx variant 6, except under SEQIDNO: T30V and substitution of I94V 1, but also have additional amino acid substitution A173V; A332N; F414V; and V560L.
[0086] 在设及上述方面中任一个的另一个方面，本发明提供了0化变体，当借助于ABTS 测定与黑曲霉的野生型(X)x相比较和/或根据SEQIDNO: 1的(X)x相比较时，对于作为电子受体的氧具有降低至少5倍的活性，所述ABTS测定包括下述步骤： a. )将75化样品酶溶液转移至含有100化憐酸盐缓冲液（pH7)的96孔平底微孔板b. )将20化反应混合物加入每个孔，导致下述浓度：0. 91U/mLHRP(辣根过氧化物酶）；2. 3mlABTS(2, 2' -联氮-双（3-乙基苯并嚷挫嘟-6-横酸)） C.)通过添加25化葡萄糖底物溶液，且随后将板W1000巧m振荡共30秒来起始反应d.)使用微孔板阅读器在414皿处动态测定ABTS的氧化。 [0086] In any of the above aspects and provided a further aspect, the present invention provides variants 0, when measured by means of ABTS wildtype Aspergillus niger (X) x is compared and / or according SEQIDNO: 1 is when (X) x as compared to having a reduced oxygen as an electron acceptor is at least 5 times the activity, the ABTS assay comprising the steps of:. a) the 75 samples of the enzyme containing solution was transferred to a 100 of formate buffer pity the liquid (pH 7) 96-well flat-bottomed microplate b) the 20 reaction mixture was added to each well, resulting in the following concentrations: 0 91U / mLHRP (horseradish peroxidase); 2 3mlABTS (2, 2.. '- azino - bis (3-ethyl-benzo cried setback beep -6- cross acid)) C.) by addition of 25 glucose substrate solution, and then the plates were shaken W1000 m clever reaction was initiated 30 seconds D.) was measured using a microplate reader dynamic oxidation of ABTS at 414 at the dish.
[0087] 在设及上述方面中任一个的另一个方面，本发明提供了0化变体，由此当通过介质测定进行测定时，与根据SEQIDNO: 1的(X)x相比较时，所述(X)x变体显示出对于除氧外的介质增加至少1. 5倍的活性，所述介质测定包括下述步骤： a. )将75化酶溶液样品转移至96孔平底微孔板b. )加入100化介质溶液（19. 05mlN，N-双（2-径乙基）-4-亚硝基苯胺）;5% (w/w) 聚乙締化咯烧酬，pH7和20化25ml憐钢酸C.)通过添加25化葡萄糖底物溶液，且随后将板W1000巧m振荡共1分钟来起始反应d.)使用微孔板阅读器在700nm处监控憐钢酸还原的动力学。 When [0087] In another aspect is provided, and any one of the above aspects, the present invention provides variants 0, whereby when measured by the measurement medium, and in accordance with SEQIDNO: when compared (X) x 1, and the said (X) x variants showed increased to the medium outside the oxygen activity of at least 1.5-fold, the assay medium comprising the steps of:. a) the enzyme solution was 75 samples were transferred to 96 well flat bottom microtiter plate . b) medium was added a solution of 100 (19. 05mlN, N- bis (2-diameter ethyl) -4-nitroaniline); 5% (w / w) of polyglycolide associated pay slightly burned, pH7 and 20 25ml of steel pity acid C.) by addition of 25 glucose substrate solution, and then the plates were shaken W1000 m clever reaction was started one minute d.) monitored using a microplate reader at 700nm pity steel reduced acid dynamics.
[0088] 在设及上述方面中任一个的另一个方面，本发明提供了0化变体，当通过介质测定进行测定时，所述(X)x变体显示出至少99. 9%的葡萄糖特异性，和/或< 4%的半乳糖特异性，和/或< 0. 3%的麦芽糖特异性，和/或< 6%的木糖特异性，和/或< 0. 1%的麦芽S 糖特异性，所述介质测定包括下述步骤： a. )将75化酶溶液样品转移至96孔平底微孔板b. )加入100化介质溶液（19. 05mlN，N-双（2-径乙基）-4-亚硝基苯胺）;5% (w/w) 聚乙締化咯烧酬，pH7和20化25ml憐钢酸C.)通过添加25化各自的糖底物溶液，且随后将板W1000巧m振荡共1分钟来起始反应d.)使用微孔板阅读器在700nm处监控憐钢酸还原的动力学。 [0088] In another aspect is provided, and any one of the above aspects, the present invention provides variants 0, when measured by the assay medium, the (X) x variant exhibits at least 99.9% of glucose specificity, and / or <4% galactose-specific and / or <0.3% maltose specificity, and / or <6% xylose specificity, and / or <0.1% malt specific sugar S, said assay medium comprising the steps of:.. a) the enzyme solution sample was transferred to 75 well flat bottom microtiter plate 96 b) medium was added a solution of 100 (19. 05mlN, N- bis (2- diameter ethyl) -4-nitroaniline); 5% (w / w) of polyglycolide associated pay slightly burned, pH7 and 20 of steel 25ml Rei acid C.) by addition of a solution of the respective sugar substrate 25, and then shaking the plates of 1 m W1000 clever initial reaction D min.) using a microplate reader to monitor the kinetics Rei acid is reduced in the steel at 700nm.
[0089] 在本公开内容的上下文中，表达"对于葡萄糖特异性或对于底物葡萄糖特异性"指示：当通过如下文材料与方法部分中概述的介质测定进行测定时，本文提供的(X)x对于底物葡萄糖100%的活性。 [0089] In the context of the present disclosure, the expression "specific for glucose or glucose to the substrate specificity of" indicates: When measured by dielectric measurement described below in Materials and Methods outlined provided herein (X) x for the substrate glucose is 100% active. 言下之意，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，本文提供的GOx对于除葡萄糖外的糖例如半乳糖、麦芽糖、木糖和麦芽=糖的残留活性< 6%。 This implies, when measured by the measuring medium such as in the Materials and Methods section entry ff) outlined herein GOx provided in addition to sugar residues such as galactose, glucose, maltose, xylose, malt sugar = activity of <6%.
[0090] 在设及上述方面中任一个的另一个方面，本发明提供了(X)x变体，当借助于介质测定与根据SEQIDNO: 1的(X)x的亚硝基苯胺介质活性相比较时，所述(X)x变体显示出对于用于电子转移的亚硝基苯胺介质〉400%、或〉500%、或〉600%的活性，所述介质测定包括下述步骤： a. )将75化酶溶液样品转移至96孔平底微孔板b. )加入100化介质溶液（19. 05mlN，N-双（2-径乙基）-4-亚硝基苯胺）;5% (w/w) 聚乙締化咯烧酬，pH7和20化25ml憐钢酸C.)通过添加25化葡萄糖底物溶液，且随后将板W1000巧m振荡共1分钟来起始反应d.)使用微孔板阅读器在700nm处监控憐钢酸还原的动力学。 [0090] In another aspect is provided, and any one of the above aspects, the present invention provides a (X) x variant, when measured by means of a medium in accordance with SEQIDNO: nitrosoaniline medium for the active phase (X) x 1 when compared to the (X) x variants showed nitrosoaniline for transfer medium for an electronic> 400%, or> 500%, or> 600% of the activity, the assay medium comprising the steps of: a .) the enzyme sample solution was transferred to a 75 well flat bottom microtiter plate 96 b) medium was added a solution of 100 (19. 05mlN, N- bis (2-diameter ethyl) -4-nitroaniline);. 5% (w / w) of polyglycolide associated pay slightly burned, pH7 and 20 of steel 25ml Rei acid C.) by addition of 25 glucose substrate solution, and then the plates were shaken W1000 m clever reaction was initiated one minute d. ) using a microplate reader at 700nm monitored Rei acid reduction kinetics steel.
[00川和当借助于ABTS测定与根据SEQIDNO: 1的(X)x的氧活性相比较时，《30%、或< 25%、或< 20%，且特别是< 15%、或《10%的氧活性，所述ABTS测定包括下述步骤： a. )将75化样品酶溶液转移至含有100化憐酸盐缓冲液（pH7)的96孔平底微孔板b. )将20化反应混合物加入每个孔，导致下述浓度：0. 91U/mLHRP(辣根过氧化物酶）；2. 3mlABTS(2, 2' -联氮-双（3-乙基苯并嚷挫嘟-6-横酸)） C.)通过添加25化葡萄糖底物溶液，且随后将板W1000巧m振荡共30秒来起始反应d.)使用微孔板阅读器在414皿处动态测定ABTS的氧化。 [Chuan and 00 when measured in accordance with the aid of ABTS SEQIDNO: When comparing the oxygen activity (X) x 1 "30%, or <25% or <20%, and in particular <15%, or" 10 % of the oxygen activity, the ABTS assay comprising the steps of:.. a) the 75 samples of the enzyme containing solution was transferred to a 100 Rei of acetate buffer (pH 7) 96-well flat-bottomed microplate b) the reaction of 20 mixture was added to each well, resulting in the following concentrations:.. 0 91U / mLHRP (horseradish peroxidase); 2 3mlABTS (2, 2 '- azino - bis (3-ethyl-benzo cried setback beep -6 - cross-acid)) C.) by addition of 25 glucose substrate solution, and then the plates were shaken W1000 Qiao m for 30 seconds starting reaction d) measured using a microplate reader at 414 dynamic oxidation of ABTS at dish. .
[0092] 在另一个方面，本发明的主题设及经分离的多核巧酸，其编码根据上述方面中任一个的(X)x变体或其活性片段。 [0092] In another aspect, the subject matter of the present invention is provided isolated and polynuclear clever acid which encodes a variant or an active fragment thereof according to any one of the above aspects (X) x.
[0093] 在另一个方面，本发明提供了根据本发明的(X)x变体的活性(功能)等价物/片段。 [0093] In another aspect, the present invention provides an active (X) x variant of the invention (functional) equivalents / fragment.
[0094] 术语"其一种或多种活性等价物/ 一个或多个片段"或同义词"其一种或多种功能等价物/ 一个或多个片段"指依照本发明的任何经修饰的和因此优化的(X)x变体，由此如在根据SEQIDNO: 1的相应序列中，至少一个氨基酸缺失或由另一个氨基酸置换，前提是此类一种或多种等价物/ 一个或多个片段仍显示出关于酶活性、酶特异性和显著降低的耗氧率和/或对于除氧外的特定介质显著增加的活性的基本特性，通过具有根据SEQIDNO: 1 的至少置换T30V和I94V的存在，并且进一步具有在选自根据SEQIDNO: 1的S53;A137; A173 ;A332 ;F414 ;和V560的位置中的至少一个另外的氨基酸置换。 [0094] The term "/ one or more fragments thereof of one or more active equivalent" or synonymous with "one or more of its functional equivalents / or one or more fragment" refers to any modified in accordance with the present invention and therefore optimization in (X) x variant, whereby as in the SEQIDNO: 1 in the sequence corresponding to the at least one amino acid deleted or substituted by another amino acid, provided that such one or more equivalents / display one or more segments remain on the activity, enzymatic specificity, and significant reduction in the oxygen consumption rate and / or the outer oxygen particular medium significantly increased the activity of the basic characteristics, in accordance with by SEQIDNO: T30V and I94V substitution presence of at least 1, and further in accordance with selected SEQIDNO: S53 1 a; A137; A173; A332; at least one additional amino acid substitution in position and V560; F414.
[0095] 在本发明的特定方面，根据本发明的(X)x变体的一种或多种功能等价物/ 一个或多个片段包含一种或多种氨基酸序列，其与根据SEQIDNO: 2至SEQIDNO: 9的序列至少70%同源，特别是至少80%同源或〉90%同源。 [0095] In a particular aspect of the invention, according to one (X) x variant of the invention, one or more functional equivalents / comprise one or more segments of one or more amino acid sequences, which according SEQIDNO: 2 to SEQIDNO: 9 sequence is at least 70% homology, particularly at least 80% homologous or> 90% homologous.
[0096] 在另一个方面，本发明的主题设及通过根据本发明的(X)x变体或其活性片段来检巧。 [0096] In another aspect, the subject matter of the present invention is provided by the subject, and according to the coincidence (X) x variant or an active fragment of the invention. 、测定或测量先体外后体内样品中的葡萄糖的方法；所述检测、测定或测量包括使先体外后体内样品与所述(X)x或其活性片段接触。 After ex vivo assay or measuring glucose in a sample method; the detecting, determining or measuring the x or active fragment comprising the sample with the ex vivo (X) in contact.
[0097] 在设及上述方面的另一个方面，所述葡萄糖检测、测定或测量使用传感器或测试条装置执行。 [0097] In another aspect is provided and the above-described aspect, the detection of glucose, determined or measured using a sensor or test strip device to perform.
[0098] 本发明还设及用于生产本发明的(X)x变体的方法，编码所述酶的核酸，载体，宿主细胞W及使用所述酶检测、测定或测量样品中的葡萄糖的方法，W及同样包含所述酶的装置。 [0098] The present invention also provided methods and (X) x of the present invention is produced for variants, nucleic acid encoding the enzyme, vectors, and host cells using the W enzymatic detection, assay or measurement of glucose in the sample method, W, and the apparatus also includes an enzyme.
[0099] 在特定方面，本发明提供了用于测定样品中的葡萄糖的装置，其包含本发明的GOx 变体中的至少一种和除氧外的电子介质。 [0099] In a particular aspect, the present invention provides an apparatus for determining glucose in a sample, which comprises at least the electron mediator GOx variants of the present invention and an external oxygen.
[0100] 在本发明的另一个特定方面，除氧外的一种或多种电子介质选自亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物。 [0100] In another particular aspect of the present invention, an electron-oxygen medium is selected from one or more sub-nitroaniline and derivatives thereof, azo compounds, thiophene winded and derivatives thereof, thiophene and cried winded derivatives, thiophene and its derivatives winded ah, ferrocene and derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof.
[0101] 设及上述方面中任一个的主题的本发明的另一个特定方面是(X)X变体，除根据沈QIDNO: 1的两个置换T30V和I94V之外，其还具有在选自沈QIDNO: 1的A173;A332; F414 ;和V560的四个位置中的至少一个另外的氨基酸置换。 Another particular aspect of the present invention [0101] provided, and any one of the above aspects of the subject matter is (X) X variants, except under heavy QIDNO: T30V substitution than two and I94V 1, further having a selected Shen QIDNO: 1 in A173; A332; at least one additional amino acid substitutions and four positions in the V560; F414.
[0102] 由本发明预期的本文提供的(X)x变体的主要应用之一是其在测试条中的用途，W 监控糖尿病患者的先体外后体内样品中的血糖水平。 [0102] One of the major applications (X) x variant provided herein is contemplated by the present invention is its use in the test strip, after ex vivo sample W diabetics to monitor blood glucose levels. 当然，可W研究许多种类的样品。 Of course, many types of samples can be studied W. 特别地，体液如血液、血清和血浆是此类样品的来源。 Particularly, body fluids such as blood, plasma and serum is a source of such samples.
[0103] 因此，在另一个方面，本发明提供了具有本发明的(X)x变体中的至少一种的酶电极，其固定在所述电极上。 [0103] Thus, in another aspect, the present invention provides at least one enzyme electrode (X) x variants with the present invention, which is fixed on the electrode.
[0104] 在另一个方面，本发明提供了用于测定葡萄糖的酶传感器，其包括本发明的酶电极作为工作电极，且因此具有本发明的(X)X变体中的至少一种。 [0104] In another aspect, the present invention provides an enzyme sensor for measuring glucose, comprising the enzyme electrode according to the present invention as a working electrode, and thus at least one (X) X variants with the present invention.
[0105] 在另外一个方面，本发明提供了用于测定样品中的葡萄糖的试剂盒，其包括根据本发明的至少一种(X)X变体和除氧外的电子介质。 [0105] In another aspect, the present invention provides a method for measuring glucose in a sample kit comprising an electronic medium in accordance with at least one outer (X) X and variations of the present invention is oxygen.
[0106] 用于测量葡萄糖的试剂盒可W使用本发明的至少一种(X)x变体进行构建。 [0106] A kit for measuring glucose of the present invention may be used W of at least one (X) x construct variants. 除本发明的(X)x变体之外，试剂盒含有测量必需的缓冲液，除氧的适当电子介质特别是亚硝基苯胺介质，和需要时的酶例如过氧化物酶，W及用于制备校准曲线的葡萄糖标准溶液和使用说明书。 In addition to (X) x variant of the invention, the kit contains the necessary measurement buffer, suitable electronic medium, in particular oxygen nitrosoaniline medium, and the need for an enzyme such as peroxidase, W, and with glucose standard solution prepared calibration curve and instructions for use.
[0107] 样品中的葡萄糖浓度可W通过测量由酶反应生成的电子量进行测定。 [0107] The glucose concentration in the sample can be measured W by the amount of electrons generated by measuring the enzyme reaction. 多种传感器系统是本领域已知的，包括碳电极、金属电极和销电极。 Multiple sensor systems are known in the art, including a carbon electrode, a metal electrode and a pin electrode. 将本发明的GOx变体固定到电极上。 The GOx variant of the present invention is fixed to the electrode. 用于固定的方法的例子包括交联、封装到大分子基质内、用透析膜包被、光学交联聚合物、导电聚合物、氧化还原聚合物及其任何组合。 Examples include a method of fixing a crosslinked, packaged into the matrix of macromolecules, was used a dialysis membrane bag, the optical cross-linked polymer, a conductive polymer, a redox polymer, and any combination thereof.
[010引测定装置可W具有与用于监控血糖水平，常规商购可得的安培测量生物传感器测试条中的任一种相似的结构。 [010 W primer may have a measurement device for monitoring blood glucose levels, a conventional commercially available amperometric biosensor test strip according to any measurement of a similar structure. 此类装置的一个例子具有置于绝缘基材上的两个电极(工作电极和参考或对电极)、试剂端口和样品接收器。 An example of such a device having two electrodes disposed on the insulating substrate (working electrode and the reference or counter electrode), a reagent and the sample receiver port. 试剂端口含有本发明的(X)x变体和除氧外的介质。 Medium outside (X) x and variants containing the oxygen scavenging agent of the present invention the port. 当样品例如血样加入样品接收器时，样品中含有的葡萄糖将与GOx变体反应，从而电子转移指示样品中的葡萄糖量。 When the blood sample is added to the sample, for example, the receiver, the glucose contained in the sample with reaction variant GOx, an electron transfer so that indicates the amount of glucose in the sample. 例如根据W0 2004/113900和US5, 997, 817,适合测定酶底物的电化学传感器的典型例子是已知的。 The example W0 2004/113900 and US5, 997, 817, a typical example of an electrochemical sensor for the measurement of enzyme substrates are known. 作为电化学传感器的替代物，可W使用光学检测技术。 As an alternative to electrochemical sensors, optical detection techniques may be W. 通常，此类光学装置基于试剂系统中发生的变色，所述试剂系统包含酶、电子介质和指示剂。 Typically, such optical apparatus discoloration agent based system, the reagent system comprises an enzyme, electron mediator and an indicator. 变色可W使用巧光、吸收或减轻（remission)测量进行定量。 W clever use of light discoloration can absorb or mitigate (the remission) measured quantitatively. 例如根据US 7, 008, 799;US6, 036, 919和US5, 334, 508,适合测定酶底物的光学装置的典型例子是已知的。 For example according to US 7, 008, 799; US6, 036, 919 and US5, 334, 508, typical examples of the optical apparatus for measuring the enzyme substrates are known.
[0109] 在另一个方面，本发明提供了含有经分离的多核巧酸的表达载体，所述经分离的多核巧酸编码根据本发明的(X)X变体或其活性片段。 [0109] In another aspect, the present invention provides an expression vector comprising the isolated polynuclear clever acid, the separated multi-core clever acid encoding according to (X) X a variant or an active fragment of the invention.
[0110] 在另一个方面，本发明提供了包含含有经分离的多核巧酸的表达载体的宿主细胞，在本文中也称为转化体，所述经分离的多核巧酸编码根据本发明的GOx变体或其活性片段。 [0110] In another aspect, the present invention provides a host cell comprising an isolated polynuclear expression vector containing a clever acids, also referred to herein as the transformant, the isolated acid encoding a clever multi-core according to the present invention GOx variant or an active fragment thereof.
[0111] 在另一个方面，本发明提供了用于生产根据本发明的(X)X变体或其活性片段的方法，该方法包括培养上述转化体。 [0111] In another aspect, the present invention provides a method for producing the (X) X a variant or active fragment thereof according to the present invention, the method comprises culturing the above transformant.
[0112] 在另一个方面，本发明提供了可通过上述方法获得的根据本发明的(X)x变体或其活性片段。 [0112] In another aspect, the present invention provides a (X) x variant or an active fragment thereof according to the present invention can be obtained by the above method.
[0113] 在另一个特定方面，本文提供的表达载体优选包含编码本发明的(X)x变体的DNA 序列之一的全部或一部分。 All or a portion of one of the DNA sequence (X) x [0113] In another particular aspect, provided herein is preferably an expression vector encoding a variant of the present invention.
[0114] 含有本文提供的(X)x变体的编码和控制序列的合适表达载体可W使用本领域已知的标准DNA技术进行构建，所述标准DNA技术中的许多在Sambrook等人，in"Molecular Cloning:ALaboratoryManual"(1989)，ColdSpringHabor，NYColdSpringHabor L油oratoryPress中描述。 [0114] Suitable expression vectors containing the coding and control sequences (X) x variants provided herein may be known in the art W using standard DNA techniques constructed, the many standard DNA techniques in Sambrook et al., In "Molecular Cloning: ALaboratoryManual" (1989), ColdSpringHabor, NYColdSpringHabor L oil oratoryPress described.
[0115] 合适的宿主细胞包括例如可得自Promega(2800WoodsHollowRoad,Madison, WI，USA)的大肠杆菌（j5： 皿10UATCC33694)，可得自StratagenedlOllNodhTorrefy PineRoad,La化lla，CA，USA)的)(L1-BlueMRF'等。合适的毕赤酵母属（巧cAia)宿主细胞包括例如，可得自Invitrogen(5791VanAllanWay,Carlsbad,CA92008，USA)的己斯德毕赤酵母（CjOa別o'is)X3 3或己斯德毕赤酵母KM71H。 [0115] Suitable host cells include, for example, available from Promega (2800WoodsHollowRoad, Madison, WI, USA) E. coli (j5: dish 10UATCC33694), available from StratagenedlOllNodhTorrefy PineRoad, La of lla, CA, USA) a) (Ll -BlueMRF 'etc. suitable Pichia (Qiao CAIA) host cells include, for example, available from Invitrogen (5791VanAllanWay, Carlsbad, CA92008, USA) hexyl de Pichia (CjOa not o'is) X3 3 or Pichia has de KM71H.
[0116] 根据本发明的(X)x变体的重组生产可W在本领域已知的宿主中进行。 [0116] The recombinant production of (X) x variant of the present invention may be W is present in a host known in the art. 合适的宿主可W选自丝状真菌菌株，例如黑曲霉、酱油曲霉（AsjOe'巧sojae)和米曲霉Uwer知合适的宿主可W选自酵母菌株，如例如己斯德毕赤酵母、酿酒酵母和多形汉逊酵母（偏nsenuia/7〇妙膨巧片3)。 Suitable host filamentous fungal strain can be selected from W, such as Aspergillus niger, Aspergillus sojae (AsjOe 'clever sojae) and Aspergillus oryzae Uwer known suitable host may be W is selected from yeast strains such as Pichia de hexyl e.g., Saccharomyces cerevisiae and Hansenula polymorpha (vinylidene nsenuia / 7〇 Miao Qiao expansion sheet 3).
[0117] 在另一个特定方面，通过在酵母特别是酿酒酵母中表达编码所述(X)x变体的多核巧酸，可获得根据本发明的GOx变体或其功能等价物。 [0117] In another particular aspect, the encoding by expressing in yeast Saccharomyces cerevisiae is particularly polynuclear (X) x variant clever acid, obtained according to the present invention GOx variants or functional equivalents thereof.
[011引表达载体可W通过本领域已知的多种方法引入宿主细胞内。 [011 W primers expression vectors can be introduced into host cells by various methods known in the art. 例如，通过聚乙二醇介导的原生质体转化方法（Sambrook等人1989,同上)，可W进行用表达载体转化宿主细胞。 For example, (1989, Sambrook et al. Supra), W can be performed by protoplast transformation procedure mediated by polyethylene glycol of a host cell transformed with an expression vector. 然而，还可W采用用于将表达载体引入宿主细胞内的其他方法，例如电穿孔、弹道DNA 注射或原生质体融合。 However, W may also be employed for other methods of introducing expression vectors into a host cell, e.g., electroporation, ballistic DNA injection or protoplast fusion.
[0119] 一旦含有根据本发明的(X)x变体的表达载体已引入适当宿主细胞内，宿主细胞就可W在允许根据本发明的所需(X)x变体表达的条件下进行培养。 [0119] Once an expression vector (X) x variant according to the invention have been introduced into an appropriate host cell, the host cell W can be cultured under conditions that allow expression (X) x variant of the present invention required . 通过例如抗生素选择或营养突变体的互补和从最小培养基中选择，可W容易地鉴定含有所需表达载体(且因此具有编码根据本发明的GOx变体的DNA序列）的宿主细胞/T; K化/556心/ "MolecularCloning:alaboratorymanual",第3版，ColdSpringHarbor,NewYork。 By selecting, for example, an antibiotic or a complement thereof selected from minimal medium and auxotrophic mutation, W may be readily identified containing the desired expression vectors (and therefore having a coding DNA sequence according to the present invention GOx variant) host cell / T; of K / 556 heart / "MolecularCloning: alaboratorymanual", 3rd edition, ColdSpringHarbor, NewYork. 本文提供的(X)x变体的表达可w通过本领域技术人员已知的不同方法进行鉴定， 如测量GOxmRNA转录物的生产，免疫检测基因产物或检测基因产物的酶促活性。 Expression (X) x variants provided herein can be identified by w various methods known to the skilled person, such as measuring the production of immunoassay detection of the enzymatic activity of the gene product or gene product GOxmRNA transcript. 特别地， 酶促测定应如条目ff)下的材料与方法部分中的介质测定下概述的应用。 Overview The application of the Materials and Methods section in particular, should such entry enzymatic assay ff) the medium was measured. 另外，当如条目gg)下所述的材料与方法部分中概述的ABTS测定进行测定时，通过如依照本发明显著降低的耗氧率，可W鉴定本文提供的(X)x变体。 Further, when the ABTS as entry gg) under outlined in Materials and Methods section when measured by the significant decrease in accordance as the oxygen consumption rate present, may be identified W (X) x assay variants provided herein.
[0120] 在本发明的另一个方面，通过在表达编码本发明的(X)x变体之一的DNA序列的宿主细胞中生产，可获得本文描述的用于本发明的(X)x变体的多肤。 [0120] In another aspect of the present invention, by producing in a host cell a DNA sequence encoding expression of one of the present invention, (X) x variant obtained (X) x used in the present invention described herein becomes multi-body skin. 通过由编码本发明的GOx 变体之一的DM序列编码的mRNA的体外翻译，还可W获得本发明的多肤。 By in vitro translation of the mRNA sequence encoded by one of the DM GOx encoding a variant of the invention, W may obtain multiple skin of the present invention. 例如，DM序列可W插入合适的表达载体内，所述表达载体依次又可W用于体外转录/翻译系统中。 For example, W can be inserted into the DM sequence appropriate expression vector, the expression vector W sequentially turn used for in vitro transcription / translation system.
[0121] 包含如上定义和描述的经分离的多核巧酸的表达载体代表本发明的另一个特定方面，所述经分离的多核巧酸可操作地连接至能够促进其在无细胞的肤合成系统中的表达的启动子序列。 The isolated expression vector multicore [0121] as defined and described herein comprising an acid behalf Coincidentally another particular aspect of the invention, the separated multi-core operatively connected to clever acid which is capable of promoting skin cell-free synthesis system promoter sequences expressed in.
[0122] 例如通过如上所述的操作产生的多肤随后可W使用多种常规蛋白质纯化技术进行分离且纯化。 [0122] generated by the operation as described above, for example, a plurality skin W may then be purified using a variety of conventional protein separation and purification techniques. 例如，可W采用层析操作例如离子交换层析、凝胶过滤层析和亲和层析。 For example, W can be employed chromatographic procedures such as ion exchange chromatography, gel filtration chromatography and affinity chromatography.
[0123] 在另一个方面，本发明提供了药物组合物，其包含根据本发明的至少一种(X)x变体或其活性片段。 [0123] In another aspect, the present invention provides a pharmaceutical composition, comprising at least one (X) x variant or an active fragment thereof according to the present invention.
[0124] 在另一个方面，根据本发明的(X)x变体保留其对于底物葡萄糖特异性的固有特性，但当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，由于除根据SEQIDNO: 1的置换T30V和I94V之外，氨基酸序列中的特定位置中的至少一个另外的氨基酸置换，最大限度的残留氧活性降低至《30%。 [0124] In another aspect, glucose retain their specificity for the substrate according to the inherent characteristics (X) x variant of the invention, but when measured by the ABTS assay Materials and Methods section in such entry gg) outlined when, due to the addition according SEQIDNO: outside T30V substitution of I94V 1 and at least one further amino acid substitution in the amino acid sequence of a particular position, the maximum residual oxygen activity is decreased to "30%. 伴随地，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，本文提供的(X)x变体显示出对于除氧外的电子介质〉400%、或〉500%、或〉600%的酶活性。 Concomitantly, when the medium measurement Materials and Methods section) in the entry ff as outlined by the time of measurement, provided herein (X) x variants showed the electron mediator other than oxygen for the> 400%, or> 500% , or> 600% activity.
[01巧]考虑到上文，本发明在本文提供的(X)x变体中组合i)G化的葡萄糖特异性特性ii) 与GDH的氧不依赖性酶活性特性经由从氧化酶活性朝向脱氨酶活性的转变iii)和任选对于除氧外的某些电子介质增加的活性， 用于实现准确的葡萄糖测量，特别是准确的血糖测量。 [Qiao 01] Considering the above, in the present invention compositions (X) x variants provided herein i) G-specific properties of the glucose ii) oxygen-dependent GDH activity is not characteristic of the via toward oxidase activity deaminase activity transition iii) and optionally for some electron mediator other than oxygen increased activity, for accurate measurement of glucose, particularly blood glucose measurement is accurate.
[0126] 在根据本发明的(X)x变体的上下文中，表达"从氧化酶活性朝向脱氨酶活性的转变"指示•当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，根据本发明的(X)x变体的氧活性从作为参考的G0X-T30V;I94V的1. 0开始朝向《0. 3、或< 0. 25、或< 0. 2,特别是< 0. 15、或《0. 1残留氧活性的减少，和•当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，根据本发明的0化变体的脱氨酶活性从作为参考的G0X-T30V;I94V的介质活性1. 0开始朝向至少1. 5 介质活性的伴随增加。 [0126] In the context of (X) x variant of the present invention, the expression ABTS Materials and Methods "from the oxidizing activity towards off transition activity Ammonia" indicates • When through such entry gg) under outlined in accordance with measurement was measured in accordance with the oxygen activity (X) x variant of the present invention from a reference G0X-T30V; I94V towards the beginning of the 1.0 "0.3 or <0.25 or <0.2, in particular <0.15, or "residual oxygen reduction activity of 0.1, and the medium was measured when • materials and methods section) in the entry ff as outlined by the time of measurement, in accordance with the present invention of the 0 variant deaminase activity from the reference G0X-T30V; I94V active medium toward the start of at least 1.0 medium 1.5 concomitant increase in activity.
[0127] 上述值代表在氧活性和介质活性方面本发明的(X)x变体的酶活性，W及作为参考的G0x-T30V;I94V的各自酶活性之间的比例馈数)。 Activity (X) x variants [0127] The value represents the oxygen activity of the active medium and aspects of the invention, W, and as a reference G0x-T30V; feed ratio between the number of the respective activity I94V). 当通过如条目ee)下的材料与方法部分中概述的化ISA进行测定时，比例（商数）基于两种(X)x活性扣/mg]表示的氧活性和介质活性)。 When measured by The ISA of the Materials and Methods section entry under ee) as outlined in the ratio (quotient) based on two (X) x activity buckle / mg] represents the oxygen activity and the active medium).
[0128] 根据本发明，根据序列SEQIDNO: 11至SEQIDNO: 17的核酸分子各自编码经修饰的多肤或其片段，其衍生自编码根据SEQIDNO: 1的多肤的SEQIDNO: 10(G化-T30V; I94V)〇 [0128] According to the invention, according to the sequence SEQIDNO: 11 to SEQIDNO: 17, each nucleic acid molecule encoding a modified multi skin or fragment thereof is derived from a coded according SEQIDNO: multiple skin 1 SEQIDNO: 10 (G of -T30V ; I94V) billion
[0129] 如本文使用的，术语"G化-T30V;I94V"指示基于根据沈QIDNO: 2的黑曲霉野生型（WT)序列的GOx，其进一步具有在其多肤序列即SEQIDNO: 1中的两个置换T30V和I94V。 [0129] As used herein, the term "G of -T30V; I94V" The indication based Shen QIDNO: Aspergillus niger wild-type (WT) sequence of the GOx 2, which further have a sequence that is a multi-skin SEQIDNO: 1 in two replacement T30V and I94V.
[0130] 如本文使用的，术语"G化双重突变体"相等地指示基于根据SEQIDNO: 2的黑曲霉WT序列的GOx，其进一步具有在其多肤序列即SEQIDNO: 1中的两个置换T30V和I94V。 [0130] As used herein, the term "G of the double mutant" The indication based equally SEQIDNO: GOx sequence of A. niger WT 2, which further have a sequence that is a multi-skin SEQIDNO: T30V substitution of two 1 and I94V. 所述(X)x双重突变体衍生自Zhu等人（2006 ;2007)。 The (X) x double mutant derived from Zhu et al. (2006; 2007).
[0131] 根据本发明，对应于沈QIDNO: 1的核酸分子（即沈QIDNO: 10)用于进一步改善根据本发明的GOx酶的动力学特性，特征在于从氧化酶活性朝向脱氨酶活性的转变。 [0131] According to the present invention, corresponding to Shen QIDNO: 1 is a nucleic acid molecule (i.e., Shen QIDNO: 10) is used to further improve the dynamics of GOx enzyme according to the present invention, wherein the oxidase activity toward deaminase activity change. 运通过本发明人经由特异性核巧酸序列修饰来实现，导致在位置S53 ;A137 ;A173 ;A332 ; F414;和V560的至少一个中的氨基酸置换，而两个置换T30V和I94V根据SEQIDNO: 1已经存在。 Operation is achieved by the present invention is opportunely modified via specific nuclear acid sequence, resulting in a position S53; A173;; A137 A332; F414; at least one amino acid substitution and V560, and substitutions T30V and I94V The two SEQIDNO: 1 already exists.
[0132] 在另一个方面，本发明提供了(X)x变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有在选自沈QIDNO: 1的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的另外两个、或=个、或四个、或五个或六个氨基酸置换，从而显示出对(X)x酶活性的协同作用。 [0132] In another aspect, the present invention provides a (X) x variants, except under SEQIDNO: two T30V substitution than 1 and I94V, further having a selected Shen QIDNO: S53 1 a; A137; A173 ; A332; F414; V560 and the other two positions in the six or more =, or four, or five, or six amino acid substitutions, thus showing a synergistic effect on (X) x activity.
[013引在本发明的上下文中，表达"对0化酶活性的协同作用"指示除根据SEQIDNO: 1 的两个置换T30V和I94V之外，在位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560中的至少两个另外的氨基酸置换，具有在减少(X)x耗氧率方面对(X)x酶活性的作用。 [013 incorporated in the present context, the expression "synergistic activity of the enzyme 0" indicates SEQIDNO The other: two outside and I94V 1 T30V substitution at positions S53; A137; A173; A332; F414; and V560 at least two additional amino acid substitutions, x has an effect on the activity of (X) decreasing (X) x oxygen consumption rates.
[0134] 进一步地，在本发明的上下文中，"对(X)x酶活性的协同作用"指示除根据SEQID NO: 1的两个置换T30V和I94V之外，在位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560中的至少两个另外的氨基酸置换，具有在减少(X)x耗氧率和/或增加对于除氧外的电子介质的GOx 介质活性方面对G化酶活性的作用。 [0134] Further, in the context of the present invention, "x synergistic effect on the (X) activity" indicates except under SEQID NO: T30V substitution and two outside I94V 1, at a position S53; A137; A173; A332; F414; and at least two additional amino acid substitutions in the V560 with reduced (X) x or oxygen consumption rate and increase the effect of Enzymes in G / GOx medium for the active electron mediator other than oxygen aspects.
[013引另外，在本发明的上下文中，"对(X)x酶活性的协同作用"指示除根据SEQIDNO: 1的两个置换T30V和I94V之外，在位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560中的至少两个另外的氨基酸置换，具有在接受除氧外的某些电子介质用于电子转移方面对(X)x酶活性的作用。 [013 cited Further, in the context of the present invention, "x synergistic effect on the activity of (X)" indicates Except under SEQIDNO: T30V substitution and two outside I94V 1, at a position S53; A137; A173; A332; F414; V560, and at least two additional amino acid substitutions in the enzyme activity with certain electron-accepting than oxygen for the electron transfer medium in terms of (X) x. 在特定方面，本发明提供了(X)x变体，除根据SEQIDNO: 1的两个置换T30V和I94V 之外，其还具有在F414和V560位置中由剩余19种蛋白源氨基酸中任一种的另外的氨基酸置换，与在位置A137 ;A173 ;和A332中由剩余19种蛋白源氨基酸中任一种的氨基酸置换组厶1=1 0 In a particular aspect, the present invention provides a (X) x variants, except under SEQIDNO: two T30V substitution than 1 and I94V, further having any of the remaining 19 kinds of amino acids in the protein source position V560 and F414 additional amino acid substitutions at positions with A137; A173; A332, and the remaining 19 kinds of proteins by any endogenous amino acid substitution of one amino group Si = 1 10
[0136] 在特定方面，本发明提供了0化变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有在F414和V560位置中由剩余19种蛋白源氨基酸中任一种的另外的组合氨基酸置换。 [0136] In a particular aspect, the present invention provides variants 0, except under SEQIDNO: I94V substitution T30V and two outside 1, further having any of the remaining 19 kinds of amino acids in the protein source position V560 and F414 in addition the combination of types of amino acid substitutions.
[0137] 本发明的另一个特定方面设及上述方面，并且提供了GOx变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有另外的组合氨基酸置换F414M或F414V和V560P 或V560L。 [0137] Another particular aspect of the present invention is provided and the above-described aspect, and provides a variant of GOx, except under SEQIDNO: two T30V substitution than 1 and I94V, further having an additional combination of amino acid substitutions and F414V, or F414M V560P or V560L.
[0138] 在进一步的特定方面，本发明提供了0化变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有另外的氨基酸置换F414M或F414V，与一个或多个氨基酸置换V560P或V560L和/ 或A13化和/ 或A173I或A173V和/ 或A332S或A332V或A332T组合。 [0138] In a further particular aspect, the present invention provides variants 0, except under SEQIDNO: two T30V substitution than 1 and I94V, which have additional amino acid substitutions or F414M F414V, with one or more amino acid V560P or V560L substitution and / or A13 and / or A173I or and / or A332S A332T or A332V, or a combination A173V.
[0139] 在进一步的特定方面，本发明提供了0化变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有另外的氨基酸置换F414M或F414V，与一个或多个氨基酸置换V560P或V560L和/ 或A13化和/ 或A173I或A173V和/ 或A332S或A332V或A332T组合。 [0139] In a further particular aspect, the present invention provides variants 0, except under SEQIDNO: two T30V substitution than 1 and I94V, which have additional amino acid substitutions or F414M F414V, with one or more amino acid V560P or V560L substitution and / or A13 and / or A173I or and / or A332S A332T or A332V, or a combination A173V.
[0140] 本发明的另一个特定方面设及上述方面，并且提供了(X)x变体，除根据SEQIDNO: 1的两个置换T30V和I94V之外，其还具有选自上述置换中任一个的至少两个组合的氨基酸置换。 [0140] Another particular aspect of the present invention is provided and the above-described aspect, and provides a (X) x variants, except under SEQIDNO: two T30V substitution than 1 and I94V, which further includes any one selected from the above permutations a combination of at least two amino acid substitutions.
[0141] 对于除氧外的电子介质的(X)x特异性可W通过如条目ff)下的材料与方法部分中概述的介质测定进行测定。 [0141] For (X) x W can be specifically measured through the medium under the Materials and Methods section, such as entry ff) outlined electron mediator other than oxygen is measured.
[0142] 在另一个特定方面，本发明提供了0化变体，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，其对于电子介质N，N-双（2-径乙基）-4-亚硝基苯胺具有〉400%、或〉450%、或〉500%、或〉550%、或甚至〉600%的介质活性。 When [0142] In another particular aspect, the present invention provides variants of 0, when the medium determination Materials and Methods section) through the entry as outlined ff was measured, for which the electronic media N, N- bis (2 - diameter ethyl) -4-nitroaniline of> 400%, or> 450%, or> 500%, or> 550%, or even to> 600% of the active medium.
[0143] 在另一个特定方面，本发明提供了0化变体，当借助于如条目gg)下的材料与方法部分中概述的ABTS测定，与GOx-WT相比较或与根据SEQIDNO: 1的G0X-T30V;I94V相比较时，对于作为电子受体的氧具有降低至少5倍的活性，或对于作为电子受体的氧具有降低至少6倍的活性，或对于作为电子受体的氧具有降低至少7倍的活性。 [0143] In another particular aspect, the present invention provides variants of 0, when the ABTS under the Materials and Methods section by means of such entries gg) outlined measured, compared with GOx-WT or according SEQIDNO: 1 is G0X-T30V; I94V when compared to having a reduced activity of at least 5 times as electron acceptor oxygen, or oxygen as the electron acceptor for having reduced activity of at least 6 times, or for oxygen as an electron acceptor having a reduced activity at least seven times.
[0144] 在另一个特定方面，本发明提供了0化变体，当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，其具有GOxWT氧化酶活性或根据SEQIDNO: 1的G0X-T30V;I94V氧化酶活性的《30%剩余氧化酶活性，或特别具有GOxWT氧化酶活性或根据SEQIDNO: 1的G0X-T30V;I94V氧化酶活性的< 20%或< 15%剩余氧化酶活性，或甚至更特别具有GOxWT氧化酶活性或根据SEQIDNO: 1的G0X-T30V;I94V氧化酶活性的< 10%剩余氧化酶活性。 [0144] In another particular aspect, the present invention provides variants 0, when measured by the ABTS assay Materials and Methods The entry under gg) outlined having oxidase activity or GOxWT according SEQIDNO: 1 the G0X-T30V; I94V oxidase activity "30% of the remaining oxidase activity, or particularly GOxWT oxidase activity or on SEQIDNO: G0X-T30V 1 a; I94V oxidase activity <20% or <15% remaining oxidase active, or even more particularly GOxWT oxidase activity or on SEQIDNO: G0X-T30V 1 a; I94V oxidase activity <10% of the remaining oxidase activity.
[0145] 在本发明的另外特定方面，如SEQIDNO: 11至SEQIDNO: 17中给出的核酸分子的功能等价物/片段是相应的RNA分子，其由所述DNA序列或与SEQIDNO: 11至SEQ IDNO: 17的序列基本上互补的序列编码。 [0145] In a further particular aspect of the present invention, as SEQIDNO: 11 to SEQIDNO: functional equivalents / 17 fragment of a nucleic acid molecule is given in the corresponding RNA molecule consisting of the DNA sequence or SEQIDNO: 11 to SEQ IDNO : 17 the sequence is substantially complementary to a sequence encoding a.
[0146] 在本发明的上下文中，术语"RNA分子"意指核糖核巧酸分子的线性聚合物，其是单链的，且充当根据SEQIDNO: 3至SEQIDNO: 9的本文提供的(X)x变体的蛋白质合成模板。 [0146] In the context of the present invention, the term "RNA molecule" means a linear polymer of ribonucleic acid molecule Qiao, which is single-stranded, and acts according SEQIDNO: 3 to SEQIDNO: 9 provided herein (X) x variant protein synthesis template body.
[0147] 在本发明的进一步特定方面，同源性程度或百分比是与SEQIDNO: 3至SEQID NO: 9至少70%、或至少80%、或至少90%、或至少95%、或至少99%或100%，条件是它们显示出与说明书自始至终概述的相同的置换，并且进一步显示出如根据本发明的(X)x变体相同的特性，所述必需特性是酶活性、对于葡萄糖的酶特异性和显著降低的耗氧率和/或对于除氧外的特定介质显著增加的活性。 [0147] In a further particular aspect of the present invention, the degree or percentage of homology is SEQIDNO: 3 to SEQID NO: 9 is at least 70%, or at least 80%, or at least 90%, or at least 95% or at least 99% or 100%, with the proviso that they exhibit the same permutation outlined throughout the specification, and according to further exhibit the same as (X) x characteristic variant of the invention, the essential characteristic of enzymatic activity, an enzyme specific for glucose and significantly reduced the rate of oxygen consumption and / or for a specific media other than oxygen significantly increased activity.
[014引序列同一性可W通过BLAST算法，基本局部比对捜索工具（BLAST) /心fsc如5：F.等人1990.J.Mol.Biol. 215:403;AltschuLS.F.等人1997.NucleicAcidRes. 么J.•义议9-进似7进行测定。 . [014 W can be incorporated by BLAST sequence identity algorithm, Basic Local Alignment Tool Dissatisfied cable (BLAST) / Heart fsc as 5: F et al 1990.J.Mol.Biol 215:. 403; AltschuLS.F et al 1997 .NucleicAcidRes. J. • What justice agenda forward like 9- 7 were measured. 本文提及的氨基酸序列同一性百分比指通过所述BLAST算法测定序列同一性，其中在其上测定同源性的区域是本发明的(X)x变体的整个序列。 Amino acid sequence identity referred to herein refers to the percentage of sequence identity is determined by the BLAST algorithm, wherein a region of homology which is measured in the entire sequence (X) x variants of the present invention. 序列同一性可W通过本领域技术人员已知的用于序列比对和比较目的的任何其他方法进行测定。 W Sequence identity may be known by the skilled person for measurement of specific sequences and any other method for comparison purposes.
[0149] 本领域技术人员应当理解本文提供的(X)x变体可W具有或不具有不同于上述置换的进一步修饰，而不改变所述(X)x变体的必需特性，即如在本发明的意义中的对于葡萄糖的特异性、显著降低的耗氧率和/或对于除氧外的特定介质显著增加的活性。 [0149] It should be understood by those skilled in the art (X) x variants provided herein may be different from W, with or without the substitution further modified without changing the essential characteristics (X) x variant, i.e., as in in the sense of the present invention on glucose specificity, significantly reduced the rate of oxygen consumption and / or for a specific media other than oxygen significantly increased activity.
[0150] 本发明的另一个特定方面是经分离的多核巧酸，其编码根据本发明的(X)x变体或其活性等价物/片段。 [0150] Another particular aspect of the present invention is a polynuclear clever acid isolated which encoded according to (X) x variant of the present invention or an active equivalent / fragment. 经分离的多核巧酸可W是编码如部分-序列表中明确列出的下述序列（即沈QIDNO: 11至沈QIDNO: 17)的DNA或RNA分子或其相应基因。 Qiao isolated multicore acid encoding a part of W is - following sequences listed in the Sequence Listing clear (i.e., Shen QIDNO: 11 to Shen QIDNO: 17) DNA or RNA molecules or their corresponding genes.
[0151] 在另一个特定方面，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，通过在30°C至47°C范围内具有至少70%特别是至少80%的残留酶活性，由本发明提供的(X)x变体显示出溫度稳定性。 [0151] In another particular aspect, when measured by the assay medium as in Materials and Methods section entry ff) outlined by between 30 ° C in a 47 ° C range with at least 70% especially at least 80% the residual enzymatic activity, (X) x variants provided by the present invention exhibit temperature stability.
[0152] 在另一个特定方面，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，通过在48°C至60°C范围内具有至少10%的残留酶活性，由本发明提供的(X)x变体显示出溫度稳定性。 [0152] In another particular aspect, when measured by the assay medium as in Materials and Methods section entry ff) outlined by the 48 ° C to 60 ° C with a range of at least 10% residual activity, (X) x variants provided by the present invention exhibit temperature stability.
[0153] 本发明的(X)x变体可多种形式提供，例如作为冷冻干燥试剂或适当的胆存溶液中的溶液。 [0153] The present invention (X) x variants may be provided in various forms, for example as freeze-dried reagent or a solution of the appropriate solution bladder storage.
[0154] 本文提供的(X)x变体占优势地预期用于在更准确的糖尿病血糖测试装置中应用。 [0154] (X) x variant provided herein is predominantly intended for a more accurate blood glucose test device application diabetes. 具体地，仅通过显著降低的耗氧率，本发明的氧不依赖性(X)x变体利用氧用于电子转移，运意指当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，对于氧的残留G化活性是《30%、或< 25%、或< 20%，特别是< 15%、或《10%。 In particular, only by significantly reducing the oxygen consumption rate, oxygen-independent invention (X) x variants with oxygen for the electron transfer, when the Materials and Methods section) by the transport means, such as entry outlined gg when the ABTS assay was measured for residual oxygen activity of G is "30%, or <25% or <20%, in particular <15% or <10%. 相应地，显著量的葡萄糖在用于更准确的血糖测量的介质相关反应中反应，而与血样中的溶解氧含量的反应显著降低。 Accordingly, significant amounts of glucose in the reaction the reaction medium used to more accurately related to blood glucose measurement, react with the dissolved oxygen content of the blood sample is significantly reduced. 当动脉、静脉或毛细血管血液是用于测量的来源，或在超过海平面的不同高度测量血糖浓度的情况下时，运对于糖尿病患者是有利的。 When the arterial, venous or capillary blood is a source of measurement, or at different height of the case of measuring blood glucose concentration than sea level, operation is advantageous for diabetics.
[0155] 在另一个方面，本发明提供了(X)x变体，其显示出显著降低的耗氧率和/或对于除氧外的电子介质显著增加的介质活性，运也导致糖尿病患者由其获益的更准确的血糖测量。 [0155] In another aspect, the present invention provides a (X) x variant which exhibits significantly reduced activity in the medium of oxygen and / or oxygen to the outer electron mediator is significantly increased, by the operation also leads to diabetes a more accurate measure their blood sugar benefit.
[0156] 定义在本说明书的上下文中，表达"氧依赖性的"和"氧依赖性"指示：当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，特征在于残留氧活性〉30%的(X)x活性。 [0156] Definitions In the context of this specification, the expression "oxygen-dependent" and "oxygen-dependent" indication: When measured by the ABTS assay Materials and Methods The entry under gg) outlined, characterized in that the residual the oxygen activity> 30% (X) x activity.
[0157] 相比之下，表达"氧不依赖性的"和"氧不依赖性"指示：当通过如条目gg)下的材料与方法部分中概述的ABTS测定进行测定时，特征在于残留氧活性《30%、或< 25%、或< 20%，特别是< 15%、或< 10%的GOx活性。 [0157] In contrast, the expression "oxygen-independent" and "oxygen-independent" indicator: When measured by the ABTS assay Materials and Methods The entry under gg) outlined, characterized in that the residual oxygen active "30%, or <25% or <20%, in particular <15% or <10% GOx activity.
[015引在本说明书的上下文中，表达"对于葡萄糖非特异性"指示：当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，对于底物葡萄糖< 100%、或< 99. 9%、或< 99.5%的(X)x活性。 [015 incorporated in the context of this specification, the expression "for glucose non-specific" indicates: When measured by dielectric measurement The Materials and Methods section entry under ff) outlined for the substrate glucose <100%, or < 99.9%, or <99.5% (X) x activity. 言下之意，当通过如条目ff)下的材料与方法部分中概述的介质测定进行测定时，"对于葡萄糖非特异性的"G化显示出对于除葡萄糖外的糖(例如半乳糖、麦芽糖、木糖和麦芽S糖）〉6%的(X)x活性。 This implies, when measured by the measuring medium such as in the Materials and Methods section entry ff) as outlined in the "non-specific for glucose" G shows the outer addition of glucose sugars (e.g. galactose, maltose, malt sugar xylose and S)> 6% (X) x activity.
[0159] 与本发明的0化变体有关的术语"经修饰的"或"修饰"指0化蛋白质，除根据SEQ IDNO: 1的两个置换T30V和I94V之外，其含有在根据SEQIDNO: 1的多肤序列中，在位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560中的至少一个另外的氨基酸置换。 [0159] 0 and the variants of the present invention is related to the term "modified" or "modification" refers to a protein 0, except under SEQ IDNO: T30V substitution than two and I94V 1, containing according SEQIDNO: multi skin in sequence, the position S53; A137; A173; A332; at least one additional amino acid substitutions and the V560; F414. 该术语还指示编码此类"经修饰的"G化变体的各自多核巧酸或其序列保守变化。 The term also indicates encoding such a "modified" G polynuclear variants of each or a clever acid sequence conserved changes.
[0160] 多核巧酸序列的"序列保守变化"是其中给定密码子位置中的一个或多个核巧酸的变化导致在该位置处编码的氨基酸不改变。 [0160] "Conservative variations sequence" polynuclear clever acid sequence in which one or more cores for a given change in coincidence acid codon position results in no change in the amino acid encoded at that position.
[0161] 术语"G化变体"指示根据本发明的葡萄糖氧化酶具有根据SEQIDNO: 1的两个氨基酸置换T30V和I94V，W及在根据SEQIDNO: 1的多肤序列中，在位置S53 ;A137 ; A173 ;A332 ;F414 ;和V560中的至少一个另外的氨基酸置换，特征在于显示出对于底物葡萄糖的酶特异性，W及显著降低的耗氧率和/或对于除氧外的特定介质显著增加的活性。 [0161] The term "G variants" indicates SEQIDNO accordance with the present invention according to the glucose oxidase: 1 two amino acid substitutions T30V and I94V, W and in accordance SEQIDNO: Multi skin in sequence, the position S53; A137 ; A173; A332; F414; and at least one additional amino acid substitution of V560, characterized in that the enzyme exhibits specificity for, W, and significantly reduced the rate of oxygen consumption of the substrate glucose and / or oxygen to the particular outer dielectric significant increased activity.
[0162] 应当理解本说明书自始至终的氨基酸序列的编号在初始S(Ser、丝氨酸)处开始，如根据SEQIDNO: 1的成熟G0X-T30V;I94V蛋白质中。 [0162] It should be understood that the amino acid sequence numbered throughout this specification starts at the initial S (Ser, serine), such as in accordance SEQIDNO: The mature G0X-T30V 1; I94V protein.
[0163] 依照本发明的术语"酶"意指整个或大部分由蛋白质或多肤组成的任何物质，其或多或少特异性地催化或促进一种或多种化学或生物化学反应。 [0163] In accordance with the present invention, the term "enzyme" means any substance or most of the whole protein or polypeptide composed of more or less specifically catalyze or facilitate one or more chemical or biochemical reactions. 术语"酶"还可W指催化多核巧酸(例如RNA或DNA)。 The term "enzyme" also refers to a catalytic polynuclear W Qiao acid (e.g. RNA or DNA).
[0164] 术语"氧化反应"在一般术语中意指设及对底物添加氧的化学或生物化学反应，W 形成加氧或氧化底物或产物。 [0164] The term "oxidation reaction" means in general terms is provided for the substrate and adding oxygen chemical or biochemical reactions, W plus oxygen or oxide formed substrate or product. 氧化反应通常伴随还原反应（因此，术语"氧化还原"反应包含氧化和还原两者)。 The reaction is generally accompanied by oxidation-reduction reaction (Thus, the term "redox" reactions containing both oxidation and reduction). 当化合物接受氧或丧失电子时，它是"氧化的"。 When the acceptance of an oxygen compound or loss of electrons, it is "oxidation." 当化合物丧失氧或获得电子时，它是"还原的"。 When the loss of an oxygen compound or gains electrons, it is "reduced." 6化通常催化伯醇基团氧化为醒。 6 of generally catalyzes oxidation of primary alcohol groups awake.
[016引如说明书自始至终使用的，术语"葡萄糖氧化酶"指定蛋白质，其催化0-D-葡萄糖氧化成D-葡糖酸-1,5-内醋（D-葡萄糖+ 02 -葡糖酸内醋+ &02)，其随后可W水解为葡糖酸。 [016 cited throughout the specification, the term "glucose oxidase" as specified protein, which catalyzes the oxidation of glucose to the 0-D- D- glucono-1,5-acetate (D- glucose + 02 - the gluconic acid vinegar + 02), which may then be hydrolyzed to gluconic acid W. 相应地，葡萄糖氧化酶是酶。 Accordingly, glucose oxidase is an enzyme. 进一步地，术语"具有葡萄糖氧化酶活性的多肤" 指具有上述活性的多肤。 Further, the term "glucose oxidase activity having a plurality of skin" means skin having the above multi-activity. 葡萄糖氧化酶可W缩写为"G化"或"EC 1.1. 3. 4"。 W glucose oxidase may be abbreviated as "G" or "EC 1.1. 3. 4". 如说明书自始至终使用的，术语"G化-WT或WT"指示真菌黑曲霉的野生型GOx。 As used throughout the specification, the term "G of -WT or WT" indicates the wild-type fungus Aspergillus niger GOx. 术语"G0X-T30V;I94V; 或T30VI94V;或双重突变体；或亲本突变体"指示具有根据SEQIDNO: 1的两个置换T30V 和I94V的(X)x变体，如Zhu等人（2006 ;2007)中所述。 The term "G0X-T30V; I94V; or T30VI94V; or double mutant; or parent mutant" indicates having According SEQIDNO: two substitutions T30v and I94V in (X) x variant 1, such as Zhu et al. (2006; 2007 ) in the.
[0166] 根据本发明的(X)x变体的确均具有共同的根据SEQIDNO: 1的两个置换T30V和I94V，W及在位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560的由根据本发明适合替换的剩余19种蛋白源氨基酸中任一种的至少一个另外的氨基酸置换。 [0166] The (X) x variant of the invention indeed have a common according SEQIDNO: T30V and two substitution of I94V 1, W, and at a position S53; A137; A173; A332; F414; and V560 by the present the remaining 19 kinds of the invention suitable for alternative protein sources of amino acids in any one of the at least one additional amino acid substitution. 说明书正文自始至终，分别的"EZ"编号可W缩写根据本发明的(X)x变体。 Throughout the foregoing description, each of the "EZ" W may be abbreviated number (X) x variant of the present invention. "EZ"代表酶。 "EZ" represents the enzyme.
[0167] 酶"葡萄糖氧化酶"因此是葡萄糖氧化酶类别的成员，其通过将氧从来源或供体添加、插入、促成或转移至底物，来催化氧化反应。 [0167] Enzyme "glucose oxidase" is thus a member of class of glucose oxidase, by adding oxygen or a source from the donor, inserting contributed or transferred to the substrate, to catalyze the oxidation reaction. 此类酶也称为氧化还原酶(oxidoreductases)或氧化还原酶（redoxenzymes),并且包含氧合酶、氨化酶或还原酶、氧化酶和过氧化物酶。 Such enzymes are also known as oxidoreductase (oxidoreductases) or oxidoreductase (redoxenzymes), and comprising oxygenase, reductase or ammonia enzymes, oxidases and peroxidases.
[0168] 在运点上，术语"氧供体"、"氧化试剂"和"氧化剂"意指物质、分子或化合物，其在氧化反应中将氧贡献给底物。 [0168] In the operation point, the term "oxygen donor", "oxidation agent" and "oxidizing agent" refers to a substance, molecule or compound, its contribution to the oxygen in the substrate oxidation reaction. 通常，氧供体被还原(接受电子)。 Typically, the oxygen donor is reduced (accepts electrons). 示例性氧供体包括例如分子氧或双氧（〇2)，并且过氧化物包括例如烷基过氧化物例如叔下基过氧化物，且最优选过氧化氨（&〇2)。 Exemplary comprises oxygen donor such as molecular oxygen or oxygen-bis (〇2), and, for example, peroxides including alkyl peroxides such as t-butyl peroxide under, and most preferably ammonia peroxide (& 〇2). "过氧化物"是具有彼此结合的两个氧原子的任何化合物。 "Peroxides" is any compound having two oxygen atoms bonded to each other.
[0169] 如所述的，葡萄糖氧化酶或如本文使用的(X)x变体的"活性"或"酶活性"设及其催化氧化反应D-葡萄糖+化一葡糖酸内醋+&化的能力的量度，并且可W表示为反应产物在其下产生的速率。 [0169] As described, "activity" or "activity" such as glucose oxidase, or (X) x variants provided herein and using catalytic oxidation of glucose + D- gluconic acid in a vinegar + measure of the ability of, and may be a reaction product W represents the rate at which generated. 例如，葡萄糖氧化酶活性可W表示为每单位时间产生的产物(葡糖酸内醋和/或&〇2)量，或每单位(例如浓度或重量）葡萄糖氧化酶。 For example, glucose oxidase activity can be generated for the W represents a product per unit time (the vinegar gluconic acid and / or & 〇2) amount, or per unit (e.g., concentration or weight) of glucose oxidase.
[0170] 一般而言，本发明的多肤在本文中也可W被称为"突变体"、"突变蛋白"或"变体" 或"经修饰的GOx"或"修饰"，意指它已由黑曲霉(X)x-WT自身W及根据SEQIDNO: 1的G化-T30V;I94V制备、改变、衍生或W某一方式不同或变化。 [0170] In general, the present invention is a multi-skin herein may also be referred to as W "mutant", "mutant protein" or "variant" or "the GOx modified" or "modification", which means Aspergillus niger by the (X) x-WT itself and W according SEQIDNO: G of -T30V 1; the preparation of different or varying I94V, altered, derived, or a W mode.
[017。 [017. 黑曲霉的(X)x-WT蛋白质包含根据SEQIDNO: 2的氨基酸的天然序列。 Aspergillus niger (X) x-WT protein comprising the SEQIDNO: 2 amino acid native sequence. 相应地， "亲本突变体"或"双重突变体"是使用本文描述的任何方法、工具或技术，本文提供的任何其他(X)x多肤由其衍生或制备的(X)x多肤。 Accordingly, "parent mutant" or "double mutant" is any other (X) x multiple skin derived or prepared from (X) x multiple skin using methods described herein any of the methods, tools or techniques provided herein. 依照本发明，"亲本突变体"或"双重突变体"是根据SEQIDNO: 1的多肤。 According to the present invention, "parent mutant" or "double mutant" is a SEQIDNO: a plurality of skin.
[017引因此，"亲本多核巧酸"是编码亲本多肤的那种，即根据SEQIDNO: 10的多核巧酸。 [017 cited Thus, "parent polynuclear clever acid" encoding the parent plurality skin kind, i.e. according SEQIDNO: 10 clever multicore acid.
[0173] 术语"突变"或"变化"或"修饰"意指遗传材料例如DNA或RNA中的任何可检测的变化，或此类变化的任何过程、机制或结果。 Any detectable change in DNA or RNA, for example, [0173] The term "mutation" or "change" or "modification" is meant genetic material, or any process of such variations, mechanism, or result. 运包括其中基因的结构(例如DNA或RNA序列） 改变的基因突变，起于任何突变过程的任何基因或DNA或RNA，W及由经修饰的基因或DNA 序列表达的任何表达产物(例如蛋白质或多核巧酸)。 Operation including mutation in which structural gene (e.g., DNA or RNA sequence) altered, any gene or DNA or RNA, W is and any expression product expressed by a modified gene or DNA sequence from any mutation process (e.g., a protein or Qiao polynuclear acid). 此类变化还包括启动子、核糖体结合位点等中的变化。 Such changes include changes in a promoter, ribosome binding site and the like.
[0174] 在本说明书的上下文中，表达"除氧外的某些介质"、"除氧外的某些电子介质"、 "某些固定的介质"和"除氧外的某些电子受体"指示选自下述的介质/电子介质：亚硝基苯胺及其衍生物、偶氮化合物、吩嗦及其衍生物、吩嚷嗦及其衍生物、吩嗯嗦及其衍生物、二茂铁及其衍生物、铁氯化钟、Ru-和Os-络合物、酿及其衍生物、吗I噪酪、紫精、四硫富瓦締及其衍生物和献菁。 [0174] In the context of the present specification, the expression of certain outer electron acceptor "certain media other than oxygen," "certain electron mediator other than oxygen," "certain fixed media" and "oxygen "indicates selected from medium / electron mediator: nitrosoaniline and derivatives thereof, azo compounds, and derivatives thereof winded thiophene, thiophene cried winded and derivatives thereof, thiophene and derivatives thereof winded ah, ferrocenium iron and its derivatives, ferric chloride, bell, and Os- Ru- complexes, stuffed and derivatives thereof, casein noise do I, viologen, tetrathiafulvalene and its derivatives offer associative phthalocyanine.
[0175] 相应地，依照本发明的表达"除氧外的某些介质"、"除氧外的某些电子介质"、"某些固定的介质"和"除氧外的某些电子受体"还指示电子介质，其通过FADH2快速还原但通过抗坏血酸缓慢还原或不还原，例如2-[4-(二甲基氨基）苯基]-二氮締甲酯胺的衍生物。 [0175] Accordingly, in accordance with the present invention, the outer expression "certain media other than oxygen," "certain electron mediator other than oxygen," "certain fixed medium" and "certain electron acceptor oxygen "also indicates an electronic medium, which is reduced by the fast or slow reduction FADH2 but not reduced by ascorbic acid, such as 2- [4- (dimethylamino) phenyl] - methyl-amine nitrous associated derivatives.
[0176] 缩写AA-氨基安替比林HRP-辣根过氧化物酶G化-葡萄糖氧化酶G化-WT或WT-真菌黑曲霉的野生型GOx GDH-葡萄糖脱氨酶ABTS- 2, 2'-联氮-双（3-乙基苯并嚷挫嘟-6-横酸） BTO-憐钢酸。 [0176] Acronym AA- amino antipyrine of HRP- horseradish peroxidase G - G of glucose oxidase -WT WT- or fungus Aspergillus niger wild-type glucose GOx GDH- deaminase ABTS- 2, 2 '- azino - bis (3-ethyl-benzo cried setback beep -6- cross acid) BTO- acid pity steel.
[0177] 附图描述图1 图1显示了本文提供的(X)x变体对氧的活性测定的原理：在(X)x和HRP的氧化半反应期间产生的&〇2用于氧化生色底物ABTS。 [0177] DESCRIPTION OF THE DRAWINGS Figure 1 shows the principle of (X) x variant provided herein is measured active oxygen: during the (X) x and semi-HRP oxidation reaction for oxidizing Health & 〇2 chromogenic substrate ABTS. ABTS的变色通过在414皿处的吸收进行监控。 ABTS discoloration by monitoring the absorption at 414 dish. 该原理构成如条目gg)下的材料与方法部分中概述的ABTS测定的基础。 This principle forms the basis ABTS as measured under the Materials and Methods section entry gg) outlined.
[017引图2 图2描述了用于检测介导的电子转移的介质测定或PM0测定(憐钢酸)。 [017 Primer Figure 2 describes a medium for the detection of electron transfer mediated assay or assay PM0 (pity steel acid). 介质化合物在G化的还原半反应期间在两步中变得还原。 Compound G of the medium during the reduction half-reaction in two steps becomes reducing. 介质将两个电子转移至氧化还原指示剂PM0,其随后变得被还原。 The medium was transferred to a two electron redox indicator PM0, which then is reduced becomes. 在还原期间的PM0变色通过在700nm处的吸收进行监控。 PM0 discoloration during the reduction is monitored by absorption at 700nm.
[0179]图3 图3显示了在根据本发明的蛋白质结构中用于氨基酸置换的六个位置的不同定位。 [0179] Figure 3 shows the different positions for the positioning of six amino acid substitutions in the protein structure according to the present invention.
[0180] 图4 图4显示了根据本发明的不同(X)x变体的耗氧率：a)根据时间的相对氧浓度的进展；b)相对耗氧率/分钟。 [0180] Figure 4 shows a different oxygen consumption rate of the present invention, (X) x variants: a) the relative concentration of oxygen according to the progress of time; b) relative to the oxygen consumption rate / min. 测定实现在条目hh)下的材料与方法部分中描述。 Is described under entry determined implemented hh) Materials and Methods section. 酶标准化至2U/ L。 Normalized to enzyme 2U / L.
[0181] 图5 图5描述了根据本发明的不同(X)x变体的米曼二氏（Michaelis-Menten)动力学。 [0181] FIG 5 depicts Miman Barre (X) x variant (Michaelis-Menten) depending on the kinetics of the present invention. 对于活性测定，用于表征的介质测定如条目ff)下的材料与方法部分中概述的应用。 For the activity assay medium for characterization applications in the Materials and Methods section, such as entry ff) outlined by. 虚线在MicrosoftExcel中应用最小二乘法进行计算。 MicrosoftExcel broken line in the least square method was calculated.
[0182] 图6 图6显示了根据本发明的不同0化变体的热稳定性特性。 [0182] Figure 6 Figure 6 shows the thermal stability characteristics of the present invention depending on the 0 variant. 测定如条目ii)下的材料与方法部分中概述的执行。 The assay performed in the Materials and Methods section entry ii) outlined. 作为另外的对照，使用去糖基化和高糖基化的GOx。 As an additional control, the use of deglycosylated and high glycosylated GOx.
[0183] 图7 图7显示了（X)x-WT、G0X-T30V;I94V和本发明的0化变体V(即除置换T30V;I94V之夕1'，4173¥;43325;。4141;¥5601')的相对耗氧率。 [0183] Figure 7 Figure 7 shows the (X) x-WT, G0X-T30V; 0 and V I94V variants of the present invention (i.e. other substitutions T30V; I94V Eve 1 ', 4173 ¥; 43325; .4141; ¥ 5601 ') relative oxygen consumption rate. 测定实现在条目化）下的材料与方法部分中描述。 Assay described in the Materials and Methods implement portion) of the entry in. 酶标准化至1. 7mg/L。 Enzyme normalized to 1. 7mg / L.
[0184] 图8 图8显示了与葡萄糖相比较，G化-WT、G化-T30V;I94V和0化变体EZ07对不同糖的残留活性。 [0184] Figure 8 Figure 8 shows a comparison with glucose, G of -WT, G of -T30V; EZ07 residual activity of different sugars and 0 I94V variants. 对于残留活性测定，用于表征的介质测定如条目ff)下的材料与方法部分中描述的应用。 Assay for residual activity, the medium for characterization applications under the Materials and Methods section, such as entry ff) measurement as described. 底物浓度在反应混合物中为181. 8ml。 Substrate concentration in the reaction mixture is 181. 8ml.
[0185] 图9 图9 显示了G0x-WT、G0x-T30V;I94V和GOx变体EZ07 的酶参数。 [0185] Figure 9 Figure 9 shows G0x-WT, G0x-T30V; EZ07 enzyme parameters of GOx and I94V variants. 应用MicrosoftExcel中的最小二乘法，根据米曼二氏动力学来计算参数。 Application of the least square method MicrosoftExcel The Miman Jakob kinetic parameters calculated. 对于活性测定，用于表征的介质测定如条目ff)下的材料与方法部分中描述的应用。 For the activity assay medium for characterization applications in the Materials and Methods section, such as entry ff) measurement as described.
[0186] 图10 图10显示了在25至67 °C之间的不同溫度下的15分钟溫育后，G化-WT、G化-T30V; 194V和(X)x变体EZ07的残留活性。 After [0186] FIG 10 FIG 10 shows a 15 min incubation at different temperatures between 25 to 67 ° C, G, of -WT, G of -T30V; 194V, and (X) x body variant residual activity EZ07 .
[0187] 序列描述多肤序列 [0187] sequence sequence descriptions plurality skin
[018引在下述实施例部分，除非指定其他商业来源，否则所有试剂、限制性酶及其他材料均得自RocheDia即osticsGermany,并且根据由供应商给出的说明书使用。 [Examples section 018 cited in the following examples, unless other commercial sources specified otherwise, all reagents, restriction enzymes and other materials were obtained from RocheDia i.e. osticsGermany, and used according to the instructions given by the supplier. 用于DNA纯化、 表征和克隆的操作和方法是本领域众所周知的幻化掌yl，in"O/rre別protocol i打曲oiecu缸r如'0知斜"""化也>，於7巧"7，并且可W如由技术人员要求的进行修改。 实施例 The method of operation and DNA purification, characterization and cloning are known in the art for the palm turned yl, in "O / rre not hit song protocol i oiecu cylinder r as' known swash 0" "" is also of> at 7 clever " 7, and W can be modified as required by the skilled person. Example
[0189] 本发明将通过下文实施例详细举例说明，尽管本发明不应限于运些具体实施例。 [0189] The present invention will be illustrated in detail below by way of illustration, some specific examples, while the present invention should not be limited to transportation.
[0190] 本发明的核屯、在于出乎意料和令人惊讶的特定(X)x变体的发现，所述特定(X)x变体显示出超过血糖测量领域中的已知(X)x的改善特性，即维持其对于底物葡萄糖的特异性，并且经由从氧化酶活性朝向脱氨酶活性的转变而显著降低耗氧率和/或对于除氧外的特定电子介质具有增加的活性。 [0190] Nuclear Tun of the present invention is to find a particular (X) X variants unexpectedly and surprisingly, the particular (X) X variants showed over the known art blood glucose measurement (X) x improve the characteristics, i.e., maintain their specificity for the substrate glucose, and the oxidase activity through the transition from the orientation of the deaminase activity is significantly reduced oxygen consumption and / or for a particular electron mediator other than oxygen having an increased .
[0191] 为了证明依照本发明的(X)x变体的(X)x特性，本发明人不依赖于大多数指示分子氧还原为过氧化氨的可用活性测定。 [0191] In order to prove (X) x characteristic in accordance with (X) x variant of the present invention, the present invention does not rely on the reduction of molecular oxygen is most indicative of excessive oxidation of ammonia available activity assays. 具体地，对于介导的电子转移，Zhu等人（2006 ; 2007)建立了基于产物的GOx检测测定（下文G0DA)倍如，么，ifo曲細，C，Zs左AsrfseK,瓜游Schwaneberg,U. (2006).Makingglucoseoxidasefitforbiofuelcellapplications bydirectedproteinevolution.BiosensorsandBioelectronics21,2046-2051;Zhu, Z.，Wang，M. 'Gautam,A.，Nazor，J.，Momeu，C.，R，P.，andU，S. (2007).Directedevolution ofglucoseoxidasefromAspergillusnigerforferrocenemethanol-mediated eiec化w皆<ms/kr.仿•〇祐cAno的巧^ Joyraai之，之如-之做7。 Specifically, for mediated electron transfer, Zhu et al. (2006; 2007) established GOx detection assay product (hereinafter G0DA) based on times, such as, what, IFO song fine, C, Zs left AsrfseK, melon travel Schwaneberg, U . (2006) .Makingglucoseoxidasefitforbiofuelcellapplications bydirectedproteinevolution.BiosensorsandBioelectronics21,2046-2051;. Zhu, Z., Wang, M 'Gautam, A., Nazor, J., Momeu, C., R, P., andU, S (2007. ) .Directedevolution ofglucoseoxidasefromAspergillusnigerforferrocenemethanol-mediated eiec of w are <ms / kr imitation • square woo cAno ^ Joyraai of clever, as the - of 7 do. 该测定事实上允许葡糖酸内醋的氧不依赖性检测，但在氧的存在下，仍然不能特异性检测通过除氧外的介质介导的电子转移，运在实时血糖分析装置的情况下是高度相关的。 This fact allows the determination of a case where the oxygen-independent gluconic acid, vinegar detected, but in the presence of oxygen, it can not detect specific mediated electron transfer medium through the outer oxygen and glucose transport in real-time analysis device They are highly correlated.
[0192] 在下述实施例中，本发明人因此测试了本发明的特定(X)x变体，分别对于其介导的电子转移、通过氧的电子转移及其葡萄糖特异性，W证明本发明的核屯、。 [0192] In the following examples, the present invention is therefore a specific test (X) x variant of the invention, for which are mediated electron transfer, electron transfer by oxygen and glucose specificity, W of the present invention is demonstrated the nuclear village. 进一步地，还测试了在热稳定性方面的特性。 Furthermore, the properties were also tested in terms of thermal stability.
[019引因此，本发明人使用众所周知的ABTS测定/"况/打，Z.，化妃r, 7.，心caiofe,瓜， Petrounia,IP和Arnold,F.比（2002).Modificationofgalactoseoxidaseto introduceglucose6-0xidaseactivity.Chembiochem3，781-783],Ay壶觀GQ'l咬形巧氧的活性。图1显示了对氧的活性测定的原理。 [019 cited Accordingly, the present invention is measured using well known ABTS / "condition / play, Z., Princess of r, 7., heart caiofe, melon, Petrounia, IP and Arnold, F. Ratio (2002) .Modificationofgalactoseoxidaseto introduceglucose6- 0xidaseactivity.Chembiochem3,781-783], Ay pot-shaped bite clever concept GQ'l active oxygen. Figure 1 shows the principle of measurement of reactive oxygen species.
[0194] G化变体的测试为了在葡萄糖特异性、降低的耗氧率和/或对于除氧外的特定介质增加的活性方面， 就其对6化特性的改善测试适当的氨基酸置换，测试本发明的特定0化变体。 [0194] Test G variants In order to glucose specificity, reduce the oxygen consumption rate and / or for a particular medium outside increased activity of oxygen, to improve its characteristics of 6 amino acid substitutions suitable test, Test 0-specific variants of the present invention.
[019引（i)对于其耗氧率，即酶促活性，在比色ABTS测定中使用氧作为电子受体，和(ii) 对于通过亚硝基苯胺介质作为示例性介质介导的电子转移， (iii) 在不同糖作为底物的存在下（即葡萄糖、半乳糖、麦芽糖、木糖和麦芽=糖)，对于通过亚硝基苯胺介质作为示例性介质介导的电子转移。 [019 primer (i) for which the oxygen consumption rate, i.e. the enzymatic activity determined colorimetrically using ABTS oxygen as an electron acceptor, and (ii) to the medium as a by-nitrosoaniline exemplary electron transfer mediators , (iii) at different sugars as a substrate is present (i.e., glucose, galactose, maltose, xylose and = malt sugar), for the medium as a by nitrosoaniline exemplary electron transfer mediators.
[0196] 对于所述介导的电子转移，使用对亚硝基苯胺化合物/5ec知。 [0196] For the dielectric mediated electron transfer using p-nitroaniline compound / 5ec art. 0. 分.化e Glucose-Dye-OxidoreduktaseinderklinischenDiagnostik.DISSERTATIONthesis， TechnischeUniversitatKaiserslautern,Kaiserslautern]。 0.5 min. Of e Glucose-Dye-OxidoreduktaseinderklinischenDiagnostik.DISSERTATIONthesis, TechnischeUniversitatKaiserslautern, Kaiserslautern]. 图2 显示了介质巧ij定，良P PMO(憐钢酸）测定的原理，其中应用亚硝基苯胺介质N，N-双（2-径乙基）-4-亚硝基苯胺。 Figure 2 shows a clever ij given medium, the principles of good P PMO (pity steel acid) determined, wherein the application medium nitrosoaniline N, N- bis (2-diameter ethyl) -4-nitroaniline.
[0197] 在上述测定中，来自介质反应的两个电子转移至PM0。 [0197] In the above assay, two electrons from the reaction medium is transferred to PM0. PM0随后变得被还原。 PM0 subsequently came to be restored. 通过在700nm处的吸收，来监控从氧化PM0的淡黄色到还原PM0的深蓝色的变色。 By absorption at 700nm, monitored from pale yellow to deep oxidation reduction PM0 to blue discoloration of PM0.
[019引为了证明依照本发明的特定(X)x变体，上述介质测定修改为酵母细胞上清液中的低(X)x活性，其显示在憐酸钟缓冲液（0. 2M，pH7. 0)中从0. 65U/L到22U/L的广泛线性检测范围。 [019 In order to demonstrate primer (X) x variant, the medium was measured in accordance with certain modifications of the present invention is low (X) x activity of yeast cell supernatants, which shows the clock pity acid buffer (0. 2M, pH7 0) from 0. 65U / L to 22U / L wide linear range. 在所述条件下达到在96孔板上10. 2%的标准差。 Under the conditions to achieve a standard deviation of 10.2% in 96 well plates. 对于标准差的计算，通过扣除阴性对照的值考虑本底(真标准差)。 For the standard deviation is calculated by deducting the value of the negative control background considerations (true standard deviation).
[0199] 葡萄糖氧化酶变体依照本发明，选择Zhu等人（2006 ;2007)中所述的双重突变体作为用于本文提供的特异性氨基酸置换的原材料。 [0199] Glucose oxidase variants according to the invention, selected Zhu et al. (2006; 2007) of the starting material for the double mutant as provided herein specific amino acid substitutions. 运种双重突变体具有固有地存在的两个置换T30V和I94V (SEQIDN0:1)。 Transport two types of double mutants having substitutions T30V and I94V (SEQIDN0: 1) inherently present. （X)x-T30V;I94V显示改善的热稳定性、改善的抑稳定性W及增加的kcat 喧X议!.5/5量_\?>].]/s) [Zhu,Z.，Wang,M.，Gautam,A.，Nazor,J.，Momeu,C.，R,R，and U，S. (2007).DirectedevolutionofglucoseoxidasefromAspergillusnigerfor ferrocenemethanol-mediatedelectrontransfer.BiotechnologyJounal2,241-248]。 (X) x-T30V; I94V display improved thermal stability, improved stability W suppressing noise and increased kcat .5 X Protocol / an amount of 5 _ \>]] / s) [Zhu, Z,!?.. wang, M., Gautam, A., Nazor, J., Momeu, C., R, R, and U, S. (2007) .DirectedevolutionofglucoseoxidasefromAspergillusnigerfor ferrocenemethanol-mediatedelectrontransfer.BiotechnologyJounal2,241-248].
[0200] 选择高糖基化的酿酒酵母菌株作为表达宿主/ZeAie，Z.，仿'沁打，乂，ZeA打erf，jf.， Haselbeck,A.矛口Kopetzki,E. (1995).GlycoproteinbiosynthesisinSaccharomyces cerevisiae:ngd29,anN-glycosylationmutantallelictoochlhavingadefectin theinitiationofouterchainformation.FEESLett370，41-45]。 [0200] Select hyperglycosylated S. cerevisiae strain as expression host / ZeAie, Z., Imitation 'play Qin, qe, Zea playing erf, jf., Haselbeck, A. Lance mouth Kopetzki, E. (1995) .GlycoproteinbiosynthesisinSaccharomyces cerevisiae: ngd29, anN-glycosylationmutantallelictoochlhavingadefectin theinitiationofouterchainformation.FEESLett370,41-45].
[0201] 在具有依照本发明的特定氨基酸置换的细胞克隆培养后，平行应用介质测定和ABTS测定。 [0201] After cell clones were cultured in accordance with the present invention specific amino acid substitutions, measured parallel application media and ABTS assay. 选择显示增加的介质活性、降低的氧活性或两者的克隆用于进一步研究。 Select media show increased activity, reduced activity, or both oxygen clones for further study. 因此， 将所选克隆和对照培养且筛选12次。 Accordingly, the selected clones and screening control cultures and 12 times. 随后，将依照本发明的改善变体的(X)x基因分离且测序。 Subsequently, (X) x in accordance with the present invention for improving the gene variants isolated and sequenced. 其后，所选变体的所述基因充当用于进一步的特定氨基酸置换的模板。 Thereafter, the selected gene variants act as a template for further specific amino acid substitutions.
[020引氨基酸位置的表征本发明人进一步表征了本文提供的(X)x变体的特定氨基酸位置，即S53 ;A137 ;A173 ;A332 ;F414 ;和V560。 [Cited amino acid position 020 of human characterize the invention are further characterized by a specific amino acid position (X) x variants provided herein, i.e., S53; A137; A173; A332; F414; and V560. 运些位置在图3中描述。 These transport position depicted in FIG.
[0203] 预表征为了进一步研究特定(X)x变体，本发明人考虑下述特性进行预表征： 1) 耗氧量2) 对于作为底物的葡萄糖的特异性3) 米曼二氏动力学(介质活性/葡萄糖亲和力）和4) 热稳定性。 [0203] To further investigate the specific pre-characterized (X) x variant, the present invention contemplates the following characteristics pre-characterized: 1) 2 oxygen consumption) for glucose as substrate specificity 3) Miman Barre power Science (medium activity / affinity for glucose), and 4) thermal stability.
[0204] 相应的细胞克隆的培养在常规摇瓶中发生。 [0204] culturing the respective cell clones occurs in conventional shake flasks. 将上清液浓缩且在0. 2M憐酸钟，pH 7中缓冲。 The supernatant was concentrated and buffer acid pity bell 7, pH in 0. 2M.
[0205] 1)耗氧率如下文材料与方法部分中的条目gg)下概述的，通过生色底物ABTS的氧化间接测定耗氧量。 Gg outlined under entry packet in the Materials and Methods section) and indirect measurement of oxygen consumption by the oxidation of a chromogenic substrate ABTS [0205] 1) the oxygen consumption rate is as follows. 为了证明该数据，使用光学氧探针直接测量耗氧量。 To prove this data, an oxygen probe is measured directly using an optical oxygen consumption. 6化变体在反应方法中标准至2 U/L。 6 variants in the reaction process to a standard 2 U / L.
[0206] 2)特异性如上文已概述的，G化对于底物葡萄糖的特异性在酶促葡萄糖测定中起主要作用，因为存在紧靠葡萄糖在临床上相关的糖，例如四种糖：半乳糖、麦芽糖、木糖和麦芽=糖。 [0206] 2) As already outlined specificity, G of the substrate glucose to play a major role in the specificity of an enzymatic glucose assay, glucose because there against clinically related sugar, for example, four kinds of sugars: Semi lactose, maltose, xylose and = malt sugar. 因此， 为了证明本文提供的(X)x变体的特异性，选择所述糖且与葡萄糖相比较。 Accordingly, in order to prove specificity (X) x variants provided herein, selecting and compared to the sugar and glucose. 对于运些研究，使用介质测定，应用181.8ml每种糖。 For transport of these studies, the medium was measured, the application 181.8ml each sugar.
[0207] 3)介质活性/葡萄糖亲和力关于介质活性的更详细研究和葡萄糖亲和力的测定，本发明人应用本文公开的变体关于米曼二氏动力学的测试。 [0207] 3) activity assay medium / glucose medium affinity for a more detailed study of the activity and the affinity for glucose, the present invention is applied herein disclosed variants tested on two Miman's dynamics.
[020引4)热稳定性应用如条目ff)下的材料与方法部分中概述的介质测定，分析本发明的所选(X)x变体的热稳定性。 Medium measurement section under Materials and Methods [020 Primer 4) thermal stability of applications such as entry ff) outlined, thermal stability analysis selected according to the invention (X) x variant. 作为另外的对照，还分析在黑曲霉中表达的高糖基化的(X)x和去糖基化的GOx-WT。 As an additional control, further analysis of expression in Aspergillus niger highly glycosylated (X) x and the deglycosylated GOx-WT.
[0209] G化变体G0X-EZ07的最终表征对于最终表征，各自的G0X-EZ07表达酿酒酵母菌株在10L发酵罐中进行培养，随后纯化所获得的G0X-EZ07。 [0209] G variants G0X-EZ07 final characterization of the final characterization respective G0X-EZ07 expression of the Saccharomyces cerevisiae strain is cultured in 10L fermentor, then purified G0X-EZ07 obtained. 最终酶制剂的纯度在50ml憐酸钟缓冲液抑7中高于90%。 Purity of the final enzyme preparation of the acid in 50ml pity clock buffer 7 in greater than 90% inhibition. 如预表征时期中，研究耗氧量、特异性、活性和葡萄糖亲和力。 The period pre-characterized, oxygen consumption study, specificity, activity and affinity for glucose. 对于(X)x浓度的特异性测定，应用ELISA(参见条目ee下的材料与方法部分)。 Specific assay for (X) x concentration, application of ELISA (see Materials and Methods section entry under ee).
[0210] 变体G0X-EZ07也在下述方面进行标准： la)耗氧量2a)对于作为底物的葡萄糖的特异性3a)米曼二氏动力学(介质活性/葡萄糖亲和力）和4a)热稳定性。 [0210] A variant G0X-EZ07 criteria are the following aspects: la) oxygen consumption 2a) for glucose as substrate specificity 3a) Miman Barre kinetics (medium activity / affinity for glucose), and 4a) heat stability.
[0211] la)GOx-EZO?的耗氧量如1)中所述实现测量。 [0211] la) GOx-EZO? 1 as the amount of oxygen consumption) implemented in the measurement. 如条目dd)下的材料与方法部分中概述的标准方案，纯化所有测试的酶（GOx-WT ;G0x-T30V ;I94V和G0X-EZ07)，且调整至5嗦/血的蛋白质浓度。 The standard protocol in the Materials and Methods section entry dd) outlined, all purified enzymes tested (GOx-WT; G0x-T30V; I94V and G0X-EZ07), and the protein concentration adjusted to 5 winded / blood.
[0212] 检测到下述耗氧率[%/分钟]:G0x-WT: 17. 44 ;G0x-T30V;I94V: 24. 22 和本发明的GOx-EZO? :3. 86。 [0212] detected following the oxygen consumption rate [% / min]: G0x-WT: 17. 44; G0x-T30V; I94V: 24. 22 and GOx-EZO present invention: 386?. GOx-EZO?的残留氧活性与G0X-T30V;I94V相比较为15. 9%，并且与G化-WT相比较为22. 13〇/〇。 ? GOx-EZO residual oxygen activity G0X-T30V; I94V more as compared to 15.9% and more as compared to 22. 13〇 / G and the square of -WT.
[0213] 2a)G0X-EZ07 的特异性如2)中所述实现测量。 [0213] 2a) G0X-EZ07 specific claim 2) implemented in the measurement.
[0214] 3a)G0X-EZ07的米曼二氏动力学如3)中所述实现测量。 [0214] 3a) G0X-EZ07 kinetics as the Miman Barre 3) implemented in the measurement.
[0引引4a)热稳定性如4)中所述实现测量。 [0 cited primer 4a) thermal stability as 4) implemented in the measurement.
[0216] 结果本发明人发现六个不同的氨基酸位置S53 ;A137 ;A173 ;A332 ;F414 ;和V560负责降低的耗氧率和/或对于除氧外的电子介质增加的介质活性。 [0216] The present inventors have found that the results of six different amino acid positions S53; A137; A173; A332; F414; and V560 is responsible for reducing the oxygen consumption rate and / or medium for the active oxygen of the outer electron mediator increased. 进一步地，本发明人发现在六个氨基酸位置中的协同效应，其允许本领域技术人员在葡萄糖特异性、显著降低的耗氧率和/ 或对于除氧外的介质增加的活性方面设计(X)x变体。 Further, the present inventors have found that a synergistic effect six amino acid positions, which allows the person skilled in the glucose-specific, significant reduction in oxygen consumption rate and / or the outer oxygen activity of the medium increased design (X ) x variant.
[0217] 特别地，本发明人发现单独或组合的位置V560和F414负责显著降低的耗氧率和/或负责对于显著增加的介质活性。 [0217] In particular, the present inventors have found that alone or in combination responsible positions V560 and F414 significantly reduced the rate of oxygen consumption and / or media is responsible for a significant increase in activity. 另外，发现位置A173和A332负责组合的两者，即本文提供的(X)x变体的介质活性中的显著增加和伴随的耗氧率中的显著降低。 Further, the position of both the A173 and A332 was found responsible combined, significantly reduce the activity significantly increased i.e. medium (X) x variants provided herein and the accompanying oxygen consumption rate. 发现位置S53和A137负责对于除氧外的特定介质显著增加的介质活性。 I found S53 and A137 medium for the active position responsible for a specific media other than oxygen significantly increased.
[021引进一步地，本发明人发现适合由本发明提供的(X)x变体的特定氨基酸。 [021 cited Further, the present invention is found to be suitable (X) x variant of the present invention is provided by the specific amino acid.
[0219] 表1显示了用于位置173、332、414和560的合适氨基酸集合和相应的简并密码子。 [0219] Table 1 shows the codon for suitable amino acid positions 173,332,414 and 560 and the corresponding set of degenerate.
[0220] 根据本发明测试的(X)x变体的比活性通过各自的EZ编号显示于表2中。 [0220] EZ via respective numbers shown in Table 2 according to the present invention, the specific activity of the test (X) x variant. 该表是根据本发明测试的(X)x变体在经由介质测定的介质活性和经由ABTS测定的耗氧量特性方面的比较。 The table compares the measured medium for the active medium via the oxygen consumption characteristics and via the ABTS assay according to aspects of the (X) x test variants of the present invention.
[022。 [022. 结果W根据本发明的(X)x变体和作为原材料的亲本突变体即(X)x-T30V;I94V之间的比例（商数）的形式显示。 The results of W (X) x and variants of the present invention as a raw material of the present pro mutant i.e., (X) x-T30V; ratio (quotient) of the display form between I94V. 不同微量滴定板的结果是可比较的。 Results different microtiter plate are comparable. 该比例基于通过常规ELISA技术测定的比活性扣/mg]，如条目ee)下的材料与方法部分中概述的。 The ratio is based on measurement by conventional ELISA techniques specific activity buckle / mg], such as the entry ee) in the Materials and Methods section outlined. 该表还显示了依照本发明测试的氨基酸位置的确切置换。 The table also shows the exact amino acid substitutions according to the present invention, the position of the test.
[0222] 表2 :就介质活性和(残留)氧活性测试的具有EZ数字代码的本发明的特定(X)x变体。 [0222] Table 2: For the present invention has particular EZ active medium and a digital code (residual) activity test of the oxygen (X) x variant.
[0223] 结果显示不同特定变体（6203、6205、6212、6206、6207、6208、6210、6211、6215)具有根据本发明的必需特性，即降低的氧亲和力，导致与亲本突变体G0X-T30V;I94V相比较， 显著降低的耗氧率和/或增加的介质活性。 [0223] The results show different variants of a specific (6203,6205,6212,6206,6207,6208,6210,6211,6215) having the required characteristics according to the present invention, i.e. a reduced oxygen affinity, resulting in the parent body G0X-T30V mutations ; I94V comparison, a significant reduction of the oxygen consumption rate and / or increased activity medium. 变体6203、6205、6207、6208和6210经历进一步研究。 Further study of variants 6203,6205,6207,6208 and 6210 experience.
[0224] 例如，本发明人发现与亲本突变体相比较，G化-EZ07和EZ10显示对于增加的介质活性W及氧测定中的显著减少的两种显著改善。 [0224] For example, the present inventors found that compared to the parental mutant, G and EZ10 of -EZ07 show significant reduction increased to medium for the active oxygen assay and W both significantly improved.
[0225] 特别地，本发明人发现变体EZ07是依照本发明显著改善的(X)x变体，而对所述变体的相应特定氨基酸置换没有任何限制。 [0225] In particular, the present invention is found in accordance with the variant EZ07 (X) x variant of the present invention significantly improved, and the corresponding specific amino acid substitution variant without any limitation. EZ07显示在介质测定中与亲本突变体相比较的4. 4倍增加，和在ABTS测定中亲本突变体的氧活性仅0. 1倍的氧活性。 EZ07 display 4.4-fold increase compared to the parental mutant in the assay medium, the activity of oxygen and the oxygen affinity of the present mutation is only 0.1-fold in the ABTS assay. 相比之下，与亲本突变体相比较，G化变体EZ12和EZ15显示对于介质和氧活性两者的伴随增加。 In contrast, compared to the parent mutant, G and variants EZ12 EZ15 display for medium and concomitant increase of both the oxygen activity. 与亲本突变体相比较，EZ12携带在位置S53上的另外置换(参见表2)。 Compared to the parental mutant, EZ12 carry additional substitution at position S53 (see Table 2). 进一步地，发现由于氨基酸置换A137LEZ15具有介质活性中的6倍增加。 Further, since the amino acid substitutions found A137LEZ15 6x medium activity increases.
[0226] 氨基酸位置的表征位置A173和A332远离活性位点。 [0226] Characterization of the amino acid positions A173 and A332 position away from the active site. A173是表面位置；A332定位接近于底物增强通道。 A173 is a surface location; A332 augmentation channel positioned close to the substrate. 对氧活性具有影响的两个位置F414和V560定位接近于活性位点。 Having influence on the oxygen activity of the two positions V560 and F414 positioned close to the active site. F414在葡萄糖结合位点上方，并且V560紧靠葡萄糖和FAD。 F414 in the above glucose binding site, and close to the V560 glucose and FAD. 两个活性位置A137和S53均为接近FAD的表面位置。 Two active positions A137 and S53 are close to the surface position of the FAD.
[0227] 预表征所选预表征的变体的结果如下： 1)耗氧率图4显示了根据时间（a)和耗氧率/分钟（b)，在反应混合物中的氧含量的进展。 Results [0227] Characterization of selected pre-variant pre-characterized as follows: 1) Figure 4 shows the rate of oxygen consumption according to the time progress (a) and oxygen consumption rate / min (b), the reaction mixture in oxygen content. 作为对照，还分析G0X-T30V;I94V、GOx-WT和阴性对照菌株的上清液。 As a control, were also analyzed G0X-T30V; I94V, GOx-WT supernatant and negative control strains.
[0228] G化-WT和G0X-T30V;I94V显示相似的行为。 [0228] G and -WT of G0X-T30V; I94V display similar behavior. 因此，此处测试的所有(X)x变体(EZ03、EZ05、EZ07、EZ08、EZ10)均具有显著降低的耗氧率。 Thus, all tested here (X) x variant (EZ03, EZ05, EZ07, EZ08, EZ10) has significantly reduced the rate of oxygen consumption. 此外，作为本发明的令人惊讶和出乎意料的发现，所测试的变体不能明确与阴性对照区分，运指示各自的氧活性在所选条件下甚至低于测定的检测范围。 Further, as the present invention surprisingly and unexpectedly found that the tested variants can not be clearly distinguished with the negative control, indicating the respective transport the oxygen activity at the chosen assay conditions even below the detection range.
[0229] 2)特异性表3显示参考葡萄糖的残留(X)x酶活性。 [0229] 2) Table 3 shows specific reference residual glucose (X) x activity.
[0230] 表3 :本发明的(X)x变体对于不同糖的残留活性。 [0230] Table 3: (X) x variant residual activity of the present invention for different sugars. 对于残留活性的测定，应用用于表征的介质测定。 For measuring the residual activity assay medium for characterization applications. 底物浓度为各自反应混合物中181.81111。0化-胖1'和6(^-13(^;194¥活性充当参考。 Each substrate concentration of the reaction mixture 181.81111.0 - Fat 1 ', and 6 (^ - 13 (^; 194 ¥ activity serves as a reference.
[023。 [023. 与(X)x-WT和根据沈QIDNO: 1的亲本突变体G0X-T30V;I94V相比较，本发明的G化变体无一显示被麦芽糖或麦芽=糖的显著干扰。 And (X) x-WT and Shen according QIDNO: 1 of the present mutant parent G0X-T30V; I94V comparison, G variants of the present invention is a display without significantly interfering = malt sugar or maltose. EZ07和EZ10对于所有测试的糖具有最佳结果。 EZ07 and EZ10 with the best results of all tests for sugar.
[0232] 3)介质活性/葡萄糖亲和力米曼二氏动力学在图5中描绘。 [0232] 3) medium for the active / s affinity for glucose kinetics Miman two is depicted in FIG. 5. 表4显示Vm。 Table 4 shows Vm. 、和Km的计算值。 , And calculated values ​​of Km.
[023引表4 :不同(X)x变体的酶参数。 [Cited 023 Table 4: Different (X) enzyme variant parameter x. 应用MicrosoftExcel中的最小二乘法，根据米曼二氏动力学来计算参数。 Application of the least square method MicrosoftExcel The Miman Jakob kinetic parameters calculated.
[0234] 表4显示EZ07 -方面具有与亲本突变体G0X-T30V;I94V相比较高大约7. 5倍的VmJ直，但另一方面具有高几乎两倍的Km值。 [0234] Table 4 shows the EZ07 - having regard to the parent mutant G0X-T30V; about 7.5-fold higher compared VmJ I94V straight, but on the other hand has a higher Km value almost twice. 然而，高Km值是高VmJ直的结果。 However, a high Km value is high VmJ straight results. 在图5 中，明确可见(X)x-EZ07在低底物浓度下的表现可与WT和亲本突变体T30V、I94V相比较。 In FIG. 5, clearly visible (X) x-EZ07 performance at low substrate concentrations and can body T30V parent WT and mutant, I94V compared. G化-EZ10达到相似结果，但更低的Vm。 G of -EZ10 achieve similar results, but lower Vm. 、值。 ,value.
[0235] 4)热稳定性在图6中，对于八个不同溫度图解残留活性。 [0235] 4) Thermal stability in FIG. 6, illustrating eight different temperatures residual activity.
[0236] 根据本发明测试的(X)x变体、G化-WT和G0X-T30V;I94V亲本突变体显示相似结果。 [0236] The (X) x test variants of the present invention, G and -WT of G0X-T30V; I94V parent mutant showed similar results. 对于去糖基化的GOx-WT可W观察到热稳定性中的显著下降；然而，高糖基化的分子显示更高的热稳定性。 For the deglycosylated GOx-WT W may be observed a significant decrease in thermal stability; however, highly glycosylated molecules show a higher thermal stability.
[0237] 结论在所有预表征研究期间的特定测试1)至4)的结论中，G化-EZ07和GOx-EZlO变体显示最显著的结果。 1) to 4) Conclusion [0237] Conclusion particular test during all the pre-characterizing studied, G and GOx-EZlO of -EZ07 variant shows the most significant results. 根据预表征结果实现氧活性中的显著下降和介质活性中的显著增加。 To achieve a significant increase in the oxygen activity in the medium, and a significant decrease in activity in accordance with the pre-characterization results. G化-EZ07和GOx-EZlO的特异性几乎未改变，仅检测到对木糖的轻微活性变化。 G and GOx-EZlO of -EZ07 specific almost unchanged, only slight activity in the detection of xylose. 因为EZ07 的活性比EZ10的活性高大约1. 7倍，所W选择G0X-EZ07用于最终表征。 Since the active EZ07 about 1.7 times higher than the activity EZ10, W is selected for the final G0X-EZ07 characterization.
[023引应强调的是上述结果使用本发明的未纯化的(X)x变体生成。 [023 cited above results should be emphasized that the use of unpurified present invention (X) x variant generator. 相应地，本领域技术人员认识到在使用相似的纯化的(Xk变体后，结果可W改变。另外，在应用化ISA的细胞上清液中的特定蛋白质测定可W受杂质影响。 Accordingly, those skilled in the art recognize purified using similar (Xk variant, W results change. Further, a specific application of the protein in the cell supernatant was measured in ISA W may be affected by the influence of impurities.
[0239] 变体G0X-EZ07的最终表征对于最终表征，选择纯化的G0X-EZ07变体。 [0239] Characterization of the final variant of the final G0X-EZ07 characterized, purified G0X-EZ07 selected variants.
[0240] la)G0X-EZ07 的耗氧量G0x-WT、G0x-T30V;I94V和G0X-EZ07的相对耗氧量在图7中描绘。 [0240] la) the oxygen consumption G0X-EZ07 G0x-WT, G0x-T30V; I94V and relative oxygen consumption G0X-EZ07 depicted in FIG. 7.
[0241] 在所选条件下，与GOx-WT相比较，G化-EZ07的耗氧率降低超过10倍，运确认预表征研究的数据。 [0241] Under selected conditions, as compared with GOx-WT, G -EZ07 rate of oxygen consumption is reduced more than 10 times, transport studies confirm the data pre-characterized.
[0242] 2a)GOxEZ07 的特异性图8显示了GOx-WT、G0X-T30V;I94V和G0X-EZ07参考葡萄糖（100%)的残留活性。 [0242] FIG specificity 2a) GOxEZ07 8 shows GOx-WT, G0X-T30V; I94V and G0X-EZ07 reference glucose (100%) of residual activity. 图8显示了与G0X-T30V;I94V和(X)x-WT相比较，在(X)x-EZ07的特异性中无显著变化。 Figure 8 shows the G0X-T30V; I94V and (X) x-WT compared to no significant change in (X) x-EZ07 of specificity.
[0243] 3a)G化变体EZ07的米曼二氏动力学图9比较了(X)x-WT、G化-T30V;I94V和G0X-EZ07在介质测定中的动力学。 [0243] 3a) G variants Miman Barre EZ07 kinetics of FIG 9 compares (X) x-WT, G of -T30V; G0X-EZ07 kinetics and I94V in the assay medium.
[0244] 根据图9,与(X)x-WT相比较，G化变体EZ07显示在介质测定中641. 5%的残留活性和在ABTS中17. 5%的残留活性，导致降低37倍的氧化酶活性。 [0244] According to FIG. 9, as compared with (X) x-WT, G variants EZ07 displayed 641.5% residual activity and 17.5% residual activity in ABTS, resulting in 37-fold decrease in the assay medium oxidase activity.
[0245] 4a)G0X-EZ07 的热稳定性图10指示了当与GOx-WT和G0X-T30V;I94V相比较时，G化-EZ07的热稳定性通过各自置换得到维持。 [0245] 4a) the thermal stability of FIG G0X-EZ07 10 indicates when and GOx-WT and G0X-T30V; when compared I94V, G -EZ07 of thermal stability by replacing each maintained.
[0246]G化变体的概括在亲本突变体G0X-T30V、I94V中鉴定了六个氨基酸位置，其显示对本文提供的(X)x变体的葡萄糖特异性、介导的电子转移的酶活性和氧活性的作用。 [0246] G variants are summarized in the enzyme mutant of a parent G0X-T30V, I94V six amino acid positions were identified, which display specific glucose (X) x variants provided herein, mediated electron transfer and the role of reactive oxygen species. 所有位置均个别地饱和。 All positions are individually saturated. 为了研究协同效应，使在活性位点周围聚簇的位置（S53 ;A137 ;A173 ;A332 ;F414 ;V560)同时饱和。 To study the synergistic effect of the active site around the cluster position (S53; A137; A173; A332; F414; V560) simultaneously saturated. 选择与亲本突变体特性相比较改善的介质活性和/或降低的耗氧率的最显著变体用于预表征。 Medium for the active mutant of a parent selects characteristic improved compared and / or variants of the most significant decrease in the oxygen consumption rate for the pre-characterized. 事实证明变体G0X-EZ07 (具有另外的置换A173V;A332S;F414I;和V560T)显示在本发明的意义中，对于所测试的特性葡萄糖特异性、介质活性和耗氧率的最显著结果。 Proved variant G0X-EZ07 (having additional substitution A173V; A332S; F414I; and V560T) displayed in the sense of the present invention, characteristic for the tested results of the most significant glucose-specific, medium activity and oxygen consumption rate.
[0247] 因此，G化-EZ07就其介质活性、氧化酶活性、葡萄糖特异性W及其热稳定性特性进行深入研究。 [0247] Thus, G-depth study of its -EZ07 medium activity, oxidase activity, the glucose-specific W, and thermal stability properties. 该变体显示在对于介导的电子转移的比活性中的6. 4倍增加（G化-WT: 7. 4 ;G0x-EZ07: 47. 5U/mg)和ABTS测定中5. 7 倍的活性降低（GOx-WT: 451. 1U/mg; G化-EZ07: 78. 86U/mg)，导致降低36. 5倍的氧化酶活性。 The variant shown in 6.4 times greater than for the active electron transfer mediated increases (G of -WT: 7. 4; G0x-EZ07: 47. 5U / mg) and assay ABTS 5.7 fold activity is reduced (GOx-WT: 451. 1U / mg; G of -EZ07: 78. 86U / mg), results in a reduction of 36.5 times oxidase activity. （X)x-EZ07没有热稳定性或特异性中的显著变化。 (X) x-EZ07 no significant change in the thermal stability or specificity of.
[024引材料与方法所有化学制品均为分析级别且购自Sigma-Al化ich(Taufkirchen，德国）、Applichem(Darmsta化，德国）或CarlRoth(Karlsruhe,德国）。 [024 primer Materials and Methods All chemicals were of analytical grade and purchased from Sigma-Al of ich (Taufkirchen, Germany), Applichem (Darmsta of, Germany) or CarlRoth (Karlsruhe, Germany). 所有酶均购自化wElnglandBiol油s (UK)和Sigma-Al化ich(Taufkirchen，德国）。 All enzymes were purchased from oil of wElnglandBiol s (UK) and Sigma-Al of ich (Taufkirchen, Germany). pYES2穿梭载体和大肠杆菌菌株。 pYES2 shuttle vector and E. coli strains. 化领勾自InvitrogenCKarlsruhe，德国）。 Of the collar hook from InvitrogenCKarlsruhe, Germany). 酿酒酵母菌株7087(ngd29皿nl)由Rochedia即ostics提供[Lehle,L. ,Eiden,A. ,Lehnert,K. ,Haselbeck,A.和Kopetzki,E. (1995).Glycoprotein biosynthesisinSaccharomycescerevisiae:ngd29,anN-glycosylationmutant allelictoochlhavingadefectintheinitiationofouterchainformation.FEES Zeff 口，如-必7。 S. cerevisiae strain 7087 (ngd29 dish nl) by Rochedia i.e. ostics [Lehle, L, Eiden, A, Lehnert, K, Haselbeck, A and Kopetzki, E (1995) .Glycoprotein biosynthesisinSaccharomycescerevisiae:..... Ngd29, anN- glycosylationmutant allelictoochlhavingadefectintheinitiationofouterchainformation.FEES Zeff mouth, such as - 7 will. 使用NanoDrop光度计（NanoDropTechnologies,Wilmington,DE，USA) 定量DM。 Using a NanoDrop photometer (NanoDropTechnologies, Wilmington, DE, USA) quantitative DM. 测序和寡核巧酸合成通过化rofinsMWG化e;ron(Ebersberg，德国)完成。 Qiao acid sequencing and oligonucleotide synthesis by chemical rofinsMWG of e; ron (Ebersberg, Germany) is completed. 使用的引物在表5 (支持信息）中提及。 The primers used in Table 5 (Supporting Information) mentioned.
[0249]aa)多重和单一位点饱和诱变对于多重位点饱和诱变，应用Omnichange方案/"化如乂，捕ira化•6,AK，Marienhagen,J.和Schwaneberg,U. (2011) .OmniChange:TheSequenceIndependent MethodforSimultaneousSite-SaturationofFiveCodons.PLoSONE6，e26222]AW MlPCR反应混合物含有1ng/Pl质粒模板、lxPhusion缓冲液（NewElnglandBiol油s， 化anlrfud，德国）、1UPhusion聚合酶和200剛dNTP。将反应混合物分成四个相等体积， 并且加入250nM祀向特异性位点的正向引物和反向引物用于片段扩增。在用于载体主链扩增的混合物中，加入另外的dNTPW达到300剛的最终浓度。对于片段1，将祀向A173的引物P7和P8加入主混合物中。同时对于A332饱和，使用P9和P10。通过使用P11和P12 祀向F414位点。将V560位点包括在载体主链中，因为它通过P13和P14进行扩增。用于片段扩增的PCR程序为98°C45秒（1个循环）；98°C15秒；65°C30秒；72°C30秒（20个循环） [0249] aa) multiple and single site-saturation mutagenesis For multiple site-saturation mutagenesis, application Omnichange programs / "technology as qe, catching ira of • 6, AK, Marienhagen, J., And Schwaneberg, U. (2011) .OmniChange: TheSequenceIndependent MethodforSimultaneousSite-SaturationofFiveCodons.PLoSONE6, e26222] AW MlPCR reaction mixture containing 1ng / Pl plasmid template, lxPhusion buffer (NewElnglandBiol oil s, of anlrfud, Germany), 1UPhusion polymerase and dNTP reaction mixture just 200 divided into four. of equal volume, and a final concentration of 250nM was added to the sacrificial site specific forward primer and reverse primer used for amplification of fragments in the mixture used for amplification of the vector backbone, additional dNTPW 300 Gang. for fragment 1, the Si forward primer P7 A173 and P8 added to the main mixture while for A332 saturated, using P9 and P10.. the V560 sites include the vector backbone by using P11 and P12 worship to the F414 site, because it was amplified by P13 and P14 for PCR amplification program for the fragment 98 ° C45 seconds (1 cycle);. 98 ° C15 seconds; 65 ° C30 seconds; 72 ° C30 seconds (20 cycles) ；72°C5分钟（1个循环)。对于载体主链扩增，应用4分钟的延伸时间。将所有片段进行柱纯化，并且在NanoDrop光度计（NanoDropTechnologies,Wilmington,DE，USA)上测量浓度。随后对PCR产物实施过夜化wl消化(各5U)。 ; 72 ° C5 min (1 cycle) for amplification of the vector backbone, the application time of 4 minutes extend all fragments subjected to column purification, and measuring the concentration in the NanoDrop photometer (NanoDropTechnologies, Wilmington, DE, USA)... then wl embodiment of overnight digestion (each 5U) PCR product.
[0巧0] 片段的消化和连接：将插入片段的浓度调整至0. 06pmol/rt。 [Qiao 0 0] and digested fragments are connected: the concentration of the insert was adjusted to 0. 06 pmol/rt. 同时，将载体主链浓度调整至0. 02pmol/rt(还考虑用化消化的稀释因子)。 Meanwhile, the vector backbone was adjusted to a concentration of 0. 02 pmol/rt (also contemplated by the dilution factor of the digestion). 将1W切割溶液（50%(500ml TrispH9. 0、30% (lOOmM舰的乙醇溶液）和20%地20)加入4W每种片段。反应随后在70°C下溫育5分钟。将切割的片段混合在一起，并且在转化化学感受态大肠杆菌D册6用于文库扩增前，在室溫下（RT)溫育5分钟。对于酵母转化，使用质粒分离试剂盒"NucleoSpin® Plasmid" (MAC肥RY-NAGEL，D化en，德国），从大肠杆菌D册6中分离基因文库。 The cutting 1W solution (50% (500ml TrispH9. 0,30% (lOOmM ship ethanol solution) and 20 to 20%) was added 4W each fragment. The reaction followed by incubation 70 ° C for 5 minutes. The cleaved fragments were mixed together, and transformed in E. coli D register 6 for the front chemically competent library amplification, at room temperature (RT) and incubated for 5 min. for yeast transformation using a plasmid isolation kit "NucleoSpin® plasmid" (MAC fertilizer RY-NAGEL, D of the en, Germany), isolated from the E. coli gene library book D 6.
[0巧1] 对于个别位点饱和诱变（SSM)，已遵循两步PCR方案/如W3片乂游化wseA D. (2001).Molecularcloning:alaboratorymanual,第2卷（ColdSpringHarborZaAora妃化e^s6パ。50MlPCR反应混合物含有1ng/Pl质粒模板、lxPhusion缓冲液(化wElnglandBiol油s，Rrankfu;rt，德国）、1UPhusion聚合酶和300 剛dNTP。将反应混合物分成两个相等体积，并且将400nM祀向位点的正向引物和反向引物加入分开的反应混合物中。PCR程序为98°C45秒（1个循环）;98°C15秒；65°C30秒；72°C30秒（5个循环）； 72°C5分钟（1个循环)。在第一步后，将两个反应合并，并进行相同程序且对于扩增循环多15次重复。使用下述引物：P15/P16用于位置173，P17/P18用于位置332，P19/P20用于位置414和P21/22用于位置560。将所有PCR产物进行柱纯化，并且随后实施化消化。 [Qiao 1 0] for the individual site-saturation mutagenesis (SSM), has followed a two-step PCR protocol / qe travel of sheet W3 as wseA D. (2001) .Molecularcloning: alaboratorymanual, Volume 2 (ColdSpringHarborZaAora Princess of e ^ s6 the reaction mixture was SUPER .50MlPCR containing 1ng / Pl plasmid template, lxPhusion buffer. (oil of wElnglandBiol s, Rrankfu; rt, Germany), 1UPhusion polymerase and dNTP reaction mixture immediately 300 into two equal volumes, and the Si 400nM forward primer and reverse primer sites added to the reaction mixture in a separate program of .PCR 98 ° C45 seconds (1 cycle); 98 ° C15 seconds; 65 ° C30 seconds; 72 ° C30 seconds (5 cycles); . 72 ° C5 min (1 cycle) after the first step, two reactions were combined, and the same procedure and for 15 repetitions of multiple cycles of amplification using the following primers:. P15 / P16 to position 173, P17 / P18 to position 332, P19 / P20 and a position 414 P21 / 22 is used to position 560. All PCR product was subjected to column purification, and then the embodiment of digestion.
[0巧2] bb)基因克隆和酵母细胞转化根据重组介导的无连接酶方法/"WofenAy/斯足.乂，防，足.7.，ificAse^s，51游化奶口打，C. (1997).Recombination-mediatedPCR-directedplasmidconstructioninvivo in乃3別.尬化searcA之5，必7-必之7,执行突变的GOx基因克隆到pYES2载体主链内。首先，2嗦pYES2-G0xT30V、I94V双重突变体通过Sall/BamHI消化（10U/嗦DNA，6小时，37°C)进行线性化，并且随后通过采用NucleoSpin®ExtractII-试剂盒(MAC肥RY-NAGEL，D化en，德国）的凝胶提取进行纯化。用于文库扩增的引物W运样的方式进行设计，使得整个引物序列与线性化的载体主链互补。 [Qiao 0 2] bb) cloning the transformed yeast cells and non-recombinant ligase mediated method / "WofenAy / Si foot. Qe, anti-foot .7., IficAse ^ s, 51 opening travel of milk play, C . (1997) .Recombination-mediatedPCR-directedplasmidconstructioninvivo in 3 is the other. searcA embarrassed of the 5, 7 it will 7- will, GOx performed mutant gene was cloned into the pYES2 vector backbone. first, 2 winded pYES2-G0xT30V, I94V double mutants were linearized by Sall / BamHI digested (10U / repetitious DNA, 6 hours, 37 ° C), and then by using NucleoSpin®ExtractII- kit (MAC fertilizer RY-NAGEL, D of the en, Germany) condensate extraction gel purified. W library amplification primers sample transport way for the design, so that the entire linear primer sequence complementary to the vector backbone.
[0巧3] 其次，将线性化的PYES2载体和扩增的基因文库W1:3 (250 ng/750 ng)的比例混合。 [Qiao 0 3] Next, the linearized vector and PYES2 amplified gene library W1: 3 (250 ng / 750 ng) of proportions. 运种DM混合物用于采用乙酸裡方法的酿酒酵母7087的转化/仿'6戊Z?.游Schiestl,RH (2007).High-efficiencyyeasttransformationusingtheLiAc/SS carrier仿忠曲6曲3^/.絶化re化otocois之，*57-%7。 DM conversion transported species to employ a mixture of acetic acid in the method of the Saccharomyces cerevisiae 7087 / imitation '6-pentyl Z ?. travel Schiestl, RH (2007) .High-efficiencyyeasttransformationusingtheLiAc / SS carrier faithful imitation song 6 ^ 3 /. Of absolute re of the otocois, * 57- 7%. 转化体在含有2%葡萄糖的SC-U 选择性平板上生长。 Transformants were grown on SC-U selective plates containing 2% glucose. 对于环状质粒的转化，使用300 ng DM。 For transformation circular plasmid, using 300 ng DM.
[0254]cc)在96孔板中的培养和表达使用牙签，将在含有2%葡萄糖的SC-U选择性琼脂平板上生长的单一菌落转移到每孔含有100化SC-U培养基（1%葡萄糖）的96孔微量滴定板（PS-F形底，greinerbio-one) 内。 [0254] cc) cultured in 96 well plates and expression using a toothpick, a single SC-U colonies on selective agar plates containing 2% glucose grown transferred to each well containing 100 of SC-U medium (1 % glucose) in 96 well microtiter plates (PS-F-bottom, greinerbio-one) within. 预培养物的培养在板振荡器（InforsGmbH,Eisenach,德国）中在下述条件下发生：900 rpm，30°C，70%湿度，24小时。 Preculture culture (InforsGmbH, Eisenach, Germany) under the following conditions occurs plate shaker: 900 rpm, 30 ° C, 70% humidity, for 24 hours. 将一定量的预培养物从每个孔转移到深孔板（riplate-矩形， ritter)中的500化SC-U自身诱导培养基（0. 5%葡萄糖/ 2%半乳糖)。 The amount of pre-culture was transferred from each well to a deep well plate (riplate- rectangle, ritter) 500 of the SC-U induction medium itself (0.5% glucose / 2% galactose). 主要培养物在与预培养物相同的条件下进行培养，但共72小时。 The main culture was cultured under the same conditions as preculture, but a total of 72 hours. 板在4000巧m和RT下离屯、20分钟。 Tun from the plate at 4000 m clever and RT, 20 min. 所得的上清液用于活性测定。 The resulting supernatant was used for the activity assay.
[0巧引dd)重组GOx的生产和纯化预培养：使用Sc7087/pYES2-G0x的过夜培养物，将500mLSynY培养基（2%葡萄糖)接种至0D600 = 0. 2,培养在化摇瓶中在30°C和250巧m下发生12小时。 [0 Qiao primer dd) Recombinant production and purification of GOx preculture: Use Sc7087 / pYES2-G0x overnight culture, the 500mLSynY medium (2% glucose) was inoculated into 0D600 = 0. 2, cultured in shake flasks of 12 hours at 30 ° C and occurs Qiao 250 m. 主要培养：用500 mL预培养物接种10LSynY培养基（1%葡萄糖)。 Main culture: 500 mL with a pre-culture was inoculated 10LSynY medium (1% glucose). 对于培养，使用10L发酵罐，应用下述条件：溫度：30°C;揽拌器：400巧m巧流：1vvm;时间：48小时。 For culture, a 10L fermenter, applying the following conditions: Temperature: 30 ° C; embrace stirrer: 400 m Qiao Qiao flow: 1 vvm; Time: 48 hours.
[0巧6] 细胞分离和缓冲液更换：通过微量过滤回收含(X)x的上清液。 [Qiao 0 6] Cell isolation and buffer exchange: containing supernatant was recovered by filtration (X) x by trace. 为此，使用具有一个滤器盒（SART0C0NSlice,Hy化osart0. 45 化，SartoriusStedimBiotechGmbH,GSttingen，德国）的交叉流过滤单元Sartojet(SartoriusStedimBiotechGmbH, GSttingen，德国）。 For this purpose, having a filter box (SART0C0NSlice, Hy of osart0. 45 of, SartoriusStedimBiotechGmbH, GSttingen, Germany), cross-flow filtration unit Sartojet (SartoriusStedimBiotechGmbH, GSttingen, Germany). 进料压力调整至2.0己，并且截留液压力调整至1.0己。 Feed pressure was adjusted to 2.0 hexyl, and the retentate pressure was adjusted to 1.0 hexyl. 在下述条件下应用超滤盒（SART0C0NSlice,Hy化osart10kDa，SartoriusStedimBiotechGmbH, GSttingen，德国)，相同过滤系统用于缓冲液更换和样品浓度：缓冲液50mlNa&PCVpH6; 缓冲液更换体积：7 ;进料压力：2. 0己；截留液压力：1. 0己；最终体积0. 5L。 Application of ultrafiltration cartridge under the following conditions (SART0C0NSlice, Hy of osart10kDa, SartoriusStedimBiotechGmbH, GSttingen, Germany), the same filtration system for buffer exchange and sample concentration: Buffer 50mlNa & PCVpH6; buffer exchange volume: 7; feed pressure: 2 0 hexyl; retentate pressure: 10 hexyl; final volume of 0. 5L.
[0巧7]对于酶纯化，使用AKTApilot系统和FinLinePilot35 柱（GEHealthcare,Munich,德国），应用阴离子交换层析。 [Qiao 0 7] For enzyme purification, and use AKTApilot system FinLinePilot35 column (GE Healthcare, Munich, Germany) using anion exchange chromatography. 使柱充满220血树脂（化actogelTSKDEAE-650S Merck),选择62. 5cm/h的流速。 The column was filled with blood 220 resin (of actogelTSKDEAE-650S Merck), select 62. 5cm / h flow rate. 平衡：440血50ml憐酸钢缓冲液抑6 ;样品负荷：0. 5L 截留液；洗涂：440mL50ml憐酸钢缓冲液抑6;洗脱：95% 50ml憐酸钢缓冲液抑6 / 5% 1M化C1 (不连续梯度)。 Balance: 440 Rei acid 50ml blood suppression steel buffer 6; Sample load:. 0 5L retentate; washcoated: 440mL50ml steel pity acid buffer suppression 6; elution: 95% 50ml Rei acid buffer suppression steel 6/5% 1M of C1 (discontinuous gradient). 将含有GOx的级分合并，随后发生针对50ml憐酸钟缓冲液抑7 的最终缓冲液更换（Amicon®叫trace]_-30kMillipore，Cork，爱尔兰)。 The fractions containing the GOx were combined, subsequent clock for 50ml buffer Rei acid suppression final buffer exchange 7 (Amicon® called trace] _- 30kMillipore, Cork, Ireland).
[0巧引ee)蛋白质浓度的标准化酶联免疫吸附测定法（ELISA)用于(X)x浓度的特定测定。 [0 Qiao primer ee) x concentration measured specific protein concentration normalized enzyme-linked immunosorbent assay (ELISA) to (X). 链霉抗生物素蛋白包被的微量滴定板（Roche-材料编号11643673001)的每个孔填充有100化一抗溶液（2嗦/ 血；R油bitPAKGOD-BiotinAcrisR1083B)，使板在室溫下溫育1小时。 Avidin streptavidin-coated microtiter plate (Roche- material No. 11643673001) filled with 100 per well of antibody solution (2 repetitious / blood; R & lt oil bitPAKGOD-BiotinAcrisR1083B), the plates at room temperature for 1 hour. 在后续洗涂步骤（4次350化9g/L化Cl_0.1%Tween20)后，将100血酶样品吸取到孔内，随后为第二溫育步骤。 After a subsequent wash-coating step (4 times 350 of 9g / L of Cl_0.1% Tween20), 100 enzyme sample blood drawn into the hole, followed by a second incubation step. 重复洗涂步骤，并且使板填充有100化二抗溶液（10嗦/mL;R油bitPAK G0D-HRPAcrisR1083HRP)。 Repeat wash coating step, and the plate is filled with 100 of secondary antibody solution (10 repetitious / mL; R oil bitPAK G0D-HRPAcrisR1083HRP). 在第S溫育和洗涂步骤后，使板填充有100化ABTS/H2O2溶液（11684302001RocheDia即osticsGmbH,Mannheim,德国）。 At S incubation and washing step after coating, the plates are filled with 100 of ABTS / H2O2 solution (11684302001RocheDia i.e. osticsGmbH, Mannheim, Germany). 在测定在405nm处的吸收之前，使板在室溫下溫育30分钟。 Prior to the measurement of the absorption at 405nm, the plates were incubated at room temperature for 30 minutes. 来自黑曲霉的葡萄糖氧化酶用作内部标准（G7141-50KU SIGMA-ALDRICH)。 From Aspergillus niger glucose oxidase as an internal standard (G7141-50KU SIGMA-ALDRICH).
[0巧引ff)介质测定对于液体处理，使用来自（Transfe巧etteS-8)和Elppendorf(Researchpro)的多通道移液管。 [0 Qiao primer ff) for measuring the fluid treatment medium, using from (Transfe Qiao etteS-8) and Elppendorf (Researchpro) multichannel pipette. 将75化样品（制备的上清液/酶溶液）转移至96孔平底微孔板（greiner bio-one)。 75 of sample (supernatant / enzyme solution prepared) were transferred to 96 well flat bottom microtiter plates (greiner bio-one). 加入100化介质溶液（19. 05mlN，N-双（2-径乙基)-4-亚硝基苯胺/Seeder, 0. (2005).DieGlucose-Dye~OxidoreduktaseinderklinischenDiagnostik. DISSERTATIONthesis，TechnischeUniversitatKaiserslautern,Kaiserslautern], Roche-材料编号100088314 ;5% (w/w)聚乙締化咯烧酬，Roche- 10003476964;pH7) 和20化25ml憐钢酸（Roche,Genisys-nr. : 11889893001)。 100 medium was added a solution of (19. 05mlN, N- bis (2-diameter ethyl) -4-nitroaniline / Seeder, 0. (2005) .DieGlucose-Dye ~ OxidoreduktaseinderklinischenDiagnostik. DISSERTATIONthesis, TechnischeUniversitatKaiserslautern, Kaiserslautern], Roche - numbers 100088314; 5% (w / w) of polyglycolide associated pay slightly burned, Roche- 10003476964; pH7) and 25ml of 20 steel pity acid (Roche, Genisys-nr: 11889893001).. 通过添加25化底物溶液且随后将板WlOOO巧m振荡1分钟，来起始反应。 25 by addition of substrate solution and the plate is then shaken for 1 m WlOOO Qiao minute reaction was initiated. 使用微孔板阅读器TecanSun;rise(Tecan TradingAG，瑞±)，在700nm处监控憐钢酸还原的动力学。 Using a microplate reader TecanSun; rise (Tecan TradingAG, Switzerland ±), to monitor the kinetics of the reduction of acid pity steel at 700nm. 对于动力学分析，由根据底物浓度标绘的初速度数据测定Vmax和KM值，其中线性关联系数〉0. 99。 For kinetic analysis, determination of KM and Vmax values ​​starting from the initial velocity of substrate concentration plot data, wherein the linear correlation coefficient of> 0.99.
[0260]gg)ABTS测定对于液体处理，使用来自（Transfe巧etteS-8)和Elppendorf(Researchpro)的多通道移液管。 [0260] gg) ABTS assay for liquid handling, from the use of (Transfe Qiao etteS-8) and Elppendorf (Researchpro) multichannel pipette. 将75化样品（制备的上清液/酶溶液）转移至含有100化憐酸盐缓冲液（pH 7)的96孔平底微孔板（greinerbio-one)。 75 of sample (prepared supernatant / enzyme solution) was transferred to a 96 well flat bottom microplate (greinerbio-one) containing 100 of pity formate buffer (pH 7) a. 将20化反应混合物加入每个孔，导致下述浓度：0. 91U/mLHRP;2. 3mlABTS。 The 20 reaction mixture was added to each well, resulting in the following concentrations: 0 91U / mLHRP; 2 3mlABTS... 通过添加25化底物溶液且随后将板W1000巧m振荡30秒，来起始反应。 25 by addition of substrate solution and the plate is then shaken for 30 m clever W1000 seconds to initiate the reaction. 使用微孔板阅读器化UOstar0mega(BMGLABTECH)，在414皿处动态测定ABTS的氧化。 Using a microplate reader of UOstar0mega (BMGLABTECH), dynamic oxidation of ABTS at 414 measured at the dish. 对于动力学分析，由根据底物浓度标绘的初速度数据测定Vm。 For kinetic analysis, the initial velocity measurement Vm according to the substrate concentration plotted data. 、和Km值， 其中线性关联系数>0.99。 , And Km values, wherein the linear correlation coefficient of> 0.99.
[0261] hh)耗氧量测定对于耗氧量的直接测定，使用光学氧探针"FiboxS-Minisensor(kygenMeter"(PrecisionSensingGmbH,Regensburg,德国）。1540化反应混合物由840化憐酸盐缓冲液（0.2M/pH7)、175化底物溶液和525化0化溶液组成。动态测定耗氧量（％/分钟）。 [026引ii)热稳定性使100化酶溶液在相应溫度下溫育15分钟，且随后在冰上冷却5分钟。 [0261] hh) for direct measurement of oxygen consumption was measured oxygen consumption, using an optical oxygen probe "FiboxS-Minisensor (kygenMeter" (PrecisionSensingGmbH, Regensburg, Germany) .1540 reaction mixture consisted of 840 Rei formate buffer ( 0.2M / pH7), 175 and 525 of substrate solution composed of a solution 0 of the dynamic determination of oxygen consumption (% / min). [026 primer ii) thermal stability of 100 enzyme solution was incubated at the respective temperatures 15 minutes, and then cooled on ice for 5 minutes. 75化用于活性测定。 75 of assay for activity. 研究下述溫度：30-、35-、40-、45-、50-、55-、60-和65°C。 Study of the following temperatures: 30-, 35-, 40-, 45-, 50-, 55-, 60- and 65 ° C.
[0263]表5 :在实验部分中使用的引物 [0263] Table 5: Primers used in the experimental section
1. 一种根据SEQIDNO: 1的葡萄糖氧化酶，其选自： a) 根据SEQIDNO: 1的葡萄糖氧化酶，除两个氨基酸置换T30V和I94V之外，还具有在选自所述SEQIDNO: 1中的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个另外的氨基酸置换；或b) 显示出与根据a)的葡萄糖氧化酶至少70%，特别是至少80%或> 90%的氨基酸序列同一性的葡萄糖氧化酶，条件是除所述两个氨基酸置换T30V和I94V之外，还具有在选自根据SEQ ID NO: 1的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的至少一个另外的氨基酸置换， 并且条件是根据b)的葡萄糖氧化酶显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80%或> 90%的酶活性，并且显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80% 或> 90%的对于葡萄糖的酶特异性， 并且条件是根据b)的葡萄糖氧化酶显示出根据SEQ ID NO: 1的所述葡萄糖氧化酶对于作为电子受体的氧 1. A process according SEQIDNO: 1 glucose oxidase, which is selected from: a) The SEQIDNO: 1 glucose oxidase, in addition to two amino acid substitutions T30V and I94V, but also has the selected SEQIDNO: 1 in the S53; at least one additional six amino acid substitutions in position and V560;; A137; A173; A332; F414 or b) exhibits according to a) glucose oxidase at least 70%, especially at least 80%, or> amino acid sequence identity to 90% of the glucose oxidase, with the proviso that the two amino acid substitutions in addition to and I94V T30V but also selected in accordance with SEQ ID NO: S53 1 a; A137; A173; A332; F414; and at least one additional amino acid substitution in the six position V560, and provided that exhibits according to b) glucose oxidase according to a) glucose oxidase at least 70%, especially at least 80% or> 90% of the enzyme activity, and shows according to a) glucose oxidase at least 70%, especially at least 80% or> 90% for the glucose-specific enzyme, and shows that according to b) according to the glucose oxidase SEQ ID NO: 1 of the glucose oxidase for oxygen as an electron acceptor 低至少5倍的活性，或显示出根据SEQ ID NO: 1的所述葡萄糖氧化酶对于除氧外的电子介质增加至少1. 5倍的活性，或两者；或c) 根据a)或b)的葡萄糖氧化酶的活性片段，条件是当与根据a)或b)的葡萄糖氧化酶相比较时，根据c)的活性片段具有如在a)或b)下概述的所述氨基酸置换， 并且条件是根据c)的葡萄糖氧化酶显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80%或> 90%的酶活性，并且显示出根据a)的葡萄糖氧化酶至少70%，特别是至少80% 或> 90%的对于葡萄糖的酶特异性， 并且条件是根据c)的葡萄糖氧化酶显示出根据SEQIDNO: 1的所述葡萄糖氧化酶对于作为电子受体的氧降低至少5倍的活性，或显示出根据SEQIDNO: 1的所述葡萄糖氧化酶对于除氧外的电子介质增加至少1. 5倍的活性，或两者。 At least 5-fold lower activity, or a display showing the SEQ ID NO: 1 of the glucose oxidase electron mediator other than oxygen for at least 1.5-fold increase in activity, or both; or c) according to a) or b ) active fragment glucose oxidase, with the proviso that when the time according to a) or b) compared to glucose oxidase, according to c) as an active fragment having the amino acid outlined under a) or b) substituted, and with the proviso that exhibits according to a) glucose oxidase at least 70%, especially at least 80% or> 90% of the enzymatic activity according to c), glucose oxidase, and shows according to a) glucose oxidase at least 70%, particularly at least 80% or> 90% for the glucose-specific enzyme, and shows that according to c) according to the glucose oxidase SEQIDNO: at least 5 times the glucose oxidase is reduced to oxygen as an electron acceptor activity, or exhibit according SEQIDNO: activity, or both said glucose oxidase is 1 to increase the electron mediator other than oxygen for at least 1.5-fold.
2. 根据权利要求1的葡萄糖氧化酶，其中•通过ABTS测定法来测定对于作为电子受体的氧的所述活性，其包括下述步骤： a) 将75ML样品酶溶液转移至含有100ML磷酸盐缓冲液（pH7)的96孔平底微孔板b) 将20 ML反应混合物加入每个孔，导致下述浓度：0.91 U/mL HRP ;2. 3 mM ABTS (2, 2' -联氮-双（3-乙基苯并噻唑啉-6-磺酸)） c) 通过添加25ML葡萄糖底物溶液，且随后将板以1000rpm振荡共30秒来起始所述反应d) 使用微孔板阅读器在414 nm处动态测定ABTS的氧化•通过介质测定法来测定对于除氧外的电子介质的所述活性，其包括下述步骤： a) 将75ML酶溶液样品转移至96孔平底微孔板b) 加入100ML介质溶液（19. 05mMN，N-双（2-羟乙基）-4-亚硝基苯胺）；5% (w/w) 聚乙烯吡咯烷酮，pH7和20ML25mM磷钼酸c) 通过添加25ML葡萄糖底物溶液，且随后将板以1000rpm振荡共1分钟来起始所述反应d) 使用微孔板阅读 100ML to phosphate containing a) The enzyme solution was transferred 75ML sample: 2. glucose oxidase according to claim 1, wherein the determined activity towards oxygen as electron acceptor • by ABTS assay, comprising the steps of buffer (pH 7) 96-well flat-bottomed microplate b) 20 mL the reaction mixture was added to each well, resulting in the following concentrations: 0.91 U / mL HRP; 2 3 mM ABTS (2, 2 '- azino - bis. (3-ethyl benzo thiazoline-6-sulfonic acid)) C) by adding 25ML glucose substrate solution, and then the plates were shaken at 1000rpm for 30 seconds to initiate the reaction d) using a microplate reader a) the enzyme solution 75ML sample was transferred to 96 well flat bottom microplates b: ABTS oxidation is determined • the active oxygen of the outer electron mediator by the assay medium, comprising the steps of dynamically measured at 414 nm ) was added a solution 100ML medium (19. 05mMN, N- bis (2-hydroxyethyl) -4-nitroaniline); 5% (w / w) polyvinylpyrrolidone, pH7 20ML25mM phosphomolybdic acid, and c) by adding 25ML glucose substrate solution, and then the plates were shaken at 1000rpm for 1 minute initiate the reaction d) the use of a microplate reader 在700 nm处监控磷钼酸还原的动力学。 Monitoring the kinetics of reduction of phosphomolybdic acid at about 700 nm.
3. 根据权利要求1或2的葡萄糖氧化酶，其中所述除氧外的电子介质选自亚硝基苯胺及其衍生物、偶氮化合物、吩嗪及其衍生物、吩噻嗪及其衍生物、吩噁嗪及其衍生物、二茂铁及其衍生物、铁氰化钾、Ru-和Os-络合物、醌及其衍生物、吲哚酚、紫精、四硫富瓦烯及其衍生物和酞菁。 The glucose oxidase as claimed in claim 1 or 2, wherein the electronic medium is selected from oxygen outside the sub-nitroaniline and derivatives thereof, azo compounds, phenazine and derivatives thereof, phenothiazine and derivatives It was phenoxazine and its derivatives, ferrocene and derivatives thereof, potassium ferricyanide, and Os- Ru- complexes, and quinone derivatives, indoxyl, viologen, tetrathiafulvalene and derivatives and phthalocyanine.
4. 根据前述权利要求中任一项的葡萄糖氧化酶，除根据SEQIDNO: 1的所述两个置换T30V和I94V之外，其还具有在选自根据SEQIDNO: 1的S53 ;A137 ;A173 ;A332 ;F414 ;和V560的六个位置中的另外两个、或三个、或四个、或五个或六个氨基酸置换。 According to one of the preceding claims of any of claims glucose oxidase, except under SEQIDNO: 1, other than the two T30V substitution and I94V, further having a selected according SEQIDNO: S53 1 a; A137; A173; A332 ; F414; V560 and the other two positions in six or three, or four, or five, or six amino acid substitutions.
5. 根据前述权利要求中任一项的葡萄糖氧化酶，其中关于所述一个或多个另外置换的氨基酸选自Ala、Arg、Asn、Asp、Cys、Gin、Glu、Gly、His、lie、Leu、Lys、Met、Phe、Pro、Ser、 Thr、Trp、Tyr和Val，条件是所述取代氨基酸是如根据SEQIDNO:I的各自位置中存在的其他氨基酸。 5. a glucose oxidase according to any of the preceding claims, wherein the amino acid with respect to the one or more additional substitutions selected from Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu , Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, with the proviso that the amino acid substitutions are as according SEQIDNO: another amino acid I is present in the respective positions.
6. 根据前述权利要求中任一项的葡萄糖氧化酶，其中关于一个或多个另外置换的所述氨基酸选自： 用于位置S53的Phe;和/或用于位置A137的Ser、Leu;和/或用于位置A173的lie、Thr、Val;和/或用于位置A332的Ser、Asn、Val、Thr;和/或用于位置F414 的Arg、Asn、Asp、Cys、Gly、His、lie、Met、Ser、Thr、Tyr、Val;和/ 或用于位置V560 的Ala、lie、Leu、Met、Pro、Thr、Tyr、Val。 6. glucose oxidase to any one of the preceding claims, wherein said amino acid with respect to one or more additional substitutions selected from: a position S53 of Phe; and / or Ser in position A137, Leu; and / A173 or the location of lie, Thr, Val; and / or a position A332 of Ser, Asn, Val, Thr; and / or Arg in position F414, Asn, Asp, Cys, Gly, His, lie , Met, Ser, Thr, Tyr, Val; and / or a position V560 is Ala, lie, Leu, Met, Pro, Thr, Tyr, Val.
7. 根据前述权利要求中任一项的葡萄糖氧化酶，其中除根据SEQIDNO:I的所述置换T30V和I94V之外，所述位置F414和/或V560中的至少一个另外置换的氨基酸置换与所述位置A137和/或A173和/或A332中的至少一个氨基酸置换组合。 The glucose oxidase in any one of the preceding claims, wherein the addition according SEQIDNO: the I and I94V substitution T30V outside, and the position of F414 / V560 at least one additional amino acid substitution or substitutions and the said position A137 and / or A173 and / or A332 in combination of at least one amino acid substitution.
8. 根据前述权利要求中任一项的葡萄糖氧化酶，除所述置换T30V和I94V之外，其还具有根据SEQIDNO: 4的另外的氨基酸置换A173I;A332S;和F414L。 According to one of the preceding claims of any of claims glucose oxidase, in addition to the replacement T30V and I94V addition, in accordance with further SEQIDNO: 4 additional amino acid substitution A173I; A332S; and F414L.
9. 根据前述权利要求中任一项的葡萄糖氧化酶，除所述置换T30V和I94V之外，其还具有根据SEQIDNO: 3的另外的氨基酸置换A173V;A332S;F414I;和V560T。 According to one of the preceding claims in any one of claims glucose oxidase, in addition to the replacement T30V and I94V addition, in accordance with further SEQIDNO: 3 additional amino acid substitution A173V; A332S; F414I; and V560T.
10. 根据前述权利要求中任一项的葡萄糖氧化酶，除所述置换T30V和I94V之外，其还具有根据SEQIDNO: 6的另外的氨基酸置换A173V;A332N;F414V;和V560L。 10. a glucose oxidase according to any of the preceding claims, in addition to the replacement T30V and I94V addition, in accordance with further SEQIDNO: amino acid substitution A173V additional 6; A332N; F414V; and V560L.
11. 根据前述权利要求中任一项的葡萄糖氧化酶，当通过介质测定法进行测定时，其显示出至少99. 9%的葡萄糖特异性，和/或〈4%的半乳糖特异性，和/或〈0. 3%的麦芽糖特异性，和/或〈6%的木糖特异性，和/或〈0. 1%的麦芽三糖特异性，所述介质测定法包括下述步骤： a) 将75ML酶溶液样品转移至96孔平底微孔板b) 加入100ML介质溶液（19. 05mMN，N-双（2-羟乙基）-4-亚硝基苯胺）；5% (w/w) 聚乙烯吡咯烷酮，pH7和20ML25mM磷钼酸c) 通过添加25ML各自的糖底物溶液，且随后将板以1000rpm振荡共1分钟来起始所述反应d) 使用微孔板阅读器在700nm处监控磷钼酸还原的动力学。 Glucose oxidase according to any one of the preceding claims when measured by the assay medium, which exhibits at least 99.9% of the glucose specificity, and / or <4% galactose-specific, and . / or <03% maltose specificity, and / or <6% xylose specificity, and / or <01% maltotriose specifically, the assay medium comprising the steps of: a ) 75ML the enzyme solution sample was transferred to 96 well flat bottom microplates b) medium was added 100ML solution (19. 05mMN, N- bis (2-hydroxyethyl) -4-nitroaniline); 5% (w / w ) polyvinylpyrrolidone, pH7 20ML25mM phosphomolybdic acid, and c) by adding the respective saccharide 25ML substrate solution, and then the plates were shaken at 1000rpm for 1 minute at 700nm in the starting reaction d) using a microplate reader kinetic monitoring phosphomolybdic acid reduction.
12. 根据前述权利要求中任一项的葡萄糖氧化酶，当借助于介质测定与根据SEQID NO: 1的所述GOx的亚硝基苯胺介质活性相比较时，其显示出对于用于电子转移的亚硝基苯胺介质> 400%、或> 500%、或> 600%的活性，所述介质测定法包括下述步骤： a) 将75ML酶溶液样品转移至96孔平底微孔板b) 加入100ML介质溶液（19. 05mMN，N-双（2-羟乙基）-4-亚硝基苯胺）；5% (w/w) 聚乙烯吡咯烷酮，pH7和20ML25mM磷钼酸c) 通过添加25ML葡萄糖底物溶液，且随后将板以1000rpm振荡共1分钟来起始所述反应d) 使用微孔板阅读器在700nm处监控磷钼酸还原的动力学， 和当借助于ABTS测定法与根据SEQIDNO: 1的所述GOx的氧活性相比较时，彡30%、 或〈25%、或〈20%，且特别是〈15%、或彡10%的氧活性，所述ABTS测定法包括下述步骤： a) 将75ML样品酶溶液转移至含有100ML磷酸盐缓冲液（pH7)的96孔平底微孔板b) 将20ML反 12. Glucose oxidase according to any one of the preceding claims, when measured in accordance with the medium by means of SEQID NO: nitrosoaniline when compared to the activity of the medium 1 of GOx, which shows a electron transfer to nitrosoaniline medium> 400%, or> 500%, or> 600% of the activity, the assay medium comprising the steps of: a) 75ML enzyme solution sample was transferred to 96 well flat bottom microplates b) was added 100ML medium solution (19. 05mMN, N- bis (2-hydroxyethyl) -4-nitroaniline); 5% (w / w) polyvinylpyrrolidone, pH7 20ML25mM phosphomolybdic acid, and c) by adding glucose substrate 25ML solution, and then the plates were shaken at 1000rpm for 1 minute initiate the reaction d) using a microplate reader to monitor the kinetics of reduction of phosphomolybdic acid at 700nm, and when and by means of ABTS assay according SEQIDNO: when the oxygen activity of the GOx 1 compared Pie 30%, or <25% or <20%, and in particular <15%, or 10% oxygen San activity, the ABTS assay comprising the steps of : a) the enzyme solution was transferred to a sample 75ML 100ML containing phosphate buffer (pH 7) 96-well flat-bottomed microplate b) the inverse 20ML 混合物加入每个孔，导致下述浓度：0.91U/mLHRP;2. 3mMABTS (2, 2' -联氮-双（3-乙基苯并噻唑啉-6-磺酸)） c) 通过添加25ML葡萄糖底物溶液，且随后将板以1000rpm振荡共30秒来起始所述反应d) 使用微孔板阅读器在414nm处动态测定ABTS的氧化。 Mixture was added to each well, resulting in the following concentrations:. 0.91U / mLHRP; 2 3mMABTS (2, 2 '- azino - bis (3-ethyl benzo thiazoline-6-sulfonic acid)) C) by adding 25ML glucose substrate solution, and then the plates were shaken at 1000rpm for 30 seconds to initiate the reaction d) Determination of the dynamic oxidation of ABTS at 414nm using a microplate reader.
13. -种经分离的多核苷酸，其编码根据前述权利要求任一项的葡萄糖氧化酶或其活性片段。 13. - species isolated polynucleotide encoding a glucose oxidase or an active fragment thereof according to any one of the preceding claims.
14. 一种通过根据权利要求1-13的葡萄糖氧化酶或其活性片段来检测、测定或测量先体外后体内样品中的葡萄糖的方法，所述检测、测定或测量包括使离体样品与所述葡萄糖氧化酶或其活性片段接触。 14. A detected by glucose oxidase or active fragment thereof according to claim 1-13, the ex vivo assay or measuring glucose in a sample method for the detection, measurement or assay comprises contacting the body sample with isolated said glucose oxidase or an active fragment thereof.
15. 权利要求14的方法，其中所述葡萄糖检测、测定或测量使用传感器或测试条装置执行。 15. The method of claim 14, wherein the glucose detection, determined or measured using a sensor or test strip device executed.
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