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May 2020
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Chromatographic Assays for the Enzymatic Degradation of Chitin
几丁质酶解的色谱分析   

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Abstract

Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis.


Graphical abstract:



Overview of chromatographic methods for assessing the enzymatic degradation of chitin


Keywords: Chitin (几丁质), Chitinase (几丁质酶), Lytic polysaccharide monooxygenase (溶解性多糖单氧酶), LPMO (溶解性多糖单氧酶), Ascorbic acid (AscA) (抗坏血酸), N-acetylglucosamine (N-乙酰葡糖胺), Chitobiose (壳二糖), Chitobionic acid (壳二糖酸), HPLC (高效液相色谱法)

Background

Chitin, an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine, is one of the most abundant recalcitrant polysaccharides in nature, existing predominantly in two allomorphs. In α-chitin, the chains are organized in an anti-parallel fashion, which is the most common and recalcitrant form in nature; in β-chitin, the chains are organized in a parallel fashion, leading to a less recalcitrant structure with a higher water content (Gardner and Blackwell, 1975; Minke and Blackwell, 1978). Chitin is rarely found in its pure form in nature and is usually associated with minerals and proteins, and often with other polysaccharides as seen in fungal cell walls. Hence, obtaining pure chitin, e.g., from crustacean shells, requires demineralization and deproteination steps (Aye and Stevens, 2004). The efficiency of enzymatic saccharification of chitin depends on multiple factors: 1) the method used to extract chitin from the initial biomass; 2) the chitin particle size; and 3) the chitin form [e.g., Nakagawa et al. (2013)]. Thus, the choice of substrate for assaying enzymatic chitin degradation is far from trivial. It is noteworthy that the substrates used for assessing enzymatic chitin degradation, both here and in the field in general, are usually heavily processed and differ considerably from natural chitinous substrates.


Chitin may be converted to oligo- and/or mono-sugars by the coordinated action of multiple carbohydrate-active enzymes [CAZymes (Lombard et al., 2014)], which include several hydrolytic enzymes, such as chitinases (processive exo-chitinases and endo-chitinases; reviewed by Horn et al., 2006; Oyeleye et al., 2018), β-N-acetylhexosaminidases (reviewed by Slámová et al., 2010; also known as chitobiases], and oxidative metallo-enzymes called lytic polysaccharide monooxygenases [LPMOs (Vaaje-Kolstad et al., 2010)]. These enzymes differ in terms of their catalytic mechanism, substrate preferences (crystalline vs. amorphous, endo- vs. exo-attack), and product profiles (Vaaje-Kolstad et al., 2013). Chitinases cleave soluble chitooligosaccharides from chitin, the dominant product often being chitobiose. LPMOs (Chylenski et al., 2019) catalyze the oxidative cleavage of chitin chains, generating soluble and non-soluble C1-oxidized products. β-N-acetylhexosaminidases degrade soluble chitooligosaccharides produced by chitinases and LPMOs from the non-reducing end, producing N-acetylglucosamine and C1-oxidized chitobiose (chitobionic acid). In the CAZy database (Lombard et al., 2014), chitinases primarily occur in the glycoside hydrolase (GH) families GH18 and GH19. β-N-acetylhexosaminidases are found in GH family 20; however, enzymes possessing similar catalytic activity are also found in the GH families 3, 84, and 85. LPMOs are classified as Auxillary Activities (AAs), and chitin-active LPMOs have been detected in families AA10, AA11, and AA15 (Eijsink et al., 2019).


The active site of LPMOs consists of two highly conserved histidine residues that bind a single, catalytically crucial copper ion in a histidine brace (Quinlan et al., 2011; Chylenski et al., 2019). One of these histidines is the N-terminal residue (His1), which contributes to copper coordination with both its imidazole side chain and its (N-terminal) amino group. The latter implies that recombinantly produced LPMOs must be correctly processed, i.e., have an N-terminal histidine, to obtain an active enzyme (Eijsink et al., 2019). LPMOs require an oxygen-containing co-substrate (either O2 or H2O2) and a reductant for the reduction of copper (II) to copper (I). Chitin-active LPMOs appear to primarily target crystalline regions within chitin (Vaaje-Kolstad et al., 2010; Nakagawa et al., 2013), whereas chitinases are thought to prefer regions with a lower degree of substrate crystallinity. Characterization of LPMO activity faces multiple challenges related to the complex interplay among enzyme, reductant, oxidant, and substrate, as described in Eijsink et al., (2019). Importantly, LPMOs are prone to auto-catalytic inactivation (Bissaro et al., 2017; Loose et al., 2018), which obviously complicates the analysis of LPMO activity.


Proper characterization of the activity of chitinases or LPMOs, or combinations thereof, requires enzyme assays using a true polymeric substrate. While artificial chromogenic chitinase substrates are available and can be useful, measured activities toward such substrates provide only limited insight into the chitin-degrading ability of these enzymes. For chitin-active LPMOs, chromogenic artificial substrates do not exist and the very nature of these enzymes dictates that only assays using insoluble chitin are meaningful. Here, we describe methods for analyzing chitin degradation by bacterial LPMOs and chitinases. We also discuss points one should consider to successfully identify and evaluate the biochemical activities of these enzymes.

Materials and Reagents

  1. 2.0 ml microcentrifuge tubes (e.g., Axygen, catalog number: MCT-2000-C-S)

  2. Single-channel mechanical pipettes, e.g.

    0.2-2 µl (VWR, catalog number: 613-5258)

    0.5-10 µl (VWR, catalog number: 613-5259)

    2-10 µl (VWR, catalog number: 613-5260)

    20-200 µl (VWR, catalog number: 613-5263)

    100-1,000 µl (VWR, catalog number: 613-5265)

    1,000-5,000 µl (VWR, catalog number: 613-5266)

  3. Standard pipette tips, e.g.

    Volume 0.1-10 µl (VWR, catalog number: 613-0735)

    Volume 20-200 µl (VWR, catalog number: 613-0732)

    Volume 100-1,250 µl (VWR, catalog number: 613-0739)

    Volume 1,000-5,000 µl (VWR, catalog number: 613-0338)

  4. Wide-orifice pipette tip refill system, e.g.

    Volume 200 µl (VWR, catalog number: 732-3345)

    Volume 1,000 µl (VWR, catalog number: 732-3348)

  5. PD MidiTrap G-25 column (Cytiva LifeSciences, catalog number: 28918008)

  6. Blue-capped flasks 25-1,000 ml (Fisher Scientific, DWK Life Sciences)

  7. 96-well filter plate (Millipore, catalog number: MSHVN4550) with MultiScreen Vacuum Manifold (Millipore, catalog number: MSVMHTS00)

  8. Rezex RFQ-Fast Acid H+ (8%) 100 mm LC column (Phenomenex, catalog number: 00d-0223-k0)

  9. Rezex RFQ-Fast Acid H+ (8%) 50 mm LC Guard column (Phenomenex, catalog number: 03b-0223-k0)

  10. Rezex ROA-Organic Acid H+ (8%) 300 mm LC column (Phenomenex, catalog number: 00h-0138-k0)

  11. 0.3 ml polypropylene HPLC vials with caps and septa (Thermo Scientific, catalog number: 055428)

  12. Bottle-top vacuum filtration PES filter, 0.2 or 0.45 µm (e.g., VWR, catalog number: 514-0338 or 514-0339)

  13. Appropriate preparations of chitinases (e.g., Vaaje-Kolstad et al., 2013), chitobiase (e.g., Loose et al., 2014), and/or chitin-active LPMOs (e.g., Vaaje-Kolstad et al., 2010)

  14. Tris (hydroxymethyl) aminomethane (Tris-HCl) (Sigma-Aldrich, catalog number: 1.08219)

  15. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: SX0607N)

  16. α-Chitin from shrimp shell (e.g., from Chitinor AS or from Sigma, catalog number: C9213)

  17. β-Chitin from squid pen (Suppliers are France Chitin, and several Japanese and Chinese companies; the preparation process is described in Chaussard and Domard (2004))

  18. BisTris (VWR, catalog number: 0715)

  19. Ascorbic acid (Sigma-Aldrich, catalog number: A5960)

  20. Copper (II) sulfate (Sigma-Aldrich, catalog number: 451657)

  21. TraceSelect water (Fisher Scientific, catalog number: 95305)

  22. N-acetylglucosamine (GlcNAc), purity ≥99% (Sigma-Aldrich, catalog number: A8625)

  23. Diacetyl-chitobiose (GlcNAc2), purity >95% (Megazyme, catalog number: O-CHI2)

  24. Triacetyl-chitotriose (GlcNAc3), purity >95% (Megazyme, catalog number: O-CHI3)

  25. Tetraacetyl-chitotetraose (GlcNAc4), purity >95% (Megazyme, catalog number: O-CHI4)

  26. Pentaacetyl-chitopentaose (GlcNAc5), purity >95% (Megazyme, catalog number: O-CHI5)

  27. Hexaacetyl-chitohexaose (GlcNAc6), purity >95% (Megazyme, catalog number: O-CHI6)

  28. Chitooligosaccharide oxidase from Fusarium graminearum as described by Heuts et al. (2008)

  29. Sulfuric acid (Sigma-Aldrich, catalog number: 258105)

  30. Hydrochloric acid, 37% (Merck, catalog number: 100317)

  31. Acetonitrile (VWR, catalog number: 83640.400)

  32. Ascorbic acid stock solution (see Recipes)

  33. 20 mM Tris pH 8.0 (see Recipes)

  34. 15 mM Tris pH 8.0 (see Recipes)

  35. 1 M BisTris pH 6.0 (see Recipes)

  36. 20 ml 50 mg/ml (w/v) chitin suspension (see Recipes)

  37. 50 mM H2SO4 (see Recipes)

  38. 5 mM H2SO4 (see Recipes)

Equipment

  1. Magnetic stirrer (e.g., IKA RCT Basic, catalog number: 0003810000)

  2. Stirring magnets, 25 mm (e.g., VWR, catalog number: 442-4524)

  3. Security Guard cartridge and holder (Phenomenex, catalog numbers: aj0-4490 and kj0-4282)

  4. Acquity UPLC BEH Amide column, 130 Å, 1.7 µm, 2.1 mm × 150 mm (Waters Corp., catalog number: 186004802)

  5. Acquity UPLC BEH Amide VanGuard pre-column, 130 Å, 1.7 µm, 2.1 mm × 5 mm (Waters Corp., catalog number: 186004799)

  6. pH meter (e.g., Metrohm, catalog number: 2.913.0210)

  7. 4°C refrigerator

  8. -20°C freezer

  9. Water purification system (MilliQ water)

  10. Benchtop centrifuge (e.g., Eppendorf centrifuge 5418/5418R, catalog number: EP022620304)

  11. Planetary ball-mill (e.g., Retsch, PM 100, catalog number: 20.540.0001)

  12. Grinding jar (e.g., Retsch, Zirconium Oxide 500 ml, Comfort, catalog number: 01.462.0227)

  13. Grinding balls (e.g., Retsch, Zirconium Oxide 10 mm Ø, catalog number: 22.455.0009)

  14. Stainless-steel sieve, 0.8 mm (e.g.,Thermo Fisher Scientific, catalog number: 10739122)

  15. Thermomixer C (Eppendorf, catalog number: 5382000015) with ThermoTop (Eppendorf, catalog number: 5308000003) and SmartBlock 1.5/2.0 ml (Eppendorf, catalog number: 5362000035)

  16. Vacuum pump/compressor VCP 130 (VWR, catalog number: 181-0308)

  17. UltiMateTM 3000 UHPLC system (Thermo Fisher Scientific) with the following central parts:

    1. SRD-3200 Solvent Rack with 2 degasser channels (Thermo Fisher Scientific, catalog number: 5035.9250)

    2. UltiMateTM ISO-3100BM Biocompatible Isocratic Pump (Thermo Fisher Scientific, catalog number: 5042.0011)

    3. UltiMateTM TCC-3000RS Rapid Separation Thermostatted Column Compartment (Thermo Fisher Scientific, catalog number: 5730.0000)

    4. WPS-3000 TSL Analytical Split-Loop Thermostatted Well Plate Autosampler (Thermo Fisher Scientific, catalog number: 5822.0020)

    5. VWD-3100 Variable Wavelength Detector, one channel (Thermo Fisher Scientific, catalog number: 5074.0005)

  18. UHPLC Agilent Technologies 1290 Infinity (Agilent Technologies Inc., catalog number: G4220-90301)

Software

  1. Chromeleon data system, Chromeleon 7.2.9 (Thermo Fisher Scientific, https://www.thermofisher.com/order/catalog/product/CHROMELEON7#/CHROMELEON7)

Procedure

Unless otherwise stated, all solutions are prepared in standard purified water (MilliQ water). Enzymes are produced and purified as described elsewhere (e.g., Mekasha et al., 2020) and are usually stored at 4°C.

  1. Preparation of copper-saturated LPMO

    Note: All steps are performed at room temperature; see Notes below for challenges associated with using LPMOs in enzymatic reactions.

    1. Mix a solution of Cu(II)SO4 in TraceSelect water (e.g., 50 mM) with a solution of purified LPMO (typically 50-100 μM, for example in 20 mM Tris pH 8.0) to obtain a 3-fold molar surplus of Cu(II) relative to the LPMO.

    2. Incubate for 30 min at 4°C, without stirring.

    3. To remove excess Cu(II), use a standard gravity flow size-exclusion chromatography protocol as described below (this protocol is slightly modified relative to supplier instructions, to minimize copper contamination in the final enzyme sample):

      1. Equilibrate a PD MidiTrap G-25 desalting column by applying 15 ml 20 mM Tris pH 8.0 (enzyme storage buffer) in 5-ml aliquots. Allow the buffer to enter the packed column bed before adding the next portion.

      2. Discard the flowthrough.

      3. After the final 5-ml aliquot of washing buffer has entered the column, apply 0.5 ml Cu(II)-saturated LPMO solution; allow the solution to enter the packed bed completely.

      4. Apply 0.5 ml 20 mM Tris pH 8.0 and allow the buffer to enter the packed bed completely.

      5. Discard the flowthrough.

      6. To elute the sample, place an microfuge tube under the column and apply 1.0 ml 20 mM Tris pH 8.0 to the column.

      7. Collect the flowthrough, which contains the Cu(II)-saturated LPMO.

      8. Discard the column.

      Alternatively, excess free copper may be removed by standard high-performance size-exclusion chromatography using an appropriate chromatographic system. Dialysis is not recommended.


  2. Preparation of substrate (see Notes below for challenges associated with using chitin as a substrate)

    1. Mill the chitin to the desired particle size using a Retsch PM100 planetary ball-mill as described below:

      1. Transfer chitin flakes to a 500-ml zirconium oxide grinding vessel containing zirconium oxide balls (10 × 10 mm) in (approximately) a 1:1 volume:volume ratio. Typically, with this vessel, one uses 100 ml chitin flakes and 100 ml milling balls; volumes are approximate and determined using a plastic graduated beaker. The appropriate total working volume for this vessel is 75-220 ml.

      2. Mill the chitin using a milling speed of 450 rpm for a total milling period ranging from 15 to 30 min; to avoid excess heating, apply 5-min milling periods followed by 2-min pauses.

      3. Pass the milled chitin through a Retsch 0.8-mm stainless-steel sieve and collect particles with a size ≤0.8 mm.

      4. Transfer the milled chitin (particle size ≤0.8 mm) to a clean and dry container.

      5. Store at room temperature.

    2. Prepare a homogeneous stock suspension of the milled chitin (50 mg/ml) in TraceSelect water or a suitable buffer by mixing the suspension well for 24 h using magnetic stirring (see Recipes).

    3. The suspension can be stored at 4°C. We recommend a maximum storage time of three months.


  3. Activity assays
    Chitinase assay

    1. Stock solutions needed before starting the assays:

      1. 50 mg/ml chitin suspension prepared as described above.

      2. 1 M BisTris pH 6.0, where pH is set at 40°C.

      3. Stock solution of purified chitinase, stored on ice. A typical concentration of such a stock solution would be 50 µM.

      4. MilliQ water.

    2. Prepare reactions (200 µl) as follows (volumes are based on the use of a 50 µM chitinase stock solution):

      1. Using a wide-orifice pipette tip, transfer 40 µl magnetically stirred chitin suspension (final concentration of 10 mg/ml) to 2.0-ml reaction tubes.

      2. Add 152 µl MiliQ water and 4 µl 1 M BisTris pH 6.0 (20 mM final concentration) to the chitin suspension.

      3. Incubate the tubes in a pre-heated themomixer with ThermoTop at 40°C for 10 min.

      4. Add 4 µl chitinase solution to the reaction tubes. This gives a final chitinase concentration of 1 µM, which tends to be appropriate; variations are possible and may be needed, depending on the enzyme type.

      5. Incubate the reactions in a thermomixer with ThermoTop at 40°C, 1,000 rpm for the desired duration (typically 0.5-24 h; the optimal duration of the incubation depends on multiple factors, such as the efficiency of the chitinase(s), the type of chitin, and the purpose of the experiment).


    LPMO assay
    1. The following stock solutions are needed in addition to those listed above:

      1. 100 mM ascorbic acid (see Recipes).

      2. TraceSelect water (metal-free).

      3. 50 µM stock solution of purified and Cu(II)-saturated LPMO, prepared as described above and stored on ice.

    2. Prepare reactions (200 µl) as follows:

      1. Using a wide-orifice pipette tip, transfer 40 µl magnetically stirred chitin suspension (final concentration of 10 mg/ml) to 2.0 ml reaction tubes.

      2. Add 150 µl TraceSelect water and 4 µl 1 M BisTris pH 6.0) (20 mM final concentration) to the chitin suspension.

      3. Incubate the tubes in a pre-heated thermomixer with ThermoTop at 40°C for 10 min.

      4. Add 4 µl Cu(II)-saturated LPMO (final concentration of 1 µM) to the reaction tubes.

      5. Add 2 µl 100 mM ascorbic acid (1 mM final concentration).

      6. Incubate the reactions in a thermomixer with ThermoTop at 40°C, 1,000 rpm for the desired duration (typically 0.5-24 h; the optimal duration of the incubation depends on multiple factors, such as the efficiency of the LPMO, the type of chitin, the amount and nature of the reductant, and the purpose of the experiment).


  4. Sampling

    Sampling of chitinase reactions
    1. Draw 50 µl samples from the reaction tubes under shaking using wide-orifice pipette tips to ensure obtaining a homogenous suspension of chitin particles.

    2. Immediately filter the samples using a 96-well filter plate and a multiScreen Vacuum Manifold connected to a vacuum pump/compressor. The filtrates are collected into another 96-well plate. This step should be well prepared and take less than 30 s.

    3. Quench the reactions by mixing 20 µl filtrate (containing soluble products from the reaction) with 20 µl 50 mM H2SO4 (i.e., a two-fold dilution; see Recipes) in 0.3-ml polypropylene vials; seal with caps and septa.

    4. Analyze the samples using the method described in Step E1 below.


    Sampling of LPMO reactions and further processing for quantitation

    To analyze the complete profile of soluble LPMO products, including longer oligosaccharides:

    1. Follow steps 1 and 2 in “Sampling of chitinase reactions” above. This yields filtrates (from 50 μl reaction samples) containing soluble products.

    2. Mix 13 µl filtrate with 37 µl 100% acetonitrile in a microfuge tube (i.e., a final dilution factor of approximately 4).

    3. Spin the mixtures at max speed using an Eppendorf centrifuge 5418/54 to pellet the enzyme precipitate.

    4. Transfer the supernatants to 0.3-ml polypropylene vials, seal with cap and septum.

    5. Analyze the samples using the method described in Step E2 below.


    To simplify quantitation of soluble LPMO products, further degradation of oxidized chitooligosaccharides by chitobiase (CHB) is recommended. The degradation by CHB yields the oxidized dimer, GlcNAc2ox, and the monomer, GlcNAc, as final products.

    1. Follow steps 1 and 2 in “Sampling chitinase reactions” in Section D. This yields filtrates (from 50 µl reaction samples) containing soluble products.

    2. Mix the filtrate (containing soluble LPMO products) with a solution of purified CHB, to reach a final CHB concentration of 2 µM. For example, mix 38 µl filtrate with 2 µl 40 µM CHB solution; see Loose et al. (2014) for details.

    3. Incubate for 16 h at 37°C without shaking.

    4. Spin the reaction tubes containing the CHB degradation products for a few seconds at max speed using an Eppendorf centrifuge 5418/54 to collect condensation droplets.

    5. Transfer 20 µl reaction mixtures to 0.3-ml polypropylene vials containing 20 µl 50 mM H2SO4 (giving a total dilution factor of 2.1), mix well before sealing with caps containing septa.

    6. Analyze the samples using the method described in Step E1 below.


  5. Product analysis and quantitation

    1. Analysis of native products from chitinase reactions and CHB-treated LPMO reactions (Figure 1).

      1. If analyzing a mixture of native monomers and dimers, or a mixture of oxidized dimers and native monomers, HPLC analysis of the samples may be achieved using a Rezex RFQ-Fast Acid H+ (8%) column heated to 85°C, mounted in an UltimateTM 3000 UHPLC system, with 5 mM H2SO4 as the mobile phase (see Recipes) at a flow rate of 1 ml/min. The solutes are separated isocratically over 6 min and detected using UV absorption at 194 nm.

        Note: The native trimer, tetramer, pentamer, and hexamer are also visible using this method, but the separation of these analytes is suboptimal (Figure 1).

      2. If analysis of longer chitooligosaccharides is desired, a Rezex ROA-Organic H+ (8%) column heated to 65°C may be used, with the same system settings as described in point a above, except that the flow rate should be 0.6 ml/min. With this set-up, isocratic elution over 15 min gives better separation of the trimer, tetramer, and pentamer peaks as compared with the method described in a and shown in Figure 1. See Monge et al. (2018) for examples of chromatograms.

      3. The amounts of GlcNAc1-6 and GlcNAc2ox are quantitated using GlcNAc1-6 and GlcNAc2ox standards of known concentrations, which need to be analyzed in the same round of chromatographic runs (same sample series). See Section F for preparation of the standards.

      4. Analysis of chromatograms and calculations of analyte concentrations are performed using the Chromeleon 7.2.9 software.



      Figure 1. Examples of chromatograms produced using the method described in Step E1a. Peaks for native GlcNAc1-6, indicated as A1-A6, and the peak for oxidized chitobiose, indicated as A2ox, are labeled. The chromatograms show four standards (A1, A2, A3, and A1-A6), the products of a chitinase reaction (“Jd1381”), and the products of an LPMO reaction that has been treated with CHB (dotted line, showing the native monomer and the oxidized dimer). The figure is modified from Mekasha et al. (2020). The Jd1381 chitinase also contains an LPMO domain, and since the reaction was run with a reductant, the product mixture also contained small amounts of oxidized products, which elute prior to A2, whose poorly resolved peaks overlap with those belonging to longer non-oxidized products. mAU stands for milli-absorbance units.


    2. Analysis of product profiles from LPMO-containing reactions (Figure 2).

      Note: This method can also be used to analyze the product profile of reactions with only chitinases if the separation of chitooligosaccharides using the (faster) methods described in Section E1 is considered insufficient. In this case, samples from the chitinase reactions need to be treated as described for the LPMO reactions (i.e., dilution in acetonitrile).

      1. Analyze the samples by HPLC using an Acquity UPLC BEH Amide column mounted in an UHPLC Agilent Technologies 1290 Infinity system with a gradient composed of 15 mM Tris-HCl, pH 8.0, and 100% acetonitrile, as follows: 0-5 min, 74% acetonitrile; 5-7 min, 74-62% acetonitrile; 7-8 min, 62% acetonitrile; 8-10 min, 62-74% acetonitrile; 10-12 min, 74% acetonitrile.

      2. Quantitate the amounts of oxidized products using a standard curve based on standards produced as described below. The chromatograms are analyzed using the Chromeleon 7.2.9 software.



      Figure 2. Examples of chromatograms produced using the method described in Step E2. Peaks for native GlcNAc3-6 are labeled A3-A6. Peaks representing oxidized products are labeled A2ox-A6ox. The dotted lines represent standards of native and oxidized chitooligosaccharides. Note that the α- and β-anomers of the native chitooligosaccharides are separated. The solid lines are examples of product profiles obtained after chitin degradation by a chitin-active LPMO (SmLPMO10A) in the presence or absence of a reductant, ascorbic acid (AscA). The figure is modified from Mekasha et al. (2020).


  6. Production of chitooligosaccharide standards, including GlcNAc2ox, GlcNAc3ox, GlcNAc4ox, GlcNAc5ox, or GlcNAc6ox.

    Preparation of oxidized chitooligosaccharides.

    1. Mix 5 mM GlcNAc2, GlcNAc3, GlcNAc4, GlcNAc5 or GlcNAc6, or a mixture of these, with 2 µM purified chitooligosaccharide oxidase from Fusarium graminearum (Heuts et al., 2008; Loose et al., 2014).

    2. Incubate at room temperature for 16 h without stirring; this leads to full conversion. The products in the reaction mixture are stable for months when frozen at -20°C.

    3. Prepare standard solutions in 74% acetonitrile at concentrations in the range of 0.02 mM to 0.5 mM to generate standard curves for quantitation.

    4. Spin the mixtures at max speed using an Eppendorf centrifuge 5418/54 to remove enzyme precipitate.

    5. Transfer the supernatants to 0.3-ml polypropylene vials; seal with caps and septa.

      Standard solutions for native products are prepared by diluting the chitooligosaccharide solutions made in point 1 to yield the appropriate concentrations. The required concentrations depend on the experiment and will usually be higher than those of the standards for oxidized products.

Notes

  1. Chitin-related challenges

    1. The lack of standard procedures for chitin isolation and pretreatment (e.g., milling) leads to variation among the chitins used in different laboratories, which affects the enzyme activity data obtained.

    2. Since chitin is insoluble, it is important, and not straightforward, to produce homogenous chitin suspensions. Special care should be taken to have homogenous chitin suspensions both when setting up and when sampling reactions. The use of smaller particle sizes, e.g., below 1 mm in diameter, makes it easier to obtain homogenous suspensions and to pipette accurately. It is advisable to use wide-bore pipette tips (such as VWR’s wide-orifice pipette tips mentioned in the protocols above). When working with stock suspensions, magnetic stirring tends to be sufficient to keep the suspensions homogeneous.

    3. Note that the particle size and crystallinity of the chitin affect the activity of chitin-degrading enzymes.


  2. Enzyme-related challenges

    While chitinases are “regular” hydrolytic enzymes that do not require special measures, working with LPMOs poses multiple challenges, as outlined in detail in Eijsink et al., (2019). A few key points are:

    1. The LPMO reaction is dependent on a reductant, and different reductants may lead to very different reaction kinetics [e.g., Hegnar et al. (2019)]. Ascorbic acid is most frequently used.

    2. LPMOs use H2O2, which may be added to the reaction or generated within the mixture by reactions involving O2 and the reductant (e.g., ascorbic acid). Special care should be taken to supply sufficient O2 (or H2O2) and reductant for the entire reaction time. This is not a problem when using the reaction conditions described above.

    3. Special care should be taken to avoid inactivation of LPMOs, which may occur when using excess amounts of reductant and/or adding high amounts of H2O2.

    4. LPMO activity and stability decrease if the enzyme is not fully copper-saturated.

    5. The various redox processes in LPMO reactions, such as generation of H2O2 in the reaction mixture, will be affected by the presence of free transition metals in the solution, to an extent that depends on the reductant that is being used. Reactions with ascorbic acid are strongly affected by the presence of free copper ions (Bissaro et al., 2020).

Recipes

  1. Ascorbic acid stock solution (100 mM)

    1. Transfer 17.6 mg ascorbic acid to an Eppendorf tube

    2. Keep the tube on ice and dissolve the ascorbic acid in 1 ml TraceSelect water by pipetting the mixture up and down

    3. Prepare 20-µl aliquots in Eppendorf or PCR tubes and store at -20°C

    4. Use the aliquots only once

  2. 500 ml 20 mM Tris pH 8.0

    1. Weigh 1.211 g Tris (Mw 121.14 g/mol) and dissolve in approximately 400 ml MilliQ-water

    2. Adjust the pH to 8.0 using HCl and then add MilliQ water to a total volume of 500 ml in a volumetric flask

    3. Filter through a 0.45-µm or 0.22-µm bottle-top PES filter

    4. Store at room temperature

  3. 500 ml 15 mM Tris pH 8.0

    1. Weigh 0.909 g Tris (Mw 121.14 g/mol) and dissolve in approximately 400 ml MilliQ-water

    2. Adjust the pH to 8.0 using HCl and then add MilliQ water to a total volume of 500 ml in a volumetric flask

    3. Filter through a 0.45-µm or 0.22-µm bottle-top PES filter

    4. Store at room temperature

  4. 500 ml 1 M BisTris pH 6.0

    1. Weigh 104.62g BrisTris (Mw 209.24 g/mol) and dissolve in approximately 400 ml MilliQ-water

    2. Adjust the pH to 6.0 using HCl and then add MilliQ water to a total volume of 500 ml in a volumetric flask

    3. Filter through a 0.45-µm or 0.22-µm bottle-top PES filter

    4. Store at room temperature

  5. 20 ml 50 mg/ml (w/v) chitin suspension

    1. Weigh 1.0 g chitin and add 20 ml Trace Select water or a suitable buffer

    2. Mix the suspension for 24 h by magnetic stirring at 4°C

    3. Store at 4°C

  6. 50 ml 50 mM H2SO4

    1. Add approximately 40 ml MilliQ-water to a 50-ml volumetric flask

    2. Add 136 µl H2SO4 (Mw 98.08, 1.83 g/cm3)

    3. Mix well and add MilliQ water to a total volume of 50 ml

    4. Store at room temperature

  7. 2 L 5 mM H2SO4

    1. Add approximately 1.9 L MilliQ-water to a 2-L volumetric flask

    2. Add 544 µl H2SO4 (Mw 98.08, 1.83 g/cm3)

    3. Mix well and add MilliQ water to a total volume of 2 L

    4. Store at room temperature

Acknowledgments

We thank former and current lab members for their contributions to the development of our protocols. The present protocol is derived from Mekasha et al., J Biol Chem, 2020, 295:9134-9146, which describes results from projects 221576 and 247001, funded by the Research Council of Norway.

Competing interests

The authors declare no competing interests.

Ethics

There are no ethical issues associated with this protocol.

References

  1. Aye, K. N. and Stevens, W. F. (2004). Improved chitin production by pretreatment of shrimp shells. J Chem Technol Biot 79(4): 421-425.
  2. Bissaro, B., Røhr, A. K., Müller, G., Chylenski, P., Skaugen, M., Forsberg, Z. and Eijsink, V. G. H. (2017). Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. Nature Chem Biol 13(10): 1123-1128.
  3. Bissaro, B., Kommedal, E., Røhr, Å. K. and Eijsink, V. G. H. (2020) Controlled depolymerization of cellulose by light-driven lytic polysaccharide oxygenases. Nature Commun 11(1): 890.
  4. Chaussard, G. and Domard, A. (2004) New aspects of the extraction of chitin from squid pens. Biomacromolecules 5(2): 559-564.
  5. Chylenski, P., Bissaro, B., Sørlie, M., Røhr, Å. K., Várnai, A., Horn S. J. and Eijsink, V. G. H. (2019) Lytic polysaccharide monooxygenases in enzymatic processing of lignocellulosic biomass. ACS Catal 9(6): 4970-4991.
  6. Eijsink, V. G. H., Petrovic, D., Forsberg, Z., Mekasha, S., Røhr, Å. K., Várnai, A. and Vaaje-Kolstad, G. (2019). On the functional characterization of lytic polysaccharide monooxygenases (LPMOs). Biotechnol Biofuels 12: 58.
  7. Gardner, K. H. and Blackwell, J. (1975). Refinement of the structure of β-chitin. Biopolymers 14(8): 1581-1595.
  8. Hegnar, O. A., Petrovic, D. M., Bissaro, B., Alfredsen, G., Várnai, A. and Eijsink, V. G. H. (2019). pH-Dependent relationship between catalytic activity and hydrogen peroxide production shown via characterization of a Lytic Polysaccharide Monooxygenase from Gloeophyllum trabeum. Appl Environ Microbiol 85(5): e02612-18.
  9. Heuts, D. P., Winter, R. T., Damsma, G. E., Janssen, D. B. and Fraaije, M. W. (2008). The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum. Biochem J 413(1): 175-183.
  10. Horn, S. J., Sikorski, P., Cederkvist, J. B., Vaaje-Kolstad, G., Sørlie, M., Synstad, B. and Eijsink, V. G. H. (2006). Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. Proc Natl Acad Sci USA 103(48): 18089-18094.
  11. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. and Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42(Database issue): D490-495.
  12. Loose, J. S., Forsberg, Z., Fraaije, M. W., Eijsink, V. G. H. and Vaaje-Kolstad, G. (2014) A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett 588(18): 3435-3440.
  13. Loose, J. S. M., Arntzen, M. O., Bissaro, B., Ludwig, R., Eijsink, V. G. H. and Vaaje-Kolstad, G. (2018). Multipoint precision binding of substrate protects lytic polysaccharide monooxygenases from self-destructive off-pathway processes. Biochemistry 57(28): 4114-4124.
  14. Mekasha, S., Tuveng, T. R., Askarian, F., Choudhary, S., Schmidt-Dannert, C., Niebisch, A. and Eijsink, V. G. H. (2020). A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin. J Biol Chem 295(27): 9134-9146.
  15. Minke, R. and Blackwell, J. (1978). The structure of alpha-chitin. J Mol Biol 120(2): 167-181.
  16. Monge, E. C., Tuveng, T. R., Vaaje-Kolstad, G., Eijsink, V. G. H. and Gardner, J. G. (2018). Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions. J Biol Chem 293(10): 3849-3859.
  17. Nakagawa, Y. S., Eijsink, V. G. H., Totani, K. and Vaaje-Kolstad, G. (2013). Conversion of α-chitin substrates with varying particle size and crystallinity reveals substrate preferences of the chitinases and lytic polysaccharide monooxygenase of Serratia marcescens. J Agr Food Chem 61(46): 11061-11066.
  18. Oyeleye, A. and Normi, Y. M. (2018). Chitinase: diversity, limitations, and trends in engineering for suitable applications. Biosci Rep 38(4): BSR2018032300.
  19. Quinlan, R. J., Sweeney, M. D., Lo Leggio, L., Otten, H., Poulsen, J. C., Johansen, K. S. and Walton, P. H. (2011). Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci USA 108(37): 15079-15084.
  20. Slámová, K., Bojarová, P., Petrásková, L. and Kren, V. (2010). β-N-acetylhexosaminidase: what's in a name? Biotechnol Adv 28(6): 682-693.
  21. Vaaje-Kolstad, G., Horn, S. J., Sørlie, M. and Eijsink, V. G. H. (2013). The chitinolytic machinery of Serratia marcescens-a model system for enzymatic degradation of recalcitrant polysaccharides. FEBS J 280(13): 3028-3049.
  22. Vaaje-Kolstad, G., Westereng, B., Horn, S. J., Liu, Z., Zhai, H., Sørlie, M. and Eijsink, V. G. H. (2010). An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330(6001): 219-222.

简介

[摘要]甲壳素是的不溶性线型聚合物β(1 → 4)-连接的Ñ乙酰氨基葡萄糖。可以使用称为几丁质酶的水解酶和/或称为分解多糖单加氧酶(LPMO)的氧化酶来实现几丁质链的酶促裂解。这些两组酶小号具有不同的作用模式和屈服需要用于检测和孔定量不同的分析方法不同的产品类型吨 信息。尽管可溶性发色底物很容易用于几丁质酶,但是只有通过测量对它们的聚合的,不溶性底物几丁质的活性才能获得对这些酶活性的正确了解。对于LPMO,只有使用不溶性几丁质的测定是可行且相关的。使用不溶性底物会使从底物制备到产品分析的酶分析变得复杂。在这里,我们描述了几丁质降解反应的典型设置以及用于产品分析的色谱方法。



图形概要:





色谱评估方法概述了甲壳素酶降解


[Backgrou ND ]甲壳素,的不溶性线型聚合物β(1 → 4)-连接的Ñ乙酰氨基葡萄糖,是自然界中最丰富的顽抗多糖,两个同质异形体主要存在中的一个。在α-几丁质中,链以反平行的方式组织,这是自然界中最常见,最顽强的形式。我Ñβ-甲壳质,链以平行有组织的方式,导致具有较少顽抗结构一个较高的水含量(加德纳和Blackwell ,1975;小须和Blackwell ,1978)。几丁质在自然界中很少以其纯净形式存在,通常与矿物质和蛋白质有关,并且通常与真菌细胞壁中所见的其他多糖有关。因此,例如从甲壳类动物壳中获得纯甲壳质需要脱矿质和脱蛋白步骤(Aye和Stevens ,2004)。几丁质酶促糖化的效率取决于多个因素:1)从初始生物质中提取几丁质的方法;2)甲壳素的粒径;和3)几丁质形式[例如,Nakagawa等。((2013)]。因此,用于测定酶促几丁质降解的底物的选择绝非易事。这是笔记值得Ÿ是用于评估酶降解甲壳素在这里和在外地一般的基板,是通常重处理,并从自然壳质基材有很大不同。

几丁质可以是CON verted到寡糖和/或单糖由多个碳水化合物活性enzy的协调动作MES [ CAZymes (伦巴等人,2014)] ,其包括若干水解酶,如几丁质酶(进行性外切-几丁质酶和内切几丁质酶;审查由喇叭。等人,2006; Oyeleye 。等人,2018),β- ñ - acetylhexosaminidases (综述由Slámová等人。,2010;也称为chitobiases ],和氧化金属-enzymes称为裂解多糖单加氧酶[ LPMOs(Vaaje -Kolstad等人,2010)]这些酶在它们的催化机制,底物偏好方面不同。(结晶VS 。无定形的,内-与外-attack) ,和产品型材(Vaaje -科尔斯塔等人,2013年)。几丁质酶裂解可溶性壳寡糖从几丁质,主导的产品常常是壳二糖。LPMOs(Chylenski等人,2019)催化的几丁质链的氧化裂解,生成可溶的一个第二非水溶性C1-氧化产物。β - ñ - acetylhexosaminid ASES降解可溶性壳寡糖通过从非还原性末端几丁质酶和LPMOs,产生生产Ñ乙酰氨基葡萄糖和C1-氧化壳二糖(chitobionic酸)。在CAZY数据库(伦巴等人,2014),几丁质酶主要发生在所述糖苷水解酶(GH)家族GH18和GH19。β - ñ - acetylhexosaminidases在GH家族20发现; 然而,酶具有相似的催化活性,也发现了GH家族3,84 ,和85 LPMOs被分类为Auxillary活动(AAS) ,和在家庭中AA10,AA11已经检测到几丁质活性LPMOs ,和AA15(Eijsink等等,2019)。

LPMOs的活性位点由两个高度保守的组氨酸残基组成,它们与组氨酸支架中的单个催化关键的铜离子结合(Quinlan等人,2011;Chylenski等人,2019)。这些组氨酸之一是N末端残基(His1),其与咪唑侧链及其(N末端)氨基均有助于铜的配位。后者意味着重组产生LPMOs必须被正确处理,即,具有N-末端组氨酸,以获得一个活性酶(Eijsink等人,2019)。LPMO需要含氧的共基质(O 2或H 2 O 2 )和还原剂,以将铜(II)还原为铜(I)。几丁质活性LPMOs出现到主要针对结晶区与几丁质(Vaaje -Kolstad等人,2010 ;中川等人,2013),而几丁质酶被认为倾向于区域具有较低程度的基板的结晶性。LPMO活性的表征面临多重挑战相关的复杂的相互作用中酶,还原剂,氧化剂,和底物,如在Eijsink等。,(20 19)。重要的是,LPMO很容易发生自催化失活(Bissaro等人,2017; Loose等人,2018),这显然使LPMO活性的分析变得复杂。

几丁质酶或LPMO或其组合的活性的正确表征需要使用真正的聚合物底物进行酶分析。虽然人造发色几丁质酶底物是可用的并且可能有用,但是针对此类底物的活性测定仅提供了对这些酶的几丁质降解能力的有限了解。对于几丁质活性的LPMO,不存在发色的人工底物,这些酶的本质决定了只有使用不溶性几丁质的检测才有意义。在这里,我们描述了通过细菌LPMO和几丁质酶分析几丁质降解的方法。我们还将讨论成功识别和评估这些酶的生化活性时应考虑的要点。

关键字:几丁质, 几丁质酶, 溶解性多糖单氧酶, 溶解性多糖单氧酶, 抗坏血酸, N-乙酰葡糖胺, 壳二糖, 壳二糖酸, 高效液相色谱法

 
材料和试剂
 
1. 2.0毫升麦克风ř ocentrifuge管(例如,爱思进,目录号:MCT-2000-CS)      
2.单-通道移液器机械,例如                    
0.2-2 µl (VWR,目录号:613-5258 )
0.5-10 µl (VWR,目录号:613-5259 )
2-10 µl (VWR,目录号:613-5260 )
20-200 µl (VWR,目录号:613-5263 )
100-1 ,000微升(VWR,目录号:613-5265 )
1,000-5,000 µl (VWR,目录号:613-5266)
3.标准移液器吸头,例如      
体积0.1-10 µl (VWR,目录号:613-0735 )
V olume 20-200微升(VWR,目录号:613-0732 )
V olume 100-1,250微升(VWR,CATA日志号码:613-0739 )
V olume 1 ,000-5 ,000微升(VWR,目录号:613-0338)
4.宽-孔移液管尖端笔芯小号ystem ,例如      
体积200微升(VWR,目录号:732-3345 )
V olume 1000微升(VWR,目录号:732-3348)
5. PD MidiTrap G-25色谱柱(Cytiva LifeSciences ,目录号:28918008)      
6.蓝-帽PED烧瓶25-1,000毫升(Fisher Scientific公司,DWK Life Sciences)上      
7.带有MultiScreen真空歧管(Millipore,目录号:MSVMHTS00)的96孔滤板(Millipore,目录号:MSHVN4550)      
8. Rezex RFQ-Fast Acid H +(8%)100毫米LC色谱柱(Phenomenex,目录号:00d-0223-k0)      
9. Rezex RFQ-Fast Acid H +(8%)50 mm LC保护柱(Phenomenex,目录号:03b-0223-k0)      
10. Rezex ROA-有机酸H +(8%)300毫米LC色谱柱(Phenomenex,目录号:00h-0138-k0)   
11. 0.3 ml带盖和隔垫的聚丙烯HPLC小瓶(Thermo Scientific,目录号:055428)   
12.瓶顶真空过滤PES过滤器,0.2或0.45 µm(例如,VWR,目录号:514-0338或514-0339)   
13.几丁质酶的合适制剂(例如,Vaaje -Kolstad等人,2013),chitobiase (例如,松散等人,2014),和/或几丁质活性LPMOs(例如,Vaaje -Kolstad等人,2010)   
14.的Tris (羟甲基)氨基甲烷È (的Tris-HCl)(Sigma-Aldrich公司,目录号:1.08219)   
15.氢氧化钠(NaOH)(Sigma-Aldrich,目录号:SX0607N)   
16.虾壳中的α- C角蛋白(例如,来自Chitinor AS或来自Sigma,目录号:C9213)   
17.鱿鱼圈中的β- C基因(供应商是法国几丁质,以及几家日本和中国公司;其制备过程在Chaussard和Domard (2004)中进行了描述)   
18. BisTris (VWR,目录号:0715)   
19.抗坏血酸(Sigma-Aldrich,目录号:A5960)   
20.硫酸铜(II)(Sigma-Aldrich,目录号:451657)   
21. TraceSelect水(Fisher Scientific,目录号:95305)   
22. N-乙酰氨基葡萄糖(GlcNAc ),纯度≥99%(Sigma-Aldrich,目录号:A8625)   
23.二乙酰基壳二糖葡糖胺(GlcNAc 2 ),纯度> 95%(Megazyme的,目录号:-O-CH1 2)   
24.三乙酰基-chitotriose糖胺(GlcNAc 3 ),纯度> 95%(Megazyme的,目录号:O形CHI3)   
25.四乙酰基壳聚糖四糖(GlcNAc 4 ),纯度> 95%(Megazyme ,目录号:O-CHI4)   
26.五乙酰基- chitopentaose糖胺(GlcNAc 5 ),纯度> 95%(Megazyme的,目录号:O形CHI5)   
27.六乙酰基-chitohexaose糖胺(GlcNAc 6 ),纯度> 95%(Megazyme的,目录号:O形CHI6)   
28.如Heuts等人所述,来自禾谷镰刀菌的壳寡糖氧化酶。,(2008年)   
29.硫酸(西格玛奥德里奇,目录号:258105)   
30.盐酸37%(默克,目录号:100317)   
31.乙腈(VWR,目录号:83640.400)   
32.抗坏血酸储备溶液(请参阅食谱)   
33. 20 mM Tris pH 8.0(请参阅食谱)   
34. 15 mM Tris pH 8.0(请参阅食谱)   
35. 1 M BisTris pH 6.0(请参阅食谱)   
36. 20毫升50毫克/毫升(w / v)几丁质悬浮液(请参阅食谱)   
37. 50 mM H 2 SO 4 (请参阅食谱)   
38. 5 mM H 2 SO 4 (请参阅食谱)   
 
设备
 
电磁搅拌器(例如,IKA RCT Basic,目录号:0003810000)
搅拌磁铁,25毫米(例如,VWR,目录号:442-4524)
Security Guard墨盒和支架(Phenomenex,目录号:aj0-4490和kj0-4282)
Acquity UPLC BEH Amide色谱柱,130Å ,1.7 µm,2.1 mm × 150 mm(Waters Corp.,目录号:186004802)
Acquity UPLC BEH Amide VanGuard预柱,130Å ,1.7 µm,2.1 mm × 5 mm(Waters Corp.,目录号:186004799)
pH计(例如,瑞士万通,目录号:2.913.0210)
4 °C冰箱
-20 °C冷冻室
净水系统(MilliQ水)
台式离心机(例如,Eppendorf离心机5418 / 5418R,目录号:EP022620304)
行星球磨机(例如,Retsch公司,PM 100,目录号:20.540.0001)
研磨罐(例如,Retsch公司,氧化锆500毫升,舒适,目录号:01.462.0227)
研磨球(例如,Retsch公司,氧化锆10mM邻,目录号:22.455.0009)
不锈钢-钢筛,0.8毫米(例如,热电费舍尔科学entific,目录号:10739122)
带有ThermoTop (Eppendorf,目录号:5308000003)和SmartBlock 1.5 / 2.0 ml(Eppendorf,目录号:5362000035)的Thermomixer C(Eppendorf ,目录号:5382000015)
真空泵/压缩机VCP 130(VWR,目录号:181-0308)
UltiMate TM 3000 UHPLC系统(Thermo Fisher Scientific),具有以下主要部件:
SRD-3200带有2个脱气机通道的溶剂架(Thermo Fisher Scientific ,目录号5035.9250)
UltiMate TM ISO-3100BM生物相容性等度泵(Thermo Fisher Scientific ,目录号5042.0011)
UltiMate TM TCC-3000RS快速分离恒温柱箱(Thermo Fisher Scientific ,目录号5730.0000)
WPS-3000 TSL分析型分体式恒温孔板自动进样器(Thermo Fisher Scientific ,目录号5822.0020 )
VWD-3100可变波长检测器,一个通道(Thermo Fisher Scientific ,目录号:5074.0005)
UHPLC安捷伦科技1290 Infinity(Agilent Technologies Inc.,目录号:G4220-90301)
 
软件
 
Chromeleon数据系统,Chromeleon 7.2.9(Thermo Fisher Scientific,https://www.thermofisher.com/order/catalog/product/CHROMELEON7#/CHROMELEON7)
 
程序
 
除非另有说明,否则所有溶液均在标准纯净水(MilliQ水)中制备。如其他地方所述(例如,Mekasha等人,2020年),酶的产生和纯化通常在4°C下进行。
的制备铜-饱和LPMO
注:LL步骤进行在室温下; 有关在酶促反应中使用LPMO的挑战,请参见下面的注释。
混合铜的溶液(II)SO 4在TraceSelect水(例如,50毫摩尔)与(纯化LPMO的溶液通常为50 - 100μ中号,例如在20mM Tris pH 8.0)中,以获得3倍相对于LPMO的Cu(II的摩尔过剩)。
在4 °C下孵育30分钟,无需搅拌。
要除去过量的Cu(II),请使用以下所述的标准重力流大小-排阻色谱法(相对于供应商的说明,该法略有修改,以最大程度地减少最终酶样品中的铜污染):
平衡一个PD MidiTrap通过施加在515毫升的20mM Tris pH 8.0中(酶储存缓冲液)G-25脱盐柱-米升等分试样。在添加下一部分之前,让缓冲液进入填充柱床。
                                          丢弃流通液。
最后5之后-米升的等份洗涤缓冲液已进入柱,应用0.5毫升的Cu(II)-饱和LPMO溶液; 让溶液完全进入填充床。
施加0.5 ml 20 mM Tris pH 8.0,并使缓冲液完全进入填充床。
丢弃流通液。
到ELUT Ë样品,放置一个微量离心管中的柱下和应用1.0毫升的20mM Tris pH 8.0中到柱上。
收集包含Cu(II)-饱和LPMO的流出物。
丢弃色谱柱。
可替代地,可以通过使用合适的色谱系统的标准高性能尺寸-排阻色谱法去除过量的游离铜。不建议透析。             
 
底物的制备(有关使用几丁质作为底物的挑战,请参见下面的注释)
如下所述,使用Retsch PM100行星式球磨机将几丁质磨成所需的粒径:
转移几丁质片到500 -毫升的氧化锆研磨收容容器的氧化锆球(10 ×在(约)1 10毫米):1体积:体积比。通常情况下,使用此容器时,需要使用100毫升的几丁质薄片和100毫升的研磨球。体积是近似的,并使用塑料刻度烧杯确定。该容器中的适当的总工作容积为75 - 220毫升。
使用450 rpm的研磨速度研磨甲壳质,整个研磨时间为15到30分钟;以避免过度加热,应用5 -分钟研磨周期接着用2 -分钟暂停。
通过通过碾磨几丁质Retsch公司0.8 -毫米的不锈钢-钢筛并收集颗粒与尺寸≤0.8毫米。
将研磨后的几丁质(粒径≤0.8毫米)转移到干净干燥的容器中。
存放在室温下。
通过在磁力搅拌下将混悬液充分混合24小时,从而制备出在TraceSelect水或合适的缓冲液中均质的研磨甲壳质(50 mg / ml)的均质悬浮液。
悬浮液可以在4°C下保存。我们建议最长存储时间为三个月。
 
活性测定
几丁质酶测定
1.开始测定之前所需的储备溶液:      
如上所述制备的50mg / ml甲壳质悬浮液。
1 M BisTris pH 6.0 ,其中pH设置为40 °C 。
储备溶液的纯化几丁质酶,在冰上储存。这种储备溶液的典型浓度为50 µM。
MilliQ水。
2.如下准备反应(200 µl)(量基于使用50 µM几丁质酶原液的使用量):      
使用宽-孔移液管尖端,转移40μl的磁力搅拌悬浮液中几丁质至2.0(10毫克/毫升终浓度)-毫升反应管中。
向甲壳素悬浮液中加入152 µl MiliQ水和4 µl 1 M BisTris pH 6.0(终浓度20 mM)。
将试管在与ThermoTop一起在40°C下预热的混合器中孵育10分钟。
甲DD4μl的几丁质酶溶液到反应管中。这样最终的几丁质酶浓度为1 µM,这很合适。根据酶的类型,可能会有变化,也可能需要变化。
孵育在具有恒温反应ThermoTop在40℃,1,000rpm下所需的持续时间(一般为0.5 - 24小时;温育最佳持续时间取决于多种因素,如几丁质酶(一个或多个的效率),类型几丁质的,并在实验的目的)。
 
LPMO检测
1.下列储备溶液需要除了第OSE上面列出:      
100 mM抗坏血酸(请参阅食谱)。
TraceSelect水(不含金属)。
如上所述制备的50 µM纯化的和Cu(II)饱和的LPMO储备液,并保存在冰上。
2.如下准备反应(200 µl):      
使用宽-孔移液管尖端,转移40μl的磁力搅拌悬浮液几丁质(10毫克/毫升终浓度)至2.0毫升反应管中。
向甲壳质悬浮液中加入150 µl TraceSelect水和4 µl 1 M BisTris pH 6.0(终浓度20 mM)。
将试管在带有ThermoTop的预热热混合器中于40°C孵育10分钟。
向反应管中加入4 µl饱和Cu(II)的LPMO(终浓度为1 µM)。
加入2 µl 100 mM抗坏血酸(终浓度1 mM)。
孵育在具有恒温反应ThermoTop在40℃,1,000rpm下所需的持续时间(一般为0.5 - 24小时;温育最佳持续时间取决于多种因素,如LPMO,几丁质的类型的效率,量和还原剂的性质,和本实验的目的)。
 
采样
几丁质酶反应的采样
1.绘制将50μl来自样品下使用宽摇动反应管-孔移液管尖端,以确保获得homogeno我们悬浮壳多糖粒子。      
2.立即使用96孔滤板和连接到真空泵/压缩机的MultiScreen真空歧管过滤样品。将滤液收集到另一个96孔板中。此步骤应做好充分准备,并且少于30秒。      
3.淬火通过与20μl(从反应含有可溶性产物)混合20微升滤液中的反应的50mMħ 2 SO 4 (即,两倍稀释;见配方)在0.3 -毫升聚丙烯小瓶; 用盖帽和隔垫密封。      
4. ANALY ž E使用S中描述的方法将样品TEP E1下面。      
 
LPMO反应,并进一步处理抽样孔定量吨通货膨胀
要分析可溶性LPMO产品(包括更长的寡糖)的完整概况,请执行以下操作:
1.按照步骤小号1和“采样2的上述几丁质酶的反应”。这产生的滤液(50个μ升反应样品)含有可溶性产品。      
2.在微量离心管中将13 µl滤液与37 µl 100%乙腈混合(即,最终稀释系数约为4)。      
3.使用Eppendorf离心机5418/54以最大速度旋转混合物以沉淀酶沉淀物。      
4.转移上清至0.3 -毫升聚丙烯小瓶中,密封帽和隔膜。      
5.分析使用S中描述的方法将样品TEP E2以下。      
 
为了简化孔定量吨的可溶性LPMO产品,氧化的进一步降解通货膨胀壳寡糖通过chitobiase (CHB)的建议。CHB的降解产生氧化的二聚体GlcNAc 2ox和单体GlcNAc作为最终产物。
1.按照D部分“采样几丁质酶反应”中的步骤s 1和2进行操作。这将产生包含可溶产物的滤液(来自50 µl反应样品)。      
2.将滤液(包含可溶性LPMO产品)与纯化的CHB溶液混合,以使CHB最终浓度达到2 µM。例如,混合38微升滤液用2微升40 μ中号CHB溶液; 参见Loose等。,(2014年)以获取详细信息。      
3.在37°C下孵育16小时,不要摇晃。      
4.使用Eppendorf离心机5418/54以最大速度将装有CHB降解产物的反应管旋转几秒钟,以收集冷凝液滴。      
5.转移20微升的反应混合物至0.3 -毫升含有20微升50毫米高的聚丙烯小瓶2 SO 4(产生2.1的总稀释倍数),用含有隔片盖密封前充分混合。      
6.分析使用S中描述的方法将样品TEP下面E1。      
 
产品分析和孔定量牛逼通货膨胀
1.从几丁质酶反应和CHB天然产物的分析-处理LPMO反应(图1 )。      
如果要分析天然单体和二聚体的混合物,或氧化的二聚体和天然单体的混合物,则可以使用加热到85°C的Rezex RFQ-Fast Acid H +(8%)色谱柱进行样品的HPLC分析。一个终极TM 3000 UHPLC系统,具有5毫米高2 SO 4作为流动相(见配方)以1ml / min的流速。在6分钟内等度分离溶质,并使用194 nm的UV吸收进行检测。
注:原生的三聚体,四聚体,五聚体,和六聚体也是可见使用这种方法,但这些分析物的分离是不理想的(图1 )。
如果需要分析更长的壳寡糖,可以使用加热至65°C的Rezex ROA-Organic H +(8%)色谱柱,系统设置与上述第a点相同,但流速应为0.6 ml /分钟 与此设置中,等度洗脱在15分钟内提供更好的分离三聚体,tetrame - [R ,和五聚体的峰为相比与该方法中所描述一个并且在所示图1 。参见Monge等。(2018)中的色谱图示例。
的GlcNAc的量1-6和GlcNAc的20倍是孔定量达ED使用的GlcNAc 1-6和GlcNAc的20倍已知浓度,这需要在同一轮色谱运行(相同样品系列)的要被分析的标准。见F节准备的的标准。
使用Chromeleon 7.2.9软件进行色谱图分析和分析物浓度计算。
 
Figu ř ë 1 。È色谱xamples产生使用方法具中所述d步骤中E1a。标记了天然GlcNAc 1-6的峰(表示为A1-A6)和氧化的壳二糖的峰(表示为A2 ox )。色谱图显示四个标准(A1,A2,A3和A1-A6),几丁质酶反应(“产物JD 1381”),以及一个LPMO反应的那公顷产品小号一直与CHB处理(虚线,显示天然单体和氧化的二聚体)。该图改编自Mekasha等人。(2020)。所述Jd1381几丁质酶还包含一个LPMO域,并且由于反应用运行一个还原剂,产物混合物还含有少量的氧化产物的,其之前A2,洗脱其解决较差的峰与重叠那些属于较长的非氧化产物。mAU代表毫吸光度单位。
 
2.分析来自含LPMO的反应的产物概况(图2 )。      
注意:如果认为使用E1节中描述的(更快)方法分离壳寡糖不足,则该方法也可用于仅与几丁质酶反应的产物分析。在第是情况下,从几丁质酶反应样品需要如所描述的被处理用于所述LPMO反应(即,在乙腈中稀释)。
使用通过HPLC分析样品一个Ñ的Acquity UPLC BEH酰胺柱安装在UHPLC安捷伦科技的1290 Infinity系统具有15毫摩尔Tris-HCL,pH 8.0中和100%的乙腈组成的梯度,如下:0 - 5分钟,74 %乙腈; 5 - 7分钟,74 - 62%乙腈; 7 - 8分钟,62%乙腈; 8 - 10分钟,62 - 74%乙腈; 10 - 12分钟,74%乙腈。
孔定量泰特氧化产物的使用基于如下所述制备标准品的标准曲线的量。的色谱分析使用的变色龙7.2.9软件。
 
图2 。使用S tep E2中描述的方法生成的色谱图示例。天然GlcNAc 3-6的峰标记为A3-A6。代表氧化产物的峰标记为A2ox-A6ox。虚线代表天然和氧化壳寡糖的标准品。注意,天然壳寡糖的α-和β-异头物是分开的。实线是在存在或不存在还原剂抗坏血酸(AscA )的情况下,通过几丁质活性LPMO (Sm LPMO10A)对几丁质进行降解后获得的产品概况的示例。该图改编自Mekasha等人。(2020)。
 
壳寡糖标准品的生产,包括GlcNAc 2ox ,GlcNAc 3ox ,GlcNAc 4ox ,GlcNAc 5ox或GlcNAc 6ox 。
Prepar一个和灰Ø xidized chitooligosacc harides 。
1.混合的5mM的GlcNAc 2 ,GlcNAc的3 ,GlcNAc的4 ,GlcNAc的5或GlcNAc,6,或这些的混合物,用2μM纯化ç hitooligosaccharide从氧化酶镰孢菌(Heuts等人,2008;松散。等人,2014 )。      
2.在室温下孵育16小时,无需搅拌;这导致完全转换。在-20℃冷冻时,反应混合物中的产物可稳定数月       ℃。
3.在74%的乙腈制备标准溶液在浓度在0.02毫米的范围内,以0.5毫米到发电机密封Ë为孔定量标准曲线吨通货膨胀。      
4.使用Eppendorf离心机5418/54以最大速度旋转混合物,以去除酶沉淀物。      
5.转移上清至0.3 -毫升聚丙烯小瓶; 用盖帽和隔垫密封。      
              通过稀释第1点中制备的壳寡糖溶液以产生适当的浓度,可以制备天然产品的标准溶液。所需浓度的D-在th epend Ë实验和将通常大于Th OSE的标准小号为氧化产物。
 
笔记
 
几丁质相关的挑战
1.缺乏STANDAR为d程序几丁质的分离和预处理(例如,研磨)导致的变化之间在不同的实验室中使用的几丁质,这会影响酶的活性数据获得。      
2.由于几丁质是不溶的,因此生产均质的几丁质悬浮液很重要,而不是简单。在建立反应和取样反应时,均应特别注意均质的几丁质悬浮液。使用较小的粒径,例如,直径小于1mm,使得更容易获得均质的悬浮液并精确地移液。可取的是使用宽-bore移液管头(如VWR的宽-在协议上述孔移液管尖端)。当使用储备悬浮液时,磁力搅拌往往足以保持悬浮液均匀。      
3.请注意,几丁质的粒径和结晶度会影响几丁质降解酶的活性。      
 
酶相关挑战
几丁质酶是不需要特殊措施的“常规”水解酶,但与LPMO协同工作会带来多重挑战,这在Eijsink等人的文章中有详细介绍。,(2019)。一些关键点是:
1.该LPMO反应依赖于还原剂,和不同的还原剂可能导致非常不同的反应动力学[例如,Hegnar等。(2019)]。最常使用抗坏血酸。      
2. LPMOs使用https 2 ö 2 ,这可以通过涉及ö反应中加入到反应或在混合物中生成的2和还原剂(例如,抗坏血酸)。应特别注意在整个反应时间内提供足够的O 2 (或H 2 O 2 )和还原剂。当使用上述反应条件时,这不是问题。      
3.特别应该注意到LPMOs的避免失活,使用过量的红眼时可能出现的坦和/或添加量高h的2 ö 2 。      
4. LPMO活性和稳定性降低,如果酶不完全的铜-饱和的。      
5. LPMO反应中的各种氧化还原过程(例如反应混合物中生成H 2 O 2)将受到溶液中游离过渡金属的存在的影响,其程度取决于所使用的还原剂。与抗坏血酸的反应受游离铜离子的强烈影响(Bissaro等,2020)。      
 
菜谱
 
抗坏血酸原液(100 mM)
将17.6 mg抗坏血酸转移到Eppendorf管中
保持在冰上的管中并溶解在1ml的抗坏血酸TraceSelect用移液管水吨荷兰国际集团的混合物向上和向下
制备20 -在Eppendorf或PCR管并存储微升等分试样在-20 ℃下
等份仅使用一次
500 ml 20毫米Tris pH 8.0
权衡1.211克的Tris(MW121.14克/ mol)和溶解在约imately 400毫升的MilliQ -水
使用HCl将pH调节至8.0,然后在容量瓶中加入MilliQ水至总体积为500 ml
通过0.45 - µm或0.22 - µm的瓶子过滤-顶部PES过滤器
室温保存
500毫升15 mM Tris pH 8.0
权衡0.909克的Tris(MW121.14克/摩尔)并溶解在约imately 400毫升的MilliQ -水
使用HCl将pH调节至8.0,然后在容量瓶中加入MilliQ水至总体积为500 ml
通过0.45 - µm或0.22 - µm的瓶子过滤-顶部PES过滤器
室温保存
500毫升1 M BisTris pH 6.0
称重104.62克BrisTris (MW209.24克/ mol)和溶解在约imately 400毫升的MilliQ -水
使用HCl将pH调节至6.0,然后在容量瓶中加入MilliQ水至总体积为500 ml
通过0.45 - µm或0.22 - µm的瓶子过滤-顶部PES过滤器
室温保存
20 ml 50 mg / ml(w / v)几丁质悬浮液
称取1.0克几丁质,然后加入20毫升Trace Select水或合适的缓冲液
中号IX中的悬浮液24小时,通过磁力搅拌在4 ℃下
储存在4 °C
50毫升50 mM H 2 SO 4
添加约imately 40毫升的MilliQ -水到50 -毫升容量瓶
甲DD 136微升水中2 SO 4 (MW 98.08,1.83克/厘米3 )
混合均匀并加入MilliQ水至总体积为50 ml
室温保存
2 L 5毫米H 2 SO 4
添加约imately 1.9大号的MilliQ -水的2 -大号容量瓶
甲DD 544微升水中2 SO 4 (MW 98.08,1.83克/厘米3 )
混合均匀并加入MilliQ水至2 L的总体积
室温保存
 
致谢
 
我们感谢前任和现任实验室成员为他们捐款的发展换货我们的协议。本协议源自Mekasha等人。,J Biol Chem,2020,295:9134-9146,其描述了由挪威研究理事会资助的项目221576和247001的结果。
 
利益争夺
 
作者宣称没有利益冲突。
 
伦理
 
Ť这里有与此协议相关联没有道德问题。
 
参考
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mekasha, S., Tuveng, T. R., Vaaje-Kolstad, G. and Eijsink, V. G. H. (2021). Chromatographic Assays for the Enzymatic Degradation of Chitin. Bio-protocol 11(9): e4014. DOI: 10.21769/BioProtoc.4014.
  2. Mekasha, S., Tuveng, T. R., Askarian, F., Choudhary, S., Schmidt-Dannert, C., Niebisch, A. and Eijsink, V. G. H. (2020). A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin. J Biol Chem 295(27): 9134-9146.
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