The production and recovery analysis of a promising recombinant phytase from Yersinia intermedia expressed in Escherichia coli and its potential use in biotechnology
DOI:
https://doi.org/10.5327/fst.00433Palavras-chave:
downstream process, fermentation, animal nutrition, enzymes, cell disruptionResumo
As fitases recombinantes produzidas em Escherichia coli tornaram-se uma alternativa ao uso de fósforo inorgânico em dietas monogástricas, reduzindo custos de produção e solo e água poluição. A melhoria da biodisponibilidade do fósforo fítico, minerais como Cu 2+ , Zn 2+ , Mn 2+ , Mg 2+ , proteínas, carboidratos e os benefícios do inositol são bem vistos pelos
especialistas na formulação de rações com níveis adequados do aditivo enzimático. Depois de ser produzido em alta densidade celular em E. coli BL21 (λDE3) em um biorreator de bancada Bioflo 3000 (5L) em meio complexo, foi recuperada a fitase recombinante de Yersinia intermedia através da coleta de células, ruptura celular em um homogeneizador de alta pressão e coleta de proteínas em duas frações em uma centrífuga. Da Análise de Variância (ANOVA), ao nível de significância de 5% nível, a influência do número de passagens e da concentração de ração na proteína total foi avaliado o conteúdo após lise mecânica, operada entre 500 e 15.000 psi. Para fração sobrenadante, o fator concentração de alimentação foi significativo e para precipitado fração, número de passagens e concentração de alimentação. O gel SDS-PAGE indica o potencial para recuperação de corpos de inclusão na fração precipitada após lise mecânica. A intensidade da banda fitase em comparação com outras proteínas no sobrenadante do lisado celular e frações precipitadas demonstraram que E. coli recombinante tem grande potencial para proteínas expressão em alta densidade celular.
Downloads
Referências
Belkova, M., Köszagová, R., & Nahálka, J. (2022). Active inclusion
bodies: The unexpected journey. The Journal of Microbiology, Biotechnology and Food Sciences, 12(1), e5951. https://doi.org/10.55251/
jmbfs.5951
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(1-2), 248-254.
https://doi.org/10.1016/0003-2697(76)90527-3
Brasil (2004). Ministry of Agriculture, Livestock and Food Supply.
Secretariat of Rural Support and Cooperatives. Regulatory Instruction No. 13, November 30, 2004. Amended by Normative
Instruction No. 44, December 15, 2015. Retrieved from https://
sistemasweb.agricultura.gov.br/sislegis/action/detalhaAto.
do?method=visualizarAtoPortalMapa&chave=133040692
Castellanos-Mendoza, A., Castro-Acosta, R. M., Olvera, A., Zavala,
G., Mendoza-Vera, M., García-Hernández, E., Alagón, A., Trujillo-Roldán, M. A., & Valdez-Cruz, N. A. (2014). Influence of pH
control in the formation of inclusion bodies during production of
recombinant sphingomyelinase-D in Escherichia coli. Microbial
Cell Factories, 13, 137. https://doi.org/10.1186/s12934-014-0137-9
De Souza, T. P. P., Cantão, L. X. S., Rodrigues, M. Q. R. B., Gonçalves,
D. B., Nagem, R. A. P., Rocha, R. E. O., Godoi, R. R., Lima, W. J. N.,
Galdino, A. S., Minardi, R. C. de M., & Lima, L. H. F. de. (2024).
Glycosylation and charge distribution orchestrates the conformational ensembles of a biotechnologically promissory phytase
in different pHs - a computational study. Journal of Biomolecular
Structure & Dynamics, 42(10), 5030-5041. https://doi.org/10.108
/07391102.2023.2223685
de Souza, T. P. P., da S Mariano, R. M., Vieira, M. S., Andrade, S. F.
V., Godoi, R. R., Goncalves, A. F. A., Naves, L. P., Lima, W. J. N.,
Goncalves, D. B., Campos-da-Paz, M., & Galdino, A. S. (2018).
Biofactories for the production of recombinant phytases and
their application in the animal feed industry. Recent Patents on
Biotechnology, 12(2), 113-125. https://doi.org/10.2174/18722083
Doran, P. M. (1995). Bioprocess Engineering Principles. Academic Press.
European Commission (2002). Regulation (Ec) No 178/2002 of the European Parliament and of the Council of 28 January 2002. General
principles and requirements of food law, establishing the European
Food Safety Authority and laying down procedures in matters of
food safety. European Commission. Retrieved from https://eur-lex.
europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32002R0178
Food and Drug Administration (2010). Center for Food Safety and
Applied Nutrition. Guidance for Industry Enzyme Preparations:
Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices. Center
for Food Safety and Applied Nutrition. Retrieved from https://
www.fda.gov/media/79379/download#:~:text=Enzyme%20preparations%20can%20be%20regulated,GRAS%20through%20
GRAS%20affirmation%20petitions
Glitsoe, V., Ruckebusch, J.-P., & Knap, I. (2015). Innovation in enzyme development. DSM. Retrieved from https://www.dsm.com/
content/dam/dsm/anh/en_na/poultry/resources/DSM-Whitepaper_Enzymes_CTA2.pdf
Greiner, R., & Konietzny, U. (2012). Update on characteristics of commercial phytases. International Phytase Summit 2012. Retrieved
from https://www.openagrar.de/servlets/MCRFileNodeServlet/
Document_derivate_00011870/A1312.pdf
Herrmann, K. R., Brethauer, C., Siedhoff, N. E., Hofmann, I., Eyll, J.,
Davari, M. D., Schwaneberg, U., & Ruff, A. J. (2022). Evolution
of E. coli Phytase Toward Improved Hydrolysis of Inositol Tetraphosphate. Frontiers of Chemical Engineering in China, 4, 838056.
https://doi.org/10.3389/fceng.2022.838056
Huang, H., Luo, H., Yang, P., Meng, K., Wang, Y., Yuan, T., Bai, Y., &
Yao, B. (2006). A novel phytase with preferable characteristics
from Yersinia intermedia. Biochemical and Biophysical Research Communications, 350(4), 884-889. https://doi.org/10.1016/j.
bbrc.2006.09.118
Jatuwong, K., Suwannarach, N., Kumla, J., Penkhrue, W., Kakumyan, P.,
& Lumyong, S. (2020). Bioprocess for production, characteristics,
and biotechnological applications of fungal phytases. Frontiers in
Microbiology, 11, 188. https://doi.org/10.3389/fmicb.2020.00188
Jung, G., Denèfle, P., Becquart, J., & Mayaux, J. F. (1988). High-
-cell density fermentation studies of recombinant Escherichia
coli strains expressing human interleukin-1 beta. Annales de
l’Institut Pasteur. Microbiology, 139(1), 129-146. https://doi.
org/10.1016/0769-2609(88)90100-7
Kim, J., & Kim, K. H. (2017). Effects of minimal media vs. complex
media on the metabolite profiles of Escherichia coli and Saccharomyces cerevisiae. Process Biochemistry, 57, 64-71. https://doi.
org/10.1016/j.procbio.2017.04.003
Lamm, R., Jäger, V. D., Heyman, B., Berg, C., Cürten, C., Krauss, U.,
Jaeger, K.-E., & Büchs, J. (2020). Detailed small-scale characterization and scale-up of active YFP inclusion body production
with Escherichia coli induced by a tetrameric coiled coil domain.
Journal of Bioscience and Bioengineering, 129(6), 730-740. https://
doi.org/10.1016/j.jbiosc.2020.02.003
Food Sci. Technol, Campinas, 45, e00433, 2025
Lemuchi, M. O., Vieira, M. S., Granjeiro, P. A., Silva, J. A. da, Lima, W.
J. N., Gonçalves, D. B., Galdino, A. S., Comar, M., Jr, & Taranto,
A. G. (2013). Uso de modelagem comparativa na determinação
estrutural de fitase de Yersinia. Biochemistry and Biotechnology
Reports, 2(1), 25. https://doi.org/10.5433/2316-5200.2013v2n1p25
Nezhad, N. G., Raja Abd Rahman, R. N. Z., Normi, Y. M., Oslan, S.
N., Shariff, F. M., & Leow, T. C. (2020). Integrative structural and
computational biology of phytases for the animal feed industry.
Catalysts, 10(8), 844. https://doi.org/10.3390/catal10080844
Peternel, S., & Komel, R. (2010). Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells. Microbial Cell
Factories, 9, 66. https://doi.org/10.1186/1475-2859-9-66
Rao, D. E. C. S., Rao, K. V., & Reddy, V. D. (2008). Cloning and
expression of Bacillus phytase gene (phy) in Escherichia coli
and recovery of active enzyme from the inclusion bodies. Journal of Applied Microbiology, 105(4), 1128-1137. https://doi.
org/10.1111/j.1365-2672.2008.03833.x
Rizwanuddin, S., Kumar, V., Naik, B., Singh, P., Mishra, S., Rustagi, S.,
& Kumar, V. (2023). Microbial phytase: Their sources, production,
and role in the enhancement of nutritional aspects of food and feed
additives. Journal of Agriculture and Food Research, 12, 100559.
https://doi.org/10.1016/j.jafr.2023.100559
Rothwell, S. A., Doody, D. G., Johnston, C., Forber, K. J., Cencic, O.,
Rechberger, H., & Withers, P. J. A. (2020). Phosphorus stocks and
flows in an intensive livestock dominated food system. Resources,
Conservation and Recycling, 163, 105065. https://doi.org/10.1016/j.
resconrec.2020.105065
Santos, C. A., Beloti, L. L., Toledo, M. A. S., Crucello, A., Favaro, M.
T. P., Mendes, J. S., Santiago, A. S., Azzoni, A. R., & Souza, A. P.
(2012). A novel protein refolding protocol for the solubilization
and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.
Protein Expression and Purification, 82(2), 284-289. https://doi.
org/10.1016/j.pep.2012.01.010
Simpson, R. J. (2006). SDS-PAGE of Proteins. CSH Protocols, 2006(1),
pdb.prot4313. https://doi.org/10.1101/pdb.prot4313
van Hee, P., Middelberg, A. P. J., van der Lans, R. G. J. M., & van der
Wielen, L. A. M. (2004). Quantification of solid cell material by
detection of membrane-associated proteins and peptidoglycan.
Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 807(1), 111-119. https://doi.org/10.1016/j.
jchromb.2004.01.052
Vieira, M. S., Pereira, V. V., da Cunha Morales Álvares, A., Nogueira,
L. M., Lima, W. J. N., Granjeiro, P. A., Gonçalves, D. B., Campos-
-da-Paz, M., de Freitas, S. M., & 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. https://repositorio.ufmg.
br/handle/1843/10.2174/2212798410666181205114153
Withers, P. J. A., Rodrigues, M., Soltangheisi, A., de Carvalho, T. S., Guilherme, L. R. G., Benites, V. de M., Gatiboni, L. C., de Sousa, D. M.
G., Nunes, R. de S., Rosolem, C. A., Andreote, F. D., Oliveira, A. de,
Jr, Coutinho, E. L. M., & Pavinato, P. S. (2018). Transitions to sustainable management of phosphorus in Brazilian agriculture. Scientific
Reports, 8(1), 2537. https://doi.org/10.1038/s41598-018-20887-z
Wong, H. H., O’Neill, B. K., & Middelberg, A. P. (1997). Cumulative
sedimentation analysis of Escherichia coli debris size. Biotechnology and Bioengineering, 55(3), 556-564. https://doi.org/10.1002/
(sici)1097-0290(19970805)55:3%3C556::aid-bit13%3E3.0.co;2-e
Yang, D., Park, S. Y., Park, Y. S., Eun, H., & Lee, S. Y. (2020). Metabolic
engineering of Escherichia coli for natural product biosynthesis.
Trends in Biotechnology, 38(7), 745-765. https://doi.org/10.1016/j.
tibtech.2019.11.007
Yang, Y., & Sha, M. A. (2019). A beginner’s guide to bioprocess modes-batch, fed-batch, and continuous fermentation. Eppendorf Inc, 408, 1-16.
Yee, L., & Blanch, H. W. (1992). Recombinant protein expression in high
cell density fed-batch cultures of Escherichia coli. Bio/technology,
(12), 1550-1556. https://doi.org/10.1038/nbt1292-1550
