Diversidade de sequências e propriedades catalíticas de fitases
DOI:
https://doi.org/10.33448/rsd-v11i10.32765Palavras-chave:
Conservação de sequências de enzimas; Ácido fítico; Alimentação animal; Estabilidade de enzimas; Estrutura de enzimas.Resumo
O ácido fítico é um fator antinutricional de cereais que compõem rações, e o uso de fitases aumenta a biodisponibilidade de nutrientes ligados a esta molécula. No entanto, a aplicação dessas enzimas depende de sua estabilidade térmica e atividade em pH ácido. Por isso, nesse estudo criamos um banco de dados com 59 sequências de fitases e analisamos as interações que estabilizam suas estruturas a fim de compreender se estas contribuem para as propriedades bioquímicas observadas. As sequências foram alinhadas e agrupadas a 30% de similaridade, gerando 5 clusters, o que evidencia a alta variabilidade entre elas. Uma análise comparativa estrutural das fitases do cluster 3 revelou domínios catalíticos conservados, assim como oito resíduos de cisteína ao longo da sequência primária, formando pontes dissulfeto para estabilização da estrutura tridimensional proteica. Entretanto, o número de interações de hidrogênio, Van der Waals e iônicas e pontes dissulfeto não foi determinante para as características bioquímicas apresentadas por essas enzimas. A fitase KM873028, do cluster 3, foi selecionada para estudos de caracterização, mas a sua expressão em Pichia pastoris gerou uma proteína com propriedades distintas daquelas derivadas da mesma sequência expressa em sistema procarioto. É provável que as diferenças observadas estejam associadas à localização das interações nas estruturas, resíduos de aminoácidos não conservados encontrados ao redor do sítio catalítico, além de modificações pós-traducionais inerentes aos sistemas de expressão escolhidos. Essas possibilidades evidenciam a relevância das escolhas estratégicas relacionadas à expressão da enzima visando sua produção e viabilização industrial.
Referências
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.
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Copyright (c) 2022 Elizabeth Bárbara Epalanga Pires ; Marcelo Depólo Polêto; Pedro Marcus Pereira Vidigal; Matheus Italo Bomfim Aragão; Tarley Araújo Barros ; Rafael Locatelli Salgado; Valéria Monteze Guimarães ; Monique Renon Eller
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