AiM  Vol.9 No.1 , January 2019
Isolation of Pectinase Producing Bacteria from the Rhizosphere of Andrographis paniculata Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains
Abstract: Pectinases, the enzymes which break down pectic substances, have a wide range of applications in food, agriculture and environmental sectors. In the present study, attempts were made to isolate highly efficient pectinase producer from the rhizosphere of a medicinal plant, Andrographis paniculata Nees, known as the “King of bitters”. The total heterotrophic bacterial count of the rhizosphere soil of A. paniculata Nees ranged from 1.53 × 109 to 2.52 × 109 cfu/g. A total of 65 bacterial colonies were randomly selected from the nutrient agar plates, purified and assessed for pectinase activity. Out of the 65 isolates, 62 (95.38%) showed varying degree of pectinase activity in plate assay using pectin as a sole source of carbon. Among the pectinase producing strains, JBST36 showed best pectinase activity which is followed by the JBST22 and JBST27. Morphological characterization, biochemical tests and 16S rRNA gene sequencing were performed to identify the three most potential strains. Based on the morphological, biochemical and molecular data, JBST22 was identified as Bacillus flexus and the other two were identified as Bacillus subtilis. Furthermore, nucleotide sequences of the 16S rRNA gene of these 3 strains were compared and a phylogenetic tree was constructed. The study reveals that there are at least 66 base differences in the 16S rRNA gene sequences of B. flexus JBST22 and the B. subtilis JBST36.
Cite this paper: Kabir, M. and Tasmim, T. (2019) Isolation of Pectinase Producing Bacteria from the Rhizosphere of Andrographis paniculata Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains. Advances in Microbiology, 9, 1-13. doi: 10.4236/aim.2019.91001.

[1]   Pires, T.D.C.R. and Finardi-Filho, F. (2005) Extraction and Assay of Pectic Enzymes from Peruvian Carrot (Arracacia xanthorriza Bancroft.) Food Chemistry, 89, 85-92.

[2]   Whitaker, J.R. (1990) New and Future Uses of Enzymes in Food Processing. Food Biotechnology, 4, 669-697.

[3]   Banu, A.R., Devi, M.K., Gnanaprabhal, G.R., Pradeep, B. and Palaniswamy, M. (2010) Production and Characterization of Pectinase Enzyme from Penicillium chrysogenum. Indian Journal of Science and Technology, 3, 377-381.

[4]   De Vries, J.A., Voragen, A.G.J., Rombouts, F.M. and Pilnik, W. (1986) Structural Studies of Apple Pectins with Pectolytic Enzymes. Chemistry and Function of Pectins, 38-48.

[5]   Cao, J., Zheng, L. and Chen, S. (1992) Screening of Pectinase Producer from Alkalophilic Bacteria and Study on Its Potential Application in Degumming of Ramie. Enzyme and Microbial Technology, 14, 1013-1016.

[6]   Sharma, H.S.S. (1987) Screening of Polysaccharide-Degrading Enzymes for Retting Flax Stem. International Biodeterioration, 23, 181-186.

[7]   Tanabe, H., Yoshihara, K., Tamura, K., Kobayashi, Y., Akamatsu, I., Niyomwan, N. and Footrakul, P. (1987) Pretreatment of Pectic Wastewater from Orange Canning Process by an Alkalophilic Bacillus sp. Journal of Fermentation Technology, 65, 243-246.

[8]   Chesson, A. and Codner, T.L.R. (1978) The Maceration of Vegetable Tissue by a Strain of Bacillus subtilis. Journal of Applied Microbiology, 44, 347-364.

[9]   Silley, P. (1986) The Production and Properties of a Crude Pectin Lyase from Lachnospira multiparas. Letters in Applied. Microbiology, 2, 29-31.

[10]   Sorensen, J. (1997) The Rhizosphere as a Habitat for Soil Microorganisms. In: Elsas, J.D.V., Trevors, J.T. and Wellington, E.M.H., Eds., Modern Soil Microbiology, Mercel Dekker, New York, 21-45.

[11]   Badri, D.V., Weir, T.L., van der Lelie, D. and Vivanco, J.M. (2009) Rhizosphere Chemical Dialogues: Plant-Microbe Interactions. Current Opinion in Biotechnology, 20, 642-650.

[12]   Dennis, P.G., Miller, A.J. and Hirsch, P.R. (2010) Are Root Exudates More Important than Other Sources of Rhizodeposits in Structuring Rhizosphere Bacterial Communities? FEMS Microbiology Ecology, 72, 313-327.

[13]   Doornbos, R.F., van Loon, L.C. and Bakker, P.A.H.M. (2012) Impact of Root Exudates and Plant Defense Signaling on Bacterial Communities in the Rhizosphere. A Review. Agronomy for Sustainable Development, 32, 227-243.

[14]   Micallef, S.A., Channer, S., Shiaris, M.P. and Colon-Carmona, A. (2009) Plant Age and Genotype Impact the Progression of Bacterial Community Succession in the Arabidopsis Rhizosphere. Plant Signaling & Behavior, 4, 777-780.

[15]   Joselin, J. and Jeeva, S. (2014) Andrographis paniculata: A Review of Its Traditional Uses, Phytochemistry and Pharmacology. Medicinal and Aromatic Plants, 3, 169.

[16]   Akbar, S. (2011) Andrographis paniculata: A Review of Pharmacological Activities and Clinical Effects. Alternative Medicine Review, 16, 66-77.

[17]   Boopathi, C.A. (2000) Andrographis SPP.: A Source of Bitter Compounds for Medicinal Use. Ancient Science of Life, 19, 164-168.

[18]   Jarukamjorn, K. and Nemoto, N. (2008) Pharmacological Aspects of Andrographis paniculata on Health and Its Major Diterpenoid Constituent Andrographolide. Journal of Health Sciences, 54, 370-381.

[19]   Niranjan, A., Tewari, S.K. and Lehri, A. (2010) Biological Activities of Kalomegh (Andrographis paniculata Nees) and Its Active Principles—A Review. Indian Journal of Natural Products and Resources, 1, 125-135.

[20]   WHO (2004) WHO Monographs on Selected Medicinal Plants. Vol. 2, World Health Organization, Geneva.

[21]   Gerhardt, P., Murray, R.G.E., Wood, W.A. and Kreig, N.R. (1994) Methods for General and Molecular Bacteriology. ASM, Washington DC.

[22]   Hankin, L., Zucker, M. and Sands, D.C. (1971) Improved Solid Medium for the Detection and Enumeration of Pectolytic Bacteria. Applied and Environmental Microbiology, 22, 205-209.

[23]   Fernandes-Salomao, T.M., Amorim, A.C.R., Alves, V.M.C., Coelho, J.L.C., Silva, D.O. and Araujo, E.F.D. (1996) Isolation of Pectinase Hyperproducing Mutants of Penicillium expansum. Revista de Microbiologia, 27, 15-18.

[24]   Brooks, G.F., Caroll, K.C., Butel, J.S. and Morse, S.A. (2007) Jawetz, Melnick & Adelberg’s Medical Microbiology. The McGraw-Hill Co., New York.

[25]   Cappuccino, J.G. and Sherman, N. (1996) Microbiology a Laboratory Manual. The Benjamin Cummings Publishing Co. Inc., San Francisco.

[26]   Holt, J.G., Krieg, N. Sneath, R.P.H.A., Staley, J.T. and Williams, S.T. (1994) Bergey’s Manual of Determinative Bacteriology. Williams & Wilkins, Maryland.

[27]   Sanchez-Gonzalez, M., Blanco-Gamez, A., Escalante, A., Valladares, A.G., Olvera, C. and Parra, R. (2011) Isolation and Characterization of New Facultative Alkaliphilic Bacillus flexus Strains from Maize Processing Waste Water (Nejayote). Letters in Applied Microbiology, 52, 413-419.

[28]   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic Local Alignment Search Tool. Journal of Molecular Biology, 215, 403-410.

[29]   Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Research, 22, 4673-4680.

[30]   Tamura, K. and Nei, M. (1993) Estimation of the Number of Nucleotide Substitutions in the Control Region of Mitochondrial DNA in Humans and Chimpanzees. Molecular Biology and Evolution, 10, 512-526.

[31]   Kumar, S., Stecher, G. and Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33, 1870-1874.

[32]   Geetanjali, P., Jain, P., and Kumar, T. (2016) Evaluation of Antimicrobial Potential of Rhizospheric Soil Fungi Isolated from Tinosporac ordifolia, Menthe arvensis and Ocimum tenuiflorum Medicinal Plants. International Research Journal of Pharmacy, 7, 37-40.

[33]   Tariq, A. and Latif, Z. (2012) Isolation and Biochemical Characterization of Bacterial Isolates Producing Different Levels of Polygalacturonases from Various Sources. African Journal of Microbiological Research, 6, 7259-7264.

[34]   Soares, M.M., da Silva, R. and Gomes, E. (1999) Screening of Bacterial Strains for Pectinolytic Activity: Characterization of the Polygalacturonase Produced by Bacillus sp. Revista de Microbiologia, 30, 299-303.

[35]   Chen, K., Neimark, H., Rumore, P. and Steinman, C.R. (1989) Broad-Range DNA Probes for Detecting and Amplifying Eubacterial Nucleic Acids. FEMS Microbiology Letters, 57, 19-24.

[36]   Relman, D.A. (1999) The Search for Unrecognized Pathogens. Science, 284, 1308-1310.

[37]   Woo, P.C.Y., Lau, S.K.P., Teng, J.L.L., Tse, H. and Yuen, K.Y. (2008) Then and Now: Use of 16S rDNA Gene Sequencing for Bacterial Identification and Discovery of Novel Bacteria in Clinical Microbiology Laboratories. Clinical Microbiology and Infection, 14, 908-934.