Sequence diversity and catalytic properties of phytases




Enzyme sequence conservation; Phytic acid; Animal feed; Enzyme stability; Enzyme structure.


Phytic acid is an antinutritional factor in cereal feeds, and the use of phytases increases the bioavailability of nutrients bound to this molecule. However, the application of these enzymes depends on their thermal stability and activity at acidic pH. Therefore, in this study we created a database composed of 59 phytase sequences and analyzed the interactions that stabilize their structures in order to understand whether they contribute to the biochemical properties observed. The sequences were aligned and grouped at 30 % similarity, generating 5 clusters, which highlights the high variability among them. A comparative structural analysis of the cluster 3 phytases revealed conserved catalytic domains, as well as eight cysteine residues along the primary sequence, forming disulfide bonds for stabilizing the three-dimensional structure. However, the number of Van der Waals, ionic, and hydrogen interactions, and disulfide bonds was not determinant for the biochemical characteristics presented by these enzymes. The phytase KM873028, from cluster 3, was selected for characterization studies, but its expression in Pichia pastoris generated a protein with properties distinct from those derived from the same sequence expressed in a prokaryotic system. It is likely that the differences observed are associated with the location of the interactions in the structures, non-conserved amino acid residues found around the catalytic site, and post-translational modifications inherent to the expression systems. These possibilities highlight the relevance of strategic choices related to enzyme expression aiming at its production and industrial feasibility.


Abdulla, J. M., Rose, S. P., Mackenzie, A. M., & Pirgozliev, V. R. (2017). Feeding value of field beans (Vicia faba L. var. minor) with and without enzyme containing tannase, pectinase and xylanase activities for broilers. Archives of Animal Nutrition, 71(2), 150-164.

Acquistapace, I. M., Thamopson, E. J., Kuhn, I., Bedford, M. R., Brearley, C. A., & Hemmings, A. M. (2022) Insights to the structural basis for the stereospecificity of the Escherichia coli phytase, AppA. International journal of molecular science. 23(11):6346.

Akbarzadeh, A., Dehnavi, E., Aghaeepoor, M., & Amani, J. (2015). Optimization of recombinant expression of synthetic bacterial phytase in Pichia pastoris using response surface methodology. Jundishapur journal of microbiology, 8(12), e27553.

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), 403-410.

Arredondo, M. A., Casas, G. A., & Stein, H. H. (2019). Increasing levels of microbial phytase increases the digestibility of energy and minerals in diets fed to pigs. Animal Feed Science and Technology, 248, 27-36.

Azeke, M. A., Egielewa, S. J., Eigbogbo, M. U., & Ihimire, I. G. (2011). Effect of germination on the phytase activity, phytate and total phosphorus contents of rice (Oryza sativa), maize (Zea mays), millet (Panicum miliaceum), sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Journal of food science and technology, 48(6), 724-729.

Bohn, L., Meyer, A. S., & Rasmussen, S. (2008). Phytate: impact on environment and human nutrition. A challenge for molecular breeding. Journal of Zhejiang University Science B, 9(3), 165-191.

Böhm, K., Herter, T., Müller, J. J., Borriss, R., & Heinemann, U. (2010). Crystal structure of Klebsiella sp. ASR1 phytase suggests substrate binding to a preformed active site that meets the requirements of a plant rhizosphere enzyme. The FEBS Journal, 277(5), 1284-1296.

Cowieson, A. J., Wilcock, P., & Bedford, M. R. (2011). Super-dosing effects of phytase in poultry and other monogastrics. World's Poultry Science Journal, 67(2), 225-236.

Dassa, J., Marck, C., & Boquet, P. L. (1990). The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase. Journal of Bacteriology, 172(9), 5497-5500.

Fu, D., Huang, H., Luo, H., Wang, Y., Yang, P., Meng, K., ... & Yao, B. (2008). A highly pH-stable phytase from Yersinia kristeensenii: cloning, expression, and characterization. Enzyme and Microbial Technology, 42(6), 499-505.

Fu, D., Li, Z., Huang, H., Yuan, T., Shi, P., Luo, H., Meng, K., Yang, P & Yao, B. (2011). Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site. Applied microbiology and biotechnology, 90(4), 1295-1302.

Gu, W., Huang, H., Meng, K., Yang, P., Fu, D., Luo, H., & Zhan, Z. (2009). Gene cloning, expression, and characterization of a novel phytase from Dickeya paradisiaca. Applied biochemistry and biotechnology, 157(2), 113-123.

Haefner, S., Knietsch, A., Scholten, E., Braun, J., Lohscheidt, M., & Zelder, O. (2005). Biotechnological production and applications of phytases. Applied microbiology and biotechnology, 68(5), 588-597.

Hamed, R. (2018). Physiological parameters of the gastrointestinal fluid impact the dissolution behavior of the BCS class IIa drug valsartan. Pharmaceutical Development and Technology, 23(10), 1168-1176.

Han, N., Miao, H., Yu, T., Xu, B., Yang, Y., Wu, Q., Zhang, R & Huang, Z. (2018). Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation. BMC Biotechnology, 18(1), 1-8.

Hesampour, A., Siadat, S. E. R., Malboobi, M. A., Mohandesi, N., Arab, S. S., & Ghahremanpour, M. M. (2015). Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis. Applied biochemistry and biotechnology, 175(5), 2528-2541.

Huber, K., Zeller, E., & Rodehutscord, M. (2015). Modulation of small intestinal phosphate transporter by dietary supplements of mineral phosphorus and phytase in broilers. Poultry Science, 94(5), 1009-1017.

Huang, Y., Niu, B., Gao, Y., Fu, L., & Li, W. (2010). CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics, 26(5), 680-682.

Huang, H., Luo, H., Wang, Y., Fu, D., Shao, N., Wang, G., ... & Yao, B. (2008). A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Applied microbiology and biotechnology, 80(3), 417-426.

Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., & Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596 (7873), 583-589.

Katoh, K., Rozewicki, J., & Yamada, K. D. (2019). MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in bioinformatics, 20(4), 1160-1166.

Kim, Y. O., Kim, H. W., Lee, J. H., Kim, K. K., & Lee, S. J. (2006). Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae. Biotechnology letters, 28(1), 33-38.

Kryukov, V. S., Glebova, I. V., & Zinoviev, S. V. (2021) Reevaluation of phytase action mechanism in animal nutrition. Biochemistry (Mosc.), 86(1), S152-S165.

Kumar, K., Patel, K., Agrawal, D. C., & Khire, J. M. (2015). Insights into the unfolding pathway and identification of thermally sensitive regions of phytase from Aspergillus niger by molecular dynamics simulations. Journal of Molecular Modeling, 21(6), 1-13.

Li, J., Li, X., Gai, Y., Sun, Y., & Zhang, D. (2019). Evolution of E. coli phytase for increased thermostability guided by rational parameters. Journal of Microbiology and Biotechnology, 29(3): 419-428.

Liao, Y., Li, C. M., Chen, H., Wu, Q., Shan, Z., & Han, X. Y. (2013). Site-directed mutagenesis improves the thermostability and catalytic efficiency of Aspergillus niger N25 phytase mutated by I44E and T252R. Applied biochemistry and biotechnology, 171(4), 900-915.

Luo, H., Huang, H., Yang, P., Wang, Y., Yuan, T., Wu, N., ... & Fan, Y. (2007). A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris. Current Microbiology, 55(3), 185-192.

McCormick, K., Walk, C. L., Wyatt, C. L., & Adeola, O. (2017). Phosphorus utilization response of pigs and broiler chickens to diets supplemented with antimicrobials and phytase. Animal Nutrition, 3(1), 77-84.

Mitchell, D. B., Vogel, K., Weimann, B. J., Pasamontes, L., & van Loon, A. P. (1997). The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology, 143(1), 245-252.

Mulvenna, C. C., McCormack, U. M., Magowan, E., McKillen, J., Bedford, M. R., Walk, C. L., Oster, M., Reyer, H., Wimmers, K., Fornara, D. A., & Ball, M. E. E. (2022) The growth performance, nutrient digestibility, gut bacteria and bone strength of broilers offered alternative, sustainable diets varying in nutrient specification and phytase dose. Animals (Basel.), 12(13):1669.

Noorbatcha, I. A., Sultan, A. M., Salleh, H. M., & Amid, A. (2013). Understanding thermostability factors of Aspergillus niger PhyA phytase: a molecular dynamics study. The protein journal, 32(4), 309-316.

Navone, L., Vogl, T., Luangthongkam, P., Blinco, J. A., Luna-Flores, C. H., Chen, X., Hellens, J. V., Mahler, S & Speight, R. (2021). Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris. Biotechnology for biofuels, 14(1), 1-14.

Nayeem, A., Chiang, S. J., Liu, S. W., Sun, Y., You, L., & Basch, J. (2009). Engineering enzymes for improved catalytic efficiency: a computational study of site mutagenesis in epothilone-B hydroxylase. Protein Engineering, Design & Selection, 22(4), 257-266.

Niu, C., Yang, P., Luo, H., Huang, H., Wang, Y., & Yao, B. (2017). Engineering of Yersinia phytases to improve pepsin and trypsin resistance and thermostability and application potential in the food and feed industry. Journal of agricultural and food chemistry, 65(34), 7337-7344.

Pal Roy, M., Mazumdar, D., Dutta, S., Saha, S. P., & Ghosh, S. (2016). Cloning and expression of phytase appA gene from Shigella sp. CD2 in Pichia pastoris and comparison of properties with recombinant enzyme expressed in E. coli. PloS one, 11(1), e0145745.

Pearson, W. R. (2013). An introduction to sequence similarity (“homology”) searching. Current protocols in bioinformatics, Cap3, Unid 3.1.

Piovesan, D., Minervini, G., & Tosatto, S. C. (2016). The RING 2.0 web server for high quality residue interaction networks. Nucleic acids research, 44(1), 367-374.

Pires, E. B. E., de Freitas, A. J., Salgado, R. L., Guimarães, V. M., Pereira, F. A., & Eller, M. R. (2019). Production of fungal phytases from agroindustrial byproducts for pig diets. Scientific Reports, 9(1), 1-9.

Pramanik, K., Kundu, S., Banerjee, S., Ghosh, P. K., & Maiti, T. K. (2018). Computational-based structural, functional and phylogenetic analysis of Enterobacter phytases. 3 Biotech, 8(6), 1-12.

Selim, S., Abdel-Megeid, N. S., Khalifa, H. K., Fakiha, K. G., Majrashi, K. A., & Hussein, E. (2022) Efficacy of various feed additives on performance, nutrient digestibility, bone quality, blood constituents, and phosphorus absorption and utilization of broiler chickens fed low phosphorus diet. Animals (Basel.), 12(14):1742.

Shao, N., Huang, H., Meng, K., Luo, H., Wang, Y., Yang, P., & Yao, B. (2008). Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074. Journal of microbiology and biotechnology, 18(7), 1221-1226.

Shivange, A. V., Hoeffken, H. W., Haefner, S., & Schwaneberg, U. (2016). Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution. BioTechniques, 61(6), 305-314.

Shivange, A. V., Roccatano, D., & Schwaneberg, U. (2016). Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution. Applied microbiology and biotechnology, 100(1), 227-242.

Singh, B., Sharma, K. K., Kumari, A., Kumar, A., & Gakhar, S. K. (2018). Molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould Sporotrichum thermophile reveals insights into molecular catalysis and biochemical properties. International journal of biological macromolecules, 115, 501-508.

Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38(7), 3022-3027.

Tan, H., Wu, X., Xie, L., Huang, Z., Peng, W., & Gan, B. (2016a). Identification and characterization of a mesophilic phytase highly resilient to high-temperatures from a fungus-garden associated metagenome. Applied microbiology and biotechnology, 100(5), 2225-2241.

Tan, H., Miao, R., Liu, T., Cao, X., Wu, X., Xie, L., & Gan, B. (2016b). Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges S. Journal of Microbiology and Biotechnology, 26(10), 1717-1722.

Vieira, M. S., Pereira, V. V., da Cunha Morales Álvares, A., Nogueira, L. M., Lima, W. J., Granjeiro, P. A., & Galdino, A. S. (2019). Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli. Recent Patents on Food, Nutrition & Agriculture, 10(2), 131-139.

Taussky, H. H., & Skorr, E. (1953). A microcolorimetric method for the determination of inorganic phosphorus. The Journal of Biological Chemistry, 202(2), 675–685.

Wang, H., Chen, H., Li, Q., Yu, F., Yan, Y., Liu, S., & Tan, J. (2022). Enhancing the thermostability of transglutaminase from Streptomyces mobaraensis based on the rational design of a disulfide bond. Protein Expression and Purification, 195, 106079.

Yang, W., Yang, Y., Zhang, L., Xu, H., Guo, X., Yang, X., & Cao, Y. (2017). Improved thermostability of an acidic xylanase from Aspergillus sulphureus by combined disulphide bridge introduction and proline residue substitution. Scientific reports, 7(1), 1-9.

Yao, M. Z., Wang, X., Wang, W., Fu, Y. J., & Liang, A. H. (2013). Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation. Biotechnology letters, 35(10), 1669-1676.

Yi, Z., & Kornegay, E. T. (1996). Sites of phytase activity in the gastrointestinal tract of young pigs. Animal Feed Science and Technology, 61(1-4), 361-368.

Zhang, G. Q., Dong, X. F., Wang, Z. H., Zhang, Q., Wang, H. X., & 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(11), 4125-4131.

Zhang, Z., Yang, J., Xie, P., Gao, Y., Bai, J., Zhang, C.,Liu, L., Wang, Q & Gao, X. (2020). Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering. Enzyme and Microbial Technology, 142, 109679.

Zhao, W., Xiong, A., Fu, X., Gao, F., Tian, Y., & Peng, R. (2010). High level expression of an acid-stable phytase from Citrobacter freundii in Pichia pastoris. Applied biochemistry and biotechnology, 162(8), 2157-2165.

Zinin, N. V., Serkina, A. V., Gelfand, M. S., Shevelev, A. B., & Sineoky, S. P. (2004). Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus. FEMS microbiology letters, 236(2), 283-290.




How to Cite

PIRES , E. B. E.; POLÊTO, M. D.; VIDIGAL, P. M. P.; ARAGÃO , M. Ítalo B.; BARROS , T. A.; SALGADO, R. L.; GUIMARÃES , V. M.; ELLER, M. R. Sequence diversity and catalytic properties of phytases. Research, Society and Development, [S. l.], v. 11, n. 10, p. e427111032765, 2022. DOI: 10.33448/rsd-v11i10.32765. Disponível em: Acesso em: 2 oct. 2022.



Agrarian and Biological Sciences