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 AiM  Vol.6 No.1 , January 2016
Production and Partial Characterization of an Extracellular Phytase Produced by Muscodor sp. under Submerged Fermentation
Abstract: In most of the raw materials of plant origin used in animal feed, a portion of the phosphorus is stored as phytic acid or phytate. Phytate is the main storage form of phosphorus in vegetables but is not readily assimilated into food at low concentrations of the enzyme phytase. In addition to making phosphorous unavailable, phytate binds divalent cations such as calcium, copper, magnesium, iron, manganese and zinc, preventing the absorption of these nutrients in the gut of the animal. Phytase promotes the hydrolysis of the phytate phosphorus-releasing molecule, thereby increasing its bioavailability in feed. Phytase is distributed in plant and animal tissues and it is synthesized by some species of bacteria and fungi. The addition of this enzyme in the diet of animals is essential to promote greater uptake of phosphorus and also contributes to a decrease in the levels of phosphorus excreted by animals, thus reducing the pollution caused by excess phosphorus in the environment. This work aimed to select a fungus that stands out in the production of phytase among 100 isolates from Brazilian caves belonging to the genera Aspergillus, Penicillium and Cladosporium and 13 endophytic fungi of the aerial part of the coffee plant. For selection, the fungi were cultured in medium containing phytic acid as a sole source of phosphorus. After seven days at 25 °C, we evaluated growth and enzyme production by the presence of the phytic acid halo degradation (Enzymatic Index-EI) surrounding the colonies. Forty-seven species produced phytase, and the fungi Penicillium minioluteum (CF279) and Muscodor sp. (UBSX) showed higher degradation halos, 2.41 and 4.46, respectively. Considering the Muscodor sp. as the main source of phytase, high enzymatic levels were obtained when the fungus was grown under submerged fermentation with initial pH of 5.0 using wheat bran as additional carbon source for 144 h, at 125 rpm and 30 °C. Additionally, the enzyme was stable at pH 5.0 and 40 °C, and inhibited (14% - 88%) by all compounds analyzed. Then, this is the first study that reports the production of phytase by the endophytic fungus Muscodor sp.
Cite this paper: Alves, N. , Guimarães, L. , Piccoli, R. and Cardoso, P. (2016) Production and Partial Characterization of an Extracellular Phytase Produced by Muscodor sp. under Submerged Fermentation. Advances in Microbiology, 6, 23-32. doi: 10.4236/aim.2016.61003.
References

[1]   Zuo, R., Chang, J., Yin, Q., Chen, L., Chen, Q., Yang, X., Zheng, Q., Ren, G. and Feng, H. (2010) Phytase Gene Expression in Lactobacillus and Analysis of Its Biochemical Characteristics. Microbiological Research, 165, 329-335.
http://dx.doi.org/10.1016/j.micres.2009.06.001

[2]   Ravindran, V., Selle, P.H., Ravindran, G., Morel, P.C.H., Kies, A.K. and Bryden, W.L. (2001) Microbial Phytase Improves Performance, Apparent Metabolizable Energy, and Ileal Amino Acid Digestibility of Broilers Fed a Lysine-Deficient Diet. Poultry Science, 80, 338-344.
http://dx.doi.org/10.1093/ps/80.3.338

[3]   Sandberg, A.S. and Andlid, T. (2002) Phytogenetic and Microbial Phytases in Human Nutrition. International Journal of Food Science & Technology, 37, 823-833.
http://dx.doi.org/10.1046/j.1365-2621.2002.00641.x

[4]   Turner, B.L. and Leytem, A.B. (2004) Phosphorus Compounds in Sequential Extracts of Animal Manures: Chemical Speciation and a Novel Fractionation Procedure. Environmental Science & Technology, 38, 6101-6108.
http://dx.doi.org/10.1021/es0493042

[5]   Singh, B., Kaur, P. and Satyanarayana, T. (2006) Fungal Phytases for Improving the Nutritional Status of Foods and Combating Environmental Phosphorus Pollution. In Chauhan, A.K., Verma, A. (eds.) (2006) Microbes: Health and Environment. IK International Publishers, New Delhi, 289-326.

[6]   Kim, T., Mullaney, E.J., Porres, J.M., Roneker, K.R., Crowe, S., Rice, S., Ko, T., Ullah, A.H.J., Daly, C.B., Welch, R. and Lei, X.G. (2006) Shifting the pH Profile of Aspergillus niger PhyA Phytase To Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive. Applied and Environmental Microbiology, 72, 4397-4403.
http://dx.doi.org/10.1128/AEM.02612-05

[7]   Cousins, B. (1009) Enzimas na nutricao de aves. In: Concórdia, S.C., Ed., Simpósio internacional ACAV EMBRAPA sobre nutricao de aves, EMBRAPA-CNPSA, Concórdia, 118-132.

[8]   Dvoráková, J., Volfová, O. and Kopecky, J. (1998) Characterization of Phytase Produced by Aspergillus niger. Folia Microbiologica, 42, 349-352.
http://dx.doi.org/10.1007/BF02816948

[9]   Lee, S.H., Cho, J., Bok, J., Kang, S., Choi, Y. and Lee, P.C. (2015) Characterization, Gene Cloning, and Sequencing of a Fungal Phytase, phyA, from Penicillium oxalicum PJ3. Preparative Biochemistry and Biotechnology, 45, 336-347.
http://dx.doi.org/10.1080/10826068.2014.923446

[10]   Xavier Salmon, D.N., Piva, L.C., Binati, R.L., Rodrigues, C., Vandenbergue, L.P., Soccol, C.R. and Spier, M.R. (2012) A Bioprocess for the Production of Phytase from Schizophyllum commune: Studies of Its Optimization, Profile of Fermentation Parameters, Characterization and Stability. Bioprocess and Biosystems Engineering, 35, 1067-1079.
http://dx.doi.org/10.1007/s00449-012-0692-6

[11]   Rani, R. and Ghosh, S. (2011) Production of Phytase under Solid-State Fermentation Using Rhizopus oryzae: Novel Strain Improvement Approach and Studies on Purification and Characterization. Bioresource Technology, 102, 10641-10649.
http://dx.doi.org/10.1016/j.biortech.2011.08.075

[12]   Zhang, G.Q., Dong, X.F., Wang, Z.H., Zhang, Q., Wang, H.X. and Tong, J.M. (2010) Purification, Characterization, and Cloning of a Novel Phytase with Low pH Optimum and Strong Proteolysis Resistance from Aspergillus ficuum NTG-23. Bioresource Technology, 101, 4125-4131.
http://dx.doi.org/10.1016/j.biortech.2010.01.001

[13]   Sato, V.S., Jorge, J.A., Oliveira W.P., Souza, C.R.F. and Guimaraes, L.H.S. (2014) Phytase Production by Rhizopus microsporus Var. Microsporus Biofilm: Characterization of Enzymatic Activity after Spray Drying in Presence of Carbohydrates and Nonconventional Adjuvants. Journal of Microbiology and Biotechnology, 24, 177-187.
http://dx.doi.org/10.4014/jmb.1308.08087

[14]   Pandey, A., Szakacs, G., SoccoL, C.R., Rodriguez-Leon, J.A. and Soccol, V.T. (2001) Production, Purification and Properties of Microbial Phytases. Bioresource Technology, 77, 203-214.

[15]   Silva, L.T., Moreira, K.A., Porto, T.S. and Porto, A.L.F. (2010) Producao de fitase a partir de resíduos agroindustriais em fermentacao no estado sólido utilizando fungos do gênero Aspergillus. Jepex 2010, Universidade Federal Rural de Pernambuco, Recife.

[16]   Gulati, H.K., Chadha, B.S. and Saini, H.S. (2007) Production of Feed Enzymes (Phytase and Plant Cell Wall Hydrolyzing Enzymes) by Mucorindicus MTCC 6333: Purification and Characterization of Phytase. Folia Microbiologica, 52, 491-497.
http://dx.doi.org/10.1007/BF02932109

[17]   Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.
http://dx.doi.org/10.1016/0003-2697(76)90527-3

[18]   Strobel, G. (2006) Muscodor albus and Its Biological Promise. Journal of Industrial Microbiology & Biotechnology, 33, 514-522.
http://dx.doi.org/10.1007/s10295-006-0090-7

[19]   Mitchell, A.M., Strobel, G.A., Hess, W.M., Vargas, P.N. and Ezra, D. (2008) Muscodor crispans, a Novel Endophyte from Ananas ananassoides in the Bolivian Amazon. Fungal Diversity, 31, 37-43.
http://dx.doi.org/10.1099/mic.0.032540-0

[20]   Ehrlich, K.C., Montalbano, B.G., Mullaney, E.J., Dischinger, H.C. and Ullah, A.H. (1994) An Acid-Phosphatase from Aspergillus ficuumhas Homology to Penicillium chrysogenum phoA. Biochemical and Biophysical Research Communications, 204, 63-68.

[21]   Mullaney, E.J., Daly, C.B. and Ullah, A.H. (2000) Advances in Phytase Research. Advances in Applied Microbiology, 47, 157-199.
http://dx.doi.org/10.1016/S0065-2164(00)47004-8

[22]   Bhavsar, K., Kumar, V.R. and Khire, J.M. (2011) High Level Phytase Production by Aspergillus niger NCIM 563 in Solid State Culture: Response Surface Optimization, Up-Scaling, and Its Partial Characterization. Journal of Industrial Microbiology & Biotechnology, 38, 1407-1417.
http://dx.doi.org/10.1007/s10295-010-0926-z

[23]   Roopesh, K., Ramachandran, S., Nampoothiri, K.M., Szakacs, G. and Pandey, A. (2006) Comparison of Phytase Production on Wheat Bran and Oilcakes in Solid-State Fermentation by Mucor racemosus. Bioresource Technology, 97, 506-511.
http://dx.doi.org/10.1016/j.biortech.2005.02.046

[24]   Casey, A. and Walsh, G. (2003) Purification and Characterization of Extracellular Phytase from Aspergillus niger ATCC 9142. Bioresource Technology, 86, 183-188.
http://dx.doi.org/10.1016/S0960-8524(02)00145-1

[25]   Spier, M.R., Fendrich, R., Almeida, P., Noseda, M., Greiner, R., Konietzny, U., Woiciechowski, A., Soccol, V. and Soccol, C.R. (2010) Phytase Produced on Citric Byproducts: Purification and Characterization. World Journal of Microbiology and Biotechnology, 27, 267-274.
http://dx.doi.org/10.1007/s11274-010-0455-y

[26]   Vohra, A. and Satyanarayana, T. (2003) Phytases: Microbial Sources, Production, Purification, and Potential Biotechnological Applications. Critical Reviews in Biotechnology, 23, 29-60.
http://dx.doi.org/10.1080/713609297

[27]   Papagianni, M., Psomas, S.K., Batsilas, L., Paras, S.V., Kyriakidis, D.A. and Liakopouloukyriakides, M. (2001) Xanthan Production by Xanthomonas campestris in Batch Cultures. Process Biochemistry, 37, 73-80.
http://dx.doi.org/10.1016/S0032-9592(01)00174-1

[28]   Pasamontes, L., Haiker, M., Wyss, M., Tessier, M. and Van Loon, A.P. (1997) Gene Cloning, Purification, and Characterization of a Heat-Stable Phytase from the Fungus Aspergillus fumigatus. Applied and Environmental Microbiology, 63, 1696-1700.

[29]   Howson, S.J. and Davis, R.P. (1983) Production of Phytate-Hydrolyzing Enzyme by Some Fungi. Enzyme and Microbial Technology, 5, 377-382.
http://dx.doi.org/10.1016/0141-0229(83)90012-1

[30]   Greaves, M.P., Anderson, G. and Webley, D.M. (1967) The Hydrolysis of Inositol Phosphates by Aerobacter aerogenes. Biochimica et Biophysica Acta, 132, 412-418.
http://dx.doi.org/10.1016/0005-2744(67)90160-X

[31]   Quan, C.S., Fan, S.D., Zhang, L.H., Wang, Y.J. and Ohta, Y. (2002) Purification and Properties of a Phytase from Candida krusei WZ-001. Journal of Bioscience and Bioengineering, 94, 419-425.

[32]   Yu, P., Wang, X.T. and Liu, J.W. (2015) Purification and Characterization of a Novel Cold-Adapted Phytase from Rhodotorula mucilaginosa Strain JMUY14 Isolated from Antarctic. Journal of Basic Microbiology, 55, 1029-1039.
http://dx.doi.org/10.1002/jobm.201400865

[33]   Shivanna, G.B. and Venkateswaran, G. (2014) Phytase Production by Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 through Submerged and Solid-State Fermentation. The Scientific World Journal, 2014, 1-6.
http://dx.doi.org/10.1155/2014/392615

[34]   Promdonkoy, P., Tang, K., Sornlake, W., Harnpicharnchai, P., Kobayashi, R.S., Ruanglek, V., Upathanpreecha, T., Vesaratchavest, M., Eurwilaichtr, L. and Tanapongpipat, S. (2009) Expression and Characterization of Aspergillus Thermostable Phytases in Pichiapastoris. FEMS Microbiology Letters, 290, 18-24.
http://dx.doi.org/10.1111/j.1574-6968.2008.01399.x

[35]   Soni, S.K., Magdum, A. and Khire, J.M. (2010) Purification and Characterization of Two Distinct Acidic Phytases with Broad pH Stability from Aspergillus niger NCIM 563. World Journal of Microbiology and Biotechnology, 26, 2009-2018.
http://dx.doi.org/10.1007/s11274-010-0385-8

[36]   Tadashi, N., Satoshi, K., Tadanori, Y., Hideharu, A., Yoko, K., Seiji, S. and Keiichi, Y. (2001) Phytase Having a Low Michaelis Constant for Phytic Acid from Monascus. United States Patent No. 6261592.
http://www.freepatentsonline.com/6261592.html

[37]   Boyce, A. and Walsh, G. (2007) Purification and Characterization of an Acid Phosphatase with Phytase Activity from Mucor hiemalis Wehmer. Journal of Biotechnology, 132, 82-87.
http://dx.doi.org/10.1016/j.jbiotec.2007.08.028

[38]   Casey, A. and Walsh, G. (2004) Identification and Characterization of a Phytase of Potential Commercial Interest. Journal of Biotechnology, 110, 313-322.
http://dx.doi.org/10.1016/j.jbiotec.2004.03.001

[39]   Woodzinski, R.J. and Ullah, A.H.J. (1996) Biochemical Characterization of Cloned Aspergillus fumigatus Phytase (PhyA). Advances in Applied Microbiology, 42, 263-302.

[40]   Ullah, A.H.J., Sethumadhavan, K. and Mullaney, E.J. (2008) Unfolding and Refolding of Aspergillus niger PhyB Phytase: Role of Disulfide Bridges. Journal of Agricultural and Food Chemistry, 56, 8179-8183.
http://dx.doi.org/10.1021/jf8013712

[41]   Konietzny, U., Greiner, R. and Jany, K.D. (1995) Purification and Characterization of a Phytase from Spelt. Journal of Food Biochemistry, 18, 165-183.
http://dx.doi.org/10.1111/j.1745-4514.1994.tb00495.x

[42]   Maenz, D.D., Engele-Schaan, C.M., Newkirk, R.W. and Classen, H.L. (1999) The Effect of Minerals and Mineral Chelators on the Formation of Phytase-Resistant and Phytase-Susceptible Forms of Phytic Acid in Solution and in a Slurry of Canola Meal. Animal Feed Science and Technology, 81, 177-192.
http://dx.doi.org/10.1016/S0377-8401(99)00085-1

[43]   Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N. and Apajalahti, J. (1998) Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64, 2079-2085.

 
 
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