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A novel and sensitive plate assay for screening of tannase-producing bacteria

A novel and sensitive plate assay for screening of tannase-producing bacteria Ann Microbiol (2010) 60:177–179 DOI 10.1007/s13213-010-0022-7 SHORT COMMUNICATION A novel and sensitive plate assay for screening of tannase-producing bacteria Rakesh Kumar & Ashwani Kumar & Ravinder Nagpal & Jitender Sharma & Anju Kumari Received: 25 September 2009 /Accepted: 13 January 2010 /Published online: 8 February 2010 # Springer-Verlag and the University of Milan 2010 Abstract In the present investigation, a new method for been used extensively in the food, beverage, pharmaceutical isolation and selective screening of tanninolytic, i.e. tannase- and chemical industries. Several microorganisms have been producing, bacteria was developed and compared with the reported to produce tannase capable of hydrolysing tannins to earlier prevalent method. Tannase-producing bacteria were gallic acid during fermentation (Lekha and Lonsane 1997; screened on agar plates using a newly developed plate assay Mukharjee and Banerjee 2003). Gallic acid, the hydrolysed method in which tannase cleaves the tannin–protein complex product of tannin, is also employed for dye making, formed by addition of tannic acid to the medium, and forms pharmaceuticals, and in the leather and chemical industries a greenish brown zone around the bacterial colony due to the (Hadi et al. 1994). The association of tannase-producing degradation of tannic acid. Using conventional methods, the bacteria with advanced stage colon cancer has also been zone formed was not clearly visible and pigmentation reported (Noguchil et al. 2007), allowing the possibility of development took 3–4 days; however, the new method bacterial tannase as an indicator for colon cancer to be yielded clearer and more sensitive results within a shorter explored. Although various methods for the estimation of incubation time of 48–72 h. tannase, such as gas chromatography (Jean et al. 1981), detection on polyacrylamide gels (Aoki et al. 1979), . . . Keywords Tannins Bacterial tannase Tannase activity spectrophotometric methods (Iibuchi et al. 1967;Sharmaet Plate-assay al. 2000), colorimetric methods (Mondal et al. 2001), etc. have been reported, there are very few rapid methods for the isolation and screening of tannase-producing bacteria. The Introduction plate assay method devised by Osawa and Walsh (1993)is the most widely used method, but it is time consuming and Tannase (tannin acyl hydrolase) degrades tannic acid to gallic not highly sensitive. Hence, in the present investigation, an acid and glucose. Because of this property, the enzyme has effort was made to develop a rapid, reproducible, visual and easier screening method for detection of tannase-producing bacteria, so as to facilitate the isolation process. R. Kumar J. Sharma Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India Material and methods A. Kumar (*) R. Nagpal Department of Biotechnology, Isolation of tannase-producing bacteria was carried out as JMIT Institute of Engineering & Technology, per the method of Osawa and Walsh (1993) and our newly Radaur-135133, developed method. Soil samples were collected from a goat Yamuna Nagar, Haryana, India farm, garbage, and a poultry farm from the outskirts of the e-mail: ashwanindri@rediffmail.com city of Kurukshetra, India. Thereafter, the isolation of A. Kumari tannase-producing bacteria was carried out on nutrient agar Department of Microbiology, plates supplemented with tannic acid (2%; filter sterilised). Chaudhary Charan Singh Haryana Agricultural University, Addition of tannic acid to nutrient agar forms a tannin– Hissar, Haryana, India 178 Ann Microbiol (2010) 60:177–179 protein complex; cleavage of this complex by bacteria producing tannase forms a zone around the colonies. After a prolonged incubation of 3–4 days, a greenish brown zone around the colonies appears. In our newly developed method, the nutrient agar plates supplemented with tannic acid were flooded with FeCl solution (0.01 M FeCl in 3 3 0.01 N HCl) and kept for 5–10 min at room temperature. FeCl reacts with tannic acid and forms a brown colour; thus a clear zone is formed on a dark brown background. Result and discussion Tannase-producing bacteria were isolated by serial-dilution and plating on nutrient agar medium supplemented with Fig. 2 Plate showing tannase-producing bacteria after 4 days incubation tannic acid on the basis of zone formation and greenish brown coloration after prolonged incubation. The zone formed during this procedure was not very clear (Fig. 1), 48 h of incubation. The bacteria isolated were screened and was not differentiable between tannase-producing and using both methods, and the newly developed method was non-tannase-producing bacteria. Therefore, a longer incu- found to be quicker and more sensitive. bation time of up to 4 days was required for confirmation of The isolated bacterial strains were further tested for tannase production (Fig. 2). To overcome this, an additional tannase production using Mondal’s colorimetric method for staining method that stains the plate was implemented. The tannase (Mondal et al. 2001), and were found to produce resulting zone was clearer and easily observable, even in more than 10 IU/ml tannase (IU: µmol tannic acid −1 −1 cases of low levels of tannase production. The method degraded min ml ). The newly isolated bacterial strains described by Osawa and Walsh (1993) was not highly are being further used for tannase production and purification, sensitive since zone formation in nutrient agar was not and their application will be exploited in various industries. clearly visible even after a longer incubation time. However, in the present technique, FeCl reacted with the tannin–protein complex, yielding a brown to black colour, Conclusion providing a dark background allowing clear visibility of zones of tannin degradation (Fig. 3). In the Osawa and From the results observed, the new method developed in Walsh (1993) method, a long incubation time (3–4 days) the present study can be advocated as more rapid, sensitive, was required; however, in the present method, colonies of easily visible and reproducible than existing methods, and tannase-producing bacteria could be screened out within hence it will certainly be helpful in the rapid screening and Fig. 1 Isolation of tannase-producing bacteria on nutrient agar Fig. 3 Isolation of tannase-producing bacteria using the staining supplemented with tannic acid method developed in this study Ann Microbiol (2010) 60:177–179 179 isolation of tannase-producing bacteria. However, more in Jean D, Pourrat H, Pourrat A, Carnat A (1981) Assay of tannase (tannin acyl hydrolase E.C. 3.1.1.20) by gas chromatography. vitro and in vivo trials are needed, and are already in Anal Biochem 110:369–372 progress in author’s laboratory, in order to validate the Lekha PK, Lonsane BK (1997) Production and application of tannin method for large-scale application. acyl hydrolase: state of the art. Adv Appl Microbiol 44:215–260 Mondal KC, Banerjee D, Jana M, Pati BR (2001) Colorimetric assay method for determination of tannin acyl hydrolase (E.C. 3.1.1.20) activity. Anal Biochem 295:168–171 References Mukharjee G, Banerjee R (2003) Production of gallic acid: biotech- nological route (Part I). Chim Oggi 21:59–62 Noguchil N, Ohashil T, Shiratori T, Naruil K, Hagiwara T, Ko M, Aoki K, Tanaka T, Shinke R, Nishira H (1979) Detection of tannase in Watanabe K, Miyahara T, Taira S, Moriyasu F, Sasatsul M (2007) polyacrylamide gels. J Chromatogr 17:446–448 Hadi TA, Banerjee R, Bhatacharya BC (1994) Optimization of tannase Association of tannase producing Staphylococcus lugdunensis biosynthesis by a newly isolated Rhizopus oryzae. Bioprocess with colon cancer and characterization of novel tannase gene. J Eng 11:239–243 Gastroenterol 42:346–351 Iibuchi S, Minoda Y, Yamada K (1967) Studies on tannin acyl Osawa R, Walsh TP (1993) Visual reading method for detection of hydrolase. Part II. A new method determining the enzyme bacterial tannase. Appl Environ Microbiol 18:74–78 activity using the change of ultraviolet absorption. Agric Biol Sharma S, Bhatt TK, Dawra RK (2000) A spctrophotometric method Chem 31:513–518 for assay of tannase using rhodanine. Anal Biochem 279:85–89 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

A novel and sensitive plate assay for screening of tannase-producing bacteria

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Publisher
Springer Journals
Copyright
Copyright © 2010 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-010-0022-7
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Abstract

Ann Microbiol (2010) 60:177–179 DOI 10.1007/s13213-010-0022-7 SHORT COMMUNICATION A novel and sensitive plate assay for screening of tannase-producing bacteria Rakesh Kumar & Ashwani Kumar & Ravinder Nagpal & Jitender Sharma & Anju Kumari Received: 25 September 2009 /Accepted: 13 January 2010 /Published online: 8 February 2010 # Springer-Verlag and the University of Milan 2010 Abstract In the present investigation, a new method for been used extensively in the food, beverage, pharmaceutical isolation and selective screening of tanninolytic, i.e. tannase- and chemical industries. Several microorganisms have been producing, bacteria was developed and compared with the reported to produce tannase capable of hydrolysing tannins to earlier prevalent method. Tannase-producing bacteria were gallic acid during fermentation (Lekha and Lonsane 1997; screened on agar plates using a newly developed plate assay Mukharjee and Banerjee 2003). Gallic acid, the hydrolysed method in which tannase cleaves the tannin–protein complex product of tannin, is also employed for dye making, formed by addition of tannic acid to the medium, and forms pharmaceuticals, and in the leather and chemical industries a greenish brown zone around the bacterial colony due to the (Hadi et al. 1994). The association of tannase-producing degradation of tannic acid. Using conventional methods, the bacteria with advanced stage colon cancer has also been zone formed was not clearly visible and pigmentation reported (Noguchil et al. 2007), allowing the possibility of development took 3–4 days; however, the new method bacterial tannase as an indicator for colon cancer to be yielded clearer and more sensitive results within a shorter explored. Although various methods for the estimation of incubation time of 48–72 h. tannase, such as gas chromatography (Jean et al. 1981), detection on polyacrylamide gels (Aoki et al. 1979), . . . Keywords Tannins Bacterial tannase Tannase activity spectrophotometric methods (Iibuchi et al. 1967;Sharmaet Plate-assay al. 2000), colorimetric methods (Mondal et al. 2001), etc. have been reported, there are very few rapid methods for the isolation and screening of tannase-producing bacteria. The Introduction plate assay method devised by Osawa and Walsh (1993)is the most widely used method, but it is time consuming and Tannase (tannin acyl hydrolase) degrades tannic acid to gallic not highly sensitive. Hence, in the present investigation, an acid and glucose. Because of this property, the enzyme has effort was made to develop a rapid, reproducible, visual and easier screening method for detection of tannase-producing bacteria, so as to facilitate the isolation process. R. Kumar J. Sharma Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India Material and methods A. Kumar (*) R. Nagpal Department of Biotechnology, Isolation of tannase-producing bacteria was carried out as JMIT Institute of Engineering & Technology, per the method of Osawa and Walsh (1993) and our newly Radaur-135133, developed method. Soil samples were collected from a goat Yamuna Nagar, Haryana, India farm, garbage, and a poultry farm from the outskirts of the e-mail: ashwanindri@rediffmail.com city of Kurukshetra, India. Thereafter, the isolation of A. Kumari tannase-producing bacteria was carried out on nutrient agar Department of Microbiology, plates supplemented with tannic acid (2%; filter sterilised). Chaudhary Charan Singh Haryana Agricultural University, Addition of tannic acid to nutrient agar forms a tannin– Hissar, Haryana, India 178 Ann Microbiol (2010) 60:177–179 protein complex; cleavage of this complex by bacteria producing tannase forms a zone around the colonies. After a prolonged incubation of 3–4 days, a greenish brown zone around the colonies appears. In our newly developed method, the nutrient agar plates supplemented with tannic acid were flooded with FeCl solution (0.01 M FeCl in 3 3 0.01 N HCl) and kept for 5–10 min at room temperature. FeCl reacts with tannic acid and forms a brown colour; thus a clear zone is formed on a dark brown background. Result and discussion Tannase-producing bacteria were isolated by serial-dilution and plating on nutrient agar medium supplemented with Fig. 2 Plate showing tannase-producing bacteria after 4 days incubation tannic acid on the basis of zone formation and greenish brown coloration after prolonged incubation. The zone formed during this procedure was not very clear (Fig. 1), 48 h of incubation. The bacteria isolated were screened and was not differentiable between tannase-producing and using both methods, and the newly developed method was non-tannase-producing bacteria. Therefore, a longer incu- found to be quicker and more sensitive. bation time of up to 4 days was required for confirmation of The isolated bacterial strains were further tested for tannase production (Fig. 2). To overcome this, an additional tannase production using Mondal’s colorimetric method for staining method that stains the plate was implemented. The tannase (Mondal et al. 2001), and were found to produce resulting zone was clearer and easily observable, even in more than 10 IU/ml tannase (IU: µmol tannic acid −1 −1 cases of low levels of tannase production. The method degraded min ml ). The newly isolated bacterial strains described by Osawa and Walsh (1993) was not highly are being further used for tannase production and purification, sensitive since zone formation in nutrient agar was not and their application will be exploited in various industries. clearly visible even after a longer incubation time. However, in the present technique, FeCl reacted with the tannin–protein complex, yielding a brown to black colour, Conclusion providing a dark background allowing clear visibility of zones of tannin degradation (Fig. 3). In the Osawa and From the results observed, the new method developed in Walsh (1993) method, a long incubation time (3–4 days) the present study can be advocated as more rapid, sensitive, was required; however, in the present method, colonies of easily visible and reproducible than existing methods, and tannase-producing bacteria could be screened out within hence it will certainly be helpful in the rapid screening and Fig. 1 Isolation of tannase-producing bacteria on nutrient agar Fig. 3 Isolation of tannase-producing bacteria using the staining supplemented with tannic acid method developed in this study Ann Microbiol (2010) 60:177–179 179 isolation of tannase-producing bacteria. However, more in Jean D, Pourrat H, Pourrat A, Carnat A (1981) Assay of tannase (tannin acyl hydrolase E.C. 3.1.1.20) by gas chromatography. vitro and in vivo trials are needed, and are already in Anal Biochem 110:369–372 progress in author’s laboratory, in order to validate the Lekha PK, Lonsane BK (1997) Production and application of tannin method for large-scale application. acyl hydrolase: state of the art. Adv Appl Microbiol 44:215–260 Mondal KC, Banerjee D, Jana M, Pati BR (2001) Colorimetric assay method for determination of tannin acyl hydrolase (E.C. 3.1.1.20) activity. Anal Biochem 295:168–171 References Mukharjee G, Banerjee R (2003) Production of gallic acid: biotech- nological route (Part I). Chim Oggi 21:59–62 Noguchil N, Ohashil T, Shiratori T, Naruil K, Hagiwara T, Ko M, Aoki K, Tanaka T, Shinke R, Nishira H (1979) Detection of tannase in Watanabe K, Miyahara T, Taira S, Moriyasu F, Sasatsul M (2007) polyacrylamide gels. J Chromatogr 17:446–448 Hadi TA, Banerjee R, Bhatacharya BC (1994) Optimization of tannase Association of tannase producing Staphylococcus lugdunensis biosynthesis by a newly isolated Rhizopus oryzae. Bioprocess with colon cancer and characterization of novel tannase gene. J Eng 11:239–243 Gastroenterol 42:346–351 Iibuchi S, Minoda Y, Yamada K (1967) Studies on tannin acyl Osawa R, Walsh TP (1993) Visual reading method for detection of hydrolase. Part II. A new method determining the enzyme bacterial tannase. Appl Environ Microbiol 18:74–78 activity using the change of ultraviolet absorption. Agric Biol Sharma S, Bhatt TK, Dawra RK (2000) A spctrophotometric method Chem 31:513–518 for assay of tannase using rhodanine. Anal Biochem 279:85–89

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Annals of Microbiology – Springer Journals

Published: Feb 8, 2010

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