Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method

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Aug 2016



α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor. We have shown that Thermofluor can also be applied to predict binding of substrates and inhibitors to a yeast α-glucosidase. The methods described here in detail were used in Viigand et al., 2016.

Keywords: Maltase (麦芽糖酶), Isomaltase (异麦芽糖), Maltase assay (麦芽糖酶测定), Methylotrophic yeast (甲醇营养型酵母), Ogataea polymorpha (多型汉逊酵母), Glucose liquicolor (葡萄糖liquicolor), Differential scanning fluorometry (差示扫描荧光法)


Maltases (EC and isomaltases (EC are α-glucosidases belonging to family 13 of glycoside hydrolases according to the CAZy classification (Lombard et al., 2014). Maltase MAL1 of a methylotrophic yeast Ogataea polymorpha is nonselective–it hydrolyses maltose- and isomaltose-like α-glucosidic sugars producing D-glucose as one of the reaction products. Thus, activity of maltase on its substrates can be determined according to glucose release. The Glucose liquicolor-aided method described in this work allows rapid and convenient assay of the activity, substrate specificity and thermostability of the maltase. Importantly, this activity-based method can be adapted to other enzymes that produce glucose as a reaction product. A high-throughput Thermofluor method is mostly used in protein crystallography to measure (thermal) stability of the protein (Boivin et al., 2013; Ericsson et al., 2006). We used Thermofluor 1) to evaluate thermostability of the maltase protein and 2) to study its substrate specificity (Viigand et al., 2016). Substrate specificity assay of glycoside hydrolases and other sugar-acting enzymes using Thermofluor is cost-efficient–it requires very low amounts of the protein as well as ligand sugars that can be very expensive. Regarding substrates of α-glucosidases, one gram of isomaltose from Sigma-Aldrich costs almost 1,000 euros, 10 milligrams of nigerose 143 euros and 1 mg of kojibiose almost 200 euros.

Materials and Reagents

  1. For both methods
    1. 1.5 ml microtubes (Corning, Axygen®, catalog number: MCT-150-C )
    2. 0.2 μm cellulose acetate membrane filter (Sartorius, catalog number: 11107-47-N )
    3. Sucrose (Sigma-Aldrich, catalog number: 16104 )
    4. MilliQ quality water (MQ)
    5. Crushed ice
    6. In-house laboratory purified C-terminally His-tagged maltase MAL1 (from Ogataea polymorpha) and its inactive mutant protein (Asp199Ala) overexpressed in Escherichia coli (prepared as in Viigand et al., 2016)

  2. For classical activity assay
    1. Special PS (polystyrene) micro photometer cuvette, 2 ml (LP ITALIANA, catalog number: 112117 )
    2. Glucose liquicolor kit (GOD-PAP Method, Enzymatic Colorimetric Test for Glucose) (Human, catalog number: 10260 )
    3. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 795488 )
    4. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: RES20765 )
    5. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    6. Tris base (Roche Molecular Systems, catalog number: 10708976001 )
    7. Hydrochloric acid 37% (HCl) (AppliChem, catalog number: A0659 )
    8. 1 M Tris-HCl buffer (pH 8.3) (see Recipes)
    9. Maltase buffer (see Recipes)

  3. For Thermofluor method
    1. LightCycler® 480 Multiwell Plate 96 (white) with sealing foils (Roche Molecular Systems, catalog number: 04729692001 )
    2. 5,000x SYPRO Orange Protein Gel Stain (Sigma-Aldrich, catalog number: S5692 )
    3. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
    4. Sodium hydroxide (NaOH) (AppliChem, catalog number: 131687.1211 )
    5. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-M )
    6. 0.5 M HEPES buffer (pH 7.0) (see Recipes)
    7. 4x Thermofluor buffer (see Recipes)


  1. Pharmaceutical balance PS2100/C/2 (RADWAG Balances and Scales, model: PS 2100/C/2 )
  2. JENWAY pH meter model 3510 (Cole-Parmer Instrument, model: Jenway 3510 )
  3. Pipettes (FisherbrandTM Elite Pipette Kit) (Fisher Scientific, catalog number: 14-388-100 )
  4. Refrigerator-freezer (Electrolux, model: EN2900AOW )
  5. Ultrospec 3100 pro UV/Visible spectrophotometer (GE Healthcare, Amersham Biosciences, model: Ultrospec 3100 pro , catalog number: 80-2112-37)
  6. ThermoBlock TDB-120 (Biosan, model: TDB-120 )
  7. Digital timer/clock (Fisher Scientific, catalog number: S01619 )
  8. Plate sealer spatula for microtiter plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5701 )
  9. LightCycler® 480 Instrument II (Roche Molecular Systems, model: LightCycler® 480 Instrument II , catalog number: 05015278001)
  10. Refrigerated centrifuge 4K-15 (Sigma Laborzentrifugen, model: 4K-15 , catalog number: 10742) with the swing-out rotor for 4 buckets (Sigma Laborzentrifugen, catalog number: 11150 ) and bucket, aluminium, for microtiter plates (Sigma Laborzentrifugen, catalog number: 13220

Part I. A classical thermostability assay

Note: A classical thermostability assay of the maltase is based on determination of residual catalytic activity of the enzyme after its incubation at various temperatures. Therefore, we first give the protocol for the measurement of maltase activity.


  1. Maltase activity assay: The principle of the method
    Maltase (MAL1) of Ogataea (Hansenula) polymorpha hydrolyses α-glycosidic linkages in numerous di- and trisaccharides (Figure 1A) typically releasing one molecule of glucose per molecule of the cleaved substrate. In case of some substrates, for example maltose and isomaltose, two glucose molecules are produced. Maltase activity is determined at a chosen temperature (we routinely use 37 °C) by monitoring the initial velocity of substrate hydrolysis according to the release of glucose. The method described here is a modification of the method of Hackel (1975) for determination of invertase activity in baker’s yeast, and was also used by us in Viigand et al., 2016. In the protocol presented here, sucrose is used as the substrate for the enzyme. The Km of sucrose hydrolysis reaction of MAL1 is ~25 mM (Viigand et al., 2016). We use two times higher concentration (50 mM) of sucrose for the maltase activity assay. The amount of glucose released from sucrose in maltase reaction is determined using a commercial Glucose liquicolor kit (Figure 1B).
    A standard reaction mixture (1 ml in total) contains the maltase buffer (see Recipes), 50 mM sucrose and 3.6 μg of maltase protein. Maltase was purified using immobilized metal affinity chromatography. Our data shows that the protein’s catalytic activity is not reduced after months of storage at 4 °C.

    Figure 1. Schematic overview of MAL1 substrates, their expected binding to the enzyme (A) and the maltase assay via determination of released glucose with Glucose liquicolor reagent (B). A. Monosaccharidic composition and linkage types of O. polymorpha maltase substrates and their predicted binding to subsites -1, +1 and +2 on maltase protein are indicated. Glucose residue of the non-reducing end of di- or trisaccharidic substrates is considered to bind at -1 subsite. Cleavage of the glycosidic linkage by maltase is executed between the residues bound at -1 and +1 subsites releasing the hydrolysis products into the reaction medium. The arrow on the background shows increase of the affinity of the enzyme towards the depicted substrate. The figure is based on Figure 1 of Viigand et al. (2016). B. Scheme of maltase activity measurement using Glucose liquicolor reactive. Free glucose (green circle) released from a maltase substrate (e.g., sucrose) is oxidised by the glucose oxidase to gluconic acid and hydrogen peroxide. Peroxidase uses hydrogen-peroxide to oxidize 4-aminoantipyrine in the presence of phenol to red-violet quinoneimine. The absorbance of the quinoneimine is measured at 500 nm wavelength. The colour of the product measured in Glucose liquicolor assay is also shown in Table 1 (see the cuvettes).
    1. Two parallel samples (two repeats) of 850 μl of maltase buffer and 50 μl (3.6 μg) of maltase protein (appropriately diluted in maltase buffer) are pre-incubated in 1.5 ml microtubes for 5 min on a thermoblock (Figure 2) adjusted to 37 °C to warm up the mixture.
      Note: The maltase protein stock solution should be kept on ice during the experiment.

      Figure 2. The Biosan thermoblock

    2. Reaction is initiated by adding 100 μl of 500 mM sucrose stock solution (in maltase buffer) to the pre-heated mixture of the buffer and maltase protein, mixed and the timer is started. Incubation is conducted for required time (usually from 2 to 20 min, depending on catalytic activity of the maltase protein) and two parallel samples (50 μl) are withdrawn from each tube at each time point during the reaction. In case of the maltase preparation with a high catalytic activity, sampling should be for example at 2, 4, 6 and 8 min from the start of the reaction.
      Note: Initial velocity of the reaction should be measured–meaning that the amount of released glucose should increase linearly during the reaction time. Maltase activity measurement should be repeated if the reaction slows down. In repeated experiment, we recommend using a lower amount of maltase protein.
    3. 50 μl samples withdrawn from the reaction mixture are pipetted into 1.5 ml microtubes containing 150 μl of stopping solution of 200 mM Tris-HCl buffer with pH 8.3 and heated for 5 min at 96 °C in the thermoblock.
      Note: Preparation of 1 M Tris-HCl buffer is shown in Recipes section 1. Dilution to obtain 200 mM buffer was made in MQ water.
    4. The stopped reaction samples are cooled on ice.
    5. 800 μl of Glucose liquicolor reagent is added to each stopped sample and thoroughly shaken by hand.
    6. The microtubes with the samples are incubated at 37 °C with open lids for 5 min. The solution turns purple due to conversion of glucose into quinoneimine (Figure 1B).
    7. Absorbance of purple quinoneimine is measured using special PS micro photometer cuvettes with path-length of 1 cm (Table 1, column on the right) with a spectrophotometer at 500 nm wavelength against the reference (blank control).
      1. For reference, 900 μl maltase buffer is mixed with 100 μl of 500 mM sucrose. 50 μl is withdrawn from this mixture and combined with 150 μl of stopping solution and 800 μl of Glucose liquicolor reagent and incubated at 37 °C with open lid for 5 min.
      2. The most suitable range for OD500 measurement is from 0.1 to 1.0 that ensures linearity between the OD500 measurement and glucose concentration of the solution.

  2. A maltase thermostability assay
    1. 50 μl of maltase protein preparation is added to 850 μl of maltase buffer (final concentration of maltase protein 0.0036 mg/ml [3.6 µg/ml]). At least duplicate samples should be prepared for each temperature studied.
    2. The mixture (900 μl) is incubated on a thermoblock at various temperatures (in this experiment from 30 °C to 50 °C) for 30 min.
    3. After the thermal treatment the samples are cooled on ice.
      Note: We recommend to collect all thermally inactivated samples (keeping them on ice) before proceeding with their further analysis (see the next step).
    4. The cooled samples are warmed to 37 °C (~5 min) on a thermoblock and the reaction is started by adding the maltase substrate (100 μl of 500 mM sucrose; final concentration of sucrose in the reaction mixture 50 mM).
    5. Continue as described in Procedure A: Maltase activity assay, step 3.

Data analysis

  1. Maltase activity assay
    Calculate maltase activity (E) according to the formula: OD500 (1 min)/(ε x [c]). OD500 (1 min) is absorbance (optical density) change of the reaction mixture per 1 min measured at 500 nm wavelength; [c] is concentration of the maltase protein (mg/ml) in the reaction mixture (see step B1) and (ε) is the extinction coefficient withdrawn from glucose calibration curve (see section Notes: Calibration curve for glucose concentration determination).
    The maltase activity is expressed as the amount of maltase substrate in μmol (sucrose in current example) that is hydrolysed per min per mg of protein. The activity is expressed as μmol/(min x mg) or U/mg.
    Note: The activity measured according to glucose production should be divided by two if two glucose molecules are released due to hydrolysis of the maltase substrate (for example maltose or isomaltose; Figure 1A).
    An example of calculation:
    1. Data from the experiment:
      1. 0.0036 mg/ml of maltase protein reacted with 50 mM sucrose.
      2. Samples were withdrawn at every 2 min during 6 min of the reaction, stopped and free glucose in the samples was determined by using Glucose liquicolor method (see Table 1, first column ‘OD500’).
        Note: During the sampling time (6 min), reaction velocity stayed ~ constant (see Table 1, third column ‘OD500 [1 min]’).
    2. Calculations:
      1. Average OD500 per 1 min was 0.731/6 = 0.122.
      2. 0.292 is used as extinction coefficient (ε) (see section Notes: Calibration curve for glucose concentration determination).
      3. Maltase activity E = 0.122/(0.292 x 0.0036) = 115.9 μmol/(min x mg) or 115.9 U/mg.
        115.9 μmol of sucrose were hydrolysed by 1 mg of maltase protein during 1 min.

        Table 1. Example of calculation of maltase activity

  2. A maltase thermostability assay
    The melting temperature (Tm) of the enzyme is considered the temperature which results in 50% reduction of its catalytic activity after a 30-min incubation. The calculated Tm of the maltase protein is ~44.4 °C (Figure 3).

    Figure 3. Thermostability of O. polymorpha maltase MAL1 evaluated according to residual catalytic activity. Mean values and standard deviations were calculated from at least three independent measurements.

Part II. A Thermofluor (differential scanning fluorometry; DSF) assay of maltase thermostability

A catalytically inactive mutant of the O. polymorpha maltase MAL1, where nucleophile Asp199 is substituted with Ala, is used in this experiment. This substitution enables to study thermostability of MAL1 in the presence of its substrates (see Figure 1A) without consecutive catalytic reaction (Viigand et al., 2016). A real-time PCR equipment is used enabling online monitoring of protein denaturation during its gradual heating in the presence of a fluorescent dye, SYPRO Orange. The dye binds to hydrophobic amino acids of the protein which become exposed at denaturation of the protein due to heating. The fluorescence intensity is plotted as a function of temperature generating a sigmoidal curve (see Figure 5). Inflection point (Tm) of this curve corresponds to the temperature at which 50% of the protein is unfolded. In this protocol, we used sucrose as a ligand sugar to evaluate its stabilizing effect on the maltase protein. In Viigand et al. (2016) we used several additional potential substrates (e.g., maltose, maltulose and palatinose) and inhibitors (e.g., glucose) of the maltase to predict which substrates and how strongly may bind the enzyme.


  1. A multiwell plate 96 of the LightCycler® 480 is cooled on crushed ice or in the freezer before the experiment.
  2. The LightCycler® 480 should be warmed up before starting the analysis.
  3. The following ingredients are added one by one, final volume of the mixture is 20 μl per sample:

    1. *All stock solutions are dissolved in MQ water.
    2. All components should be mixed as fast as possible to minimize evaporation. The Multiwell plate should be kept on crushed ice and protected from the light. Control samples contain MQ water instead of sucrose.
  4. The 96-well plate is covered with a LightCycler® 480 Multiwell Plate 96 (white) sealing foils and sealed tightly with a spatula by pressing the spatula to each well.
  5. The sealed plate is centrifuged for 30 sec at 10 °C (a refrigerated centrifuge 4K-15) at 200 x g.
    Note: The rotor of the centrifuge must be balanced with a similar plate for example with one from a previous experiment. Mild centrifugation removes the bubbles and ensures that all liquid is remaining at the bottom of the well.
  6. The program to be used for the thermostability assay is: LightCycler® 480 SW 1.5.
    1. We use wavelengths of 465 nm for excitation and 580 nm for emission as in Layton and Hellinga (2010).
    2. Program: Melting curves; temperature is raised from 25 °C to 95 °C; 30 fluorescence measurements are taken per 1 degree of temperature increase.
    3. The experiment takes about 45 min.
  7. Data are collected by exporting the .txt file. Further analysis of the raw data is described in ‘Data analysis’.

Data analysis

Data analysis of the Thermofluor experiment

  1. All data are exported as a .txt file (Figure 4).

    Figure 4. Data from a Thermofluor experiment saved in a .txt file (a screenshot)

  2. Further analysis is carried out using Microsoft Excel. The fluorescence emission values (F1, F2, F3 etc.) recorded for each well during the assay are copied horizontally below the row showing temperature values (T1, T2, T3 etc.) corresponding to respective fluorescence (Figure 5, upper panel).

    Figure 5. Organization of Thermofluor data for Excel-based analysis (upper panel) and an illustrative fluorescence vs. temperature graph (lower panel). Catalytically inactive maltase protein is gradually heated without ligands (no ligand) and in the presence of 300 mM sucrose. Fluorescence signal is plotted as a function of temperature to get a sigmoidal melting curve of the protein (lower panel of the figure). Fluorescence of the samples increases when the protein starts to unfold. Maximal fluorescence intensity is observed at complete unfolding of the protein. After that the SYPRO Orange signal decreases due to dye-protein dissociation as described in Boivin et al. (2013). Presence of enzyme’s substrate, sucrose, increases thermostability of the protein by 5.8 °C–the melting temperature Tm increases from 59.5 °C (no ligand added; shown in yellow) to 65.3 °C (in the presence of sucrose; shown in blue).

  3. Though the Tm value of a protein can be roughly derived from the raw data graph as in Figure 5, further data analysis described in Niesen et al. (2007) and Boivin et al. (2013) is required for more accurate Tm determination.
  4. For that, we processed the raw data from Thermofluor assay, removing most of fluorescence values and keeping only those corresponding to temperature increase by ~0.5 °C.
  5. The first derivative was then calculated from the processed data. The first derivative (m) was expressed as:

    where, Δy is the change in fluorescence intensity and Δx is the change in temperature increase corresponding to respective fluorescence change.
  6. The temperature which corresponds to the peak of the first derivative curve represents the Tm value.
  7. The Thermofluor Tm data presented in Figure 5 and in Viigand et al. (2016) were obtained using the latter approach.
Part III. Comparison of activity-based assay and the Thermofluor method

Tm of the catalytically inactive mutant Asp199Ala of MAL1 in the absence of sugar ligands determined by us in Thermofluor assay was 59.5 °C. Respective value of the wild-type (catalytically active) MAL1 was 51.0 (Viigand et al., 2016). Thus, the inactive mutant was more stable than the wild-type enzyme. Tm of the wild-type MAL1 determined using a classical activity-based assay was 44.4 °C (Figure 3) being lower than the Tm detected from the Thermofluor assay. As described in this paper, in a classical thermostability assay the protein is heated at selected temperatures during 30 min and after that the residual catalytic activity is measured. In the case of Thermofluor assay, the protein is heated gradually (1.5 °C per min) and subsequent denaturation is monitored online. These data show that the maltase protein tolerates short-term gradual heating better than the extended stepwise one. When the maltase mutant Asp199Ala was assayed using Thermofluor in the presence of enzyme’s substrate (see Figure 5 of this protocol and Viigand et al., 2016) or inhibitors (Viigand et al., 2016), the Tm of the protein was increased.


Calibration curve for glucose concentration determination

  1. 1-5.55 mM glucose solutions in maltase buffer are prepared from the glucose standard (1 mg/ml; 5.55 mM) of the Glucose liquicolor kit.
  2. Duplicate samples of 50 μl of glucose calibration curve solutions (1-5.55 mM) are mixed with 150 μl of 200 mM Tris-HCl buffer (pH 8.3) and 800 μl of Glucose liquicolor reactive in 1.5 ml microtubes.
  3. Samples are incubated at 37 °C with open lids for 5 min.
  4. Optical density of the purple product (quinoneimine) is measured at 500 nm wavelength with a spectrophotometer against the reference. Reference contains maltase buffer instead of the glucose solution and it is treated similarly as glucose-containing solutions of the calibration curve.
  5. The obtained values are plotted as in Figure 6.

    Figure 6. Calibration curve for glucose concentration determination using the Glucose liquicolor kit. Quinoneimine formation was measured at 500 nm wavelength for different glucose concentrations: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 5.55 mM.

  6. Slope of the linear trendline of Figure 6 (functions: Set Intercept and Display equation on chart) is used in maltase activity calculations as an extinction coefficient (ε) to calculate the amount of glucose released in the maltase reaction.


  1. 1 M Tris-HCl buffer (pH 8.3)
    1. Dissolve 24.228 g of Tris base in 150 ml of MQ water
    2. Adjust pH to 8.3 with 37% HCl
    3. Complete to 200 ml with MQ water and filter through a 0.2 μm cellulose acetate membrane
    4. Store at room temperature
  2. Maltase buffer–100 mM potassium-phosphate buffer (pH 6.5) with 0.1 mM EDTA
    1. Prepare 1 M stock solution for potassium phosphate dibasic (K2HPO4) and potassium phosphate monobasic (KH2PO4) salts in MQ water
    2. Prepare 0.5 M EDTA solution in MQ water
    3. For 400 ml 100 mM maltase buffer (pH 6.5) mix 14 ml of 1 M K2HPO4 (alkaline component) and 26 ml of 1 M KH2PO4 (acidic component). The buffer is prepared using pre-calculated amounts of acidic and alkaline components of the same molarity to obtain the desired pH value of the buffer (Gomori, 1955)
    4. Add 80 μl 0.5 M EDTA
    5. Complete to 400 ml with MQ water
    6. Filter through a 0.2 μm cellulose acetate membrane filter and store at 4 °C
  3. 0.5 M HEPES buffer (pH 7.0)
    1. Dissolve 5.96 g of HEPES in 40 ml of MQ water
    2. Adjust pH to 7.0 with 10 N NaOH
    3. Add MQ water to a final volume of 50 ml
    4. Filter through a 0.2 μm cellulose acetate membrane filter and store at 4 °C
  4. 4x Thermofluor buffer
    1. Prepare 0.5 M HEPES stock-buffer (pH 7.0) in MQ water
    2. Prepare 5 M NaCl stock-solution in MQ water
    3. Mix 800 μl of 0.5 M HEPES buffer, 120 μl of 5 M NaCl and 80 μl of MQ water to final volume of 1 ml
    4. Prepare new buffer solution for each experiment


This work was financed by ERC grants GLOMR9072 (ETF9072) and GLTMR1050P (PUT1050). Brief description of the methods is presented in a paper Viigand, K., Visnapuu, T., Mardo, K., Aasamets, A. and Alamäe, T. (2016). Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases. Yeast 33(8): 415-432. We thank Dimitri Lubenets for kind assistance in the Thermofluor assay.


  1. Boivin, S., Kozak, S. and Meijers, R. (2013). Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91(2): 192-206.
  2. Gomori, G. (1955). Preparation of buffers for use in enzyme studies. Meth Enzymol 1: 143-146.
  3. Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N. and Nordlund, P. (2006). Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357(2): 289-298
  4. Hackel, R. A. (1975). Genetic control of invertase formation in Saccharomyces cerevisiae. I. Isolation and characterization of mutants affecting sucrose utilization. Mol Gen Genet 140(4): 361-370.
  5. Layton, C. J. and Hellinga, H. W. (2010). Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes. Biochemistry 49(51): 10831-10841.
  6. 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: D490-D495.
  7. Niesen, F. H., Berglund, H. and Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2(9): 2212-2221.
  8. Viigand, K., Visnapuu, T., Mardo, K., Aasamets, A. and Alamae, T. (2016). Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases. Yeast 33 (8): 415-432.


α-葡糖苷酶(包括麦芽糖酶和异麦芽糖酶)是从一组α-葡糖苷底物释放葡萄糖的酶。 可以使用该特征来测定其催化活性,底物特异性和热稳定性。 蛋白质的热稳定性也可以使用高通量差示扫描荧光测定法(也称为Thermofluor)来测定。 我们已经表明,Thermofluor也可以应用于预测底物和抑制剂与酵母α-葡萄糖苷酶的结合。 这里详细描述的方法用于Viigand等人,2016。
【背景】麦芽糖酶(EC和异麦芽糖酶(EC是根据CAZy分类属于糖苷水解酶家族13的α-葡糖苷酶(Lombard等,2014)。甲基营养酵母多形汉酵母的麦芽糖酶MAL1是非选择性的,它将产生D-葡萄糖的麦芽糖和异麦芽糖状α-葡萄糖苷水解为反应产物之一。因此,麦芽糖酶对其底物的活性可以根据葡萄糖释放来确定。该工作描述的葡萄糖液色辅助方法可以快速方便地测定麦芽糖酶的活性,底物特异性和热稳定性。重要的是,这种基于活性的方法可以适用于产生葡萄糖作为反应产物的其它酶。高通量Thermofluor方法主要用于蛋白质晶体学测量(热)稳定性蛋白质(Boivin等,2013; Ericsson等,2006)。我们使用Thermofluor 1)来评估麦芽糖酶蛋白的热稳定性,2)研究其底物特异性(Viigand等,2016)。使用Thermofluor的糖苷水解酶和其他糖作用酶的底物特异性测定是经济有效的 - 它需要非常少量的蛋白质以及可能非常昂贵的配体糖。关于α-葡萄糖苷酶的底物,来自Sigma-Aldrich的1克异麦芽糖花费近1000欧元,10毫克的黑皮郎143欧元和1毫克的曲曲糖几乎200欧元。

关键字:麦芽糖酶, 异麦芽糖, 麦芽糖酶测定, 甲醇营养型酵母, 多型汉逊酵母, 葡萄糖liquicolor, 差示扫描荧光法


  1. 对于这两种方法
    1. 1.5ml微管(Corning,Axygen ,目录号:MCT-150-C)
    2. 0.2μm醋酸纤维素膜过滤器(Sartorius,目录号:11107-47-N)
    3. 蔗糖(Sigma-Aldrich,目录号:16104)
    4. MilliQ质量水(MQ)
    5. 碎冰
    6. 在大肠杆菌中过表达的室内实验室纯化的C末端His标记的麦芽糖酶MAL1(来自多形态Ogataea多形核霉素)及其无活性突变蛋白(Asp199Ala)(如Viigand 等,,2016)

  2. 对于古典活动分析
    1. 特殊PS(聚苯乙烯)微型光度计比色皿,2ml(LP ITALIANA,目录号:112117)
    2. 葡萄糖液色试剂盒(GOD-PAP方法,葡萄糖酶比色法)(人类,目录号:10260)
    3. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:795488)
    4. 磷酸氢二钾(K 2 O 3 HPO 4)(Sigma-Aldrich,目录号:RES20765)
    5. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    6. Tris碱(Roche Molecular Systems,目录号:10708976001)
    7. 盐酸37%(HCl)(AppliChem,目录号:A0659)
    8. 1M Tris-HCl缓冲液(pH 8.3)(参见食谱)
    9. 麦芽糖酶缓冲液(参见食谱)

  3. 对于Thermofluor方法
    1. LightCycler ® 480具有密封箔的多孔板96(白色)(Roche Molecular Systems,目录号:04729692001)
    2. 5,000x SYPRO橙蛋白凝胶染色剂(Sigma-Aldrich,目录号:S5692)
    3. HEPES缓冲液(Sigma-Aldrich,目录号:H3375)
    4. 氢氧化钠(NaOH)(AppliChem,目录号:131687.1211)
    5. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434-M)
    6. 0.5 M HEPES缓冲液(pH 7.0)(见配方)
    7. 4x Thermofluor缓冲液(见配方)


  1. 药物平衡PS2100/C/2(RADWAG天平和秤,型号:PS 2100/C/2)
  2. JENWAY pH计型号3510(Cole-Parmer仪器,型号:Jenway 3510)
  3. 移液器(Fisherbrand TM Elite Pipette Kit)(Fisher Scientific,目录号:14-388-100)
  4. 冰箱冷柜(伊莱克斯,型号:EN2900AOW)
  5. Ultrospec 3100 pro紫外/可见分光光度计(GE Healthcare,Amersham Biosciences,型号:Ultrospec 3100 pro,目录号:80-2112-37)
  6. ThermoBlock TDB-120(Biosan,型号:TDB-120)
  7. 数字计时器/时钟(Fisher Scientific,目录号:S01619)
  8. 用于微量滴定板的板式密封刮刀(Thermo Fisher Scientific,Thermo Scientific TM,目录号:5701)
  9. LightCycler ® 480仪器II(Roche Molecular Systems,型号:LightCycler 480仪器II,目录号:05015278001)
  10. 将4K-15(Sigma Laborzentrifugen,型号:4K-15,目录号:10742)与4个桶的摆出转子(Sigma Laborzentrifugen,目录号:11150)和用于微量滴定板的桶,铝(Sigma Laborzentrifugen,目录号:13220) 




  1. 麦芽糖酶活性测定法:方法的原理
    Ogataea(Hansenula)polymorpha的Maltase(MAL1)在许多二糖和三糖中水解α-糖苷键(图1A),通常每分子的裂解底物释放一分子葡萄糖。在一些底物的情况下,例如麦芽糖和异麦芽糖,产生两个葡萄糖分子。通过根据葡萄糖的释放监测底物水解的初始速度,在选定的温度(我们常规使用37℃)测定麦芽糖酶活性。这里描述的方法是Hackel(1975)用于测定面包酵母中的转化酶活性的方法的修改,并且也被我们用于Viigand等人,2016中。在本文提出的方案中,蔗糖用作酶的底物。 MAL1的蔗糖水解反应的K 约为25mM(Viigand等人,2016)。我们使用两倍高浓度(50mM)的蔗糖进行麦芽糖酶活性测定。使用市售的葡萄糖液色试剂盒测定从麦芽糖酶反应中释放的葡萄糖的量(图1B)。

    图1. MAL1底物的示意图,它们预期与酶(A)的结合和通过用葡萄糖液色试剂(B)测定释放的葡萄糖的麦芽糖酶测定。A.单糖组成和连接类型 0。指出多发性硬化症麦芽糖酶底物及其预测与麦芽糖酶蛋白质-1,+1和+2的结合。二 - 或三糖底物的非还原性末端的葡萄糖残基被认为在-1个位点处结合。在-1和+1位上结合的残基之间进行通过麦芽糖酶切割糖苷键,从而将水解产物释放到反应介质中。背景上的箭头显示酶对所描绘的底物的亲和力的增加。该图基于Viigand等人的图1。 (2016)。 B.使用葡萄糖液色反应的麦芽糖酶活性测定方案。从麦芽糖酶底物(例如蔗糖)释放的游离葡萄糖(绿色圆圈)被葡萄糖氧化酶氧化成葡萄糖酸和过氧化氢。过氧化物酶使用过氧化氢在苯酚存在下氧化4-氨基安替比林至红紫色醌亚胺。在500nm波长处测量醌亚胺的吸光度。在葡萄糖液色测定中测量的产品的颜色也显示在表1中(参见比色杯)。
    1. 将850μl麦芽糖酶缓冲液和50μl(3.6μg)麦芽糖酶蛋白(适当稀释在麦芽糖酶缓冲液中)的两个平行样品(两个重复)在1.5ml微管中在温度调节(图2)上预温育5分钟37°C加热混合物。

      图2. Biosan热块

    2. 通过将100μl500mM蔗糖储备溶液(在麦芽糖酶缓冲液中)加入缓冲液和麦芽糖酶蛋白质的预热混合物中开始反应,混合并开始计时。根据需要的时间进行孵育(通常为2至20分钟,取决于麦芽糖酶蛋白的催化活性),并且在反应期间的每个时间点从每个管中取出两个平行的样品(50μl)。在具有高催化活性的麦芽糖酶制备的情况下,采样应当例如在从反应开始起2,4,6和8分钟。
      注意:应测量反应的初始速度 - 意味着释放的葡萄糖的量在反应时间内应线性增加。如果反应减慢,应重复测定麦芽糖酶活性。在反复实验中,我们建议使用较少量的麦芽糖酶蛋白。
    3. 将从反应混合物中取出的50μl样品移液到含有150μlpH 8.3的200mM Tris-HCl缓冲液的150μl终止液的1.5ml微量管中,并在96℃加热5分钟。 注意:制备1M Tris-HCl缓冲液显示在食谱部分1中。在MQ水中稀释得到200mM缓冲液。//
    4. 停止的反应样品在冰上冷却
    5. 将800μl葡萄糖液色试剂加入每个停止的样品中,并用手彻底摇动。
    6. 将带有样品的微管在37℃下用敞开的盖孵育5分钟。由于葡萄糖转化为醌亚胺,溶液变紫色(图1B)
    7. 紫色醌亚胺的吸光度使用分光光度计在距离参考(空白对照)500nm波长的分光光度计下,使用路径长度为1cm的特殊PS微量光度计比色皿(表1,右图)测量。 注意:
      1. 为了参考,将900μl麦芽糖酶缓冲液与100μl500mM蔗糖混合。从该混合物中取出50μl,并与150μl终止溶液和800μl葡萄糖液色试剂混合,并在37℃下用开放的盖孵育5分钟。
      2. OD 500测量的最合适的范围为0.1至1.0,其确保OD 500测量与溶液的葡萄糖浓度之间的线性。

  2. 麦芽糖酶热稳定性分析
    1. 将50μl麦芽糖酶蛋白质制剂加入到850μl麦芽糖酶缓冲液(终浓度为0.0036mg/ml [3.6μg/ml]的麦芽糖酶蛋白质)中。每个研究温度应至少重复一次样品。
    2. 将混合物(900μl)在各种温度(在该实验中在30℃至50℃)下在热块上温育30分钟。
    3. 热处理后,样品在冰上冷却 注意:我们建议在进行进一步分析之前收集所有热灭活的样品(保持在冰上)(参见下一步)。
    4. 将冷却的样品在热块上温热至37℃(〜5分钟),并通过加入麦芽糖酶底物(100μl500mM蔗糖;反应混合物中的最终浓度为50mM)开始反应。 >
    5. 按照步骤A所述继续进行:Maltase活性测定,步骤3


  1. 麦芽糖酶活性分析
    根据以下公式计算麦芽糖酶活性(E):OD 500(1分钟)/(εx [c])。 OD 500(1分钟)是在500nm波长下测量的每1分钟反应混合物的吸光度(光密度)变化; [c]是反应混合物中麦芽糖酶蛋白质的浓度(mg/ml)(参见步骤B1),(ε)是从葡萄糖校准曲线取出的消光系数(参见注释:葡萄糖浓度测定的校准曲线)。 br /> 麦芽糖酶活性表示为每毫克蛋白质每分钟水解的麦芽糖酶底物的量(以当前实例为单位的μmol)(蔗糖)。活性表达为μmol/(min x mg)或U/mg。
    1. 实验数据:
      1. 0.0036mg/ml麦芽糖酶蛋白与50mM蔗糖反应
      2. 在反应6分钟内每2分钟取出样品,停止,并使用葡萄糖液色法测定样品中的游离葡萄糖(参见表1第一列"OD 500")。 /> 注意:在采样时间(6分钟)内,反应速度保持不变(参见表1,第三列"OD 500"[1分钟])。 >
    2. 计算:
      1. 平均OD 500/1分钟为0.731/6 = 0.122。
      2. 0.292用作消光系数(ε)(参见注释:葡萄糖浓度测定的校准曲线)。
      3. 麦芽糖酶活性E = 0.122 /(0.292×0.0036)=115.9μmol/(min x mg)或115.9U/mg。

  2. 麦芽糖酶热稳定性分析
    酶的解链温度(℃)认为是30分钟孵育后其催化活性降低50%的温度。计算出的麦芽糖酶蛋白质的T 约为44.4℃(图3)。

    图3. O的热稳定性。多形态麦芽糖酶MAL1根据残留催化活性进行评估。平均值和标准偏差由至少三次独立测量计算。


O的催化无活性突变体。在这个实验中使用多形核霉素麦芽糖酶MAL1,其中亲核试剂Asp199被Ala取代。该取代使得能够在其底物(参见图1A)的存在下研究MAL1的热稳定性,而没有连续的催化反应(Viigand等人,2016)。使用实时PCR设备,可在荧光染料SYPRO Orange的存在下逐渐加热期间在线监测蛋白质变性。染料结合蛋白质的疏水性氨基酸,由于加热而在蛋白质变性时暴露于其中。将荧光强度绘制为产生S形曲线的温度的函数(参见图5)。该曲线的拐点对应于50%蛋白质展开的温度。在该方案中,我们使用蔗糖作为配体糖来评估其对麦芽糖酶蛋白质的稳定作用。在Viigand等人中。 (2016),我们使用了几种额外的潜在底物(例如麦芽糖,麦芽酮糖和帕拉金糖)和麦芽糖酶的抑制剂(例如,葡萄糖)来预测哪些底物和如何强烈地可能结合酶。


  1. 在实验之前,将LightCycler 480的多孔板96在碎冰上或冷冻器中冷却。
  2. 在开始分析之前,应该将LightCycler ® 480加热。
  3. 一个接一个地加入以下成分,混合物的最终体积为每个样品20μl:

    1. 所有储备溶液都溶解在MQ水中。
    2. 所有组分应尽可能快地混合,以减少蒸发。多孔板应保存在粉碎的冰上,防止光照。对照样品含有MQ水而不是蔗糖。
  4. 96孔板用LightCycler ®480多孔板96(白色)密封箔覆盖,并用刮刀将刮刀按压到每个孔中。
  5. 将密封的板在10℃下离心30秒(冷冻离心机4K-15),200g×g。
  6. 用于热稳定性测定的程序是:LightCycler ® 480 SW 1.5。
    1. 我们使用波长为465 nm的激发和580 nm的发射,如Layton和Hellinga(2010)
    2. 程序:熔化曲线;温度从25℃升至95℃;每1度升温30次进行荧光测量
    3. 实验大约需要45分钟
  7. 通过导出.txt文件来收集数据。原始数据的进一步分析在"数据分析"中有所描述



  1. 所有数据导出为.txt文件(图4)。


  2. 使用Microsoft Excel进行进一步分析。在测定期间记录的每个孔的荧光发射值(F1,F2,F3等等)水平地复制在显示温度值(T1,T2,T3等) 。)对应于各自的荧光(图5,上图)

    图5.用于基于Excel的分析(上图)和说明性荧光与温度图(下图)的Thermofluor数据的组织。无催化无活性的麦芽糖酶蛋白在没有配体(无配体)的情况下逐渐加热,在300mM蔗糖存在下。将荧光信号绘制为温度的函数,以获得蛋白质的S形熔解曲线(图的下图)。当蛋白质开始展开时,样品的荧光增加。在蛋白质完全展开时观察到最大荧光强度。之后,如Boivin等人所述,SYPRO Orange信号由于染料 - 蛋白质解离而降低。 (2013年)。酶底物蔗糖的存在使蛋白质的热稳定性提高了5.8℃,其熔解温度T m从59.5℃(无添加的配体,黄色显示)升至65.3℃(在存在蔗糖;蓝色显示)
  3. 尽管如图5所示,尽管蛋白质的T mI值可以粗略地从原始数据图中得出,但是在Niesen等人中描述的进一步的数据分析。 (2007)和Boivin等人。 (2013)需要更准确的T< m>确定。
  4. 为此,我们从Thermofluor测定中处理了原始数据,除去大部分荧光值,只保留与温度升高相对应的温度约为0.5°C。
  5. 然后从处理的数据计算一阶导数。一阶导数(m)表示为:

  6. 对应于一阶导数曲线的峰值的温度表示T 值。
  7. 图5中以及Viigand等人提供的Thermofluor T sub数据。 (2016)使用后一种方法获得。

在我们在Thermofluor测定中测定的不存在糖配体的情况下,MAL1的催化无活性突变体Asp199Ala的T m是59.5℃。野生型(催化活性)MAL1的相应值为51.0(Viigand等人,2016)。因此,无活性突变体比野生型酶更稳定。使用基于经典活性的测定法确定的野生型MAL1的T sub亚基是44.4℃(图3)低于从Thermofluor测定中检测到的T sub。如本文所述,在经典的热稳定性测定中,蛋白质在30分钟内在选定的温度下加热,之后测量残留的催化活性。在Thermofluor测定的情况下,蛋白质逐渐加热(1.5℃/分钟),随后在线监测变性。这些数据表明,麦芽糖酶蛋白耐受短期逐渐加热比延伸逐步加热。当在酶底物存在下使用Thermofluor测定麦芽糖酶突变体Asp199Ala时(参见本方案的图5和Viigand等人,2016)或抑制剂(Viigand等人, ,2016),蛋白质的T m 增加。


  1. 葡萄糖浓度测定的校准曲线
    1. 从葡萄糖标准品(1mg/ml; 5.55mM)制备麦芽糖酶缓冲液中的1-5.55mM葡萄糖溶液。
    2. 将50μl葡萄糖校准曲线溶液(1-5.55mM)的重复样品与150μl200mM Tris-HCl缓冲液(pH 8.3)和800μl在1.5ml微量管中反应的葡萄糖溶液混合。
    3. 样品在37℃下用开放的盖子孵育5分钟
    4. 使用分光光度计在500nm波长下测量紫色产物(醌亚胺)的光密度。参考文献包含麦芽糖酶缓冲液而不是葡萄糖溶液,并且其类似于含有葡萄糖的校准曲线的溶液进行处理。
    5. 获得的值如图6所示。

      在500nm波长下测量不同葡萄糖浓度的喹啉酮形成:0mM,1mM,2mM,3mM,4mM, 5mM和5.55mM。

    6. 在麦芽糖酶活性计算中,图6的线性趋势线(功能:Set Intercept和Display方程)的斜率作为消光系数(ε)用于计算麦芽糖酶反应中释放的葡萄糖的量。


  1. 1M Tris-HCl缓冲液(pH 8.3)
    1. 将24.228g Tris碱溶于150ml MQ水中
    2. 用37%HCl调节pH至8.3
    3. 用MQ水完成200毫升,通过0.2微米醋酸纤维素膜过滤
    4. 在室温下存放
  2. 具有0.1mM EDTA的麦芽糖酶缓冲液-100mM磷酸钾缓冲液(pH6.5)
    1. 制备1M磷酸氢二钾(K 2 H 2 HPO 4)的1M储备溶液和磷酸二氢钾(KH 2 O 3 PO 4) sub>)在MQ水中的盐
    2. 在MQ水中制备0.5M EDTA溶液
    3. 对于400ml 100mM麦芽糖酶缓冲液(pH6.5),混合14ml 1KH 2 HPO 4(碱性组分)和26ml 1M KH 2/sub> PO 4(酸性成分)。使用相同摩尔浓度的预先计算量的酸性和碱性组分制备缓冲液以获得缓冲液的所需pH值(Gomori,1955)
    4. 加入80μl0.5 M EDTA
    5. 用MQ水完成400毫升
    6. 过滤通过0.2μm醋酸纤维素膜过滤器,并保存在4°C
  3. 0.5 M HEPES缓冲液(pH 7.0)
    1. 将5.96g HEPES溶于40ml MQ水中
    2. 用10N NaOH调节pH至7.0
    3. 加入MQ水至最终体积为50 ml
    4. 过滤通过0.2μm醋酸纤维素膜过滤器,并保存在4°C
  4. 4x Thermofluor缓冲液
    1. 在MQ水中准备0.5M HEPES储备缓冲液(pH 7.0)
    2. 在MQ水中制备5 M NaCl储液
    3. 混合800μl0.5 M HEPES缓冲液,120μl5 M NaCl和80μlMQ水,最终体积为1 ml
    4. 为每个实验准备新的缓冲溶液


这项工作由ERC授权GLOMR9072(ETF9072)和GLTMR1050P(PUT1050)资助。该方法的简要描述在Viigand,K.,Visnapuu,T.,Mardo,K.,Aasamets,A。和Alamäe,T。(2016)。 Ogataea(Hansenula)polymorpha的麦芽糖酶蛋白是酵母麦芽糖酶和异麦芽糖酶的复活祖先蛋白质ancMALS的对应物。酵母33(8):415-432。感谢Dimitri Lubenets在Thermofluor测定中的帮助。


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引用:Ernits, K., Viigand, K., Visnapuu, T., Põšnograjeva, K. and Alamäe, T. (2017). Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method. Bio-protocol 7(12): e2349. DOI: 10.21769/BioProtoc.2349.