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Determination of Mannitol-2-dehydrogenase Activity
甘露醇-2-脱氢酶活性的测定   

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Abstract

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) of Zobellia galactanivorans. The ZgM2DH enzyme catalyzes the reversible conversion of mannitol to fructose using NAD+ as a cofactor. ZgM2DH activity was assayed in both directions, i.e., fructose reduction and mannitol oxidation.
Reversible reaction:
D-mannitol + NAD+ ↔ D-fructose + NADH + H+

Keywords: Mannitol degradation(甘露醇的降解), Mannitol-2-dehydrogenase(mannitol-2-dehydrogenase), Flavobacteria(黄杆菌纲), Zobellia galactanivorans(zobellia galactanivorans)

Materials and Reagents

  1. UV-Star® PS Microplate (96 Well) (Greiner Bio-One GmbH, catalog number: 655801 )
  2. Purified recombinant His-tagged ZgM2DH
    Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ZgM2DH vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare Dharmacon) onto an Äkta avant system (GE Healthcare Dharmacon). The complete purification protocol is described in details in Groisillier et al. (2015).
  3. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  4. MilliQ water
  5. 4-morpholineethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M2933 )
  6. Bis-Tris (Sigma-Aldrich, catalog number: B9754 )
  7. Examples of chemicals to be tested to assess substrate and co-factor specificity:
    1. D-(−)-fructose (Sigma-Aldrich, catalog number: F0127 )
    2. D-(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
    3. D-(+)-mannose (Sigma-Aldrich, catalog number: M4625 )
    4. D-(+)-galactose (Sigma-Aldrich, catalog number: G0750 )
    5. D-(+)-xylose (Sigma-Aldrich, catalog number: X1500 )
    6. D-mannitol (Sigma-Aldrich, catalog number: M9647 )
    7. D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
    8. D-(+)-arabitol (Sigma-Aldrich, catalog number: A3381 )
    9. β-Nicotinamide adenine dinucleotide hydrate (β-NAD) (Sigma-Aldrich, catalog number: N1636 )
    10. β-Nicotinamide adenine dinucleotide phosphate hydrate (β-NADP) (Sigma-Aldrich, catalog number: N5755 )
    11. β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (β-NADH) (Sigma-Aldrich, catalog number: N8129 )
    12. β-Nicotinamide adenine dinucleotide phosphate, reduced tetra (cyclohexylammonium) salt (β-NADPH) (Sigma-Aldrich, catalog number: N5130 )
  8. 1 M Tris-HCl (pH 7.5) (see Recipes)
  9. 10 mM NADH (see Recipes)
  10. 10 mM NAD+ (see Recipes)

Equipment

  1. Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
  2. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)

Software

  1. Hyper 32 (hyper32.software.informer.com)
  2. Microsoft Excel

Procedure

  1. The standard fructose reduction reaction mixture contains 100 mM Tris-HCl (pH 6.5), 1 mM fructose, 0.2 mM NADH and 1 to 10 µg of purified recombinant enzyme, in a final volume of volume 100 µl. The standard mannitol oxidation reaction mixture contains 100 mM Tris-HCl (pH 8.5), 1 mM mannitol, 0.5 mM NAD+ and 1 to 10 µg of purified enzyme in 100 µl. Blank corresponds to reaction mixture where substrate is substituted by MilliQ water (Table 1).

    Table 1. Composition of blank and reaction mixture for determination of ZgM2DH activity (fructose reduction)
    Stock solutions
    µl added in blank
    µl added in reaction mix (test)
    Tris-HCl (pH 6.5) (1 M)
    10
    10
    NADH (10 mM)
    2
    2
    Fructose (25 mM)
    0
    4
    ZgM2DH (1 µg/µl)
    1
    1
    MilliQ water
    87
    83

  2. The continuous assay reaction is started by adding the substrate and the activity is monitored by following changes in absorbance at 340 nm, which corresponds to the decrease or production of NADH, in a Safire2 UV spectrophotometer microplate reader. The reaction is performed at 25 °C for up to 20 min. Only the early linear part of the curves is used to calculate activity (Figure 2).


    Figure 2. Early linear parts of curves representative absorbance (340 nm) monitored as a function of time (sec) in presence of mannitol. The curves represent two series of triplicates at 0 and 0.5 mM of mannitol, respectively.

  3. Fructose reduction and mannitol oxidation activities, based on decrease or production of NADH respectively, are calculated using the formula:
    [(ΔA340nm test-ΔA340nm blank)/(t*6220*0.3*0.0001)]
    Where
    ΔA340nm= variation of absorbance during the duration of incubation
    t= duration of incubation (min)
    6220= extinction coefficient for NADH (L mol-1 cm-1)
    0.3= optical path (cm)
    0.0001= assay volume (L)
    One unit (U) of both activities (fructose reduction or mannitol oxidation) corresponds to 1 µmol of NADH reduced or oxidized per min.
  4. To calculate specific activities, divide the value obtained in the equation above by the quantity of ZgM2DH proteins added in the reaction mixture. Perform three replicates for each assay, and determine the average ± S. E. (cf. Notes) for these three replicates. This applies also to the experiments described below. As an example, typical value for specific activity of ZgM2DH was 3.95 U/mg in presence of 10 mM fructose.
  5. To determine substrate specificity, test ZgM2DH activity in presence of each substrate listed in the “Materials and Reagents” section, using concentration ranging from 1 mM to at least 100 mM.
  6. To determine the optimal pH, replace the 100 mM Tris-HCl (pH 8.5 or 6.5) buffer used in step 1 of procedure by other buffers prepared at different pHs. As an indication, it is possible to use:
    100 mM MES for pH 5.5, 6, 6.5
    100 mM Bis-Tris propane for pH 6.5, 7, 7.5, 8, 8.5, 9, 9.5
    100 mM Tris-HCl for pH 7, 7.5, 8, 8.5, 9
  7. To determine the optimal temperature, incubate the reaction mixtures used in step 1 at temperatures ranging, for instance, from 10 °C to 50 °C, with incremental of 5 or 10 °C.
  8. To assess the effects of various metal ions and chemicals on ZgM2DH activities, add metal ions or chemicals individually and at varying concentrations in the reaction mixture described in step 1. Examples are given in Table 2.

    Table 2. Metal ions and chemicals tested for potential influence on the ZgM2DH activities. Mannitol, fructose, mannitol-1-phosphate and fructose-6-phosphate were used at high concentrations to assess potential inhibitory effects on enzymatic activity.
    Compounds
    Final concentration in the reaction mix
    (mM)
    Ca2+
    10
    K+
    10
    Li+
    10
    NH4+
    10
    Mg2+
    10
    Mercaptoethanol
    10
    EDTA
    10
    Fructose
    20
    Fructose
    50
    Mannitol
    20
    Mannitol
    50
    Mannitol-1-phosphate
    20
    Fructose-6-phosphate
    20
    NaCl
    100
    NaCl
    1,000

  9. To estimate the kinetic parameters of the enzyme for a selected substrate S, run individual enzyme reactions in presence of at least five different concentrations of this substrate and a fixed concentration of NAD(H). Determine the initial reaction rate for each reaction and plot 1/V versus 1/[S] to obtain a Lineweaver-Burk plot, from which Km and Vm for S can be calculated (Figure 3).


    Figure 3. Lineweaver-Burk plot used to determine the Km (1.12 mM) and Vm (7.18 U/mg) of ZgM2DH for mannitol. [S] is the mannitol concentration (in mM) and V is the reaction rate (in U/mg of protein).

Notes

  1. Before determining Km and Vm for a given substrate S, be sure that cofactor [NAD(H)] is in excess, i.e., that V does not increase with increasing quantities of cofactor in the reaction mixture; in the same vein, before determining Km and Vm for the cofactor, be sure that S is in excess, i.e., that V does not increase with increasing quantities of S in the reaction mixture. In theory, saturating concentration is equivalent to 100 Km, but 10 Km is usually sufficient (Bisswanger, 2014). It is then necessary to adjust the ZgM2DH concentration and the incubation time to obtain a linear change of absorbance at 340 nm, i.e., a linear change of NADH.
  2. S. E. corresponds to standard error calculated in Excel.

Recipes

  1. 1 M Tris-HCl (pH 7.5)
    Stored at room temperature
  2. 10 mM NADH
    Prepared fresh in water on the day of use
  3. 10 mM NAD+
    Prepared fresh in MilliQ water on the week of use and stored at -20 °C

Acknowledgments

This protocol was performed by Groisillier et al. (2015). This work was supported by the French National Research Agency via the investment expenditure program IDEALG (ANR-1 0-BTBR-02). We also acknowledge funding from the Émergence-UPMC-2011 research program.

References

  1. Bisswanger, H. (2014). Enzymes assays. Perspective in Sciences 1: 41-55.
  2. Groisillier, A., Herve, C., Jeudy, A., Rebuffet, E., Pluchon, P. F., Chevolot, Y., Flament, D., Geslin, C., Morgado, I. M., Power, D., Branno, M., Moreau, H., Michel, G., Boyen, C. and Czjzek, M. (2010). MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms. Microb Cell Fact 9: 45.
  3. Groisillier, A., Labourel, A., Michel, G. and Tonon, T. (2015). The mannitol utilization system of the marine bacterium Zobellia galactanivorans. Appl Environ Microbiol 81(5): 1799-1812.

简介

甘露醇是在多种活生物体中发生的多元醇,其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径,包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统(PST)和基于M2DH的分解代谢途径。后者涉及两种酶,甘露醇-2-脱氢酶(EC 1.1.1.67)和果糖激酶(EC 2.7.1.4),并且已经在不同的细菌中鉴定,例如, 海洋拟杆菌(Zobellia galactanivorans)( Zg ),最近对研究大分子多糖降解有兴趣。该方案描述了半乳聚糖微生物的重组甘露醇-2-脱氢酶(M2DH)的生物化学表征。使用NAD +辅助作为辅因子,M2DH酶催化甘露醇向果糖的可逆转化。 在两个方向上测定M2DH活性,即果糖还原和甘露醇氧化。
可逆反应:
D-甘露醇+ NAD + ↔D-果糖+ NADH + H +

关键字:甘露醇的降解, mannitol-2-dehydrogenase, 黄杆菌纲, zobellia galactanivorans

材料和试剂

  1. UV-Star microplate(96孔)(Greiner Bio-One GmbH,目录号:655801)
  2. 纯化的重组His-标记的Zg M2DH
    注意:如Groisillier等人(2010)所述,该蛋白质在含有重组pFO4_ZgM2DH载体的大肠杆菌BL21(DE3 em)中产生。通过使用HisPrep FF 16/10柱(GE Healthcare Dharmacon)的亲和层析将该重组蛋白质纯化到?ktaavant系统(GE Healthcare Dharmacon)上。完整的纯化方案在Groisillier等人(2015)中有详细描述。
  3. Trizma底物(Sigma-Aldrich,目录号:T1503)
  4. MilliQ水
  5. 4-吗啉乙磺酸(MES)(Sigma-Aldrich,目录号:M2933)
  6. Bis-Tris(Sigma-Aldrich,目录号:B9754)
  7. 要测试以评估底物和辅因子特异性的化学品的实例:
    1. D - ( - ) - 果糖(Sigma-Aldrich,目录号:F0127)
    2. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
    3. D - (+) - 甘露糖(Sigma-Aldrich,目录号:M4625)
    4. D - (+) - 半乳糖(Sigma-Aldrich,目录号:G0750)
    5. D - (+) - 木糖(Sigma-Aldrich,目录号:X1500)
    6. D-甘露醇(Sigma-Aldrich,目录号:M9647)
    7. D-山梨醇(Sigma-Aldrich,目录号:S1876)
    8. D - (+) - 阿拉伯糖醇(Sigma-Aldrich,目录号:A3381)
    9. β-烟酰胺腺嘌呤二核苷酸水合物(β-NAD)(Sigma-Aldrich,目录号:N1636)
    10. β-烟酰胺腺嘌呤二核苷酸磷酸水合物(β-NADP)(Sigma-Aldrich,目录号:N5755)
    11. β-烟酰胺腺嘌呤二核苷酸,还原二钠盐水合物(β-NADH)(Sigma-Aldrich,目录号:N8129)
    12. β-烟酰胺腺嘌呤二核苷酸磷酸,还原四 (环己基铵)盐(β-NADPH)(Sigma-Aldrich,目录号: N5130)
  8. 1 M Tris-HCl(pH 7.5)(见配方)
  9. 10 mM NADH(参见配方)
  10. 10 mM NAD + (参见配方)

设备

  1. Safire2紫外分光光度计酶标仪(Tecan Trading AG)
  2. NanoDrop 2000分光光度计(Thermo Fisher Scientific)

软件

  1. Hyper 32(hyper32.software.informer.com)
  2. Microsoft Excel

程序

  1. 标准果糖还原反应混合物含有100mM Tris-HCl(pH6.5),1mM果糖,0.2mM NADH和1至10μg纯化的重组酶,最终体积为100μl。标准甘露醇氧化反应混合物在100μl中含有100mM Tris-HCl(pH8.5),1mM甘露醇,0.5mM NAD +和1至10μg纯化的酶。空白对应于反应混合物,其中底物被MilliQ水代替(表1)
    表1.用于测定 M2DH活性(果糖降低)的空白和反应混合物的组成
    库存解决方案
    μl以空白
    添加
    μl加入反应混合物(试验)
    Tris-HCl(pH6.5)(1M) 10
    10
    NADH(10mM) 2
    2
    果糖(25mM)
    0
    4
    Zg M2DH(1μg/μl)
    1
    1
    MilliQ水
    87
    83

  2. 通过加入底物开始连续测定反应,并通过在Safire2UV分光光度计酶标仪中随后在340nm处的吸光度变化监测活性,其对应于NADH的减少或产生。反应在25℃下进行最多20分钟。只有曲线的早期线性部分用于计算活动(图2)。


    图 ?2.曲线的早期线性部分代表吸光度(340nm) 在甘露醇存在下作为时间(秒)的函数进行监测。曲线分别表示在0和0.5mM甘露醇的两个系列的三次重复。

  3. 分别基于NADH的减少或产生的果糖还原和甘露糖醇氧化活性使用下式计算:
    [(ΔA340nm测试-ΔA340nm空白)/(t * 6220 * 0.3 * 0.0001)]
    其中
    ΔA340nm <=孵育期间的吸光度变化
    t =培养持续时间(min)
    6220 = NADH的消光系数(L mol -1 cm -1
    0.3 =光程(cm)
    0.0001 =测定体积(L)
    两种活性(果糖还原或甘露醇氧化)的一个单位(U)对应于每分钟还原或氧化的1μmolNADH。
  4. 为了计算比活性,将在上述方程式中获得的值除以在反应混合物中加入的MdDH蛋白的量。对每个测定进行三次重复,并确定这三个重复的平均值±S.E。(参见注释)。这也适用于下述实验。作为实例,在10mM果糖存在下,Zg M2DH的比活性的典型值为3.95U/mg。
  5. 为了确定底物特异性,使用在1mM至至少100mM的浓度范围内,在"材料和试剂"部分中列出的每种底物存在下测试Zg M2DH活性。
  6. 为了确定最佳pH,用在不同pH下制备的其他缓冲液替换在步骤1中使用的100mM Tris-HCl(pH8.5或6.5)缓冲液。作为指示,可以使用:
    100mM MES,pH 5.5,6,6.5
    100mM Bis-Tris丙烷,pH6.5,7,7.5,8,8.5,9,9.5。
    100mM Tris-HCl,pH 7,7.5,8,8.5,9mM
  7. 为了确定最佳温度,在步骤1中使用的反应混合物在例如10℃至50℃的温度范围内,以5或10℃的增量孵育。
  8. 为了评估各种金属离子和化学物质对Zg/M2DH活性的影响,在步骤1所述的反应混合物中分别加入不同浓度的金属离子或化学物质。实施例在表2中给出。

    表2.金属离子和化学物质对潜在影响Zg M2DH活性的测试。甘露醇,果糖,甘露醇-1-磷酸和果糖-6-磷酸以高的浓度以评估对酶活性的潜在抑制作用

  9. 为了估计所选底物S的酶的动力学参数,在至少五种不同浓度的该底物和固定浓度的NAD(H)的存在下进行单独的酶反应。确定每个反应的初始反应速率,并绘制1/V对1/[S],以获得Lineweaver-Burk图,其中对于S来说K sub和V sub可以计算(图3)。


    图3.用于测定K m(1.12mM)和V m(7.18U/mg)的Z meweaver-Burk图, M2DH代表甘露醇。 [S]是甘露醇浓度(以mM计),V是反应速率(以U/mg蛋白质计)。

笔记

  1. 在确定给定底物S的K m和V m之前,确保辅因子[NAD(H)]过量,即 V不随反应混合物中辅因子的量增加而增加;同样地,在确定辅因子的K sub和V sub之前,确保S是过量的,即,表明V不是随着反应混合物中S的量增加而增加。理论上,饱和浓度等于100公里,但10公里通常就足够了(Bisswanger,2014)。然后需要调整M 2 DH浓度和孵育时间,以获得在340nm处的吸光度的线性变化,即NADH的线性变化。< br />
  2. S.E对应于在Excel中计算的标准误差。

食谱

  1. 1 M Tris-HCl(pH 7.5)
    在室温下贮存
  2. 10mM NADH
    使用当天在水中新鲜制备
  3. 10 mM NAD +
    在使用一周后在MilliQ水中新鲜制备并储存在-20℃下

致谢

该方案由Groisillier等人(2015)进行。这项工作是由法国国家研究机构通过投资支出方案IDEALG(ANR-10-BTBR-02)支持的。我们还感谢émergence-UPMC-2011研究计划的资助。

参考文献

  1. Bisswanger,H.(2014)。 酶学测定。 1:41- 55.
  2. Gro?illier,A.,Herve,C.,Jeudy,A.,Rebuffet,E.,Pluchon,PF,Chevolot,Y.,Flament,D.,Geslin,C.,Morgado,IM,Power,D.,Branno, M.,Moreau,H.,Michel,G.,Boyen,C.and Czjzek,M。(2010)。 MARINE-EXPRESS:利用高通量克隆和表达策略进行海洋后基因组分析微生物细胞。 9:45。
  3. Groisillier,A.,Labourel,A.,Michel,G.and Tonon,T。(2015)。 海洋细菌Zobellia galactanivorans的甘露醇利用系统。 Appl Environ Microbiol 81(5):1799-1812。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Groisillier, A. and Tonon, T. (2015). Determination of Mannitol-2-dehydrogenase Activity . Bio-protocol 5(21): e1634. DOI: 10.21769/BioProtoc.1634.
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