ISSN : 0974 - 7435
Volume 12 Issue 2
BioTechnology An Indian Journal FULL PAPER BTAIJ, 12(2), 2016 [070-074]
An economical and high throughput alternative for endoglucanase activity determination
Gastón E.Ortiz1, Martín Blasco2, Edgardo Albertó1*
1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAMCONICET), Universidad Nacional de San Martín, San Martín, Buenos Aires, (ARGENTINA)
2Centro de Investigación y Desarrollo en Biotecnología Industrial, Instituto Nacional de Tecnología Industrial, Av. General Paz 5445, Ediûcio 51, San Martín, Buenos Aires, (ARGENTINA) E-mail: eoalberto@gmail.com
ABSTRACT
The search for new strains with good productivity of cellulolytic enzymes is always necessary to improve the processes are usually employed. Screening protocols used for these purposes require a low cost and high throughput assay for determination of endoglucanase activity. However, the test proposed by the IUPAC and other microadaptations not meet this requirement. The aim of our work is to obtain an economical and highperformance proposed by IUPAC for determination of endoglucanase activity assay adaptation. For this we reduced the assay volume and investigate the effect of this reduction on endoglucanase activity determination. As a result a miniaturized assay statistically correlated with the assay proposed by IUPAC reduced 10-fold lower scale was obtained. As a result a new economic and high performance adaptation for endoglucanase activity assay based on the IUPAC protocol was obtained. 2016 Trade Science Inc. - INDIA
KEYWORDS
Miniaturized CMCase assay; Cellulase;
Endoglucanase; Carboxymethylcellulases.
INTRODUCTION
The development of efficient enzyme mixtures for the treatment of lignocellulosic material is one important goal of modern industrial biotechnology. The screening of different enzyme activities is of fundamental importance to pursue a rational design for specific applications. Three major groups of enzyme activities have been described in Trichoderma spp. cellulose degrading machinery: endo-âglucanase (EC 3.2.1.4; endoglucanase; EG), exo-âglucanase (EC 3.2.1.91; exoglucanases; CBH) and
â-glucosidase (EC 3.2.1.21)[1]. The filter paper assay (FPA), generally applied for cellulose activity assessment, accounts for the depolymerization of cellulose considering the three activities as a whole[1,2]. Other methods allow evaluating particular enzyme activities within complex enzyme mixtures broadening the information of the enzymatic profile within the mixture. In particular, endoglucanase activity is often represented by carboxymethylcellulase activity (CMCase) due to the inability of cellobiohydrolases to attack substituted cellulose substrates[3,4]. This activity is relevant in applications pursuing viscosity reduction of cellulosic sub-
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strates[5]. The screening for particular enzyme profiles in-
volves hundreds of strains and several rounds of selection, and then efficient assay methods are required[1]. Dealing with large amounts of samples requires methodological procedures able to produce good quality results with minimal turnaround time, cost and waste.
The miniaturization of already established techniques is a viable approach to develop adequate assay procedures. The scaling down of good performance known methods to around hundred microliter volume and adaptation to a microplate format would result in numerous advantages: reduced sample volume, less reagent use, reduction of reagent waste. Furthermore, the laboratory space involved in the assay procedure would be reduced, time for pipetting shortened and then cost per assay[6,9,10]. In this context, Xiao and co-workers[9] establish a microassay protocol for endoglucanase activity determination based on 96-well plate format, but this microassay requires the use of atermocycler equipment per plate thereby limiting the processing of multiple plates in parallel.
The aim of this work is to find a new microsasay protocol for endoglucanase activity determination that allows multiple plates in parallel and studying the correlation to the IUPAC assay widely used.
MATERIALS AND METHODS
Culture conditions and enzyme: The enzyme preparations were obtained from culture supernatants from the hypercellulolyticTrichoderma reeseimutant RUT C30. Cultures were performed in a 5l lab scale bioreactor using modified Mandel’s media with microcrystalline cellulose as carbon source[2]. The reactor was inoculated with 1x106espores/mL obtained from 14 days old potato dextrose agar (PDA) cultures. Fermentation was performed at pH 4.8, 28 °C and 500 rpm agitation for 7 days. The supernatant was recovered by vacuum assisted filtration using WhatmanNº 1 filterpaper, then concentrated by tangential flow filtration (10 KDa nominal cutoffpolyethersulphone hollow fiber cartridge), and it was finally formulated in
0.1 % potassium sorbate; 10 % sodium chloride and 10% glycerol.
IUPAC CMCase assay
CMCase measurements were performed following the IUPAC method compiled by Ghose[4]. Briefly, 0.5 mL of the enzyme dilution was mixed with 0.5 mL 2% carboxymethylcellulose in 0.05 M citrate buffer, pH 4.8 and kept at 50°C for 30 min. Then, 1 mL of the mixture was assayed for glucose equivalent of reducing sugar by DNS method[10].
Unit calculation
All determinations were carried out by triplicate and all activities were calculated according to IUPAC criteria using the formulas proposed in Ghose[4]. Briefly, for enzyme formulations releasing 0.5 mg of glucose equivalents (concentrated enzyme formulations), the CMC units were calculated using the formula: CMC (units/mL) = 0.185 (units/mL)/ enzyme concentration to release 0.5 mg glucose, where concentration = (vol. enzyme in dilution)/(total volume of dilution). For preparations displaying glucose release lower than 0.5 mg (diluted enzyme formulations), units were calculated as CMC (units/ mL) = mg glucose released x 0.37 (units/mL).
Miniaturized CMCase assay
CMCase measurements were performed with a reduced volume version of the IUPAC protocol. Briefly, 50 µL of the enzyme dilution and 50 µL 2 % carboxymethylcellulose both prepared in 0.05 M citrate buffer pH 4.8 was transferred to 96 wells plated in separated wells and preheated for five minutes; then the well containing the enzyme dilution was mixed with the substrate and mixed by pipetting up and down. The enzymatic reaction was carried out according IUPAC protocol at 50°C for 30 min.
Colorimetric assay in microplate
The colorimetric assay was performed according to Miller´s protocol for IUPAC CMCase assay and the reduced version of this protocol was assayed in deep well plates. In the reduced protocol, 300 µL of DNS was added to each sample (mixed by pipetting up and down) and then the plate was held for 5 min at 100°C in water bath, finally 200 µL of
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An economical and high throughput alternative for endoglucanase activity determination
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each sample were diluted with 1 mL of deionized water and absorbance measured at 540 nm.
Experimental design and statistical treatments
Twenty enzymatic dilutions were tested for glucose equivalent release (n=3) by using the miniaturized CMCase assay and the IUPAC assay. The results from the two methods were compared by linear regression and Pearson’s correlation test. The enzymatic activity of samples was measure by using both methods in parallel. Four samples of concentrated and diluted enzymes formulations were assayed. The results of all the determinations were analyzed in the first place by a Fisher test to evaluate homoscedasticity, and then the significant difference between the two methods was analyzed by Student’s test using the Statgraphics Software (Statpoint Technologies, Inc.).
lents released in the enzymatic reactions performed by using low and high enzyme concentrations in order to obtain glucose equivalents values around the 0.5 mg recommended by the IUPAC methodology[4] (Figure 1). Twenty enzymatic dilutions were tested for glucose equivalent release (n=3) by using the miniaturized CMCase assay and the IUPAC assay. The descriptive statistical analysis accomplished to compare two sets of data showed a normal distribution of data (results not shown). When results from the two methods were compared by linear regression and Pearson’s correlation test, we founda R2= 99.06 %, a coefficient of correlation 0.995 and a standard error of estimation of 0.027, all values were estimated with a p value < 0.05. These results confirmed that there is a strong linear relationship between the two methods along the data set tested, with a confidence level greater than 95%.
RESULTS
Activity determination in both methods
Evaluation of miniaturized CMCase assay
The assessment of the scaling down of the IUPAC test consisted in a stepwise validation of the assay procedures. Firstly, we studied the glucose equiva-
To evaluate the performance of the miniaturized CMCaseassay, the enzymatic activity of samples was measuredby using both methods in parallel. Samplesof concentrated and diluted enzymes formulations were assayed (Figure 2). The p value obtained in Student’s testwas higher than 0.05 (p> 0.05) confirm-
Figure 1 : Correlation model between glucose equivalents (mg) released by IUPAC assay (x-axis) and miniaturized CMCase assay (y-axis). Each point represents the mean of three independent replicates of glucose equivalents released in the enzymatic reactions performed using the miniaturized CMCase assay and the IUPAC assay respectively. The straight line represents the linear regression showing the correlation between the two methods
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Figure 2 : Comparison of activity determined by both methods. A) Concentrated enzyme formulation. B) Diluted enzyme formulation
TABLE 1: Statistical summary of activity determinations for both methods, The p value higher than 0.05 (p> 0.05) indicates that the homoscedasticity criteria is met (Fisher’s test) and there are not significant differences between the methods (Student’s test)
Sample 1 2 3 4
Sample 1 2 3 4
Miniaturized CMCase assay (n=3)
Mean in CMC [units /mL] 122 ± 17 131 ± 9 34 ± 3 35 ± 2
Miniaturized CMCaseassay (n=3)
Mean in CMC [units /mL] 0,11 ± 0,01 0,13 ± 0,01 0,14 ± 0,01 0,08 ± 0,01
Concentrated sample
IUPAC Assay (n=3)
Mean in CMC [units /mL] 118 ± 18 116 ± 14 31 ± 5 33 ± 1
Dilutedsimple
IUPAC Assay (n=3)
Mean in CMC [units /mL] 0,11 ± 0,01 0,14 ± 0,01 0,14 ± 0,01 0,08 ± 0,01
Statistics
Fisher test (p value)
0,95 0,37 0,58 0,79
Student test (p value) 0,75 0,49 0,22 0,14
Statistics
Fisher test (p value)
1,00 0,40 1,00 1,00
Student test (p value) 0,52 0,42 0,23 1,00
ing that there were not significant differencesin the enzymatic determinationsbetween the methods(TABLE 1).
DISCUSSION
The IUPAC technique presented by Ghose[4] has been used for decades in the determination of endoglucanase activity. The precise assessment of the CMCaseactivity within a sample by using this method required several dilutions of the sample to achieve a glucose equivalent release around 0.5 mg
of glucose[1,4]. The method presented here poses an order of
magnitude reduction in the reagent volume, space usage, pipetting effort and the use of water bath equipment allows multiples plates processing in parallel (TABLE 2). Thus, allowing high throughput determination of this activity more feasible and economically. On the other hand during the method optimization it was observed that the geometric parameters of the reaction container greatly affected the activity result, in particular when using PCR multiwell plates (results not shown).
BioTechnology An Indian Journal
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An economical and high throughput alternative for endoglucanase activity determination
BTAIJ, 12(2) 2016
FULL PAPER
TABLE 2 : Procedure comparison between standard IUPAC CMCase activity assay and miniaturized assay
Assay
2% CMC in Buffer Buffer volume in blank and standard curve Enzyme Dilution or glucose standard volume Enzyme reaction at 50°C for 30 min Volume of DNS Color development Amount of H2O added prior to measurement
Read at 454 nm
Comparison of methods IUPAC assay 0.5mL 0.5 mL
Miniaturized CMCase assay
50 µL
50 µL
0.5 mL
Test tubes in water bath 3.0 mL
100°C for 5 min
20 mL 1 mL in spectrophotometer
cuvette
50 µL
96 deep-well microplate in water bath 300 µL.
100°C for 5 min 1 mL of water to 200 µL of the reaction/DNS
mixture
200 µL in standard optical 96 well plate
It is noteworthy that the correlation between the miniaturized method and the IUPAC method was observed in the whole dynamic range of the standard assay;asa consequence not only CMCunits but also international units (IU) would be determined when the glucose release is lower than the critical value (0.5 mg of glucose equivalents) as stated by Ghose[4].
CONCLUSION
The results obtained in this paper indicated that a 10 fold scale down version of IUPAC CMCase assay linearly correlateswith the classical assay even at low concentrations of enzymes. The equipment used and assay reduction in the present work,diminishthe amount of DNS used (contaminant reagent), increases the number of samples that can be processed in parallel per assay in the same work area. In conclusion, the assay reduction result in a lower cost per test and then poses it as the preferred option to carry out high-throughput screening.
ACKNOWLEDGEMENTS
This work was supported by a grant from the National Agency for Science and Technology Promotion from the National Ministry of Science and Technology of the Argentina. We thankDr. Antoine Margeot(IFP Energies nouvelles) who kindly provided theTrichoderma reesei RUT C30strain.
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