A Chromogenic Assay Suitable for High-Throughput Determination of

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A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts Marie Bøjstrup,*,† Lucia Marri,† Finn Lok, and Ole Hindsgaul Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark S Supporting Information *

ABSTRACT: Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format. KEYWORDS: limit dextrinase, enzyme assay, chromogenic substrate, malt, brewing

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INTRODUCTION Starch hydrolysis is an important process in the food industry such as in the production of maltodextrins for food additives, glucose syrups, and wort, which is a liquid obtained after mashing and used for fermentation in the production of beer or spirits.1 Starch consists of the polymers amylose, which is essentially linear polymers of α(1→4) glucose units, and amylopectin, which is a branched polymer consisting of α(1→ 4) glucose units with occasional α(1 → 6) branches. Enzymes of importance for the enzymatic hydrolysis of starch are αamylase, which is an α(1→4) endo acting enzyme, β-amylase, which is an α(1→4) exo acting enzyme releasing maltose units, limit dextrinase (LD), which is an α(1 → 6) endo debranching enzyme, and α-glucosidase, which is an α(1→4) exo acting enzyme releasing glucose units.2 The starch source for beer fermentation is traditionally barley malt. This cereal has been bred extensively through history to obtain optimal malting properties such as expression of high amount of the mentioned starch hydrolytic enzymes.3 The measurement of diastatic power (DP) has been a tool for many years in the brewing industry for prediction of the level of carbohydrate degrading enzymes in the malt.4 Several laboratories have shown that the level of DP is strongly correlated to the β-amylase content in the raw material.5,6 In the last couple of years we have observed that specifying a minimum level of DP is not a sufficient tool to predict fermentable sugars in the final wort or discriminate between batches of malt that will fulfill the requirements of the final user. Increasing evidence concerning the reliability of DP as a prediction parameter shows that the level of α-amylase and limit dextrinase (LD) is also influencing the fermentability of the raw material.7 Quality analysis after barley malting can be achieved by measuring the level of α- and β-amylase with existing assays based on the selective hydrolysis of chromogenic substrates followed by UV measurements in a spectrophotometer.8,9 The © 2015 American Chemical Society

Ceralpha assay (Megazyme International, Ireland) for measuring α-amylase activity has been approved by several organizations.10 A widely used assay for LD activity is the Limit-Dextrizyme assay (Megazyme International, Ireland), which is based on dye-cross-linked pullulan which will be selectively hydrolyzed by LD in malt extracts.11 The hydrolysis of the insoluble cross-linked substrate releases blue-colored water-soluble fragments which absorb at 590 nm. While this assay is selective for LD and although it can be adapted for high-throughput assay of large numbers of malt samples,12 this solid-phase assay is still relatively time-consuming, cumbersome, and not amenable for high-throughput, semiautomatic (e.g., robotic) methods of analysis. Furthermore, the crosslinked substrate is not very well-defined potentially causing differences in the detected hydrolysis from batch to batch. Recently it has also been shown that assays based on the insoluble substrate can be problematic for samples where the limit dextrinase inhibitor (LDI) is present.13 Limit dextrinase exists in a “free” form and a “bound” form.14 The “bound” form is a latent form of limit dextrinase which is bound to a low molecular weight proteinaceous inhibitor, LDI (12.7−12.9 kDa).14 LDI shows complete inactivation of the enzyme in 1:1 molar ratio. This complex is slowly disrupted by proteases, reducing agents, or presumably heat during the mashing.15−17 Nevertheless, the tight binding of LDI to LD, the insoluble substrate, can affect the inhibitory effect of LDI on limit dextrinase due to the possible binding of LDI to the insoluble Limit-Dextrizyme substrate.13 We recently described a novel “chromogenic substrate 1” which could be used to selectively assay limit dextrinase even in malt extracts containing various other starch hydrolytic Received: Revised: Accepted: Published: 10873

September 25, 2015 November 27, 2015 November 29, 2015 November 30, 2015 DOI: 10.1021/acs.jafc.5b04596 J. Agric. Food Chem. 2015, 63, 10873−10878

Article

Journal of Agricultural and Food Chemistry

Figure 1. Principle of the developed chromogenic assay for LD activity. The substrate 1 is selectively cleaved by LD, and the formed maltotrioside is further hydrolyzed by exogenous α- and β-glucosidases in a coupled assay to release glucose and 2-chloro-4-nitrophenol, which absorbs light at a maximum at 405 nm. previously described.18 Plates used were Costar 96-well half-area flatbottomed polystyrene plates from Sigma-Aldrich. UV measurements for the Limit-Dextrizyme assay were done at room temperature at 590 nm in 1 cm cuvettes in a WPA Biowave II UV spectrophotometer from Biochrom. Kinetic UV measurements were recorded at 40 °C at 405 nm with measurements every 20 s on a SpectraMax 340PC384 Absorbance Microplate Reader from Molecular Devices.

enzymes such as α-amylase, β-amylase, and α-glucosidases whose activity is blocked at the nonreducing end (Figure 1).18 Here, we present the selectivity and sensitivity of LD toward this substrate using 24 malt samples, and compare the results with those of the existing commercially available LimitDextrizyme method.

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MATERIALS AND METHODS

MALT EXTRACT 0.5 g of malt flour sample aliquots were weighed out. Extraction was performed in a polycarbonate tube by adding 4 mL of 100 mM sodium maleate buffer pH 5.5, or 100 mM sodium maleate buffer pH 5.5 containing 25 mM DTT11,19 to extract free or bound forms of limit dextrinase.14 The mixture was incubated 1 h at 40 °C in a water bath with mixing every 15 min. The tubes were centrifuged at 4000g for 10 min, and the supernatants were transferred to 2 mL Eppendorf tubes and centrifuged 10 min at 11000g to obtain a clear supernatant. Limit dextrinase activity was assayed on freshly prepared malt extract, by the Limit-Dextrizyme kit and the chromogenic assay described below. Excess sample was stored at −20 °C for further use.

Materials. All barley samples were malted using a manually operated micromalting system. The micromalting was performed in a Thermax incubator KB8400 at 13.5 °C during the steeping and germination phase. After the germination process the barley samples were kiln dried in a Thermax incubator for 21 h. The kiln samples were processed using a manual root removal system from Wissenshaftliche Station für Brauerei, Münich. Final malt samples were stored at 20 °C. The 17th EBC standard malt was purchased from IFBM, Nancy, France. Prior to enzyme activity analysis the malt samples were milled using a standard Foss Cyclotech mill equipped with a tungsten carbide grinding ring (Foss 10004463), nickel plated impeller (Foss 1000 2666), and a 1 mm outlet screen (Foss 10001989). The flour was stored at 20 °C. The Limit-Dextrizyme kit, the α-glucosidase (Bacillus stearothermophilus, 750 U/mL), and the β-glucosidase (Agrobacterium sp., 380 U/ mL) used were purchased from Megazyme International Ireland, Bray, Ireland. Recombinant limit dextrinase was prepared as described previously.18 Barley limit dextrinase (HvLD) gene was codon optimized for Pichia pastoris expression by GenScript for cloning into the pPinkα-HC vector. The pPinkα-HC-HvLD vector, linearized with Af lII, was transformed into PichiaPink strain 1 for expression (Invitrogen). HvLD secreted into the media was desalted through a 30 kDa cutoff spin concentrator in 20 mM sodium phosphate, 10 mM NaCl, pH 7.4. Protein was checked for purity on SDS−PAGE gel and stored at 4 °C. Sodium maleate buffer (100 mM, pH 5.5) was made from maleic acid purchased from Acros and pH adjusted with 2 M NaOH. Trizma base and 2-chloro-4-nitrophenol were purchased from Sigma-Aldrich. The chromogenic substrate 1 was prepared as

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LIMIT DEXTRINASE ACTIVITY BY LIMIT-DEXTRIZYME KIT 500 μL of malt extract was transferred to new polycarbonate tubes and the activity of limit dextrinase assayed according to Megazyme’s protocol21 (Limit-Dextrizyme kit, Lot 40801) with minor changes as described. Samples were equilibrated at 40 °C in a water bath for 5 min, and then the reaction was initiated by adding a Limit-Dextrizyme tablet. After 10 min the reaction was terminated by the addition of 5 mL of 1% w/v Trizma Base solution. Tubes were mixed on a vortex mixer and centrifuged at 4000g for 10 min. In the reference blank Trizma base is 10874

DOI: 10.1021/acs.jafc.5b04596 J. Agric. Food Chem. 2015, 63, 10873−10878

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Journal of Agricultural and Food Chemistry

Figure 2. Linearity of the chromogenic assay with modified substrate concentration. (A) Correlation between amount of added recombinant barley LD and the observed initial rate of reaction. (B) Standard curve for 2-chloro-4-nitrophenol in 40% v/v EBC malt extract, pH 5.5, 40 °C. The standard deviation indicated is on replicate measurements (n = 2).

curve for 2-chloro-4-nitrophenol was used to determine the relationship between measured absorbance and concentration of the chromophore.

added to the 17th EBC standard malt extract sample before the addition of the Limit-Dextrizyme tablet. Absorbance at 590 nm was measured in a 1 mL spectrophotometer cuvette. Two replicates of each sample were assayed. Calculation of limit dextrinase activity was based on the formula in the Megazyme protocol T-LDZ1000 07/98.21 One activity unit (U) is defined as the amount of LD that releases 1 μmol of glucose reducing equivalents per min from pullulan.

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RESULTS AND DISCUSSION In order to evaluate the newly developed chromogenic assay, the LD activity in malt samples containing varying levels of LD activity was measured. Twenty-four barley malt samples were prepared by micromalting, and the malt was milled to flour. LD was extracted from this flour with an extraction buffer with or without 25 mM DTT, and the enzymatic activity was measured directly on this extract. It has previously been shown that the observed activity of LD can be increased by varying the extraction parameters as well as adding reducing agent (DTT) to reduce the disulfide bridges and thus release the endogenous limit dextrinase inhibitor (LDI).13,20 As optimization of the assay conditions was not the aim of these experiments, we decided to follow the extraction protocol as described for the Limit-Dextrizyme assay both with and without 25 mM DTT added to the extraction buffer.21 “Bound” and “free” forms of LD were thus extracted from the flour with sodium maleate buffer (100 mM, pH 5.5, with and without 25 mM DTT) during 1 h, and the activity of LD was assayed immediately thereafter. One unit of LD can be defined as the amount of enzyme that releases 1 μmol of 2-chloro-4-nitrophenol per minute in 100 mM maleate buffer, pH 5.5, at 40 °C. A standard curve for 2chloro-4-nitrophenol was prepared at these conditions in the extraction buffer containing 40 vol % EBC malt extract in order to account for a possible interference of the extract with the chromophore. From this standard curve the change in absorbance of light at 405 nm can be related to the amount of released 2-chloro-4-nitrophenol (Figure 2B). In order to obtain better sensitivity of the chromogenic assay, a substrate concentration of 1 mM was used compared to the previously used substrate concentration 0.5 mM.18 This substrate concentration was used in combination with 100 mM sodium maleate buffer, 30 U/mL α-glucosidase, 15 U/mL β-glucosidase, pH 5.5, 40 °C. In order to investigate the linearity of the assay using this concentration of substrate, varying amounts of recombinantly expressed barley LD were added to the chromogenic substrate 1 and the initial rate of the reaction was measured by kinetic measurement of the change in UV absorption at 405 nm. As the absorption maximum for 2chloro-4-nitrophenol is 405 nm and due to the low pKa value of this chromophore, absorption can be measured even at pH 5.5, which is used in the LD assay.22 The data obtained showed a

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LIMIT DEXTRINASE ACTIVITY BY CHROMOGENIC ASSAY Enzymatic Assay. The assay was done in 50 μL total volume in each well. Two replicates were made. All experiments were performed using a 100 mM sodium maleate buffer (100 mM, pH 5.5). The amount of α-glucosidase was 30 U/mL in each well, and the amount of β-glucosidase was 15 U/mL in each well. 20 μL of malt extract prepared as described above was added. The mixtures were equilibrated for 5 min at 40 °C before addition of the substrate corresponding to 1 mM, and then kinetic UV measurements were immediately recorded at 40 °C at 405 nm with measurements every 20 s. As background was used buffer containing 1 mM substrate 1 together with the mentioned amount of α- and β-glucosidase. This blank was subtracted from the measured values. Linearity of Assay. The experiments were performed as described under enzymatic assay, the amount of α-glucosidase was 30 U/mL in each well, and the amount of β-glucosidase was 15 U/mL in each well. The amount of recombinant LD was varied from 0.6 μg/mL to 10 μg/mL. The mixtures were equilibrated for 5 min at 40 °C before addition of the substrate corresponding to 1 mM. Kinetic UV measurements were immediately recorded at 40 °C at 405 nm with measurements every 20 s for an hour. 2-Chloro-4-nitrophenol Standard Curve. A standard curve correlating the concentration of 2-chloro-4-nitrophenol to the measured absorbance was made as follows: 2-Chloro-4nitrophenol was diluted to 2 mM with sodium maleate buffer (100 mM, pH 5.5). 2−25 μL of this standard was added into wells. 20 μL of extract from EBC malt prepared as described was added and the volume adjusted to 50 μL/well with sodium maleate buffer (100 mM, pH 5.5). The absorbance at 405 nm was read after equilibration to 40 °C. Two replicates were performed, and a blank containing only buffer and EBC malt extract was subtracted from the data prior to analysis. Data Analysis. After an initial lag-phase, the rate of hydrolysis was constant and the maximum initial velocity was calculated from the slope of the graph. The obtained standard 10875

DOI: 10.1021/acs.jafc.5b04596 J. Agric. Food Chem. 2015, 63, 10873−10878

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Figure 3. Twenty-four micromalted malt samples and the 17th EBC standard malt sample were analyzed for LD activity using the Limit-Dextrizyme assay (X-axis) and the chromogenic assay using substrate 1 (Y-axis). (A) Activity measured with extraction buffer containing no DTT. (B) Activity measured with extraction buffer containing 25 mM DTT. The coefficient of determination (R2) was determined by linear regression.

Figure 4. Comparison of the existing Limit-Dextrizyme assay (grey bars) with the chromogenic substrate 1 (black bars). The data was normalized to the value obtained from the EBC standard malt. (A) Results obtained with extraction buffer containing no DTT. (B) Results obtained with extraction buffer containing 25 mM DTT. The standard deviation indicated is on replicate measurements (n = 6 in A; n = 4 in B).

It has been shown that starch hydrolysis products can interfere with the Limit-Dextrizyme assay resulting in overestimation of the level of enzyme activity.23 Maltose and linear maltodextrins, present in brewer’s wort, can significantly affect the measurements of LD activity by Limit-Dextrizyme assay, almost doubling the apparent level of activity.23 This effect has been detected in assays based on solubilization of cross-linked dyed pullulan,23,24 such as red pullulan or azurine-cross-linked pullulan, the latter used in the Limit-Dextrizyme assay from Megazyme. McDougall and colleagues showed that LD activity from germinating barley was 60 to 100% activated by the presence of 2.5−25 mM maltotriose or maltotetraose, short linear maltodextrins being the most effective.24 The apparent increase of LD activity by linear maltodextrins was not correlated to more efficient cleavage of the pullulan substrate, thus not a true enzyme activation. LD can perform transglycosylation of the hydrolyzed pullulan, reducing the viscosity of the pullulan and increasing solubilization of the dyed fragments. Considering that assays not based on cross-linked substrate should not be affected by short maltodextrins, the effect of short maltodextrins on the chromogenic substrate was tested in order to verify this. Varying amount of recombinant barley LD was added to the chromogenic substrate 1 in the presence or absence of 25 mM maltotriose and activity assayed (Figure S1A). LD activity was not affected by the presence of

linear correlation between the amount of recombinant barley LD added and the observed initial rate (Figure 2A). The activity of added α-glucosidase and β-glucosidase was therefore sufficient to ensure that the initial rate was only dependent on the amount of LD in the sample. The malt samples obtained from the micromalting were assayed for LD activity using the mentioned conditions in replicate (n = 6 for extractions without DTT, or n = 4 for DTTextracted samples), and the initial rate measured correlated to mU/g malt flour from the obtained 2-chloro-4-nitrophenol standard curve. Also, the same samples were analyzed using the Limit-Dextrizyme assay (Megazyme International, Ireland) following the protocol and calculating the activity in mU/g malt flour based on the standard curve indicated in the protocol provided.21 The results from the two assays were correlated for extraction without DTT (“free” form of LD, Figure 3A) and extraction with 25 mM DTT (“free + bound” form of LD, Figure 3B). Both correlations showed a coefficient of determination (R2) greater than 0.7. The addition of 25 mM DTT to the extraction buffer resulted as expected in a higher activity of limit dextrinase as the endogenous LDI is inactivated by reducing conditions.13 This enhancement of activity was more pronounced with the Limit-Dextrizyme assay and less for the chromogenic assay. 10876

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maltotriose, and the linear correlation observed between the amount of recombinant LD and the initial rate was totally reproduced in samples incubated with maltrotriose (Figure 2A and S1A). Moreover, the interference of starch hydrolysis products on the activities measured by the chromogenic assay was also tested in malt. Malt was tested for LD activity on the chromogenic substrate 1 in the presence of varying concentrations of maltotriose (0−25 mM) (Figure S1B). No significative change in activity was noticed at any concentration of maltotriose tested. LD activity measured in malt sample was totally retained in samples incubated with 0−25 mM maltotriose. We can conclude that assays based on the “chromogenic substrate 1” were not influenced by the level of starch hydrolysis products, as known in the literature for assays based on cross-linked dyed substrate.23,24 The more pronounced enhancement of activity by DTT activation measured with the Limit-Dextrizyme assay compared to the chromogenic substrate (Figure 3B) could be ascribed to the contribution of short starch hydrolysis products present in DTT activated samples. The results from both assays (Limit-Dextrizyme and chromogenic assay) on malt samples were normalized to the value obtained from the 17th EBC standard malt in order to be able to further compare the two assays. Both assays showed in general to have a low standard deviation on replicate measurements (Figure 4). When the extraction was performed without DTT, the chromogenic assay was more sensitive with a wider variation between the sample containing the highest activity of LD and the one with the lowest compared to the Limit-Dextrizyme assay (a relationship of 3.6 for the chromogenic assay versus 1.7 for the Limit-Dextrizyme assay; so that the wider variation in the chromogenic assay results in a slope of 1.958 in the correlation of the two assays). Furthermore, it matched very well with the existing assay with respect to determining the samples with the highest and lowest activities of LD (Figure 4). For the conditions using 25 mM DTT in the extraction buffer, the sensitivity was very similar for the two assays (a relationship of 5.3 for the chromogenic assay between the sample containing the highest activity of LD and the one with the lowest, versus 6.4 for the Limit-Dextrizyme assay; resulting in a slope of 0.3841). In view of these results the chromogenic assay tends to be more sensitive, thus better discriminating the level of activity in the different samples extracted without DTT compared to the Limit-Dextrizyme assay, which seems to leverage the activity, especially in the malt samples where the LD activity was low. This chromogenic assay, as well as assays based on similar small substrates, such as those recently developed by Megazyme International Ireland,25 have great potential to be used in the specifications of malt for use in the brewing industry. The assay is easy to perform with analytic equipment already present in the malteries and is suited for a 96-well format. The developed chromogenic assay can potentially be utilized in highthroughput screening of large numbers of barley samples, e.g., for advanced selection in barley breeding, as it is sensitive enough to clearly distinguish between malts with a range of activities of LD. Furthermore, the assay correlates very well with the commercially available Limit-Dextrizyme assay, which is further strengthening the credibility of the assay.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04596.

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Figure S1 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

M.B. and L.M. equally contributed to the paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Morten Munch Nielsen and Monica Palcic, Carlsberg Research Laboratory, are acknowledged for the donation of recombinantly expressed barley limit dextrinase. Ole Olsen, Carlsberg Research Laboratory, is acknowledged for his advice regarding limit dextrinase assay optimization.

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ABBREVIATIONS USED DTT, dithiothreitol; LD, limit dextrinase; LDI, limit dextrinase inhibitor; DP, diastatic power

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REFERENCES

(1) Handbook of starch hydrolysis products and their derivatives, 1st ed.; Kearsley, M. W., Dziedzic, S. Z., Eds.; Chapman & Hall: London, 1995. (2) Crabb, W. D.; Mitchinson, C. Enzymes involved in the processing of starch to sugars. Trends Biotechnol. 1997, 15, 349−352. (3) Lewis, M. J.; Young, T. W. Brewing, 2nd ed.; Kluwer Academic/ Plenum Publishers: New York, 2001; pp 149−162. (4) European Brewer Convention. Diastatic power of malt, method 4.12. Analytica, EBC; Verlag Hans Carl: Nuremberg, Germany, 1998. (5) Gibson, T. S.; Solah, V.; Holmes, M. R. G.; Taylor, H. R. Diastatic power in malted barley: contributions of malt parameters to its development and the potential of barley grain β-amylase to predict malt diastatic power. J. Inst. Brew. 1995, 101, 277−280. (6) Delcour, J. A.; Verschaeve, S. G. Malt diastatic activity. Part II. A modified EBC diastatic power assay for the selective estimation of beta-amylase activity. Time and temperature dependence of the release of reducing sugars. J. Inst. Brew. 1987, 93, 296−301. (7) Evans, D. E.; Li, C.; Eglinton, J. K. Improved prediction of malt fermentability by measurement of the diastatic power enzymes βamylase, α-amylase, and limit dextrinase: I. Survey of the levels of diastatic power enzymes in commercial malts. J. Am. Soc. Brew. Chem. 2008, 66, 223−232. (8) McCleary, B. V.; Sheehan, H. Measurement of cereal α-amylase: a new assay procedure. J. Cereal Sci. 1987, 6, 237−251. (9) McCleary, B. V.; Codd, R. Measurement of ß-amylase in cereal flours and commercial enzyme preparations. J. Cereal Sci. 1989, 9, 17− 33. (10) E.g., AACC International and AOAC International. (11) McCleary, B. V. Measurement of the content of limit dextrinase in cereal flours. Carbohydr. Res. 1992, 227, 257−268. (12) Evans, D. E. A more cost and labour efficient assay for the combined measurement of the diastatic power enzymes, b-amylase, aamylase and limit dextrinase. J. Am. Soc. Brew. Chem. 2008, 66, 215− 222. (13) Jensen, J. M.; Hägglund, P.; Christensen, H. E. M.; Svensson, B. Inactivation of barley limit dextrinase inhibitor by thioredoxincatalysed disulfide reduction. FEBS Lett. 2012, 586, 2479−2482. 10877

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DOI: 10.1021/acs.jafc.5b04596 J. Agric. Food Chem. 2015, 63, 10873−10878