Summary
Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.
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References
Anson, M. L. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol. 22:79; 1938.
Apte, P.; Kaklij, G.; Heble, M. Proteolytic enzymes bromelains in tissue cultures of Ananas sativus pineapple. Plant Sci. Lett. 14:52–62; 1979.
Bailey, A.; Light, N. Connective tissue In: Meat and meat products. London: Elsevier Science Publishers Ltd.; 1989:213–214.
Bais, H. P.; Sudha, G.; George, J.; Ravishankar, G. A. Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. cv. Lucknow Local. In Vitro Cell. Dev. Biol. Plant 37:293–299; 2001.
Batkin, S.; Taussig, S.; Szekerezes, R. Modulation of pulmonary metastases (Lewis lung carcinoma) by bromelain, an extract of the pineapple stem (Ananas comosus). Cancer Inv. 6:241–242; 1988.
Boehm, R.; Sommer, S.; Li, S.-M.; Heide, L. Genetic engineering of shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon. Plant Cell Physiol. 41:911–919; 2000.
Bourgaud, F.; Bouque, V.; Guckert, A. Production of flavonoids by Psoralea hairy root cultures. Plant Cell Tiss. Organ Cult. 56:96–103; 1999.
Daquinta, M.; Benegas, R. Brief review of tissue culture of pineapple. Pineapple Newsl. 3:7–9; 1997.
Engwerda, C. R.; Andrew, D.; Ladhams, A.; Mynott, T. L. Bromelain modulates T and B cell immune responses in vitro and in vivo. Cell. Immunol. 210:66–75; 2001.
Escalona, M.; Lorenzo, J. C.; González, B.; Daquinta, M.; Borroto, C.; González, J. L.; Desjardins, Y. Pineapple micropropagation in temporary immersion systems. Plant Cell Rep. 18:743–748; 1999.
Fang, Y.; Smith, M. A. L.; Pépin, M. F. Benzyladenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae. Plant Cell Tiss. Organ Cult. 54:113–122; 1998.
Flórez, J. Farmacología humana. 2nd edn. Madrid: Salvat; 1995:142–146.
Haider, G.; Kislinger, T.; Kutchan, T. M. Barbiturate induced benzophenanthridine alkaloid formation proceds by gene transcript accumulation in the California poppy. Biochem. Biophys. Res. Commun. 241:606–610; 1997.
Headon, D.; Walsh, G. The industrial production of enzymes. Biotechnol. Adv. 12:635–646; 1994.
Hernández, M.; Carvajal, C.; Santos, R.; Márquez, M.; Blanco, M.; González, J.; Chávez, M. Purification alternatives of obtained bromelain from different sources. Pineapple Newsl. 6:5; 1999.
Kelly, G. S. Bromelain: a literature review and discussion of its therapeutic applications. Altern. Med. Rev. 1:405–410; 1996.
Kim, H. K.; Oh, S.-R.; Lee, H.-K., Huh, H. Benzothiadiazole enhances the elicitation of rosmarinic acid production in a suspension culture of Agastache rugosa O. Kuntze. Biotechnol. Lett. 23:55–60; 2001.
Kleef, R.; Delohery, T.; Boubjerg, D. Selective modulation of cell adhesion molecules on lymphocytes by bromelain protease 5. Pathobiology 64:339–346; 1996.
La Valle, J.; Krinsky, D.; Hawkins, E. Natural therapeutics pocket guide. Hudson, Ohio: Lexi-Comp; 2000.
Lawrie, R. Meat science. London: Pergamon Press; 1985:195–197.
Leipner, J.; Ilen, F.; Saller, R. Therapy with proteolytic enzymes in rheumatic disorders. BioDrugs 15:779–789; 2001.
Losada, E. Biomelain (on-line). URL: http://www.alergoaragon.org/1999/ tercera 2.html. Importancia de las Enzimas en el Asma Ocupacional; 1999 (accessed August, 2001).
Lotti, T. Controlled clinical studies of bromeline in the treatment of urogenital inflammation. Drugs 46:144–146; 1993.
Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.
Massot, B.; Milesi, S.; Gountier, E.; Bourgaud, F.; Guckert, A. Optimized culture conditions for production of furanocoumarins by micropropagated shoots of Ruta graveolens. Plant Cell Tiss. Organ Cult. 62:11–19; 2000.
McBrige, 1999. Bromelain (on-line). URL: http://www.findarticles.com/ 1999/tercera 2.html. Bromelain—Health Food for Bossy, Too. (anti-inflammatory); November 1999 (accessed August, 2001).
Melis, G. Clinical experience with metoxybutropate vs. bromelain in the treatment of female pelvic inflammation. Minerva Ginecol. 42:309–312; 1990.
Metzig, C.; Grabowska, E.; Eckert, K.; Rehse, K.; Maurer, H. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo. In Vivo 13:7–12; 1999.
Miller, A. Improved sausage casing. US Patent 3 666 844; 1982.
Miyanaga, K.; Seki, M.; Furusaki, S. Analysis of pigmentation in individual cultured plant cells using an image processing system. Biotechnol. Lett. 22:977–981; 2000.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.
Nezbedová, L.; Hesse, M.; Werner, C. Chemical potenticial of Aphelandra sp. cell cultures. Plant Cell Tiss. Organ Cult. 58:133–140; 1999.
Pereira, A. M. S.; Bertori, B. W.; Camara, F. L. A.; Duarte, I. B.; Queiroz, M. E. C.; Leite, V. G. M.; Moraes, R. M.; Carvalho, D.; Franca, S. C. Co-cultivation of plant cells as a technique for elicitation of secondary metabolite production. Plant Cell Tiss. Organ Cult. 60:165–166; 2000.
Ransberger, K., Stauder, G. Process of using catabolic enzymes for induction of tumor necrosis factor (TNF). US Patent 5223406; 1993.
Sakuta, M.; Komamine, A. Cell growth and accumulation of secondary metabolites. In: Constabel, F.; Vasil, I., eds. Cell culture and somatic cell genetics of plants. London: Academic Press; 1987:97–114.
Shibli, R. A.; Smith, M. A. L.; Kuskad, M. H. Headspace ethylene accumulation effects on secondary metabolite production in Vaccinium pahalae cell culture. Plant Growth Regul. 23:201–205; 1997.
Sun, X.; Linden, J. C. Shear stress effects on plant cell suspension cultures in a rotating wall vessel bioreactor. J. Indust. Microbiol. Biotechnol. 22:44–47; 1999.
Targoni, O.; Tary, L.; Lehmann, P. Prevention of murine EAE by oral hydrolytic enzyme treatment. J. Autoimmun. 12:191–198; 1999.
Verpoorte, R.; Heijden, R.; Memelink, J. Engineering the plant cell factory for secondary metabolite production. Transgenic Res. 9:323–343; 2000.
Xie, D.; Wang, L.; Ye, H.; Li, G. Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua. Plant Cell Tiss. Organ Cult. 63:161–166; 2000.
Zhong, J.-J.; Meng, X.-D.; Zhang, Y.-H.; Liu, S. Effective release of ginseng saponin from suspension cells of Panax notoginseng. Biotechnol. Tech. 11:241–244; 1997.
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Pérez, A., Nápoles, L., Lorenzo, J.C. et al. Protease excretion during pineapple micropropagation in temporary immersion bioreactors. In Vitro Cell.Dev.Biol.-Plant 39, 311–315 (2003). https://doi.org/10.1079/IVP2003416
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DOI: https://doi.org/10.1079/IVP2003416