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Saccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose
通过小规模木质纤维素生物质样本的糖化作用测定纤维素降解成葡萄糖的过程   

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Abstract

Second generation biofuels are derived from inedible lignocellulosic biomass of food and non-food crops. Lignocellulosic biomass is mainly composed of cell walls that contain a large proportion of cellulosic and hemicellulosic polysaccharides. An interesting route to generate biofuels and bio-based materials is via enzymatic hydrolysis of cell wall polysaccharides into fermentable sugars, a process called saccharification. The released sugars can then be fermented to fuels, e.g., by use of yeast.
To test the saccharification efficiency of lignocellulosic biomass on a lab-scale, a manual saccharification protocol was established that uses only small amounts of biomass and a low concentration of enzyme. This protocol can be used for different plant species like Arabidopsis thaliana, tobacco, maize and poplar. The low enzyme concentrations make it possible to detect subtle improvements in saccharification yield and to analyze the speed of hydrolysis. Although a specific acid and alkali pretreatment were included, the saccharification step can be preceded by any other pretreatment. Because no advanced equipment is necessary, this protocol can be carried out in many laboratories to analyze saccharification yield. The protocol was initially described in Van Acker et al. (2013).

Keywords: Saccharification(糖化), Lignocellulose(木质纤维素), Biofuels(生物燃料)

Materials and Reagents

  1. pH-indicator paper (pH 1-14) (Merck Millipore Corporation, catalog number: 1109620003 )
  2. Disposable PD-10 desalting columns (VWR International, catalog number: 95017001 )
  3. Whatman® qualitative filter paper, Grade 1 (Sigma-Aldrich, catalog number: WHA1001110 )
  4. Safe-Lock tubes 2 ml (Thermo Fisher Scientific, Eppendorf, catalog number: 3706 )
  5. Corning® 15 ml centrifuge tubes (Sigma-Aldrich, catalog number: CLS430790 )
  6. Corning® 50 ml centrifuge tubes (Sigma-Aldrich, catalog number: CLS430290 )
    Note: Pricing & availability is not currently available.
  7. Nunc 96-well microplate without lid and flat bottom wells (Thermo Fisher Scientific, catalog number: 269787 )
  8. Parafilm (Bemis Flexible Packaging)
  9. Sodium acetate trihydrate (CH3COONa.3H2O) (Sigma-Aldrich, catalog number: S8625-250 G )
  10. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: 71290-100 g )
  11. Cellulase from Trichoderma reesei ATCC 26291 (Sigma-Aldrich, catalog number: C2730 )
  12. Accellerase® BG from Trichoderma reesei (Genencor, DuPont)
  13. D-glucose (C6H12O6) (Sigma-Aldrich, catalog number: G8270-1 kg )
  14. Glucose oxidase from Aspergillus niger (Sigma-Aldrich, catalog number: G6125-50KU )
  15. Peroxidase from horse radish (Roche Diagnostics, catalog number: 10814407001 )
  16. 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Roche Diagnostics, catalog number: 11112422001 )
  17. Hydrochloric acid fuming 37% (Merck Millipore Corporation, catalog number: 1003171000 )
  18. Sodium hydroxide pellets for analysis (Merck Millipore Corporation, catalog number: 1064981000 )
  19. Ethanol (Merck Millipore Corporation, catalog number: 1009901001 )
  20. Acetone (Merck Millipore Corporation, catalog number: 1000121000 )
  21. Glacial acetic acid (Merck Millipore Corporation, catalog number: 1000631000 )
  22. MilliQ-water
  23. 0.1 M acetic acid buffer solution (pH 4.5) (see Recipes)
  24. 0.1 M acetic acid buffer solution (pH 4.8) (see Recipes)
  25. Glucose oxidase (GOD) – peroxidase (POD) solution (see Recipes)

Equipment

  1. Balance (Mettler Toledo, model: XP-105 Delta Range)
  2. Thermoblock (Thermo Fisher Scientific, Eppendorf, model: Thermomixer Compact )
  3. Temperature controlled benchtop microcentrifuge (Thermo Fisher Scientific, Eppendorf, model: 5417R )
  4. Temperature controlled microplate spectrophotometer (Molecular Devices, model: Spectra Max 250 )
  5. Acid-resistant CentriVap Centrifugal Vacuum Concentrator (Labconco, catalog number: 7810016 )
  6. Oven at 37 °C
  7. Tripod with clamp and knots (VWR International, catalog numbers: 2410093 and 2410258 )
  8. Volumetric flask of 100 ml (Duran Group, catalog number: 246712556 )
  9. Rotilabo® Sealing films for microtest plates (Carl Roth GmbH + Co., catalog number: EN76.1 )
  10. Microcentrifuge (LabSource, VWR International, catalog number: 37001298 )

Procedure

  1. Preparing the enzyme mixture
    1. The enzyme mixture used in this protocol is a mix of cellulase and β-glucosidase (BG). These enzymes first need to be cleared from their stabilizing salts. This is done with a desalting Econo-Pac 10 DG column, fitted with Bio-gel® P-6DG gel, and can be used for both cellulase and BG.
    2. First remove, by pouring off, the NaN3-solution that prevents growth of fungi on the column.
    3. Bring 20 ml of acetic acid buffer solution (pH 4.8) on the column and let gravity work.
    4. Mix 1 ml of enzyme (cellulase or BG) with 2 ml acetic acid buffer solution (pH 4.8) in a Corning® 15 ml centrifuge tube. Bring this mix on the column and let it run through the column. Remove the 3 ml that comes off the column.
    5. Add 4 ml acetic acid buffer solution (pH 4.8) on the column and collect the 4 ml that comes off the column in a Corning® 15 ml centrifuge tube. This is the desalted enzyme.
    6. Clean the column by running 100 ml milliQ-water through the column. The washed column can be used again for another desalting.
    7. Close the column at the bottom and fill the column with 0.02% NaN3. Close the column with a cap on the top and store the column at room temperature.
    8. When cellulase and BG are both desalted, dilute the desalted BG 350-fold with acetic acid buffer solution (pH 4.8).
    9. Mix the desalted cellulase and the desalted, diluted BG in a 5:3 ratio.
    10. Dilute this mix 10-fold with acetic acid buffer solution (pH 4.8). This is the enzyme mixture that will be used in the saccharification assay. It is recommended that the enzyme mixture is used within two months after preparation.

  2. Preparing GOD-POD solution (Bergmeyer, 1974) and calibration curve
    The glucose oxidase (GOD)-peroxidase (POD) method measures indirectly the concentration of glucose (Figure 1). In a first step GOD converts glucose monomers into gluconic acid with the production of hydrogen peroxide. The oxidizing power of hydrogen peroxide is used by POD to oxidize a dye, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Finally, this oxidized form of ABTS will absorb at a wavelength of 405 nm.


    Figure 1. Illustration of the reaction mechanism used to measure glucose

    1. Prepare 100 ml of GOD-POD solution as described in recipes. The GOD-POD solution contains 50 mg ABTS, 44.81 mg glucose oxidase and 173 µl of 4% peroxidase. The final volume is adjusted to 100 ml by adding acetic acid buffer solution (pH 4.5).
    2. Make a dilution series of different glucose concentrations (from 20 µM till 200 µM). Proceed as described in section H and plot as in Figure 2.


      Figure 2. An example of a calibration curve

  3. Measuring enzyme activity
    For publication of saccharification results, it is recommended to report the enzyme activity. As such, the saccharification assay can be repeated with exactly the same conditions. In addition, the number of samples that can be analyzed in one single assay is limited due to practical reasons. To compare saccharification results obtained in different saccharification assays, it is necessary that these assays are performed with the same enzyme activity.
    1. Fill three safe-lock tubes with 4.5 mg of Whatman filter paper. Also include a blank, i.e. a safe-lock tube with no filter paper.
    2. Add to each safe-lock tube (with or without filter paper) 450 µl acetic acid buffer solution (pH 4.8) and 50 µl of the enzyme mixture.
    3. Incubate in a thermoblock set at 50 °C for exactly 1 h while shaking at 12.5 Hz.
    4. Boil the samples and blank for 5 min at 99 °C without shaking.
    5. Centrifuge at 23,477 x g for 5 min.
    6. Dilute the supernatants 20 times with acetic acid buffer solution (pH 4.8).
    7. Measure the glucose release in the diluted supernatant using glucose oxidase-peroxidase (GOD-POD) (see section H) and calculate the activity of the enzyme mixture [expressed in filter paper units (FPU)/ml] according to the formula described in Xiao et al. (2004) and as explained in Figure 3.


      Figure 3. An example of the calculation of the enzyme activity

  4. Acid pretreatment (see Note 1)
    1. Weigh 10 mg aliquots of biomass. The biomass can be chopped into little pieces, e.g. Arabidopsis stem pieces of 2 mm long, or the biomass can be grinded, e.g. poplar stems.
    2. Add 1 ml of 1 M HCl and incubate at 80 °C for 2 h while shaking at 12.5 Hz.
    3. Centrifuge for 5 min at 23,477 x g and remove the supernatant.
    4. Wash the pellet three times by adding each time 1 ml of milliQ-water, centrifuging for 5 min at 23,477 x g and removing the supernatant.
      Note 1: There are many different pretreatments possible. Broad categories are alkali or acid or hot water. Pretreatment can also be skipped. The best type of pretreatment is case by case dependent. An alkali pretreatment has the characteristic to breakdown part of lignin and ester bonds, whereas acid pretreatments will mainly breakdown glycosidic bonds in hemicellulose and amorphous cellulose.

  5. Alkali pretreatment
    1. Weigh 10 mg aliquots of biomass. The biomass can be chopped into little pieces, e.g. Arabidopsis stem pieces of 2 mm long, or the biomass can be grinded, e.g. poplar stems.
    2. Add 1 ml of 62.5 mM NaOH, as described in Van Acker et al. (2014) and incubate at 90 °C for 3 h while shaking at 12.5 Hz.
    3. Centrifuge for 5 min at 23,477 x g and remove the supernatant.
    4. Wash the pellet three times by adding each time 1 ml of milliQ-water, centrifuging for 5 min at 23,477 x g and removing the supernatant.

  6. Severe washing step
    This washing step removes residual chemicals used during pretreatment, but also free sugars and other solutes that are not part of the cell wall. This step should thus also be included for samples that will be saccharified without any pretreatment.
    1. Add 1 ml of 70% ethanol and incubate at 55 °C overnight.
    2. Centrifuge for 5 min at 23,477 x g and remove the supernatant.
    3. Wash the pellet three times by adding each time 1 ml of 70% ethanol, centrifuging for 5 min at 23,477 x g and removing the supernatant.
    4. Finally wash the pellet with 1 ml acetone, centrifuge for 5 min at 23,477 x g and remove the supernatant.
    5. Dry the pellet using a vacuum concentrator.
    6. Weigh the left-over biomass.

  7. Saccharification (see Note 2)
    1. Dissolve every dried (pretreated or not) sample in 1 ml acetic acid buffer solution (pH 4.8) and incubate at 50 °C while shaking (12.5 Hz).
    2. After 5 min of incubation, add 100 µl of enzyme mix to each sample.
    3. At several time points, e.g. 2 h, 6 h, 24 h, 48 h after adding the enzyme mixture, take a 20 µl aliquot of the supernatant after spinning down the samples in a benchtop microcentrifuge.
    4. Dilute this 20 µl-aliquot in an appropriate way (i.e., in a range of 10 till 40-fold dilution) with acetic acid buffer solution (pH 4.8) and boil the diluted solution for 5 min at 99 °C to stop the enzymatic reactions.
    5. Measure the glucose concentration according to step H.
      Note 2: To work very precisely, it is recommended to process only 2 samples every minute. This means adding enzyme to the first two samples in the first minute, adding enzyme to the next two samples in the second minute, etc. After exactly incubating for a certain time, e.g. 2 h, the first two samples are centrifuged and an aliquot is taken in the first minute. Samples are boiled to stop the reaction. In the second minute, the two next samples are centrifuged, an aliquot is taken and boiled to stop the reaction, etc.

  8. Measuring released glucose
    1. For each sample (resulting from step G), standard glucose concentration (see step B) or enzyme activity measurement (see step C), transfer 50 µl to a separate well of a 96-well flat bottom microplate. All measurements are performed in triplicate.
    2. Fill at least 3 wells with blanks, i.e. 50 µl acetic acid buffer (pH 4.8). Also add to these blanks 150 µl GOD-POD-solution.
    3. Add to each well 150 µl GOD-POD-solution.
    4. Close the microplate with a sealing film and incubate the microtiter plate in an oven at 37 °C for exactly 30 min.
    5. Gently remove the sealing film and read the absorbance at 405 nm using a plate spectrophotometer.
    6. Use the GOD-POD calibration curve (see step B) to calculate the glucose concentration in the samples (see Notes 3, 4 and 5).
      Note 3: For the calculations, take also into account the dilution factor and the starting volumes at the different time points.
      Note 4: Figure 4 shows an example of saccharification results.


      Figure 4. An example of saccharification results of WT (blue diamonds and lines) and lignin-deficient Arabidopsis thaliana (red squares and lines)

      Note 5: It is common to normalize the released glucose to the theoretical amount of glucose that would be recovered if cellulose is completely saccharified. As such, the cellulose-to-glucose conversion can be calculated. To determine the cellulose content in small samples, the protocol as described by Foster and colleagues4 can be followed. An anhydroglucose unit of cellulose (162.14 g/mol) gives rise to glucose (180.155 g/mol) upon saccharification. Therefore, the theoretical complete conversion of 1 mg cellulose will give rise to 1.11 mg of glucose.

Recipes

  1. 0.1 M acetic acid buffer solution (pH 4.5)
    Prepare the acetic acid buffer pH 4.5 as follows:
    32.5 ml 1 M glacial acetic acid
    2.38 g sodium acetate trihydrate
    967.5 ml milliQ-water
  2. 0.1 M acetic acid buffer solution (pH 4.8)
    Prepare the acetic acid buffer pH 4.8 as follows:
    24.1 ml 1 M glacial acetic acid
    3.52 g sodium acetate trihydrate
    975.9 ml milliQ-water
  3. GOD-POD solution (see Note 6)
    Prepare the GOD-POD solution as follows:
    50 mg (=1 tablet) ABTS
    44.81 mg glucose oxidase
    173 µl 4% peroxidase (see Note 7)
    Add to reach a total volume of 100 ml with 0.1 M acetic acid buffer (pH 4.5)
    Note 6: First dissolve one ABTS tablet in a volumetric flask of 100 ml in approximately 40 ml of 0.1M acetic acid buffer (pH 4.5). Once it is dissolved and glucose oxidase and peroxidase are added, adjust the volume to the mark of 100 ml with 0.1 M acetic acid buffer (pH 4.5).
    Note 7: 4% peroxidase = 20 mg peroxidase dissolved in 500 µl of 0.1 M acetic acid buffer (pH 4.5).

Acknowledgments

This work was supported by grants from the Multidisciplinary Research Partnership “Biotechnology for a sustainable economy” of Ghent University, the European Commission through the Directorate General Research within the 7th Framework Program RENEWALL (KBBE-2007-3-1-01) and MultiBioPro (grant agreement N° 311804) and the Agency for Innovation by Science and Technology (IWT). RV is indebted to the Research Foundation-Flanders (FWO) for a postdoctoral fellowship.

References

  1. Bergmeyer, H. U. (1974). Methods of enzymatic analysis. Academic Press.
  2. Foster, C. E., Martin, T. M. and Pauly, M. (2010). Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part II: carbohydrates. J Vis Exp (37).
  3. Van Acker, R., Leple, J. C., Aerts, D., Storme, V., Goeminne, G., Ivens, B., Legee, F., Lapierre, C., Piens, K., Van Montagu, M. C., Santoro, N., Foster, C. E., Ralph, J., Soetaert, W., Pilate, G. and Boerjan, W. (2014). Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase. Proc Natl Acad Sci U S A 111(2): 845-850.
  4. Van Acker, R., Vanholme, R., Storme, V., Mortimer, J. C., Dupree, P. and Boerjan, W. (2013). Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana. Biotechnol Biofuels 6(1): 46.
  5. Xiao, Z., Storms, R. and Tsang, A. (2004). Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 88(7): 832-837.

简介

第二代生物燃料衍生自食物和非食物作物的不可食用的木质纤维素生物质。木质纤维素生物质主要由含有大比例的纤维素和半纤维素多糖的细胞壁组成。生成生物燃料和生物基材料的有趣途径是通过将细胞壁多糖酶水解成可发酵的糖,这一过程称为糖化。释放的糖然后可以例如通过使用酵母发酵成燃料。
为了在实验室规模上测试木质纤维素生物质的糖化效率,建立了仅使用少量生物质和低浓度酶的手动糖化方案。该方案可以用于不同的植物物种,例如拟南芥(Arabidopsis thaliana),烟草,玉米和杨树。低的酶浓度使得可以检测糖化产量的微妙改进并分析水解的速度。虽然包括特定的酸和碱预处理,但是糖化步骤可以在任何其它预处理之后。由于不需要先进的设备,该方案可以在许多实验室进行以分析糖化产量。该方案最初在Van Acker等人(2013)中描述。

关键字:糖化, 木质纤维素, 生物燃料

材料和试剂

  1. pH指示剂纸(pH 1-14)(Merck Millipore Corporation,目录号:1109620003)
  2. 一次性PD-10脱盐柱(VWR International,目录号:95017001)
  3. Whatman 定性滤纸,1级(Sigma-Aldrich,目录号:WHA1001110)
  4. 安全锁管2ml(Thermo Fisher Scientific,Eppendorf,目录号:3706)
  5. 15ml离心管(Sigma-Aldrich,目录号:CLS430790)中。
  6. 50ml的离心管(Sigma-Aldrich,目录号:CLS430290)中。
    注意:可用性目前不可用。
  7. Nunc 96孔微孔板(无盖和平底孔)(Thermo Fisher Scientific,目录号:269787)
  8. 石蜡膜(Bemis软包装)
  9. 乙酸钠三水合物(CH 3 COONa 3+ 3H 2 O)(Sigma-Aldrich,目录号:S8625-250G)
  10. 叠氮化钠(NaN 3)(Sigma-Aldrich,目录号:71290-100g)
  11. 来自里氏木霉ATCC 26291(Sigma-Aldrich,目录号:C2730)的纤维素酶
  12. 来自里氏木霉(Genencor,DuPont)的Accellerase ? BG
  13. D-葡萄糖(C 6 H 12 SO 6)(Sigma-Aldrich,目录号:G8270-1kg)。
  14. 来自黑曲霉的葡萄糖氧化酶(Sigma-Aldrich,目录号:G6125-50KU)
  15. 来自辣根的辣根过氧化物酶(Roche Diagnostics,目录号:10814407001)
  16. 2,2'-联氮基 - 双(3??-乙基苯并噻唑啉-6-磺酸)(ABTS)(Roche Diagnostics,目录号:11112422001)
  17. 盐酸发烟37%(Merck Millipore Corporation,目录号:1003171000)
  18. 用于分析的氢氧化钠丸(Merck Millipore Corporation,目录号:1064981000)
  19. 乙醇(Merck Millipore Corporation,目录号:1009901001)
  20. 丙酮(Merck Millipore Corporation,目录号:1000121000)
  21. 冰醋酸(Merck Millipore Corporation,目录号:1000631000)
  22. MilliQ水
  23. 0.1M乙酸缓冲溶液(pH4.5)(参见配方)
  24. 0.1M乙酸缓冲溶液(pH 4.8)(参见配方)
  25. 葡萄糖氧化酶(GOD) - 过氧化物酶(POD)溶液(参见配方)

设备

  1. 平衡(Mettler Toledo,型号:XP-105 Delta Range)
  2. Thermoblock(Thermo Fisher Scientific,Eppendorf,型号:Thermomixer Compact)
  3. 温度控制的台式微量离心机(Thermo Fisher Scientific,Eppendorf,型号:5417R)
  4. 温度控制微孔板分光光度计(Molecular Devices,型号:Spectra Max 250)
  5. 耐酸Centrivap离心真空浓缩机(Labconco,目录号:7810016)
  6. 烤箱在37°C
  7. 夹具和结扣三脚架(VWR International,目录号:2410093和2410258)
  8. 100ml容量瓶(Duran Group,目录号:246712556)
  9. 用于微测试板的密封膜(Carl Roth GmbH + Co.,目录号:EN76.1)
  10. 微量离心机(LabSource,VWR International,目录号:37001298)

程序

  1. 准备酶混合物
    1. 在该方案中使用的酶混合物是纤维素酶和纤维素酶的混合物 β-葡糖苷酶(BG)。这些酶首先需要从他们的清除 稳定盐。这是用脱盐Econo-Pac 10 DG柱, ?装有Bio-gel P-6DG凝胶,并且可以用于纤维素酶和 BG。
    2. 首先通过倾倒去除防止真菌在柱上生长的NaN 3+溶液。
    3. 在柱上加入20ml乙酸缓冲液(pH 4.8),让重力作用
    4. 混合1毫升酶(纤维素酶或BG)与2毫升乙酸缓冲液 溶液(pH 4.8)在Corning 15ml离心管中。把这个混合 ?列,并让它运行通过列。取出3毫升 离开列。
    5. 加入4ml乙酸缓冲液(pH 4.8)在柱上,收集从柱上离开的4ml Corning 15ml离心管。这是脱盐酶。
    6. 通过运行100毫升milliQ水通过柱清洁柱。的 洗涤柱可再次用于另一次脱盐
    7. 关上 柱,并用0.02%NaN 3填充柱。关上 柱顶部带有盖子,并将柱子保存在室温下
    8. 当纤维素酶和BG都脱盐时,用乙酸缓冲溶液(pH 4.8)将脱盐BG稀释350倍。
    9. 将脱盐纤维素酶和脱盐,稀释的BG以5:3的比例混合
    10. 用乙酸缓冲液(pH 4.8)稀释该混合物10倍。 这是将在糖化中使用的酶混合物 测定。建议酶混合物在两个内使用 制备后数月。

  2. 准备GOD-POD溶液(Bergmeyer,1974)和校准曲线
    葡萄糖氧化酶(GOD) - 过氧化物酶(POD)方法间接测量葡萄糖的浓度(图1)。在第一步中,GOD将葡萄糖单体转化为葡萄糖酸,同时产生过氧化氢。过氧化氢的氧化能力被POD用于氧化染料2,2'-连氮基 - 双(3??-乙基苯并噻唑啉-6-磺酸)(ABTS)。最后,这种氧化形式的ABTS将在405nm的波长处吸收

    图1.用于测量葡萄糖的反应机制示意图

    1. 如配方中所述制备100毫升GOD-POD溶液。 GOD-POD 溶液含有50mg ABTS,44.81mg葡萄糖氧化酶和173μl4% 过氧化物酶。通过加入乙酸将最终体积调节至100ml ?缓冲液(pH4.5)
    2. 制作不同的稀释系列 葡萄糖浓度(从20μM至200μM)。按照中描述操作 ?部分H,绘图如图2所示

      图2.校准曲线示例

  3. 测量酶活性
    对于糖化结果的公布,建议报告酶活性。因此,可以在完全相同的条件下重复糖化测定。此外,由于实际原因,在一个单一测定中可以分析的样品数量受到限制。为了比较在不同糖化测定中获得的糖化结果,有必要用相同的酶活性进行这些测定。
    1. 用4.5mg Whatmann滤纸填充三个安全锁管。也 包括空白,即一个没有滤纸的安全锁管。
    2. 加 到每个安全锁管(有或没有滤纸)450μl乙酸 缓冲溶液(pH4.8)和50μl酶混合物
    3. 在设置在50℃的热块中孵育恰好1小时,同时在12.5Hz下摇动
    4. 煮沸样品并在99℃下空白5分钟,无振荡。
    5. 以23,477×g离心5分钟。
    6. 用乙酸缓冲液(pH 4.8)稀释上清液20次
    7. 使用葡萄糖测量稀释的上清液中的葡萄糖释放 ?氧化酶 - 过氧化物酶(GOD-POD)(见H部分)并计算活性 ?的酶混合物[以滤纸单位(FPU)/ml表示] 根据Xiao等人(2004)和as所述的公式 如图3所示。


      图3.酶活性计算的实例

  4. 酸预处理(见注1)
    1. 称重10mg等分的生物质。生物质可以切碎成少 片段,例如2毫米长的 拟南芥茎段,或者生物量可以是 ?研磨,例如杨树茎。
    2. 加入1ml 1M HCl,并在80℃下振荡2小时,同时以12.5Hz振荡
    3. 在23,477×g离心5分钟,除去上清液
    4. 洗涤沉淀三次,每次加入1ml milliQ-水,在23,477×g离心5分钟,除去 上清液。
      注1:可能有许多不同的预处理。广阔 类别是碱或酸或热水。也可以进行预处理 跳过。最佳类型的预处理是依情况而定的。一个 碱预处理具有破坏部分木质素的特性 和酯键,而酸预处理将主要分解 半纤维素和无定形纤维素中的糖苷键

  5. 碱性预处理
    1. 称重10mg等分的生物质。生物质可以切碎成少 片段,例如2毫米长的 拟南芥茎段,或者生物量可以是 ?研磨,例如杨树茎。
    2. 加入1ml 62.5mM NaOH,如Van Acker等人(2014)中所述,并在90℃下孵育3小时,同时以12.5Hz振动。
    3. 在23,477×g离心5分钟,除去上清液
    4. 洗涤沉淀三次,每次加入1ml milliQ-水,在23,477×g离心5分钟,除去 上清液。

  6. 严重洗涤步骤
    该洗涤步骤除去在预处理期间使用的残留化学品,但也除去不是细胞壁的一部分的游离糖和其它溶质。因此,对于不经任何预处理而糖化的样品,也应包括该步骤
    1. 加入1ml的70%乙醇,并在55℃下孵育过夜
    2. 在23,477×g离心5分钟,除去上清液。
    3. 通过加入每次1毫升70%乙醇洗涤沉淀树次, 在23,477×g离心5分钟并除去上清液
    4. 最后用1ml丙酮洗涤沉淀,在23,477×g离心5分钟,除去上清液。
    5. 使用真空浓缩器干燥沉淀。
    6. 称量剩余生物量。

  7. 糖化(见注2)
    1. 将每个干燥(预处理或未处理)样品溶解在1ml乙酸中 缓冲液(pH4.8)中,在50℃下振荡(12.5Hz)孵育
    2. 孵育5分钟后,向每个样品中加入100μl酶混合物
    3. 在几个时间点,例如在添加后2小时,6小时,24小时,48小时 酶混合物,在旋转后取上清液的20μl等分试样 在台式微量离心机中下移样品
    4. 稀释这20 以适当的方式(即在10至40倍的范围内) 稀释)与乙酸缓冲溶液(pH 4.8)并煮沸稀释 ?溶液在99℃下5分钟以终止酶反应
    5. 根据步骤H测量葡萄糖浓度。
      注意2:为了非常精确地工作,建议只处理2 样品每分钟。这意味着向前两个样品中加入酶 在第一分钟,在第二个中向接下来的两个样品添加酶 ?分钟等。在完全孵育一定时间后, 2小时, ?首先将两个样品离心,并在第一个样品中取出等分试样 分钟。将样品煮沸以终止反应。在第二分钟, 将两个下一个样品离心,取出等分试样并煮沸 停止反应等。

  8. 测量释放的葡萄糖
    1. 对于每个样品(得自步骤G),标准葡萄糖浓度 (参见步骤B)或酶活性测量(参见步骤C),转移50μl ?加入96孔平底微孔板的单独孔中。所有 测量一式三份进行。
    2. 用空白填充至少3个孔,即, 50μl乙酸缓冲液(pH 4.8)。还向这些空白中加入150μlGOD-POD溶液
    3. 向每个孔中加入150μlGOD-POD-溶液
    4. 用密封膜关闭微孔板,将微量滴定板在37℃的烘箱中孵育正好30分钟。
    5. 轻轻取出密封膜,用平板分光光度计读取405nm处的吸光度
    6. 使用GOD-POD校准曲线(见步骤B)计算样品中的葡萄糖浓度(见注3,4和5)。
      注意3: 对于计算,还要考虑稀释因子和不同时间点的起始体积。
      注意4: 图4显示了糖化结果的示例。



      1. 图4. WT的糖化结果的实例 (蓝色菱形和线)和木质素缺陷型拟南芥(红色) ?正方形和线条)

        注释5:通常将释放的葡萄糖归一化为理论值 如果纤维素完全地将被回收的葡萄糖的量 糖化。因此,纤维素 - 葡萄糖转化可以是 计算。为了测定小样品中的纤维素含量, 可以遵循由Foster和同事4描述的方案。一个 纤维素的脱水葡萄糖单元(162.14g/mol)产生葡萄糖 (180.155g/mol)糖化。因此,理论上 完全转化1mg纤维素将产生1.11mg 葡萄糖。

食谱

  1. 0.1M乙酸缓冲溶液(pH4.5) 如下制备pH4.5的乙酸缓冲液:
    32.5ml 1M冰醋酸
    2.38g三水合乙酸钠 967.5ml milliQ-水
  2. 0.1M乙酸缓冲溶液(pH 4.8) 如下制备pH 4.8的乙酸缓冲液:
    24.1ml 1M冰醋酸 3.52g三水合乙酸钠 975.9ml milliQ-水
  3. GOD-POD解决方案(见注6)
    准备GOD-POD解决方案如下:
    50 mg(= 1片)ABTS
    44.81mg葡萄糖氧化酶
    173μl4%过氧化物酶(见注7)
    加入以使用0.1M乙酸缓冲液(pH4.5)达到总体积为100ml 注释6:首先将一个ABTS片剂溶解在100ml的容量瓶中 ?在约40ml的0.1M乙酸缓冲液(pH 4.5)中。一旦是 溶解并加入葡萄糖氧化酶和过氧化物酶,调节 体积用0.1M乙酸缓冲液(pH4.5)标记为100ml 注释7:4%过氧化物酶= 20mg过氧化物酶溶于500μl0.1M乙酸缓冲液(pH4.5)中。

致谢

这项工作得到了根特大学多学科研究伙伴关系"生物技术促进可持续经济",欧洲委员会通过第七框架计划RENEWALL(KBBE-2007-3-1-01)和MultiBioPro赠款协议N°311804)和科学技术创新局(IWT)。 RV负责研究基金会佛兰德斯(FWO)的博士后研究。

参考文献

  1. Bergmeyer,H.U。(1974)。 酶法分析方法。学术出版社。
  2. Foster,C.E.,Martin,T.M.and Pauly,M。(2010)。 植物细胞壁(木质纤维素生物质)的综合组成分析第二部分:碳水化合物。 em> J Vis Exp (37)。
  3. Van Acker,R.,Leple,JC,Aerts,D.,Storme,V.,Goeminne,G.,Ivens,B.,Legee,F.,Lapierre,C.,Piens,K.,Van Montagu,MC, Santoro,N.,Foster,CE,Ralph,J.,Soetaert,W.,Pilate,G.and Boerjan,W。(2014)。 改善了从缺乏肉桂酰辅酶A还原酶的田间生长的转基因杨树中的糖化和乙醇产量。 Proc Natl Acad Sci USA 111(2):845-850。
  4. Van Acker,R.,Vanholme,R.,Storme,V.,Mortimer,J.C.,Dupree,P.and Boerjan,W。(2013)。 木质素生物合成扰动影响拟南芥中的次生细胞壁组成和糖化产量 。 Biotechnol Biofuels 6(1):46。
  5. Xiao,Z.,Storms,R。和Tsang,A。(2004)。 基于微孔板的滤纸测定法,用于测量总纤维素酶活性。 Biotechnol Bioeng 88(7):832-837。
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引用:Acker, R. V., Vanholme, R., Piens, K. and Boerjan, W. (2016). Saccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose. Bio-protocol 6(1): e1701. DOI: 10.21769/BioProtoc.1701.
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