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An Evaluation of Cellulose Degradation Affected by Dutch Elm Disease
纤维素在荷兰榆树病影响下的降解测评   

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Abstract

The pathogenic fungus Ophiostoma novo-ulmi spreads within the secondary xylem vessels of infected elm trees, causing the formation of vessel plugs due to tyloses and gels, which ultimately result in Dutch elm disease. Foliage discoloration, wilting and falling from the tree are typical external leaf symptoms of the disease followed by the subsequent death of sensitive trees. Cellulolytic enzymes produced by the fungus are responsible for the degradation of medium molecular weight macromolecules of cellulose, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels but rarely in the fibers (Ďurkovič et al., 2014). The goal of this procedure is to evaluate the extent of cellulose degradation by a highly aggressive strain of O. novo-ulmi ssp. americana × novo-ulmi. Size-exclusion chromatography (SEC) compares molecular weight distributions of cellulose between the infected and the non-infected elm trees, and reveals changes in the macromolecular traits of cellulose, including molecular weights, degree of polymerization, and polydispersity index. 13C magic angle spinning nuclear magnetic resonance (13C MAS NMR) spectra help to identify and also to quantify the loss of both crystalline and non-crystalline cellulose regions due to degradation. The procedure described herein can also be easily used for other woody plants infected with various cellulose-degrading fungi.

Keywords: Ophiostoma novo-ulmi(长喙壳从头螨), Ulmus spp.(榆属), Crystalline cellulose(结晶纤维素), Size-exclusion chromatography(尺寸排阻色谱法), Nuclear magnetic resonance(核磁共振)

Materials and Reagents

  1. Absolute ethanol (99.5%) (EMD Millipore, catalog number: 107017 )
  2. Toluene (99.5%) (Merck KGaA, catalog number: 107019 )
  3. Acetylacetone (99%) (Merck KGaA, catalog number: 109600 )
  4. Dioxane (99.5%) (Merck KGaA, catalog number: 109671 )
  5. Fuming hydrochloric acid (37%) (Merck KGaA, catalog number: 101834 )
  6. Methanol (99.8%) Merck KGaA, catalog number: 107018 )
  7. Ultrapure water
    Note: It is produced by Millipore Simplicity® 185 (UV)  ultrapure water purification system (EMD Millipore).
  8. Pyridine (99.5%) (Merck KGaA, catalog number: 109728 )
  9. Phenyl isocyanate (99%) (Merck KGaA, catalog number: 107255 )
  10. Tetrahydrofuran (99.8%) (Merck KGaA, catalog number: 109731 )

Equipment

  1. Chainsaw, bandsaw, abrasive belt machine and woodworking lathe
  2. Analytical balance (accurate to 1 mg or 0.1 mg)
  3. Desiccator and oven for drying of samples (set to 50 ± 3 °C, 70 ± 3 °C, and to 105 ± 3 °C)
  4. Polymix (Kinematica, model: PX-MFC 90D )
  5. Analysette 3 vibratory sieve shaker (Fritsch)
  6. Soxhlet extraction apparatus (Sigma–Aldrich, catalog number: 64825 )
  7. Boiling flasks (50 ml) (Sigma–Aldrich, catalog number: Z418773 )
  8. Water bath (Harry Gestigkeit Gmbh, model: W 16 )
  9. Fritted-glass filtering crucible of medium porosity (16–40 µm) (VWR International, catalog number: 511-2403 )
  10. Dropping flasks (50 ml) (Smith Scientific Limited, catalog number: 8029/50 )
  11. High performance liquid chromatography system (degasser, pump, autosampler, heater and diode-array ) (Agilent Technologies, model: 1200 series )
  12. Captiva Premium Syringe Filter (0.45 mm PTFE membrane, 15 mm) (Agilent Technologies, catalog number: 5190-5085 )
  13. PLgel (10 μm, 7.5 x 300 mm, two pieces) (Agilent Technologies, model: MIXED-B column )
  14. PLgel (10 μm, 7.5 x 50 mm) (Agilent Technologies, model : Guard-column )
  15. Solid-state NMR spectrometer (Varian, model: 400-MHz ) with the following assembly (Figure 1):
    Magnet: superconducting actively shielded magnet, B = 9.4 T, wide bore – 89 mm
    Console: three high-power linear RF channels, all with spin lock and decoupling capability, output power up to 1000 W,
    1. channel – high band RF, narrow band amplifier 375–400 MHz
    2. channel – broad band amplifier 18–240 MHz
    3. channel – broad band amplifier 10–130 MHz
    Probes:
    a: 1H – 19F / 31P – 79Br / 23Na – 15N , 4 mm T3-HXY for solids, double and triple resonance, MAS up to 18 kHz
    b: 1H – 19F / 31P – 79Br / 23Na – 15N, 7.5 mm T3-HXY for solids, double and triple resonance, MAS up to 7 kHz
    c: Double channels goniometric static HX probe
    X channel – 5, 10 mm coils, resonance from 31P to 15N
    Accessories:
    Variable temperature control unit, range –150 °C ↔ +250 °C
    Accessory for low-band measurements – down from 15N
    Accessories for sample preparation and measurements:
    a. ZrO rotors with diameters of 4 and 7.5 mm
    b. Rotor packing and cleaning tools, isopropyl alcohol, liquid nitrogen
    c. Assembly for hydration and dehydration of samples
    d. Primary and secondary standards for calibration of NMR ppm scale


    Figure 1. Solid-state 13C MAS NMR equipment

Software

  1. ChemStation for LC 3D systems (Agilent Technologies)
  2. Clarity, GPC module, version 5.0.3.180 (DataApex)
  3. VnmrJ 3.2 (Varian)
  4. Mnova 8.1, or a higher version (Mestrelab Research)

Procedure

  1. Cellulose isolation from the stems of infected trees according to Seifert's method
    1. Select elm trees affected by Dutch elm disease (Figures 2A and 2D) and saw several wood discs, approximately 4 cm thick, from the stems at breast height 130 cm above a tree base. If forking does not allow disc sampling from the stem at this height, sample wood discs at the highest possible position on the stem below forking. Distinct infection zones are characterized by discoloration of the wood (Figures 2B and 2E) where the fungal hyphae grow and spread through secondary xylem vessels (Figures 2C and 2F).


      Figure 2. Dutch elm disease (DED) and the infection zones in Dutch elm hybrids 'Groeneveld' (susceptible to DED, A-C) and 'Dodoens' (tolerant to DED, D-F), which were artificially inoculated with spores of the pathogenic fungus Ophiostoma novo-ulmi ssp. americana x novo-ulmi. A. DED symptoms in an infected susceptible tree. B. Wood disc sawn from a susceptible tree, in which the infection zones (marked by red arrows) are restricted to the annual ring for current-year. Early the following growing season after an inoculation these infected trees died. C. Evidence of fungal hyphae growth inside latewood vessels within the infection zone. Cross-section in scanning electron microscopy, scale bar = 50 μm. D. Diminished symptoms of DED in an infected tolerant tree. E. Wood disc sawn from a tolerant tree which survived the infection. Infected trees continued to grow without any physiological weakening in the following years. Discolored infection zones are marked by red arrows. F. Occasional occurrence of a fungal hypha (white arrow) growing inside earlywood vessel within the infection zone. Radial section in scanning electron microscopy, scale bar = 50 μm.

    2. Separate the annual ring sections which involve the infection zones from wood discs using a bandsaw, abrasive belt machine and woodworking lathe. See Video 1 for more detail. Disintegrated and extracted wood coming only from these sections of the annual ring is subjected to size-exclusion chromatography and 13C nuclear magnetic resonance measurements. Also, separate the identical annual ring sections from the control trees to enable comparisons of macromolecular traits of cellulose between the infected and the non-infected trees.

      Video 1. Separation of the annual ring sections which involve the infection zones

    3. Disintegrate separated wood sections into sawdust using a Polymix PX-MFC 90D mill.
    4. Sieve sawdust to a desirable fraction (size 0.50-1.00 mm) and dry in a desiccator.
    5. Extract the dry sawdust (5 g per 250 ml of the extraction solution per sample) according to the ASTM International standard procedure D1107-96 (2013) in a Soxhlet extraction apparatus with an ethanol-toluene solution (1 L absolute ethanol and 427 ml toluene) for 6 h.
    6. Dry the extracted sawdust in air on bench top overnight, then under vacuum at 50 ± 3 °C for at least 4 h.
    7. Place the dry wood sawdust (1 g), acetylacetone (6 ml), dioxane (2 ml), and hydrochloric acid (1.5 ml) into a 50 ml boiling flask.
    8. Heat the flask under reflux using a boiling water bath for 30 min, then allow the mixture to cool slowly to near room temperature and add methanol (30-40 ml) in the fume hood.
    9. Dry the filtering crucibles in an oven at 105 ± 3 °C for 2 h, then cool down in a desiccator to room temperature and weigh.
    10. Filter the mixture through the previously weighed filtering crucibles. Slowly rinse the solids using the following sequence: methanol (100 ml), followed by hot water (40 ml), dioxane (40 ml), and finally methanol (50 ml). During filtration, gently apply a vacuum to remove any liquids. Each rinse step should take approximately 2 min.
    11. Dry the crucible and acid insoluble residue (i.e., Seifert’s cellulose) at 105 ± 3 °C until a constant weight is achieved, usually a minimum of 90 min.

  2. Size-exclusion chromatography (SEC) of cellulose tricarbanilates
    1.  Put Seifert’s cellulose (50 mg), pyridine (8.0 ml) and phenyl isocyanate (1.0 ml) into a 50 ml dropping flask.
    2. Place the sealed flask in an oil bath and heat in the oven at 70 ± 3 °C for 72 h.
    3. Cool to room temperature and add methanol (2.0 ml) to eliminate any excess phenyl isocyanate.
    4. Add the yellow solution dropwise into a rapidly magnetic stirring methanol: water (7:3) mixture (150 ml).
    5. Filtrate the precipitate and wash with methanol:water (7:3) mixture (1 x 50 ml), then with water (2 x 50 ml) to a neutral reaction (pH = 7.0).
    6. Dry the cellulose tricarbanilate (CTC) in air on bench top overnight, then under vacuum at 50 ± 3 °C.
    7. Dissolve CTC (2.0 mg) in tetrahydrofuran (THF) (2.0 ml) and filtrate through a Puradisc 25 NYL syringe filter (pore size 0.45 μm) into an autosampler vial using a 5 ml syringe.
    8. Inject the sample (10 μl) into a chromatograph and analyse by SEC at the following conditions: temperature of 35 °C, mobile phase (THF) flow rate of 1.0 ml/min on two PLgel, 7.5 x 300 mm, MIXED-B columns preceded by a PLgel, 7.5 x 50 mm, Guard-column.
    9. Acquire data with Chemstation software, and then import the data from Chemstation into the Clarity software. Calculate the molecular weights (Mn, Mw, Mz, Mz+1, Mp), degree of polymerization (DPw) and polydispersity index (PDI) of cellulose samples, and compare molecular weight distributions of cellulose tricarbanilates between the infected and the non-infected trees (Figure 3).


    Figure 3. Size-exclusion chromatography of molecular weight distributions of cellulose tricarbanilates prepared from the non-infected and infected trees of the Dutch elm hybrid 'Dodoens'. Cellulolytic enzymes produced by O. novo-ulmi ssp. americana x novo-ulmi are responsible for the degradation of mostly medium molecular weight macromolecules of cellulose (DPw values of approximately 235 to 238) which results in an substantial increase in low molecular weight macromolecules (left side of the chromatogram). At the same time, however, the infected tree responds to the infection with a significant increase in the biosynthesis of high molecular weight macromolecules of cellulose (right side of the chromatogram), followed by a shift in the peak (Mp value) to the high molecular weight area. Co-occurring biosynthetic and biodegradation processes result in changes to the macromolecular traits of cellulose (Mn, Mw, Mz, Mz+1, DPw, PDI) in the infected elm trees.

  3. Solid-state 13C MAS NMR
    1. For the 13C MAS NMR measurement, start with a calibration of the chemical shift scale (ppm) and measure the 13C MAS NMR spectrum of adamantane.
    2. Fill the 4 mm ZrO rotor (volume of 52 μl) with a pulverized and compacted extracted sawdust sample.
    3. Insert the rotor into the stator of T3-HXY probe for solids, insert the probe into the magnet bore, and spin the rotor to the rate of 10 kHz.
    4. Tune 1H and 13C channels of the probe.
    5. Set up the parameters of the “onepul” pulse sequence (Figure 4) - π/2-pulse width, transmitter and decoupler offsets, spectral width, decoupling type and power, and acquisition time.


    Figure 4. Scheme of the used pulse sequence - one pulse + dipolar decoupling + sample rotation at a magic angle

    1. Measure and record the free induction decay (FID).
    2. Use Mnova 8.1 software, or a higher version, for the Fourier transformation of FIDs, as well as FID and NMR spectra processing. The software and the accompanying help manual can both be downloaded from the Mestrelab Research website: http://mestrelab.com/software/ and http://mestrelab.com/software/mnova/manuals/.
    3. Compare signal intensities of 13C MAS NMR spectra at both 83 ppm (corresponding to C4 carbon atoms of amorphous cellulose) and 89 ppm (corresponding to C4 carbon atoms of crystalline cellulose) between the infected and the non-infected trees (Figure 5). Based on peak heights, calculate crystallinity index and the percentage loss for both amorphous and crystalline regions, respectively.


    Figure 5. Solid-state 13C MAS NMR spectra of extractives-free samples from the non-infected and infected trees of the Dutch elm hybrid 'Dodoens'. Signals at 83 ppm and 89 ppm correspond to C4 carbon atoms of amorphous and crystalline cellulose, respectively. The decrease in signal intensities at both resonances reveals that losses in crystalline (26.09% drop) and non-crystalline (5.26% drop) cellulose regions have occurred in parallel.

Notes

We have found that syringyl to guaiacyl (S/G) ratio in lignin affected the cellulose degradability by O. novo-ulmi in the infected elm trees (Ďurkovič et al., 2014). Other recent studies also revealed that an S/G ratio has a significant influence on the cross-linking between lignin and other cell wall components, thus modifying the microscopic structure and topochemistry of the cell wall, the cell wall degradability during chemical and hot-water pretreatments, and the successive hydrolysis of cellulose to glucose (Li et al., 2010; Studer et al., 2011; Papa et al., 2012). Therefore, we suggest using standard analytical methods such as alkaline nitrobenzene or cupric oxidations, NMR, pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) or others to determine lignin monomer composition as quantified by the S/G ratio. Thereby, both the lignin monomer composition and the cellulose degradation data can provide a more complete view of the biodegradation process caused by cellulose-degrading fungi.

Acknowledgments

The authors thank Dr. Miloň Dvořák, Dr. Jana Krajňáková, Dr. Miroslava Mamoňová, Dr. Ingrid Čaňová, Dr. Jaroslav Ohanka and Mr. Miroslav Rusnák for their technical assistance. This work was funded by the Slovak scientific grant agency VEGA (1/0149/15). This protocol has been adapted from our previous work (Ďurkovič et al., 2014).

References

  1. ASTM D1107-96. (2013). Standard test method for ethanol-toluene solubility of wood. American Society for Testing and Materials International, West Conshohocken, PA.
  2. Ďurkovič, J., Kačík, F., Olčák, D., Kučerová, V. and Krajňáková, J. (2014). Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease. Ann Bot 114(1): 47-59.
  3. Li, X., Ximenes, E., Kim, Y., Slininger, M., Meilan, R., Ladisch, M. and Chapple, C. (2010). Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment. Biotechnol Biofuels 3: 27.
  4. Papa, G., Varanasi, P., Sun, L., Cheng, G., Stavila, V., Holmes, B., Simmons, B. A., Adani, F. and Singh, S. (2012). Exploring the effect of different plant lignin content and composition on ionic liquid pretreatment efficiency and enzymatic saccharification of Eucalyptus globulus L. mutants. Bioresour Technol 117: 352-359.
  5. Studer, M. H., DeMartini, J. D., Davis, M. F., Sykes, R. W., Davison, B., Keller, M., Tuskan, G. A. and Wyman, C. E. (2011). Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci U S A 108(15): 6300-6305.

简介

病原真菌新孢子虫在受感染的榆树的次生木质部导管内扩散,导致由于菌丝和凝胶而形成血管栓塞,最终导致荷兰榆树病。叶子变色,枯萎和从树上掉下是典型的外部叶症状的疾病,随后是敏感树的死亡。由真菌产生的纤维素分解酶负责纤维素的中等分子量大分子的降解,导致在血管中发生继发性细胞壁破裂和裂纹,但是在纤维中很少发生破裂(Jurkovic等人, ,2014)。该程序的目的是通过高侵蚀性的O应变评价纤维素降解的程度。 novo-ulmi ssp。 美国× novo-ulmi 。尺寸排阻色谱法(SEC)比较了感染的和未感染的榆树之间的纤维素的分子量分布,并揭示了纤维素的大分子性状的变化,包括分子量,聚合度和多分散指数。 13 C魔幻角旋转核磁共振( 13 C MAS NMR)光谱有助于鉴定和定量由于降解而导致的结晶和非结晶纤维素区域的损失。本文所述的方法也可以容易地用于感染各种纤维素降解真菌的其它木本植物。

关键字:长喙壳从头螨, 榆属, 结晶纤维素, 尺寸排阻色谱法, 核磁共振

材料和试剂

  1. 无水乙醇(99.5%)(EMD Millipore,目录号:107017)
  2. 甲苯(99.5%)(Merck KGaA,目录号:107019)
  3. 乙酰丙酮(99%)(Merck KGaA,目录号:109600)
  4. 二恶烷(99.5%)(Merck KGaA,目录号:109671)
  5. 发烟盐酸(37%)(Merck KGaA,目录号:101834)
  6. 甲醇(99.8%)Merck KGaA,目录号:107018)
  7. 超纯水
    185(UV)  超纯水净化系统(EMD Millipore)。
  8. 吡啶(99.5%)(Merck KGaA,目录号:109728)
  9. 苯基异氰酸酯(99%)(Merck KGaA,目录号:107255)
  10. 四氢呋喃(99.8%)(Merck KGaA,目录号:109731)

设备

  1. 锯,带锯,砂带机和木工车床
  2. 分析天平(精确到1mg或0.1mg)
  3. 用于干燥样品的干燥器和烘箱(设置为50±3℃,70±3℃和105±3℃)。
  4. Polymix(Kinematica,型号:PX-MFC 90D)
  5. Analysette 3振动筛摇床(Fritsch)
  6. 索格利特萃取装置(Sigma-Aldrich,目录号:64825)
  7. 沸腾烧瓶(50ml)(Sigma-Aldrich,目录号:Z418773)
  8. 水浴(Harry Gestigkeit Gmbh,型号:W 16)
  9. 中等孔隙率(16-40μm)的烧结玻璃过滤坩埚(VWR International,目录号:511-2403)
  10. 滴加烧瓶(50ml)(Smith Scientific Limited,目录号:8029/50)
  11. 高效液相色谱系统(脱气机,泵,自动进样器,加热器和二极管阵列)(Agilent Technologies,型号:1200系列)
  12. Captiva Premium注射器过滤器(0.45mm PTFE膜,15mm)(Agilent Technologies,目录号:5190-5085)
  13. PLgel(10μm,7.5×300mm,两片)(Agilent Technologies,型号:MIXED-B柱)
  14. PLgel(10μm,7.5×50mm)(Agilent Technologies,型号:Guard-column)
  15. 固态NMR光谱仪(Varian,型号:400-MHz),具有以下组件(图1):
    磁体:超导主动屏蔽磁体, B <= 9.4 T,宽孔 - 89 mm
    控制台:三个高功率线性射频通道,全部具有自旋锁和去耦能力,输出功率高达1000 W,
    1. 通道 - 高频带RF,窄带放大器375-400 MHz
    2. 通道 - 宽带放大器18-240 MHz
    3. 通道 - 宽带放大器10-130 MHz
    探针:
    a: 1 H 19 F 31 Na- 15 N,对于固体,4mm T3-HXY,双重和三重共振,MAS高达18kHz。
    b: 1 H 19 > Na- 15 N,对于固体,7.5mm T3-HXY,双重和三重共振,MAS高达7kHz
    c:双通道测量静态HX探头
    X通道-5,10mm线圈,从 31 P到 15 N的共振
    附件:
    可变温度控制单元,范围-150°C↔+250°C
    用于低频带测量的附件 - 从 15 N向下
    样品制备和测量附件:
    一个。 ZrO转子,直径为4和7.5毫米
    b。转子包装和清洁工具,异丙醇,液氮
    C。样品水合和脱水装配
    d。 NMR ppm标度校准的主要和次要标准


    图1.固态 13 C MAS NMR设备

软件

  1. LC 3D系统化学工作站(Agilent Technologies)
  2. Clarity,GPC模块,版本5.0.3.180(DataApex)
  3. VnmrJ 3.2(Varian)
  4. Mnova 8.1或更高版本(Mestrelab Research)

程序

  1. 根据Seifert方法从受感染树木的茎上分离纤维素
    1. 选择受荷兰榆树病影响的榆树(图2A和2D) 并从茎上看到几块木盘,大约4厘米厚 乳房高度在树基地上130厘米。 如果分叉不允许光盘 在这个高度从茎上取样,样品木圆盘在最高处 可能的位置在茎上下叉。 不同的感染区 的特征在于木材的变色(图2B和2E)   真菌菌丝生长并通过次生木质部导管扩散 (图2C和2F)。


      图2.荷兰榆树病(DED)和 感染区在荷兰榆树杂种'Groeneveld'(易受DED, A-C)和"Dodoens"(耐受DED,D-F) 用致病真菌的孢子接种新的Ophiostoma novo-ulmi ssp。 x novo-ulmi 。 树。 B.从易感树上锯下的木盘,其中感染 区域(用红色箭头标记)限于年轮 今年。早在下面的生长季节接种后 这些感染的树死了。 C.真菌菌丝生长的证据 感染区内的深木船。横截面扫描 电子显微镜,比例尺=50μm。 D.DED的症状减轻  感染的耐受树。 E.从一棵宽容的树上锯下的木盘  存活的感染。感染的树木继续生长没有任何 生理弱化在接下来的几年。变色的感染 区域由红色箭头标记。 F.偶尔发生的真菌 菌丝(白色箭头)生长在感染内的木材船内  区。在扫描电子显微镜中的径向截面,比例尺= 50 μm。

    2. 分离涉及到的年轮环节 感染区从木盘使用带锯,砂带机 和木工车床。有关详细信息,请参阅视频1。分解和 提取的木材只来自年轮的这些部分 进行尺寸排阻色谱和13 C核磁 共振测量。另外,分开相同的年轮 从控制树的部分,使大分子的比较 感染树木和非感染树木之间的纤维素性状
      视频1.涉及感染区域的年轮环节的分离
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    3. 使用Polymix PX-MFC 90D磨机将分离的木材部分分解成锯末
    4. 筛分锯屑至所需分数(尺寸0.50-1.00mm),并在干燥器中干燥
    5. 提取干锯屑(每250ml提取溶液5g) 样品),根据ASTM国际标准程序D1107-96 (2013)在具有乙醇 - 甲苯的索格利特萃取装置中 溶液(1L无水乙醇和427ml甲苯)6小时
    6. 将提取的锯屑在台面上的空气中干燥过夜,然后在50±3℃的真空下干燥至少4小时。
    7. 将干木屑(1g),乙酰丙酮(6ml),二恶烷(2ml)   和盐酸(1.5ml)装入50ml沸腾烧瓶中
    8. 热 该烧瓶在回流下使用沸水浴30分钟,然后允许   将混合物缓慢冷却至接近室温并加入甲醇 (30-40ml)在通风橱中
    9. 在105±3℃的烘箱中干燥过滤坩埚2小时,然后在干燥器中冷却至室温并称重。
    10. 将混合物过滤通过之前称重的过滤坩埚。 使用以下顺序缓慢冲洗固体:甲醇(100ml),   随后加入热水(40ml),二恶烷(40ml),最后加入甲醇 (50ml)。 在过滤期间,轻轻施加真空以除去任何液体。   每次冲洗步骤约需2分钟。
    11. 干燥 坩埚和酸不溶性残余物(即Seifert's纤维素)   3℃直至达到恒重,通常至少90分钟。

  2. 纤维素三鸟氨酸酯的尺寸排阻色谱法(SEC)
    1. 将塞夫特纤维素(50mg),吡啶(8.0ml)和异氰酸苯酯(1.0ml)放入50ml滴液烧瓶中。
    2. 将密封的烧瓶置于油浴中,在70±3℃的烘箱中加热72小时
    3. 冷却至室温,加入甲醇(2.0ml)以除去任何过量的异氰酸苯酯
    4. 将黄色溶液滴加到快速磁力搅拌的甲醇:水(7:3)混合物(150ml)中
    5. 滤出沉淀,用甲醇:水(7:3)混合物洗涤   (1×50ml),然后用水(2×50ml)至中性反应(pH = 7.0)。
    6. 在台面空气中干燥三萘胺酸纤维素(CTC)过夜,然后在50±3℃下真空干燥。
    7. 将CTC(2.0mg)溶于四氢呋喃(THF)(2.0ml)中 滤液通过Puradisc 25 NYL注射器过滤器(孔径0.45μm) 使用5 ml注射器进入自动进样器样品瓶
    8. 注入样品   (10μl)加入到色谱仪中,并通过SEC在下面进行分析 条件:温度35℃,流动相(THF)流速1.0 ml/min,在两个PLgel,7.5×300mm,在PLgel之前的MIXED-B柱上, 7.5×50mm,保护柱
    9. 使用化学工作站获取数据 软件,然后将数据从Chemstation导入Clarity 软件。计算分子量( M n , M w z , p ),度数  的聚合(DP w)和纤维素的多分散指数(PDI) 样品,并比较纤维素的分子量分布 感染和非感染树之间的三氨基甲酸酯(图 3)。


    图3.从荷兰榆叶杂种'Dodoens'的未感染和感染的树制备的纤维素三鸟氨酸酯的分子量分布的尺寸排阻色谱。由 产生的纤维素分解酶>。 novo-ulmi ssp。美国 x novo-ulmi 负责大多数中等分子量的纤维素大分子的降解(DP w 值约为235至238)导致低分子量大分子(色谱图的左侧)的显着增加。然而,同时,受感染的树对感染作出响应,纤维素的高分子量大分子(色谱图的右侧)的生物合成显着增加,随后峰的移动(M < em> p 值)到高分子量区域。共生的生物合成和生物降解过程导致 改变纤维素的大分子性状( z z + 1 ,DP ,PDI)
  3. 固态13 C MAS NMR
    1. 对于 13 C MAS NMR测量,开始校准 化学位移标度(ppm)并测量的13 C MAS NMR光谱 金刚烷
    2. 用粉碎和压实的提取的锯屑样品填充4mm ZrO转子(体积为52μl)。
    3. 将转子插入T3-HXY探头的定子,用于固体,插入   将探头插入磁铁孔,并使转子旋转至10°的速率 kHz。
    4. 调谐探头的 1 H和 13 C通道
    5. 设置 "onepul"脉冲序列的参数(图4)-π/2-脉冲宽度, 发射器和去耦器偏移,频谱宽度,去耦类型和 功率和采集时间。


    图4.使用的脉冲序列的方案 - 一个脉冲+偶极解耦+在魔角下的样品旋转

    1. 测量并记录自由感应衰减(FID)。
    2. 使用Mnova  8.1软件,或更高版本,用于傅立叶变换 FID,以及FID和NMR光谱处理。软件和 随附的帮助手册都可以从Mestrelab下载 研究网站:http://mestrelab.com/software/和 http://mestrelab.com/software/mnova/manuals/。
    3. 比较信号 在83ppm(对应于无定形纤维素的C 4+碳原子)和89ppm(对应于C 4亚烷基)的两者的13 C MAS NMR光谱的强度>结晶纤维素的碳原子) 非感染树(图5)。基于峰值高度,计算 结晶度指数和无定形和二者的百分比损失 结晶区。


    图5.来自荷兰榆树杂交'Dodoens'的未感染和感染的树的无提取物样品的固态13 C MAS NMR光谱。在83位的信号ppm和89ppm对应于C 4 4碳 无定形和结晶纤维素的原子。在两个共振的信号强度的降低揭示了结晶(26.09%下降)和非结晶(5.26%下降)纤维素区域的损失平行发生。

笔记

我们已经发现,木质素中丁香酰基与愈创木基(S/G)的比率通过O x影响纤维素的降解性。 ,,, 其他最近的研究还表明,S/G比率对木质素和其他细胞壁组分之间的交联有显着的影响,从而改变细胞壁的微观结构和表面化学,化学和热水中的细胞壁降解性预处理,以及纤维素连续水解成葡萄糖(Li等人,2010; Studer等人,2011; Papa等人, ,2012)。因此,我们建议使用标准分析方法,如碱性硝基苯或铜氧化,NMR,热解气相色谱 - 质谱(Py-GC-MS)或其他,以确定由S/G比定量的木质素单体组成。因此,木质素单体组合物和纤维素降解数据都可以提供由纤维素降解真菌引起的生物降解过程的更完整的视图。

致谢

作者感谢MiloňDvořák博士,JanaKrajňáková博士,MiroslavaMamoňová博士,IngridČaňová博士,Jaroslav Ohanka博士和MiroslavRusnák先生提供的技术援助。这项工作由斯洛伐克科学赠款资助 代理VEGA(1/0149/15)。这个协议是从我们以前的工作(Ďurkovič,,2014)。

参考文献

  1. ASTM D1107-96。 (2013年)。 木材乙醇 - 甲苯溶解度的标准测试方法。美国国际测试与材料协会, West Conshohocken,PA。
  2. Ďurkovič,J.,Kačík,F.,Olčák,D.,Kučerová,V.和Krajňáková,J.(2014)。 荷兰榆树杂交种中木材成分的主体反应和代谢分布与荷兰榆树病的对比耐受性。 Ann Bot 114(1):47-59。
  3. Li,X.,Ximenes,E.,Kim,Y.,Slininger,M.,Meilan,R.,Ladisch,M.and Chapple,C。(2010)。木质素单体组成影响液体热水预处理后的拟南芥细胞壁降解性 em> 3:27.
  4. Papa,G.,Varanasi,P.,Sun,L.,Cheng,G.,Stavila,V.,Holmes,B.,Simmons,B.A.,Adani,F.and Singh, 探索不同植物木质素含量和组成对离子液体预处理效率和酶糖化的影响 > Eucalyptus globulus L.mutants。 Bioresour Technol 117:352-359。
  5. Studer,M.H.,DeMartini,J.D.,Davis,M.F.,Sykes,R.W.,Davison,B.,Keller,M.,Tuskan,G.A。和Wyman,C.E。 天然 变体中的木质素含量会影响糖释放。 Proc Natl Acad Sci USA 108(15):6300-6305。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ďurkovič, J., Kačík, F. and Olčák, D. (2015). An Evaluation of Cellulose Degradation Affected by Dutch Elm Disease. Bio-protocol 5(14): e1535. DOI: 10.21769/BioProtoc.1535.
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