Desarrollo de una Fitasa Bacteriana Recombinante para su Aplicación en la Nutrición Acuícola
Abstract
Las fitasas son importantes aditivos de alimentos que incrementan la disponibilidad de fósforo y otros minerales
debido a la hidrólisis enzimática del ácido fítico, un factor anti-nutricional presente en la mayoría de los alimentos a
base de cereales o leguminosas. En este trabajo se desarrolló una forma recombinante de la fitasa C de Bacillus
subtilis (PhyC-R) producida en Pichia pastoris y se evaluó su potencial para su aplicación en la nutrición animal
comparando sus propiedades contra dos fitasas comerciales de origen fúngico. La PhyC-R mostró ser una proteína
glicosilada y con actividad a intervalos más amplios de pH (5.5 a 9) y temperatura (25 a 70°C) que las fitasas
comerciales. Además, mostró mayor actividad residual a 80ºC por 10 min (85 ± 2%) que las dos fitasas comerciales
(65 ± 6 y 48 ± 3 %) y mayor que la reportada y evaluada para la forma nativa. Todas las fitasas fueron estables a las
enzimas digestivas de camarón blanco del Pacífico (Litopenaeus vannamei) y tripsina porcina con valores de
actividad residual de 67 a 100%. Con la PhyC-R se logró disponer de 3.2 ± 0.2 μg P/mg de harina (16-22 % menos
que las fitasas comerciales) en soya y en harina de chícharo 4.7 ± 0.2 μg P/mg (0-15% menos que las fitasas
comerciales). PhyC-R es una alternativa viable para aplicarse como aditivo para incrementar la disponibilidad de
fósforo fítico y otros nutrientes en alimentos para camarón y en especies con características fisiológicas semejantes,
también podría ser empleada en procesos industriales en condiciones suaves (pH neutros y bajas temperaturas) para
disponer del fósforo fítico en harinas de origen vegetal.
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References
Bai DQ, Qiao XT, Wei D, Guo L, Qi HL. 2004. Effects of phytase on the performance of protein hydrolysis enzyme
in the intestine and liver of common carp. J. Chin. Feed 2: 34–38.
Barrientos L, Scott JJ, Murthy PP. 1994. Specificity of hydrolysis of phytic acid by alkaline phytase from lily pollen.
Plant Physiol. 106: 1489–1495.
Baruah K, Sahu NP, Pal AK, Debnath D. 2004. Dietary phytase: an ideal approach for a cost effective and lowpolluting
aquafeed. NAGA World Fish Center Quart. 27: 15–19.
Davis DA, Johnston WL, Arnold CR. 2000. El uso de suplementos enzimáticos en dietas para camarón. pp 452-462.
En: Civera-Cerecedo, R., Perez-Estrada C.J., Ricque Marie D. y Cruz-Suárez L.E. (eds.) Avances en
Nutrición Acuícola IV. Memorias del IV Simposio Internacional en Nutrición Acuícola. Noviembre 15-18,
La Paz, B.C.S., México.
Debnath D, Pal AK, Sahu NP. 2005. Effect of dietary microbial phytase supplementation on growth and nutrient
digestibility of Pangasius pangasius (Hamilton) fingerlings. Aquacult Res 36(2): 180–187.
Fiske CH, Subbarow Y. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400.
Fredrikson M, Alminger ML, Carlsson NG, Sandberg AS. 2001. Phytate content and phytate degradation by
endogenous phytase in pea (Pisum sativum). J. Sci. Food Agric. 81: 1139-1144.
Guerrero-Olazarán M, Rodríguez-Blanco L, Viader-Salvadó JM. 2007. Producción de una fitasa recombinante en
Pichia pastoris. CIENCIA UANL 10(4): 413-418.
Haefner S, Knietsch A, Scholten E,. Braun J, Lohscheidt M, Zelder O. 2005. Biotechnological production and
applications of phytases. Appl. Microbiol. Biotechnol. 68: 588–597.
Han Y, Wilson DB, Lei XG. 1999. Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces
cerevisiae. Appl. Environ. Microbiol. 65: 1915–1918.
Hara A, Ebina S, Kondo A, Funagua T. 1985. A new type of phytase from Typha latifolia L. Agric. Biol. Chem. 49:
–3544.
Idriss EE, Makarewicz O, Farouk A, Rosner K, Greiner R, Bochow H, Richter T, Borriss R. 2002. Extracellular
phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant growth-promoting effect.
Microbiology 148: 2097–2109.
Jackson L, Li MH, Robinson EH. 1996. Use of microbial phytase in channel catfish Ictalurus punctatus diets to
improve utilization of phytate phosphorus. J. World. Aquacult. Soc 27(3): 309–313.
Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J. 1998. Isolation, characterization, molecular gene
cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl. Environ. Microbiol. 64: 2079–
Kerovuo J, Lappalainen I, Peinikainen T. 2000. The metal dependence of Bacillus subtilis phytase. Biochem.
Biophys. Res. Commun. 268(2): 365-369.
Kim YO, Lee JK, Kim HK, Yu JH, Oh TK. 1998. Cloning of the thermostable phytase gene (phy) from Bacillus sp.
DS11 and its overexpression in Escherichia coli. FEMS Microbiol. Lett. 162: 185–191.
Lei XG, Stahl CH. 2001. Biotechnological development of effective phytases for mineral nutrition and
environmental protection. Appl. Microbiol. Biotechnol. 57: 474-481.
Li MH, Robinson EH. 1997. Microbial phytase can replace inorganic phosphorus supplements in channel catfish
Ictalurus punctatus diets. J. World Aquacult. Soc 28: 402–406.
Ling C, Weimin W, Chengtai Y, Yi Y, James DC, Amararatne Yakupitiyage B, Zhi L, Dapeng L. 2007. Application
of microbial phytase in fish feed. Enzyme Microbial Technol. 40: 497–507.
Liu BL,Rafiq A, Tzeng YM, Rob A. 1998. The induction and characterization of phytase and beyond. Enzyme
Microbial Technol. 22: 415-424.
Mayer A.F, Hellmuth K, Schlieker H, Lopez-Ulibarri R, Oertel S, Dahlems U, Strasser AWM, van Loon APGM.
An expression system matures: A highly efficient and cost-effective process for phytase production
by recombinant strains of Hansenula polymorpha. Biotechnol. Bioeng. 63: 373-381.
Miksch G, Kleist S, Friehs K, Flaschel E. 2002. Overexpression of the phytase from Escherichia coli and its
extracellular production in bioreactors. Appl. Microbiol. Biotechnol. 59: 685-694.
Oh BC, Choi WC, Park S, Kim Yo, Oh TK. 2004. Biochemical properties and substrate specificities of alkaline and
histidine acid phytases. Appl. Microbiol. Biotechnol. 63: 362–372.
Papatryphon E, Howell RA, Soares JH. 1999. Growth and mineral absorption by striped bass Morone saxatilis fed a
plant feedstuff based diet supplemented with phytase. J. World Aquacult. Soc. 30: 161–173.
Powar VK, Jagannathan V. 1982. Purification and properties of phytate-specific phosphatase from Bacillus subtilis.
J. Bacteriol. 151: 1102–1108.
Riche M, Trottier NL, Ku PK. 2001. Apparent digestibility of crude protein and apparent availability of individual
amino acids in tilapia (Oreochromis niloticus) fed phytase pretreated soybean meal diets. Fish Physiol.
Biochem. 25: 181–194.
Ricque-Marie D, Cruz-Suarez LE, Zavala-Chavez BM, Nieto-Lopez M, Guajardo C, Tapia-Salazar McCallum IM
and Newkirk R. 2004. Effect of a phytase product on protein and phosphorus digestibility in shrimp
Litopenaeus vannamei fed an air classified pea protein flour (Ppf) based diet. In: Cruz Suárez, L.E., Ricque
Marie, D., Nieto López, M.G., Villarreal, D., Scholz, U. y González, M. 2004. Avances en Nutrición
Acuícola VII. Memorias del VII Simposium Internacional de Nutrición Acuícola. 16-19 Noviembre, 2004.
Hermosillo, Sonora, México.
Robinson EH, Li MH, Manning BB. 2002. Comparison of microbial phytase and dicalcium phosphate for growth
and bone mineralization of pond-raised channel catfish, Ictalurus punctatus. J. Appl. Aquacult. 12: 81–88.
Rodriguez E, Porres JM, Han Y, Lei XG. 1999. Different sensitivity of recombinant Aspergillus niger phytase (rphyA)
and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro. Arch.
Biochem. Biophys. 365(2): 262-267.
Sajjadi M, Carter CG. 2004. Effect of phytic acid and phytase on feed intake, growth, digestibility and trypsin
activity in Atlantic salmon (Salmo salar L.). Aquacult. Nutr. 10(2): 135–142.
Schaefer A, Koppe WM. 1995. Effect of a microbial phytase on utilization of native phosphorus by carp in a diet
based on soybean meal. Water Sci. Technol. 31(1): 149–155.
Scott JJ. 1991. Alkaline phytase activity in nonionic detergent extracts of legume seeds. Plant Physiol. 95: 1298–
Storebakken T, Shearer KD, Roem AJ. 1998. Availability of protein, phosphorus and other elements in fishmeal,
soy-protein concentrate and phytase-treated soy-proteinconcentrate-based diets to Atlantic salmon, Salmo
salar. Aquaculture 161(1): 365–379.
Sugiura SH, Gabaudan J, Dong FM, Hardy RW. 2001 Dietary microbial phytase supplementation and the utilization
of phosphorus, trace minerals and protein by rainbow trout Oncorhynchus mykiss (Walbaum) fed soybean
meal-based diets. Aquacult. Res. 32: 583–592.
Tye AJ, Siu FK, Leung TY, Lim BL. 2002. Molecular cloning and the biochemical characterization of two novel
phytases from B. subtilis 168 and B. licheniformis. Appl. Microbiol. Biotechnol. 59: 190–197.
Urbano G, Lopez-Jurado M, Aranda P, Vidal-Valverde C, Tenorio E, Porres J. 2000. The role of phytic acid in
legumes: antinutrient or beneficial function? J. Physiol. Biochem .56: 283–294.
Van Etten RL, Davidson R, Stevis PE, MacArthur H, Moore DL. 1991. Covalent structure, disulfide bonding, and
identification of reactive surface and active site residues of human prostatic acid phosphatase. J Biol Chem
:2313–2319.
Van Weerd JH, Khalaf KH, Aartsen EJ, Tijssen PA. 1999. Balance trials with African catfish Clarias gariepinus fed
phytase-treated soybean meal-based diets. Aquacult. Nutr. 5(2): 135–142.
Viader-Salvadó JM, Castillo-Galván M, Guerrero-Olazarán M. 2008. Desarrollo de un proceso para la producción de
una fitasa recombinante. Memoria en extenso IV Simposio Internacional de Ciencia y Tecnología de
Alimentos. Villahermosa Tabasco, México, 24-26 de septiembre de 2008.
Vielma J, Lall SP, Koskela J. 1998. Effects of dietary phytase and cholecalciferol on phosphorus bioavailability in
rainbow trout (Oncorhynchus mykiss). Aquaculture 163(3): 309–23.
Vielma J, Ruohonen K, Gabaudan J, Vogel K. 2004. Top-spraying soybean mealbased diets with phytase improves
protein and mineral digestibility but not lysine utilization in rainbow trout, Oncorhynchus mykiss
(Walbaum). Aquacult. Res. 35(10): 955–964.
Vohra A, Satyanarayana T. 2003. Phytases: microbial sources, production, purification, and potential
biotechnological applications. Crit. Rev. Biotechnol. 23(1): 29-60.
Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, van Loon APGM. 1999.
Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolase):
Catalytic properties. Appl. Environm. Microbiol. 65: 367-373.
Yoo GY, Wang XJ, Choi SM, Han KM. 2005. Dietary microbial phytase increased the phosphorus digestibility in
juvenile Korean rockfish Sebastes schlegeli fed diets containing soybean meal. Aquaculture 243: 315–322.
Yu FN, Wang DZ. 2000. The effects of supplemental phytase on growth and the utilization of phosphorus by crucian
carp Carassius carassius. J. Fish Sci. Chin. 7(2): 106–109.
