Abstract
Due to a lack of endogenous phytase enzymes in monogastric animals, exogenous phytases are employed in the animal feed industry. Phytases catalyze the hydrolysis of phytic acid and its salts (phytate) from plant-based animal feed, to supply phosphorus to monogastric animals and decrease the anti-nutritional effects of phytic acid. This study aimed to discover the new phytase with optimum activity at acidic pH range and 40 °C for the poultry feed industry. In the current investigation, phytase-producing microorganisms were isolated from different sources and locations, demonstrating that ten of the isolates are attributed to bacterial strains, and one of which is a yeast strain. Phytase-producing microorganisms were screened based on qualitative and quantitative assays. Additionally, molecular characterization was carried out based on sequencing of amplified 16S rRNA and nuclear ribosomal transcribed spacer (ITS) genes. Then, degenerate primers were designed to amplify the histidine acid phosphatase gene from potential isolates to find the new natural variant of phytase. Quantitative assays were carried out to find pH and temperature profiles at pH ranges between 2 and 7, with a 0.5 interval, and a range of temperatures from 30 °C to 90 °C, with a 10 °C interval. The results from crude enzymes demonstrated the extracellular phytase activities of isolates with different optimum pH and temperatures ranging from 4 to 6.5 and 40 °C–60 °C, respectively. Moreover, the range of optimum phytase activities of isolates was between 194.21 mU/mL and 381 mU/mL. Sequencing analysis of 16S rDNA and nuclear ribosomal transcribed spacer (ITS) genes revealed that six out of the eleven isolates are attributed to the Acinetobacter genus, two of which are affiliated with Enterobacter genus. One is affiliated with the Pseudomonas genus; one of them is affiliated with Escherichia, and another is affiliated with Saccharomyces. Finally, a new native histidine acid phosphatase gene (PhySc) was detected and amplified from the SPA isolate by designing degenerate primers. This showed a 99.50% identity with PHO5 from Saccharomyces cerevisiae YJM993 with an accession number of CP004601.2.
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References
Alias N, Shunmugam S, Ong PY (2017) Isolation and molecular characterization of phytase producing bacteria from Malaysia hot springs. J Fundam Appl Sci 9:852–865. https://doi.org/10.4314/jfas.v9i2s.53
Amutha K, Kokila V (2014) PCR amplification , sequencing of 16S rRNA genes with universal primers and phylogenetic analysis of Pseudomonas aeruginosa. Int J Sci Res 3:257–261
Aziz G, Nawaz M, Anjum AA et al (2015) Isolation and characterization of phytase producing bacterial isolates from soil. J Anim Plant Sci 25:771–776
Bae HD, Yanke LJ, Cheng KJ, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39:17–22. https://doi.org/10.1016/S0167-7012(99)00096-2
Baumann P (1968) Isolation of Acinetobacter from soil and water. J Bacteriol 96:39–42. https://doi.org/10.1128/jb.96.1.39-42.1968
Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9:165–191. https://doi.org/10.1631/jzus.B0710640
Cheryan M, Rackis JJ (1980) Phytic acid interactions in food systems. C R C Crit Rev Food Sci Nutr 13:297–335. https://doi.org/10.1080/10408398009527293
Chu HM, Guo RT, Lin TW et al (2004) Structures of Selenomonas ruminantium Phytase in complex with Persulfated Phytate: DSP Phytase fold and mechanism for sequential substrate hydrolysis. Structure 12:2015–2024. https://doi.org/10.1016/j.str.2004.08.010
Courtois J, Manet L (1952) Recherches sur la phytase. Les phytases du colibacille. Bull Soc Chim Biol 34:265–278
Dersjant-Li Y, Awati A, Schulze H, Partridge G (2015) Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. J Sci Food Agric 95:878–896. https://doi.org/10.1002/jsfa.6998
Golovan S, Wang G, Zhang J, Forsberg CW (2000) Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can J Microbiol 46:59–71
Greiner R (2006) Phytate-degrading enzymes: regulation of synthesis in microorganisms and plants. In: Inositol phosphates: linking agriculture and the environment. pp 78–96
Greiner R, Jany K-D (1991) Characterization of a phytase from Escherichia coli. Chem Hoppe-Seyler 372
Howson SJ, Davis RP (1983) Production of phytate-hydrolysing enzyme by some fungi. Enzym Microb Technol 5:377–382. https://doi.org/10.1016/0141-0229(83)90012-1
Irawan MP, Nurachman Z (2015) Isolation and characterization of Phytase from chicken manure Bacteria. J Natur Indones 15:99–105. https://doi.org/10.31258/jnat.15.2.99-105
Irving GCJ, Cosgrove DJ (1974) Inositol phosphate phosphatases of microbiological origin. Some properties of the partially purified phosphatases of Aspergillus ficuum NRRL 3135. Aust J Biol Sci 27:361–368. https://doi.org/10.1071/BI9740361
Jatuwong K, Suwannarach N, Kumla J et al (2020) Bioprocess for production, characteristics, and biotechnological applications of fungal Phytases. Front Microbiol 11:1–18. https://doi.org/10.3389/fmicb.2020.00188
Jorquera M, Martínez O, Maruyama F et al (2008) Current and future biotechnological applications of bacterial phytases and phytase-producing bacteria. Microbes Environ 23:182–191. https://doi.org/10.1264/jsme2.23.182
Jorquera MA, Gabler S, Inostroza NG et al (2018) Screening and characterization of Phytases from Bacteria isolated from Chilean hydrothermal environments. Microb Ecol 75:387–399. https://doi.org/10.1007/s00248-017-1057-0
Kalsi HK, Singh R, Dhaliwal HS, Kumar V (2016) Phytases from Enterobacter and Serratia species with desirable characteristics for food and feed applications. 3 Biotech 6:64. https://doi.org/10.1007/s13205-016-0378-x
Kim YH, Gwon MN, Yang SY et al (2002) Isolation of phytase-producing Pseudomonas sp. and optimization of its phytase production. J Microbiol Biotechnol 12:279–285
Kumar V, Singh P, Jorquera MA et al (2013) Isolation of phytase-producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea). World J Microbiol Biotechnol 29:1361–1369. https://doi.org/10.1007/s11274-013-1299-z
Lei XG, Porres JM, Mullaney EJ, Brinch-pedersen H (2007) Phytase : Source , Structure and Application. In: Industrial Enzymes. Springer, Dordrecht, pp. 505–529
Liao Y, Li C-M, Chen H et al (2013) Site-directed mutagenesis improves the thermostability and catalytic efficiency of Aspergillus niger N25 phytase mutated by I44E and T252R. Appl Biochem Biotechnol 171:900–915. https://doi.org/10.1007/s12010-013-0380-2
Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456. https://doi.org/10.1089/cmb.2005.12.431
Lopatto E, Choi J, Colina A et al (2019) Characterizing the soil microbiome and quantifying antibiotic resistance gene dynamics in agricultural soil following swine CAFO manure application. PLoS One 14:e0220770. https://doi.org/10.1371/journal.pone.0220770
Mahmood S, Shahid MG, Nadeem M, Haq I-U (2021) Screening of phytate degrading fungi and optimization of culture conditions for phytase synthesis using agro-industrial by-products. Pakistan J Bot 53. https://doi.org/10.30848/PJB2021-2(12)
Menezes-Blackburn D, Gabler S, Greiner R (2015) Performance of seven commercial Phytases in an in vitro simulation of poultry digestive tract. J Agric Food Chem 63:6142–6149. https://doi.org/10.1021/acs.jafc.5b01996
Mrudula Vasudevan U, Jaiswal AK, Krishna S, Pandey A (2019) Thermostable phytase in feed and fuel industries. Bioresour Technol 278:400–407. https://doi.org/10.1016/j.biortech.2019.01.065
Mukesh P, Suma S, Singaracharya MA, Lakshmipathi V (2004) Isolation of phytate-hydrolysing microbial strains from traditional waste water of rice fermentation and liquid cattle feeds. World J Microbiol Biotechnol 20:531–534. https://doi.org/10.1023/B:WIBI.0000040403.23667.68
Mullaney EJ, Ullah AHJ (2003) The term phytase comprises several different classes of enzymes. Biochem Biophys Res Commun 312:179–184. https://doi.org/10.1016/j.bbrc.2003.09.176
Nasir A, Rahman SS, Hossain MM, Choudhury N (2017) Isolation of Saccharomyces cerevisiae from pineapple and orange and study of metal’s effectiveness on ethanol production. Eur J Microbiol Immunol 7:76–91. https://doi.org/10.1556/1886.2016.00035
Nezhad NG, Abd Rahman RNZ, Normi YM et al (2020) Integrative structural and computational biology of Phytases for the animal feed industry. Catalysts 10:1–24. https://doi.org/10.3390/catal10080844
Ogbonna FO, Milala MA, Abubakar M, Burah B (2017) Isolation and optimization of Phytase from Pseudomonas aeruginosa and Aspergillus niger isolated from poultry Faeces. Int J Curr Microbiol Appl Sci 6:3666–3673. https://doi.org/10.20546/ijcmas.2017.611.429
Premono ME, Moawad AM, Vlek PLG (1996) Effect of phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indones J Crop Sci 11:13–23
Qvirist LA, De FC, Strati F et al (2016) Isolation, identification and characterization of yeasts from fermented goat milk of the yaghnob valley in Tajikistan. Front Microbiol 7:1690. https://doi.org/10.3389/fmicb.2016.01690
Ravindran V (2013) Feed enzymes: the science, practice, and metabolic realities. J Appl Poult Res 22:628–636. https://doi.org/10.3382/japr.2013-00739
Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Aust J Plant Physiol 28:897–906. https://doi.org/10.1071/pp01093
Rigden DJ (2008) The histidine phosphatase superfamily: structure and function. Biochem J 409:333–348. https://doi.org/10.1042/BJ20071097
Saitou N, Nei M (1987) The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Sajidan A, Farouk A, Greiner R et al (2004) Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Appl Microbiol Biotechnol 65:110–118. https://doi.org/10.1007/s00253-003-1530-1
Sajidan WR, Sari EN et al (2015) Phytase-producing bacteria from extreme regions in Indonesia. Brazilian Arch Biol Technol 58:711–717. https://doi.org/10.1590/S1516-89132015050173
Stecher G, Tamura K, Kumar S (2020) Molecular evolutionary genetics analysis (MEGA) for macOS. Mol Biol Evol 37:1237–1239. https://doi.org/10.1093/molbev/msz312
Suberu Y, Akande I, Samuel T et al (2019) Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7. Biocatal Agric Biotechnol 20:101264. https://doi.org/10.1016/j.bcab.2019.101264
Troxell B, Petri N, Daron C et al (2015) Poultry body temperature contributes to invasion control through reduced expression of Salmonella pathogenicity island 1 genes in Salmonella enterica serovars typhimurium and enteritidis. Appl Environ Microbiol 81:8192–8201. https://doi.org/10.1128/AEM.02622-15
Tsao GT, Zheng Y, Lu J, Gong CS (1997) Adsorption of heavy metal ions by immobilized phytic acid. Appl Biochem Biotechnol 63–65:731–741. https://doi.org/10.1007/BF02920471
van Hartingsveldt W, van Zeijl CMJ, Harteveld GM et al (1993) Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus Niger. Gene 127:87–94. https://doi.org/10.1016/0378-1119(93)90620-I
Wang J, Liang S, Xiang W et al (2019) A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in Cd2+ binding in the cytosol under low heavy metal concentrations. BMC Plant Biol 19:89. https://doi.org/10.1186/s12870-019-1674-5
Woyengo TA, Nyachoti CM (2013) Review: anti-nutritional effects of phytic acid in diets for pigs and poultry - current knowledge and directions for future research. Can J Anim Sci 93:9–21. https://doi.org/10.4141/CJAS2012-017
Wyss M, Brugger R, Kronenberger A et al (1999) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65:367–373. https://doi.org/10.1128/aem.65.2.367-373.1999
Yoon SJ, Choi YJ, Ki H et al (1996) Isolation and identification of phytase-producing bacterium, Enterobacter sp. 4, and enzymatic properties of phytase enzyme. Enzym Microb Technol 18:449–454. https://doi.org/10.1016/0141-0229(95)00131-X
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NGN and TCL conceived the research, analysed the data and wrote the manuscript, and all the co-authors (RNZRA, NMY, SNO and FMS) contributed to conceptualization, experimental work, data collection and manuscript preparation.
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Nezhad, N.G., Rahman, R.N.Z.R.A., Normi, Y.M. et al. Isolation, screening and molecular characterization of phytase-producing microorganisms to discover the novel phytase. Biologia 78, 2527–2537 (2023). https://doi.org/10.1007/s11756-023-01391-w
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DOI: https://doi.org/10.1007/s11756-023-01391-w