Analytical Biochemistry 279, 85– 89 (2000) doi:10.1006/abio.1999.4405, available online at http://www.idealibrary.com on
A Spectrophotometric Method for Assay of Tannase Using Rhodanine Shweta Sharma,* T. K. Bhat,† ,1 and R. K. Dawra† *Department of Chemistry and Biochemistry, College of Basic Sciences, H.P.K.V., Palampur, H.P., India; and †Regional Station, Indian Veterinary Research Institute, Palampur 176 061, H.P., India
Received August 21, 1999
A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible. © 2000 Academic Press
Tannase (tannin acyl hydrolase, EC 3.1.1.20) catalyzes the hydrolysis of ester and depside linkages in hydrolyzable tannins like tannic acid. The products of hydrolysis are glucose and gallic acid (1). Tannase is extensively used in the food, feed, beverage, brewing, pharmaceutical, and chemical industries (1–3). There is a constant search for new sources of tannase with more desirable properties for commercial application. Purification and evaluation of the enzyme require a sensitive, reproducible, and convenient assay method. Most of the old methods for tannase assay based on the titration of gallic acid released by the action of the enzyme on tannic acid did not give correct results due to the problem of accurately determining the end point (4 –7). A number of chromogenic methods have been described for the assay of tannase that are not specific (1). Madhavakrishna et al. (4) reported a method for tannase assay based on the estimation of glucose liberated by incubation with the enzyme for 24 h, which is not suitable for routine assays of the enzyme. Iibuchi et al. (9) described a spectrophotometric method that has been used by many workers in its original and modified forms (10 –14). This method was based on the de1 To whom correspondence should be addressed. Fax: ⫹91-189433063. E-mail:
[email protected].
0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
crease in absorbance of the substrate tannic acid at 310 nm. The shortcoming of spectrophotometric methods such as that of Iibuchi et al. (9), which are based on a small difference in UV absorption of gallic acid and its methyl ester or tannic acid, was observed by Haslam and Tanner (15). They developed a spectrophotometric method which used p-nitrophenyl esters of gallic acid as substrates. Some workers have assayed tannase by measuring gallic acid using such chromatographic techniques as gas chromatography (8) or high-performance liquid chromatography (16, 17). These methods require more sophisticated instrumentation, are more time-consuming, and are not suitable for routine assays. Inoue and Hagerman (18) have described a method for the determination of gallotannins, which involves the formation of a chromogen between gallic acid obtained by the acid hydrolysis of gallotannins, and rhodanine. This was later adapted by Skene and Brooker (19) for the assay of tannase in a ruminal bacterium. We report here the optimization and adaptation of this method for the assay of tannase in a fungal system. MATERIALS AND METHODS
Chemicals Gallic acid methyl ester (methyl gallate) and gallic acid were purchased from Sigma Chemical Co., U.S.A. Rhodanine was purchased from Merck–Schuchardt, Germany. All other chemicals were of an analytical grade. Fungal tannase from Aspergillus oryzae and Aspergillus niger MTCC 2425 was used in the present studies. Lyophilized fungal tannase prepared from A. oryzae was a kind gift from Dr. J. Yamakoshi (Kikkoman Corp., Japan) and was used as a standard enzyme. Fungal extract from A. niger was prepared as described below. 85
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FIG. 1. Effect of various treatments on chromogen formation between gallic acid released by the action of A. niger tannase and rhodanine. The enzyme sample had a protein content of 56 g/ml in the mycelial extract. (a) Variation of incubation time after addition of rhodanine solution. (b) Variation of incubation temperature after addition of rhodanine solution. (c) Variation of incubation time after addition of 0.2 ml of 0.5 N KOH solution. After addition of KOH solution, the assay mixture was incubated for 0 –30 min and diluted with water and the absorbance was measured after incubation of the diluted chromogen for 10 min at 30°C. (d) Variation of incubation temperature after addition of 0.2 ml of 0.5 N KOH solution. After addition of KOH solution, the assay mixture was incubated for 10 min at 10 – 60°C and the absorbance was measured after incubation of the diluted chromogen for 10 min at 30°C. (e) After the formation of the chromogen in alkaline solution, it was diluted with 4 ml water and incubated for 0 –30 min at 30°C and the absorbance was measured. Only the parameters mentioned above were changed. The other details were the same as those described under Materials and Methods. The data are means ⫾ SD.
Culture Medium and Fungal Growth A. niger van Tieghem MTCC 2425, isolated at IVRI Palampur (20), was used in the present studies. It was maintained on potato– dextrose agar slants at 4°C and subcultured every alternate month. For inoculum preparation, the culture was grown on tannic acid agar (TAA) 2 slants at 30°C for 7 days. TAA medium contained (g/L): sodium nitrate, 3.0; dipotassium hydrogen phosphate, 1.0; magnesium sulfate, 0.5; potassium chloride, 0.5, agar agar, 20.0. The pH of the medium was adjusted to 5.0, it was autoclaved (103 kPa; 121°C) 2 Abbreviations used: TA, tannic acid; TAA, tannic acid agar; MT, Mildew test.
for 20 min, and filter-sterilized tannic acid (cellulose nitrate membrane of 25 mm diameter and 0.45 m pore size; Whatman Ltd., Maidston, England) was added to provide a final concentration of 1% (w/v). A slightly modified Mildew test (MT) medium was used for growing the fungus in the presence of tannic acid (TA) as the sole source of carbon and energy (21). The basal medium contained (g/L): sodium nitrate, 3.0; dipotassium hydrogen orthophosphate, 1.0; magnesium sulfate, 0.5; potassium chloride, 0.5. The pH of the basal medium was adjusted to 5.0 and 40 ml of the medium was taken in each of the 250-ml Erlenmeyer flasks. The medium was autoclaved (103 kPa; 121°C) for 20 min and allowed to cool to room temperature.
SPECTROMETRIC ASSAY OF TANNASE TABLE 1
Correlation of Potassium Hydroxide (KOH) Concentration with pH of Diluted Reaction Mixture and Chromogen Formation KOH (N)
pH
A 520
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
5.94 ⫾ 0.03 9.58 ⫾ 0.04 10.93 ⫾ 0.05 11.29 ⫾ 0.05 11.53 ⫾ 0.04 11.73 ⫾ 0.07 11.85 ⫾ 0.05 11.96 ⫾ 0.06 12.05 ⫾ 0.06 12.15 ⫾ 0.04
0.0 ⫾ 0.00 0.0 ⫾ 0.00 0.84 ⫾ 0.019 0.88 ⫾ 0.025 1.04 ⫾ 0.022 1.01 ⫾ 0.018 1.00 ⫾ 0.028 1.00 ⫾ 0.021 0.99 ⫾ 0.026 0.99 ⫾ 0.027
Note. The reaction mixture containing 0.25 ml enzyme sample (14 g protein), and 0.25 ml methyl gallate (2.5 mol) was incubated for 5 min at 30°C. Methanolic rhodanine solution (0.3 ml) was added for stopping the reaction and for formation of complex between gallate and rhodanine. The tubes were kept at 30°C for 5 min. This was followed by addition of 0.2 ml KOH solution (0.1–1.0 N) and the tubes were again kept at 30°C for 5 min. Then, the enzyme sample was added to the control sets. All the tubes were diluted with 4.0 ml water and kept again at 30°C for 10 min. A blank was prepared as described under Methods and Materials. The absorbance was measured at 520 nm. The data are means ⫾ SD.
The filter-sterilized (cellulose nitrate membrane of 25 mm diameter and 0.45 m pore size, Whatman Ltd.) tannic acid solution in the MT– basal medium (10 ml) was added to each flask that contained the autoclaved basal medium. The final concentration of tannic acid was 2%. The medium was inoculated with the fungal spores (3– 4 ⫻ 10 7) maintained on TAA. An uninoculated culture medium was kept as control. The inoculated and uninoculated culture media were incubated at 30°C in an orbital shaker (Orbitek, Scigenics India, Chennai, India) at 120 rpm for 120 h. The mycelial suspension was filtered through Whatman No. 1 filter paper, washed thrice with glass-distilled water and finally with 0.05 M citrate buffer (pH 5.0), and pressed against filter paper and the wet weight was noted. The mycelial mass was processed for enzymatic studies. Preparation of Enzyme Extract The procedure followed was the protocol described by Bridge (22) with slight modifications. A 10% suspension of the mycelial mass was made in 0.05 M citrate buffer, pH 5.0, and frozen overnight at ⫺20°C. Acidwashed sand, four times the weight of the mycelium, was added, and the mixture was ground in a chilled pestle–mortar kept in an ice bath. The homogenate was centrifuged at 12,000g for 30 min at 4°C using a K-24 (Janetzki) centrifuge. The supernatant (mycelial extract) was used for tannase assay. The activity of Kikkoman tannase was assayed by preparing the en-
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zyme sample (300 g protein ml ⫺1) in 0.05 M citrate buffer, pH 5.0. Calibration Curve for Gallic Acid Estimation Gallic acid (0.5 mM) in citrate buffer (0.05 M, pH 5.0) was used as standard and prepared fresh before use. Aliquots of this solution containing 5–100 nmol gallic acid were taken, and the volume was made to 0.5 ml with citrate buffer. The blank contained 0.5 ml citrate buffer. Methanolic rhodanine solution (0.667%; 0.3 ml) was added to all the tubes, and they were incubated at 30°C for 5 min. Potassium hydroxide solution (0.5 M; 0.2 ml) was added to all the tubes which were again incubated at 30°C for 5 min. Glass-distilled water (4.0 ml) was added to all the tubes, and after 5–10 min absorbance was recorded at 520 nm. Tannase Assay Tannase (tannin acyl hydrolase; EC 3.1.1.20) was assayed by the method based on chromogen formation between gallic acid (released by the action of tannase on methyl gallate) and rhodanine (2-thio-4-ketothiazolidine). The reaction conditions were optimized for the chromogen formation by variation of the pH, time, and temperature at the various steps after mixing the enzyme substrate mixture with rhodanine solution (Table 1, Fig. 1). In the final protocol, the substrate solution (0.01 M methyl gallate prepared in 0.05 M citrate buffer, pH 5.0), enzyme sample, and buffer (0.05 M citrate buffer, pH 5.0) were preincubated at 30°C for 5–10 min before the enzyme reaction was started. The reaction mixture in the blank, test, and control tubes contained 0.25 ml of substrate solution to which 0.25 ml of the buffer and 0.25 ml of the enzyme sample were added to the blank and test, respectively. The tubes were incubated at 30°C for 5 min, and 0.3 ml of methanolic rhodanine (0.667%; w/v) was added to all the tubes that were then kept at 30°C for 5 min. After this, 0.2 ml of 0.5 N potassium hydroxide was added to each tube and these were incubated at 30°C for 5 min. This was followed by addition of the enzyme sample (0.25 ml) to the reaction mixture in the control tube only. Finally, each tube was diluted with 4.0 ml distilled water and incubated at 30°C for 10 min and the absorbance was recorded against water at 520 nm using a Spectronic-21 spectrophotometer. The enzyme activity was calculated from the change in absorbance: ⌬A 520 ⫽ ( A test ⫺ A blank) ⫺ ( A control ⫺ A blank). All the assays were carried out in triplicate. One unit of the enzyme was defined as micromole of gallic acid formed per minute. RESULTS AND DISCUSSION
In the present study, the method developed could detect gallic acid up to 5 nmol (absorbance 0.023),
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FIG. 2. Dependence of tannase activity on concentration of (a) enzyme, (b) substrate, and (c) time course of incubation of enzyme substrate mixture. The enzyme sample was A. niger mycelial extract. (a) The protein content in the enzyme sample in the reaction mixture was varied from 0 to 100 g. (b) The enzyme sample had a protein content of 56 g/ml of the mycelial extract. The amount of the substrate, methyl gallate in the reaction mixture was varied from 0 to 10 mol. (c) The reaction mixture contained the enzyme sample as in b and the substrate concentration was 2.5 mol substrate. The enzyme substrate mixture was incubated at 30°C for 0 –30 min. The data are means ⫾ SD. The other details were the same as those described under Materials and Methods.
while the sensitivity of the protocol of Inoue and Hagerman (18) for gallic acid was 10 g (53 nmol). It had an R value of 0.99 ( y ⫽ 0.024512x ⫹ 0.00038) and a precision of 1.7% (relative standard deviation). The absorbance change (per mol methyl gallate hydrolyzed min ⫺1) by the modified UV method of Farias et al. (23) for tannase was 1.343. On the other hand, the absorbance change in the present method (per mol gallic acid formed min ⫺1) was observed to be 27.3, which provides 20 times higher sensitivity in this method. There was no increase in enzyme activity as reflected by the amount of chromogen formed when the reaction mixture was incubated for different time intervals after the addition of methanolic rhodanine solution (Fig. 1a). This observation indicated that the methanolic rhodanine solution served the double purpose of stopping the enzymatic reaction and providing the complexing agent for chromogen formation. Skene and Brooker (19) used liquid nitrogen for stopping the enzymatic reaction for the assay of tannase in Selenomonas ruminantium ssp. ruminantium. After addition of
methanolic rhodanine solution to the reaction mixture, an incubation temperature of 30°C was suitable for peak chromogen formation (Fig. 1b). The chromogen formation between gallate and rhodanine takes place under alkaline conditions, but excess of alkali elicits autoxidation of gallate (18). It was observed that using 0.5 N KOH was most appropriate for making reaction mixture alkaline for optimal chromogen formation (Table 1). In addition, at this concentration of KOH, maximal absorbance was observed between 2.5 and 10 min (Fig. 1c) and 30 –35°C (Fig. 1d). The time course of chromogen intensity after dilution with water showed that maximal absorbance was observed at 6 –10 min of incubation at 30°C after the final dilution of the reaction mixture (Fig. 1e). Slight variation in the optimal absorbance in different experiments (Table 1, Fig. 1) might be due to the possible difference in the amount of tannase in different batches of mycelial extracts. The chromogen was rather stable for 20 min of incubation at 30°C, but the absorbance decreased slowly afterward. These results are quite similar to the observations of Inoue and Hagerman (18) who found that the
SPECTROMETRIC ASSAY OF TANNASE
maximal absorbance of this color complex was reached 4 min after the final dilution, and the absorbance did not change over the next 10 min. A. niger tannase activity showed a linear response to enzyme sample protein content of 100 g in the reaction mixture (Fig. 2a). This was more than the 70 g ml ⫺1 observed by Skene and Brooker (19) for tannase of a ruminal bacterium. The experiments on the dependence of A. niger tannase activity on substrate concentration showed a peak activity for the enzyme at methyl gallate concentration of 1.0 mol and above (Fig. 2b). The profile of incubation of enzyme substrate mixture for different time intervals showed that enzyme activity was linear up to 5 min (Fig. 2c). Therefore complete assay could be performed in a single test tube by incubation of the enzyme substrate mixture for 5 min. In the present method, the reaction protocol of Inoue and Hagerman (18) for gallic acid estimation was adapted and modified for tannase assay in A. niger van Tieghem. In this assay protocol, there was no need of adding sulfuric acid because the substrate hydrolysis was enzymatic, unlike the acid hydrolysis of gallotannins used by Inoue and Hagerman (18) for the release of gallic acid. The activity of the tannase observed in the mycelial extract of A. niger indicated that this fungal isolate expressed esterase activity that cleaved methyl gallate and released gallate. This activity is characteristic of tannin acyl hydrolase (1, 3, 19), and this was corroborated by a similar type of activity of the standard tannase preparation from A. oryzae (Kikkoman tannase). The present method is an improvement on the method described by Skene and Brooker (19) for a bacterial tannase and has a number of convenient features for monitoring the enzyme activity. Gallic acid, instead of a standard solution in 0.2 N sulfuric acid, can be prepared in the same buffer in which substrate and enzyme solutions are made. There is no need for snap-freezing with liquid nitrogen for stopping the enzyme reaction, and the same can be performed simply by using methanolic rhodanine reagent. In addition, the complete reaction can be carried out in the same tube without having to take aliquots of enzyme reaction mixture for development of color. In the present study, tannic acid was not used as the substrate because we experienced problems of higher blanks possibly due to gallic acid present as an impurity in the tannic acid preparation. This was shown by the presence of gallic acid spots in the chromatograms when different tannic acid preparations were analyzed by thin-layer chromatography (26). Gallic acid is a competitive inhibitor of tannase (1, 23) and the presence of gallic acid as an impurity in commercial preparations of tannic acid is likely to add artifacts in the assays. These problems with tannic acid as substrate led
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Yamada et al. (25) to switch to methyl gallate, which was later used as a substrate by other investigators working on this enzyme (7, 8, 16, 19, 23). The present method may find applications in the purification and characterization of tannases from different sources, and for studying the relationships between the substrate and activity modulators of the enzyme. REFERENCES 1. Lekha, P. K., and Lonsane, B. K. (1997) Adv. Appl. Microbiol. 44, 215–260. 2. Cantarelli, C., Brenna, O., Giovanelli, G., and Rossi, M. (1989) Food Biotech. 3, 203–213. 3. Lane, R. W., Yamakoshi, J., Kikuchi, M., Mizusawa, K., Henderson, L., and Smith, M. (1997) Food Chem. Toxicol. 35, 207–212. 4. Madhavakrishna, W., Bose, S. M., and Nayudamma, Y. (1960) Bull. CLRI 7, 1–11. 5. Nishira, H. (1961) Hakko Kogaku Zasshi 39, 137–146. 6. Haslam, E., and Stangroom, J. E. (1966) Biochem. J. 99, 28 –31. 7. Farias, G. M., Elkins, J. R., and Griffin, G. J. (1992) Eur. J. Forest Pathol. 22, 392– 402. 8. Jean, D., Pourrat, H., Pourrat, A., and Carnat, A. (1981) Anal. Biochem. 110, 369 –372. 9. Iibuchi, S., Minoda, Y., and Yamada, K. (1967) Agric. Biol. Chem. 31, 513–518. 10. Iibuchi, S., Minoda, Y., and Yamada, K. (1968) Agric. Biol. Chem. 32, 803– 809. 11. Sanderson, G. W., and Coggon, P. (1974) U.S. Patent 3,812,266. 12. Aoki, K., Shinke, R., and Nishira, H. (1976) Agric. Biol. Chem. 40, 79 – 85. 13. Rajakumar, G. S., and Nandy. S.C. (1983) Appl. Environ. Microbiol. 46, 525–527. 14. Chatterjee, R., Dutta, A., Banerjee, R., and Bhattacharya, B. C. (1996) Bioprocess Eng. 14, 159 –162. 15. Haslam, E., and Tanner, R. J. N. (1970) Phytochemistry 9, 2305– 2309. 16. Barthomeuf, C., Regerat, F., and Pourrat, H. (1994) J. Ferment. Bioeng. 77, 320 –323. 17. Niehaus, J. U., and Gross, G. G. (1997) Phytochemistry 45, 1555–1560. 18. Inoue, K. H., and Hagerman, A. E. (1988) Anal. Biochem. 169, 363–369. 19. Skene, I. K., and Brooker, J. D. (1995) Anaerobe 1, 321–327. 20. Bhat, T. K., Makkar, H. P. S., and Singh, B. (1996) Lett. Appl. Microbiol. 22, 257–258. 21. Bhat, T. K., Makkar, H. P. S., and Singh, B. (1997) Lett. Appl. Microbiol. 25, 22–23. 22. Bridge, P. (1996) in Protein Purification Protocols. Methods in Molecular Biology (Doonan, S., Ed.), Vol. 59, Humana Press, Totowa, NJ. 23. Farias, G. M., Gorbea, C., Elkins, J. R., and Griffin, G. J. (1994) Physiol. Mol. Plant Pathol. 44, 51– 63. 24. Bajpai, B., and Patil, S. (1996) W. J. Microbiol. Biotechnol. 12, 217–220. 25. Yamada, H., Adachi, O., Watanabe, M., and Sato, N. (1968) Agric. Biol. Chem. 32, 1070 –1078. 26. Sharma, S. (1998) Biochemical Studies on Microbial Biotransformation of Polyphenols, M.Sc. thesis, Himachal Pradesh Agricultural University (H.P.K.V.), Palampur, H.P., India.
