YABIO 11667
No. of Pages 3, Model 5G
20 March 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
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2
Notes & Tips
6 4 7 5
Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method
8
Gastón E. Ortiz a, María E. Guitart b, Edgardo Albertó a, Héctor M. Fernández Lahore b, Martin Blasco a,⇑
9 10
11 1 7 3 2 14 15 16 17 18 19 20 21 22 23 24 25 26
a b
Instituto de Investigaciones Biotecnológicas–Instituto Tecnológico Chascomús (IIB–INTECH, UNSAM–CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina Downstream Processing Laboratory, Jacobs University Bremen, D-28759 Bremen, Germany
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 23 February 2014 Accepted 26 February 2014 Available online xxxx Keywords: Microplate Pectinases Endo-polygalacturonases Assay Ruthenium red High throughput
a b s t r a c t Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 lg of polygalacturonic acid, and 45 lg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities. Ó 2014 Elsevier Inc. All rights reserved.
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37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Pectinases are used in various industrial applications related to the processing of natural products, mainly for extraction purposes [1]. The activity profile within the mixtures (endo- and exo-polygalacturonases and modifying enzymes as rhamnogalacturonases, etc.) generally defines the suitability of an enzymatic product for the different applications [2,3]. Because development and study of enzyme preparations require multiple parallel determinations of different activities in the mixture, suitable methods are demanded in this field [4–7]. To our knowledge, there is no method that accomplishes this requirement for polygalacturonase (PGase,1 EC 3.2.1.15) activity. This activity is generally determined by using viscosity reduction in pectic substances over time using a viscometer [8] or by increasing reducing sugar ends or metabolites released from pectic substances [1]. As an alternative, Torres and coworkers [9] presented a quantitative method based on the ruthenium red (RR) precipitation of polygalacturonic acid (PGA). Among the drawbacks of the methods currently used, the viscosity determination method is time-consuming and allows few determinations at a time, whereas the reducing sugar-based method could display interference due to other enzymatic activities acting in the same conditions as PGase or compounds such as amino acids [10]. The RR method ⇑ Corresponding author. Fax: +54 1140061500x2143. E-mail address: [email protected] (M. Blasco). 1 Abbreviations used: PGase, polygalacturonase; RR, ruthenium red; PGA, polygalacturonic acid; PCR, polymerase chain reaction; BCP, buffer citrate phosphate; SSF, solid-state fermentation; SmF, submerged fermentation.
presented by Torres and coworkers [9] requires a large amount of reagents. In this context, a reduced scale assay based on RR suitable for high-throughput screening and activity determination is presented and compared with the viscosity method [8]. The scaled down RR assay follows the same general procedures as Torres and coworkers [9] with some modifications that have shown to increase the assay quality. Briefly, it involves one reaction step, followed by precipitation of high-molecular-weight PGA by RR and finally the optical density determination of the remaining RR for quantifying purposes. The microplate assay was carried out in 96-well polymerase chain reaction (PCR) plates sealed with adhesive film as follows. Mixing was performed after each step involving reagent addition by orbital shaking (5 mm) at 2500 rpm for 20 s, followed by centrifugation in a swinging bucket microplate centrifuge at 4 °C and 3200g for 30 s. The calibration curves were prepared from 0.5% PGA sodium salt stock solution in buffer citrate phosphate (BCP: 50 mM, pH 5.0). This solution was obtained by dissolving 0.500 g of PGA sodium salt (Sigma–Aldrich, Germany) in citric acid solution and then adjusting the pH to 5.0 with dibasic sodium phosphate. The calibration curve of PGA was prepared at final concentrations equivalent to 33.75, 30.00, 22.50, 18.75, 15.00, 11.25, 7.50, 3.75, and 0 lg of PGA per 16 ll of BCP, and 16 ll of the mixture was placed in an ice-cooled microplate by triplicate. The reaction
http://dx.doi.org/10.1016/j.ab.2014.02.028 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
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YABIO 11667
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U=mL ¼
SB SA ; SLP t V
ð1Þ
where SB is the absorbance of the enzyme sample blank obtained from a mixture of 8 ll enzyme dilution and 8 ll PGA solution without incubation, SA is the PGA–enzyme reaction absorbance, and SLP
A 0.8 0.7
B
30 μg RR 60 μg RR A535nm = - 0.0077*PGA + 0.3561 A535nm = - 0.0079*PGA + 0.6994 2 2 R =0.9901 R =0.9868 15 μg RR 45 μg RR A535nm = - 0.0088*PGA + 0.5538 A535nm= 0.0002*PGA^2 -0.0118*PGA + 0.1945 R2=0.9912 R2=0.9938
300 Tube
Microplate
250
0.6 0.5
60 μg RR
0.4 0.3
45 μg RR
0.2 30 μg RR
Endo- PGase RR activity (kU/ml)
89
is the slope of the least-squares adjusted calibration curve obtained from A535nm versus micrograms (lg) of PGA in 16 ll, t is the time (in min) taken for the reaction (typically 20 min), and V is the volume (in ml) of enzyme dilution used in the reaction (0.0080 ml). The activity in the nonincubated reaction (SB) was determined to detect the presence of interferences in the sample and used as blank for activity calculations. For screening procedures involving samples from the same medium and strain (i.e., mutant screening), this value could be considered as constant after a short validation step to save space in the plate and then to increase the throughput of the assay. The microplate assay was compared with the tube assay by analyzing the commercial pectinase mixtures (Fig. 1B): pectinases from Aspergillus niger (CAS 9032-75-1, Sigma–Aldrich), Pectinex 3XL (Novozymes, Denmark), and Fructozyme P (Erbslöh Geisenheim, Germany) at 3200-, 800-, and 400-fold dilutions, respectively. The RR method in tube scale was performed following Torres and coworkers’ directions [9] except that absorbance determinations were performed by using 200 ll of the supernatant in 96-well plates. The enzyme activity values obtained by using microplate or tube scale assays presented no significant differences (P < 0.01) when analyzed by using a paired t test. The suitability of microplate assay in screening procedures was evaluated by assessing the activity of crude extracts from four strains of Aspergillus sojae grown in solid-state fermentation (SSF) and submerged fermentation (SmF) (Fig. 1C).The former samples were obtained from 6-day-old wheat bran (WB) and sugar beet pellet (SBP) medium as described by Heerd and coworkers [11]. The latter (SmF) samples were obtained from orange peel (OP) and sugar beet syrup (SBS) supplemented with ammonium sulfate
0.1
200 150 100 50
15 μg RR 0
0 0
3.75
7.50 11.25 15.00 18.75 22.50 26.25 30.00 33.75 37.50
Sigma
PGA (μg)
C
D
SmF medium (CP)
0.0100
6000
SmF medium (OP & SBS)
0.0090
5000
SSF (WB & SBP)
4000 3000 2000 1000 0
Ratio (viscosity units / RR units)
88
mixture was performed by placing 8 ll of 0.50% PGA and 8 ll of the enzyme dilution in BCP and then incubated for 20 min at 40.0 °C in a thermocycler or water bath. After the incubation, the plate was placed back in ice, 40 ll of 1.125 mg/ml RR aqueous solution was added to each well, and the plate was sealed and mixed for 30 s. The mixture was diluted by adding 100 ll of 8 mM NaOH solution, mixed for 30 s, and centrifuged at 4 °C and 3200g for 10 min. For quantification, a 25-ll aliquot of the supernatant was transferred to the corresponding well of a clear flat-bottom 96-well microplate containing 175 ll of water, and absorbance was read at 535 nm (A535nm) with a microplate reader. Alternatively, for this last step, the measurements could be performed at 492 nm by using 50 ll of supernatant and 150 ll of water. The calibration curve dependence on the amount of RR was used to adjust the amount of RR for the assay. It was observed that 45 lg of RR (40 ll of 1.125 mg/ml) resulted in good linearity and sensitivity (Fig. 1A), and then this concentration was used in the assays. Furthermore, calibration curves of both methods were analyzed and indicated that dynamic range and sensitivity were equivalent (results not shown). The enzyme unit was defined as the amount of enzyme required to hydrolyze 1 lg of PGA in smaller fragments unable to precipitate with the dye per minute under the assay conditions. Activities were calculated according to the equation
A535 nm
87
Endo - PGase RR activity (U/ml)
86
Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx
0.0080 0.0070 0.0060
0.0062 0.0059
0.0057
0.0050 0.0040 0.0030
M5/6
M3
5595 Strain
5595 -1D
Sigma N=432
Fructozyme P N=432
Total N=864
Fig.1. (A) Calibration curves for PGA using microplate assay. Linearity and sensitivity of calibration curves of PGA are shown using different amounts of RR (n = 3): d, 15 lg; N, 30 lg; j, 45 lg; , 60 lg. (B) Commercial samples of PGase activity determination (n = 6) using Torres and coworkers’ assay (gray bars) and microplate assay (white bars). (C) Activity determination by microplate-based PGase activity of samples from different cultivation methods (SmF and SSF) for four strains of Aspergillus sojae using different medium composition (CP, citric pectin; OP, orange peels; SBS, sugar beet pellets; WB, wheat bran; SBS, sugar beet syrup). (D) Box plot for each distribution; the median (within the box) represents the conversion factor between viscosity and RR units, whereas Total is the distribution resulting from the combination of Sigma and Fructozyme P distributions.
Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
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Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx Table 1 Comparison between tube and microplate assays.
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Parameter
Torres et al. [9]
This work
Scaling down fold
Amount of RR per reaction Amount of PGA per reaction Reaction volume Water amount for measurement Enzyme dilution volume Ratio RR/PGA Enzyme reaction at 40 °C for 20 min Read at 535 nm
200 lg 187 lg 100 ll 6 ml Variable 1.07 Test tubes 1 ml in 10-mm spectrophotometer cuvette
45 lg 40 lg 16 ll 175 ll 8 ll 1.12 96-well PCR microplate 200 ll in standard optical 96-well plate
4.44 4.67 6.25 34.28 – – – –
medium (SmF–OP–SBS: 10 g/L OP, 61.9 g/L SBS, and 8.5 g/L ammonium sulfate) and from Czapek containing 1% citric pectin (CP) medium named SmF (CP). Shorter incubation times were assayed, and it was observed that PGase activity quantification could be achieved by 4-day SmF and SSF without processing (i.e., purification, concentration) of the sample. During the scaling down, it was observed that the addition of RR directly to the enzyme reaction increases the lineal range of the method by reducing the amount of soluble RR obtained at high concentrations of PGA, thereby increasing the dynamic range and confidence at low enzyme activities. Furthermore, the use of 8 mM NaOH instead of water during the dilution of RR–PGA complex results in a better performance of the assay, reducing background at high PGA concentrations. It is recommended not to increase the initial PGA concentration due to the presence of soluble RR products. This situation was observed previously and could be related to the presence of pectin esters in the PGA solution [12]. Due to the importance of unit conversion for product comparison and yield calculations, the RR method was compared with the viscosity method. The viscosity units for commercial samples were obtained following Gusakov and coworkers’ directions [8]. Measurements were performed at 40.0 °C using a capillary viscometer of 0.60 mm internal diameter, 0.03 K constant, 1.2 to 18 mm2 s1 measuring range (kinematic viscosity) (516 13, SI Analytics, Germany). The conversion factor between the viscosity method and the RR method was calculated as the ratio between viscosity units and RR units (Fig. 1D). For this purpose, several dilutions of the enzyme Fructozyme P and Sigma pectinases were assayed in the linear range for both methods. To obtain all possible combinations of ratios, the activity values from Sigma and Fructozyme P were arranged in a Punnett square, where the first row and first column contain the activity values from the RR method and the viscosity method, respectively. The resulting distribution was analyzed statistically using the software G-Stat (version 2.01), and the median of this distribution (0.0059 RR units/viscosity units) was considered as the conversion factor. To establish the contribution of each formulation to the final value, the Sigma and Fructozyme P distributions were calculated separately as described earlier. The viscosity activity determination method indicated that the Fructozyme P preparation resulted in 350 ± 40 U/ml activity (n = 10, mean ± standard deviation, 1:5 to 1:40 dilutions for determination), whereas the Sigma pectinases resulted in 1800 ± 200 U/ ml (1:10 to 1:100 dilutions for determination). The dynamic range, calculated over the basis of enzyme dilutions, was approximately one order of magnitude (51 to 401 and 101 to 1001 for Fructozyme P and Sigma pectinases, respectively). On the other hand, the RR method displayed a lower range of dilutions, resulting in linear correlation compared with the viscosity determination (4401 to 8301 and 24001 to 38401 for Fructozyme P and Sigma pectinases, respectively). Besides, it is noteworthy that the amount of
enzyme required for the RR method is 10 to 20 times less than that required for the viscosity-based method. Considering that most of industrial processes involving pectinases are related to viscosity reduction, the conversion factor obtained for RR to viscosity units could be used as reference for activity determination using a viscometer, although validation for particular enzyme mixtures should be accomplished. The scaled down assay presented here reduces the reagent volume, space usage, and pipetting effort and increases the throughput of the assay (Table 1). The assay reduction resulted in a lower cost per test, making it the preferred option to carry out high-throughput screening of PGase activity. Considering the difference in the limit of detection for viscometer-based determination and the time required for each determination, this method should be considered as a first choice in PGase activity screening.
197
Acknowledgments
213
This work was supported by an IDB OC/AR loan with a PICT Q1 start-up 2010-1312 grant from the FONCyT–MinCyT of Argentina Q2 (issued to E. Albertó) and the PGSYS program (issued to H. Fernández-Lahore).
214
References
218
[1] R.S. Jayani, S. Saxena, R. Gupta, Microbial pectinolytic enzymes: a review, Process. Biochem. 40 (2005) 2931–2944. [2] D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari, Applications of pectinases in the commercial sector: a review, Bioresour. Technol. 77 (2001) 215–227. [3] N. Sharma, M. Rathore, M. Sharma, Microbial pectinase: sources, characterization, and applications, Rev. Environ. Sci. Biotechnol. 12 (2013) 45–60. [4] X.-J. Wang, J.-G. Bai, Y.-X. Liang, Optimization of multienzyme production by two mixed strains in solid-state fermentation, Appl. Microbiol. Biotechnol. 73 (2006) 533–540. [5] X. Zhou, H. Chen, Z. Li, CMCase activity assay as a method for cellulase adsorption analysis, Enzyme Microb. Technol. 35 (2004) 455–459. [6] Z. Xiao, R. Storms, A. Tsang, Microplate-based carboxymethylcellulose assay for endoglucanase activity, Anal. Biochem. 342 (2005) 176–178. [7] B.C. King, M.K. Donnelly, G.C. Bergstrom, L.P. Walker, D.M. Gibson, An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts, Biotechnol. Bioeng. 102 (2009) 1033–1044. [8] A. Gusakov, A.V. Markov, S.G. Grishutin, M.V. Semenova, E.G. Kondratyeva, A.P. Sinitsyn, Viscometric method for assaying of total endodepolymerase activity of pectinases, Biochemistry 67 (2002) 676–682. [9] S. Torres, J.E. Sayago, R.M. Ordoñez, M.I. Isla, A colorimetric method to quantify endo-polygalacturonase activity, Enzyme Microb. Technol. 48 (2011) 123–128. [10] R.S.S. Teixeira, A.S. da Silva, V.S. Ferreira-Leitão, E.P. da Silva Bon, Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements, Carbohydr. Res. 363 (2012) 33–37. [11] D. Heerd, S. Yegin, C. Tari, M. Fernandez-Lahore, Pectinase enzyme complex production by Aspergillus spp. in solid-state fermentation: a comparative study, Food Bioprod. Process. 90 (2012) 102–110. [12] J. Luft, Ruthenium red and violet: I. Chemistry, purification, methods of use for electron microscopy, and mechanism of action, Anat. Rec. 171 (1971) 347– 368.
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Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
No. of Pages 3, Model 5G
20 March 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method
8
Gastón E. Ortiz a, María E. Guitart b, Edgardo Albertó a, Héctor M. Fernández Lahore b, Martin Blasco a,⇑
9 10
11 1 7 3 2 14 15 16 17 18 19 20 21 22 23 24 25 26
a b
Instituto de Investigaciones Biotecnológicas–Instituto Tecnológico Chascomús (IIB–INTECH, UNSAM–CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina Downstream Processing Laboratory, Jacobs University Bremen, D-28759 Bremen, Germany
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 23 February 2014 Accepted 26 February 2014 Available online xxxx Keywords: Microplate Pectinases Endo-polygalacturonases Assay Ruthenium red High throughput
a b s t r a c t Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 lg of polygalacturonic acid, and 45 lg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities. Ó 2014 Elsevier Inc. All rights reserved.
28 29 30 31 32 33 34 35 36
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Pectinases are used in various industrial applications related to the processing of natural products, mainly for extraction purposes [1]. The activity profile within the mixtures (endo- and exo-polygalacturonases and modifying enzymes as rhamnogalacturonases, etc.) generally defines the suitability of an enzymatic product for the different applications [2,3]. Because development and study of enzyme preparations require multiple parallel determinations of different activities in the mixture, suitable methods are demanded in this field [4–7]. To our knowledge, there is no method that accomplishes this requirement for polygalacturonase (PGase,1 EC 3.2.1.15) activity. This activity is generally determined by using viscosity reduction in pectic substances over time using a viscometer [8] or by increasing reducing sugar ends or metabolites released from pectic substances [1]. As an alternative, Torres and coworkers [9] presented a quantitative method based on the ruthenium red (RR) precipitation of polygalacturonic acid (PGA). Among the drawbacks of the methods currently used, the viscosity determination method is time-consuming and allows few determinations at a time, whereas the reducing sugar-based method could display interference due to other enzymatic activities acting in the same conditions as PGase or compounds such as amino acids [10]. The RR method ⇑ Corresponding author. Fax: +54 1140061500x2143. E-mail address: [email protected] (M. Blasco). 1 Abbreviations used: PGase, polygalacturonase; RR, ruthenium red; PGA, polygalacturonic acid; PCR, polymerase chain reaction; BCP, buffer citrate phosphate; SSF, solid-state fermentation; SmF, submerged fermentation.
presented by Torres and coworkers [9] requires a large amount of reagents. In this context, a reduced scale assay based on RR suitable for high-throughput screening and activity determination is presented and compared with the viscosity method [8]. The scaled down RR assay follows the same general procedures as Torres and coworkers [9] with some modifications that have shown to increase the assay quality. Briefly, it involves one reaction step, followed by precipitation of high-molecular-weight PGA by RR and finally the optical density determination of the remaining RR for quantifying purposes. The microplate assay was carried out in 96-well polymerase chain reaction (PCR) plates sealed with adhesive film as follows. Mixing was performed after each step involving reagent addition by orbital shaking (5 mm) at 2500 rpm for 20 s, followed by centrifugation in a swinging bucket microplate centrifuge at 4 °C and 3200g for 30 s. The calibration curves were prepared from 0.5% PGA sodium salt stock solution in buffer citrate phosphate (BCP: 50 mM, pH 5.0). This solution was obtained by dissolving 0.500 g of PGA sodium salt (Sigma–Aldrich, Germany) in citric acid solution and then adjusting the pH to 5.0 with dibasic sodium phosphate. The calibration curve of PGA was prepared at final concentrations equivalent to 33.75, 30.00, 22.50, 18.75, 15.00, 11.25, 7.50, 3.75, and 0 lg of PGA per 16 ll of BCP, and 16 ll of the mixture was placed in an ice-cooled microplate by triplicate. The reaction
http://dx.doi.org/10.1016/j.ab.2014.02.028 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
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90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
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U=mL ¼
SB SA ; SLP t V
ð1Þ
where SB is the absorbance of the enzyme sample blank obtained from a mixture of 8 ll enzyme dilution and 8 ll PGA solution without incubation, SA is the PGA–enzyme reaction absorbance, and SLP
A 0.8 0.7
B
30 μg RR 60 μg RR A535nm = - 0.0077*PGA + 0.3561 A535nm = - 0.0079*PGA + 0.6994 2 2 R =0.9901 R =0.9868 15 μg RR 45 μg RR A535nm = - 0.0088*PGA + 0.5538 A535nm= 0.0002*PGA^2 -0.0118*PGA + 0.1945 R2=0.9912 R2=0.9938
300 Tube
Microplate
250
0.6 0.5
60 μg RR
0.4 0.3
45 μg RR
0.2 30 μg RR
Endo- PGase RR activity (kU/ml)
89
is the slope of the least-squares adjusted calibration curve obtained from A535nm versus micrograms (lg) of PGA in 16 ll, t is the time (in min) taken for the reaction (typically 20 min), and V is the volume (in ml) of enzyme dilution used in the reaction (0.0080 ml). The activity in the nonincubated reaction (SB) was determined to detect the presence of interferences in the sample and used as blank for activity calculations. For screening procedures involving samples from the same medium and strain (i.e., mutant screening), this value could be considered as constant after a short validation step to save space in the plate and then to increase the throughput of the assay. The microplate assay was compared with the tube assay by analyzing the commercial pectinase mixtures (Fig. 1B): pectinases from Aspergillus niger (CAS 9032-75-1, Sigma–Aldrich), Pectinex 3XL (Novozymes, Denmark), and Fructozyme P (Erbslöh Geisenheim, Germany) at 3200-, 800-, and 400-fold dilutions, respectively. The RR method in tube scale was performed following Torres and coworkers’ directions [9] except that absorbance determinations were performed by using 200 ll of the supernatant in 96-well plates. The enzyme activity values obtained by using microplate or tube scale assays presented no significant differences (P < 0.01) when analyzed by using a paired t test. The suitability of microplate assay in screening procedures was evaluated by assessing the activity of crude extracts from four strains of Aspergillus sojae grown in solid-state fermentation (SSF) and submerged fermentation (SmF) (Fig. 1C).The former samples were obtained from 6-day-old wheat bran (WB) and sugar beet pellet (SBP) medium as described by Heerd and coworkers [11]. The latter (SmF) samples were obtained from orange peel (OP) and sugar beet syrup (SBS) supplemented with ammonium sulfate
0.1
200 150 100 50
15 μg RR 0
0 0
3.75
7.50 11.25 15.00 18.75 22.50 26.25 30.00 33.75 37.50
Sigma
PGA (μg)
C
D
SmF medium (CP)
0.0100
6000
SmF medium (OP & SBS)
0.0090
5000
SSF (WB & SBP)
4000 3000 2000 1000 0
Ratio (viscosity units / RR units)
88
mixture was performed by placing 8 ll of 0.50% PGA and 8 ll of the enzyme dilution in BCP and then incubated for 20 min at 40.0 °C in a thermocycler or water bath. After the incubation, the plate was placed back in ice, 40 ll of 1.125 mg/ml RR aqueous solution was added to each well, and the plate was sealed and mixed for 30 s. The mixture was diluted by adding 100 ll of 8 mM NaOH solution, mixed for 30 s, and centrifuged at 4 °C and 3200g for 10 min. For quantification, a 25-ll aliquot of the supernatant was transferred to the corresponding well of a clear flat-bottom 96-well microplate containing 175 ll of water, and absorbance was read at 535 nm (A535nm) with a microplate reader. Alternatively, for this last step, the measurements could be performed at 492 nm by using 50 ll of supernatant and 150 ll of water. The calibration curve dependence on the amount of RR was used to adjust the amount of RR for the assay. It was observed that 45 lg of RR (40 ll of 1.125 mg/ml) resulted in good linearity and sensitivity (Fig. 1A), and then this concentration was used in the assays. Furthermore, calibration curves of both methods were analyzed and indicated that dynamic range and sensitivity were equivalent (results not shown). The enzyme unit was defined as the amount of enzyme required to hydrolyze 1 lg of PGA in smaller fragments unable to precipitate with the dye per minute under the assay conditions. Activities were calculated according to the equation
A535 nm
87
Endo - PGase RR activity (U/ml)
86
Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx
0.0080 0.0070 0.0060
0.0062 0.0059
0.0057
0.0050 0.0040 0.0030
M5/6
M3
5595 Strain
5595 -1D
Sigma N=432
Fructozyme P N=432
Total N=864
Fig.1. (A) Calibration curves for PGA using microplate assay. Linearity and sensitivity of calibration curves of PGA are shown using different amounts of RR (n = 3): d, 15 lg; N, 30 lg; j, 45 lg; , 60 lg. (B) Commercial samples of PGase activity determination (n = 6) using Torres and coworkers’ assay (gray bars) and microplate assay (white bars). (C) Activity determination by microplate-based PGase activity of samples from different cultivation methods (SmF and SSF) for four strains of Aspergillus sojae using different medium composition (CP, citric pectin; OP, orange peels; SBS, sugar beet pellets; WB, wheat bran; SBS, sugar beet syrup). (D) Box plot for each distribution; the median (within the box) represents the conversion factor between viscosity and RR units, whereas Total is the distribution resulting from the combination of Sigma and Fructozyme P distributions.
Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
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No. of Pages 3, Model 5G
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Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx Table 1 Comparison between tube and microplate assays.
146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196
Parameter
Torres et al. [9]
This work
Scaling down fold
Amount of RR per reaction Amount of PGA per reaction Reaction volume Water amount for measurement Enzyme dilution volume Ratio RR/PGA Enzyme reaction at 40 °C for 20 min Read at 535 nm
200 lg 187 lg 100 ll 6 ml Variable 1.07 Test tubes 1 ml in 10-mm spectrophotometer cuvette
45 lg 40 lg 16 ll 175 ll 8 ll 1.12 96-well PCR microplate 200 ll in standard optical 96-well plate
4.44 4.67 6.25 34.28 – – – –
medium (SmF–OP–SBS: 10 g/L OP, 61.9 g/L SBS, and 8.5 g/L ammonium sulfate) and from Czapek containing 1% citric pectin (CP) medium named SmF (CP). Shorter incubation times were assayed, and it was observed that PGase activity quantification could be achieved by 4-day SmF and SSF without processing (i.e., purification, concentration) of the sample. During the scaling down, it was observed that the addition of RR directly to the enzyme reaction increases the lineal range of the method by reducing the amount of soluble RR obtained at high concentrations of PGA, thereby increasing the dynamic range and confidence at low enzyme activities. Furthermore, the use of 8 mM NaOH instead of water during the dilution of RR–PGA complex results in a better performance of the assay, reducing background at high PGA concentrations. It is recommended not to increase the initial PGA concentration due to the presence of soluble RR products. This situation was observed previously and could be related to the presence of pectin esters in the PGA solution [12]. Due to the importance of unit conversion for product comparison and yield calculations, the RR method was compared with the viscosity method. The viscosity units for commercial samples were obtained following Gusakov and coworkers’ directions [8]. Measurements were performed at 40.0 °C using a capillary viscometer of 0.60 mm internal diameter, 0.03 K constant, 1.2 to 18 mm2 s1 measuring range (kinematic viscosity) (516 13, SI Analytics, Germany). The conversion factor between the viscosity method and the RR method was calculated as the ratio between viscosity units and RR units (Fig. 1D). For this purpose, several dilutions of the enzyme Fructozyme P and Sigma pectinases were assayed in the linear range for both methods. To obtain all possible combinations of ratios, the activity values from Sigma and Fructozyme P were arranged in a Punnett square, where the first row and first column contain the activity values from the RR method and the viscosity method, respectively. The resulting distribution was analyzed statistically using the software G-Stat (version 2.01), and the median of this distribution (0.0059 RR units/viscosity units) was considered as the conversion factor. To establish the contribution of each formulation to the final value, the Sigma and Fructozyme P distributions were calculated separately as described earlier. The viscosity activity determination method indicated that the Fructozyme P preparation resulted in 350 ± 40 U/ml activity (n = 10, mean ± standard deviation, 1:5 to 1:40 dilutions for determination), whereas the Sigma pectinases resulted in 1800 ± 200 U/ ml (1:10 to 1:100 dilutions for determination). The dynamic range, calculated over the basis of enzyme dilutions, was approximately one order of magnitude (51 to 401 and 101 to 1001 for Fructozyme P and Sigma pectinases, respectively). On the other hand, the RR method displayed a lower range of dilutions, resulting in linear correlation compared with the viscosity determination (4401 to 8301 and 24001 to 38401 for Fructozyme P and Sigma pectinases, respectively). Besides, it is noteworthy that the amount of
enzyme required for the RR method is 10 to 20 times less than that required for the viscosity-based method. Considering that most of industrial processes involving pectinases are related to viscosity reduction, the conversion factor obtained for RR to viscosity units could be used as reference for activity determination using a viscometer, although validation for particular enzyme mixtures should be accomplished. The scaled down assay presented here reduces the reagent volume, space usage, and pipetting effort and increases the throughput of the assay (Table 1). The assay reduction resulted in a lower cost per test, making it the preferred option to carry out high-throughput screening of PGase activity. Considering the difference in the limit of detection for viscometer-based determination and the time required for each determination, this method should be considered as a first choice in PGase activity screening.
197
Acknowledgments
213
This work was supported by an IDB OC/AR loan with a PICT Q1 start-up 2010-1312 grant from the FONCyT–MinCyT of Argentina Q2 (issued to E. Albertó) and the PGSYS program (issued to H. Fernández-Lahore).
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References
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Please cite this article in press as: G.E. Ortiz et al., Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.028
