EMAN RESEARCH PUBLISHING | Journal | <p>Phytase Production from a Novel <em>Klebsiella</em> sp. on Wheat Bran for Animal Feed Digestion</p>
MicroBio Pharmaceuticals and Pharmacology | Online ISSN 2209-2161

Phytase Production from a Novel Klebsiella sp. on Wheat Bran for Animal Feed Digestion

Dilruba Akter a, Md. Murad Khan b, Md. Mahmuduzzaman Mian a, Shakila Nargis Khan a, Md. Mozammel Hoq a

+ Author Affiliations

Microbial Bioactives 1(1) 014-021 https://doi.org/10.25163/microbbioacts.11004A0423100718

Submitted: 03 March 2018  Revised: 23 May 2018  Published: 02 April 2019 

The present findings will be very useful for the development of a bioprocess of the enzyme for its large-scale production at the pilot and finally at the commercial level.


Background: The present study was aimed to isolate phytase producing bacteria and optimize the physicochemical parameters of their phytase production. Materials and methods: Four bacterial isolates (Phs4, Phs5, Phs6, and Phs8), based on clear zone formation on phytase screening medium, were selected and tested for finding out the highest phytase producing strain. The production of phytase was then optimized and its biochemical properties were determined to judge the applicability of phytase as a digestive aid in animal feed.  Results: The 4 bacterial isolates (Phs4, Phs5, Phs6 and Phs8) were identified by morphological, cultural, biochemical and molecular characterization as Burkholderia cepacia, Escherichia coli, Klebsiella pneumoniae and Klebsiella sp. respectively. Of these isolates, Phs8 (Klebsiella sp.) was found to produce maximum phytase in shake culture in a basal medium containing Na-phytate at 37oC and pH 5.5 after 72 hours of incubation. The omission of Na-phytate from the medium almost completely abolished the phytase production capacity of the isolate and thus signified its important role as an inducer. Among the different complex carbon sources, viz., glucose, wheat bran, rice bran and chickpea, maximum phytase production (94 unit/ ml) was obtained with wheat bran under comparable cultivation conditions. The phytase works best at a temperature of 37oC and pH of 4.0 with a wide temperature stability (more than 80% activity up to 80oC) and wide pH stability (more than 80% activity within a range of 3-8). Although Zn2+, Co2+, and Fe2+ slightly increased the phytase activity Cu2+ and Mg2+ strongly inhibited the enzyme. Conclusion: The present findings will be very useful for the development of a bioprocess of the enzyme for its large-scale production at the pilot and finally at the commercial level.

Keywords: Phytase; animal feed; Klebsiella sp.; enzyme activity.

Significance: Economic production of bacterial phytase as a poultry feed supplement

Abbreviations: PSM, phytase screening medium; PPM, phytase production medium; LB, Luria-Bertani; RPM, rotation per minute; PCR, polymerase chain reaction; dNTPs, deoxyribonucleotides; EDTA, ethylenediaminetetraacetic acid; EtBr, ethidium bromide; KIA, Kligler iron agar; MIU, motility indole urease test; VP, Voges–Proskauer test.



Aly, M. M., Tork, S., Al-Garni, S. M., & Kabli, S. A. (2015). Production and Characterization of Phytase from Streptomyces luteogriseus R10 Isolated from Decaying Wood Samples. International Journal of Agriculture and Biology, 17(3), 515-522. doi:http://dx.doi.org/10.17957/ijab/

Arpana, M., Gulab, S., Varsha, G., Anita, Y., KamalRai, A., SanjeevKumar, G., & NeerajKumar, A. (2011). Isolation and biochemical characterization of acido-thermophilic extracellular phytase producing bacterial strain for potential application in poultry feed. Jundishapur Journal of Microbiology, 2011(4), 273-282.

Aziz, G., Nawaz, M., Anjum, A., Yaqub, T., Nazir, J., Khan, S., & Aziz, K. (2015). Isolation and characterization of phytase producing bacterial isolates from soil. Journal of Animal & Plant Sciences, 25(3), 771-776.

Badone, F. C., Amelotti, M., Cassani, E., & Pilu, R. (2012). Study of low phytic acid1-7 (lpa1-7), a new ZmMRP4 mutation in maize. Journal of Heredity, 103(4), 598-605. doi:http://dx.doi.org/10.1093/jhered/ess014

Bae, H., Yanke, L., Cheng, K.-J., & Selinger, L. (1999). A novel staining method for detecting phytase activity. Journal of Microbiological Methods, 39(1), 17-22. doi:http://dx.doi.org/10.1016/s0167-7012(99)00096-2

Bilgiçli, N., & Ibanoglu, S. (2007). Effect of wheat germ and wheat bran on the fermentation activity, phytic acid content and colour of tarhana, a wheat flour–yoghurt mixture. Journal of Food Engineering, 78(2), 681-686. doi:https://doi.org/10.1016/j.jfoodeng.2005.11.012

Cappuccino, J. G., & Sherman, N. (2008). Microbiology: A Laboratory Manual (Vol. 9): Pearson/Benjamin Cummings Boston, MA.

Cho, J. S., Lee, C. W., Kang, S. H., Lee, J. C., Bok, J. D., Moon, Y. S., . . . Choi, Y. J. (2003). Purification and characterization of a phytase from Pseudomonas syringae MOK1. Current Microbiology, 47(4), 0290-0294. doi:http://dx.doi.org/10.1007/s00284-002-3966-4

Das, K., Bandyopadhyay, D., & Sen, S. K. (2013). Optimization of fermentation conditions for phytase production by the novel isolate Klebsiella sp. The Bioscan, 8(4), 1315-1320.

El-Toukhy, N. M., Youssef, A. S., & Mikhail, M. G. (2013). Isolation, purification and characterization of phytase from Bacillus subtilis MJA. African Journal of Biotechnology, 12(20), 2957-2967.

Elkhalil, E., Männer, K., Borriss, R., & Simon, O. (2007). In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens. British Poultry Science, 48(1), 64-70. doi:http://dx.doi.org/10.1080/00071660601148195

Escobin-Mopera, L., Ohtani, M., Sekiguchi, S., Sone, T., Abe, A., Tanaka, M., . . . Asano, K. (2012). Purification and characterization of phytase from Klebsiella pneumoniae 9-3B. Journal of Bioscience and Bioengineering, 113(5), 562-567. doi:http://dx.doi.org/10.1016/j.jbiosc.2011.12.010

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39(4), 783-791. doi:https://doi.org/10.1111/j.1558-5646.1985.tb00420.x

Gontia, I., Tantwai, K., Rajput, L. P. S., & Tiwari, S. (2012). Transgenic plants expressing phytase gene of microbial origin and their prospective application as feed. Food Technology and Biotechnology, 50(1), 3.

Greiner, R., & Carlsson, N.-G. (2006). Myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate. Canadian Journal of Microbiology, 52(8), 759-768. doi:http://dx.doi.org/10.1139/w06-028

Greiner, R., Konietzny, U., & Jany, K.-D. (1993). Purification and characterization of two phytases from Escherichia coli. Archives of Biochemistry and Biophysics, 303(1), 107-113. doi:http://dx.doi.org/10.1006/abbi.1993.1261

Gupta, R. K., Gangoliya, S. S., & Singh, N. K. (2015). Reduction of phytic acid and enhancement of bioavailable micronutrients in food grains. Journal of Food Science and Technology, 52(2), 676-684. doi:http://dx.doi.org/10.1007/s13197-013-0978-y

Harland, B. F., & Morris, E. R. (1995). Phytate: a good or a bad food component? Nutrition Research, 15(5), 733-754. doi:http://dx.doi.org/10.1016/0271-5317(95)00040-p

Haros, M., Bielecka, M., Honke, J., & Sanz, Y. (2007). Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697. International Journal of Food Microbiology, 117(1), 76-84. doi:http://dx.doi.org/10.1016/j.ijfoodmicro.2007.02.021

Hegsted, D. (1968). Present knowledge of calcium, phosphorus, and magnesium. Nutrition Reviews, 26(3), 65-70. doi:http://dx.doi.org/10.1111/j.1753-4887.1968.tb00862.x

Hill, J. E., Kysela, D., & Elimelech, M. (2007). Isolation and assessment of phytate-hydrolysing bacteria from the DelMarVa Peninsula. Environmental Microbiology, 9(12), 3100-3107. doi:http://dx.doi.org/10.1111/j.1462-2920.2007.01420.x

Hong, S. W., Chu, I. H., & Chung, K. S. (2011). Purification and biochemical characterization of thermostable phytase from newly isolated Bacillus subtilis CF92. Journal of the Korean Society for Applied Biological Chemistry, 54(1), 89-94. doi:http://dx.doi.org/10.3839/jksabc.2011.012

Jones, G. (2013). How to select the best phytase for your feed formulation. Retrieved from https://www.wattagnet.com/articles/17645-how-to-select-the-best-phytase-for-your-feed-formulation

Jorquera, M., MARTíNEZ, O., Maruyama, F., Marschner, P., & de la Luz Mora, M. (2008). Current and future biotechnological applications of bacterial phytases and phytase-producing bacteria. Microbes and Environments, 23(3), 182-191. doi:http://dx.doi.org/10.1264/jsme2.23.182

Kalsi, H. K., Singh, R., Dhaliwal, H. S., & Kumar, V. (2016). Phytases from Enterobacter and Serratia species with desirable characteristics for food and feed applications. 3 Biotech, 6(1), 1-13. doi:http://dx.doi.org/10.1007/s13205-016-0378-x

Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N., & Apajalahti, J. (1998). Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64(6), 2079-2085.

Kerovuo, J., & Tynkkynen, S. (2000). Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. Letters in Applied Microbiology, 30(4), 325-329. doi:http://dx.doi.org/10.1046/j.1472-765x.2000.00660.x

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111-120. doi:https://doi.org/10.1007/BF01731581

Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35(6), 1547-1549. doi:https://doi.org/10.1093/molbev/msy096

Maathuis, F. J. (2009). Physiological functions of mineral macronutrients. Current Opinion in Plant Biology, 12(3), 250-258. doi:http://dx.doi.org/10.1016/j.pbi.2009.04.003

Mittal, A., Singh, G., Goyal, V., Yadav, A., & Aggarwal, N. K. (2012). Production of phytase by acido-thermophilic strain of Klebsiella sp. DB-3FJ711774. 1 using orange peel flour under submerged fermentation. Innovative Romanian Food Biotechnology, 10, 18.

Mullaney, E. J., & Ullah, A. H. (2003). The term phytase comprises several different classes of enzymes. Biochemical and Biophysical Research Communications, 312(1), 179-184. doi:http://dx.doi.org/10.1016/j.bbrc.2003.09.176

Musapuor, A., Afsharmanesh, M., & Shahrbabak, H. M. (2006). Use of microbial phytase for decrease of pollutant due to environmental poultry excreta phosphorus. International Journal of Agriculture and Biology, 8(1), 35-37.

Pandey, A., Szakacs, G., Soccol, C. R., Rodriguez-Leon, J. A., & Soccol, V. T. (2001). Production, purification and properties of microbial phytases. Bioresource Technology, 77(3), 203-214. doi:http://dx.doi.org/10.1016/s0960-8524(00)00139-5

Quan, C.-S., Tian, W.-J., Fan, S.-D., & Kikuchi, J.-I. (2004). Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1. Journal of Bioscience and Bioengineering, 97(4), 260-266. doi:http://dx.doi.org/10.1016/s1389-1723(04)70201-7

Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406-425. doi:https://doi.org/10.1093/oxfordjournals.molbev.a040454

Sajidan, A., Farouk, A., Greiner, R., Jungblut, P., Müller, E.-C., & Borriss, R. (2004). Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Applied Microbiology and Biotechnology, 65(1), 110-118. doi:http://dx.doi.org/10.1007/s00253-003-1530-1

Selle, P. H., Cowieson, A. J., Cowieson, N. P., & Ravindran, V. (2012). Protein–phytate interactions in pig and poultry nutrition: a reappraisal. Nutrition Research Reviews, 25(1), 1-17. doi:http://dx.doi.org/10.1017/s0954422411000151

Singh, B., Kunze, G., & Satyanarayana, T. (2011). Developments in biochemical aspects and biotechnological applications of microbial phytases. Biotechnology and Molecular Biology Reviews, 6(3), 69-87.

Soni, S. K., & Khire, J. M. (2007). Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions. World Journal of Microbiology and Biotechnology, 23(11), 1585-1593. doi:http://dx.doi.org/10.1007/s11274-007-9404-9

Spier, M. R., Greiner, R., Rodriguez-León, J. A., Woiciechowski, A. L., Pandey, A., Soccol, V. T., & Soccol, C. R. (2008). Phytase production using citric pulp and other residues of the agroindustry in SSF by fungal isolates. Food Technology & Biotechnology, 46(2), 178-182. doi:http://dx.doi.org/10.1080/00986445.2010.493115

Unno, Y., Okubo, K., Wasaki, J., Shinano, T., & Osaki, M. (2005). Plant growth promotion abilities and microscale bacterial dynamics in the rhizosphere of Lupin analysed by phytate utilization ability. Environmental Microbiology, 7(3), 396-404. doi:http://dx.doi.org/10.1111/j.1462-2920.2004.00701.x

Wang, X., Upatham, S., Panbangred, W., Isarangkul, D., Summpunn, P., Wiyakrutta, S., & Meevootisom, V. (2004). Purification, characterization, gene cloning and sequence analysis of a phytase from Klebsiella pneumoniae subsp. pneumoniae XY-5. Science Asia, 30, 383-390. doi:http://dx.doi.org/10.2306/scienceasia1513-1874.2004.30.383

Zhang, R., Yang, P., Huang, H., Shi, P., Yuan, T., & Yao, B. (2011). Two types of phytases (histidine acid phytase and β-propeller phytase) in Serratia sp. TN49 from the gut of Batocera horsfieldi (Coleoptera) larvae. Current Microbiology, 63(5), 408. doi:http://dx.doi.org/10.1007/s00284-011-9995-0

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