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Insights into the reaction mechanism of the coagulation of soy proteins induced by subtilisin Carlsberg
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
The various aspects of nutrigenomics can and should help in all of the above issues. Examples will be given of approaches related to target discovery, mechanism of action, inter-individual differences and health claim substantiation. doi:10.1016/j.jbiotec.2010.08.163 [F.11] Performance of a Novel dioxygenase of pleurotus sapidus to generate terpene-derived flavours U. Krings ∗ , R.G. Berger Institut für Lebensmittelchemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167 Hannover, Germany Keywords: Pleurotus sapidus; Dioxygenase; Biotransformation; Flavours Due to their unique capability to degrade all major components of wood and other recalcitrant organic wastes basidomycetes express a broad diversity of highly efficient degrading enzymes. Hydrolases (esterases, lipases, and peptidases) as well as carotene bleaching oxidoreductases of basisomycetes has been recently described for application in the food and feed industry (Scheibner et al., 2008; Linke et al., 2008; Linke et al., 2009; Linke et al., 2005). One of the most challenging, but also potentially most rewarding fields of current biotechnology of flavours is the transformation of abundant terpene hydrocarbons to oxyfunctionalised terpenoid derivatives. Because of their large genome cells of basidiomycetes often generated a complex mixture of products including terpenoids with distinctive flavour attributes (Schrader, 2007). Recently, a labelling study revealed a dioxygenase of Pleurotus sapidus as the active principle for the allylic oxidation of the sesquiterpene valencene to yield the valuable flavour compound nootkatone (Kruegener et al., 2010). Within this study further terpene precursors were applied to characterise the substrate specificity and to reveal the catalytic mechanism of the novel dioxygenase of P. sapidus. Either cell homogenates or freeze dried mycelium were re-suspended in a buffer and incubated agitated with pre-selected mono- and sequiterpenes. After 16 h of incubation resulting products of the dioxygenase were analyzed by means of HRGC–MS amongst them attractive flavour compounds such as carvone and verbenone. Deduced from the results obtained so far it can be concluded that the dioxygenase preferably accepted mono- and sesquiterpenes with a cyclic isoprenyl moiety as an indispensable structure element. The catalytic reaction starts with the abstraction of an hydrogen radical which might occur at all allylic positions but products indicated a distinct preference towards cyclic methylene groups. The novel fungal dioxygenase of P. sapidus offers a powerful tool for the selective oxidation of cyclic terpenes at moderate conditions and thereby open access to natural flavour compounds or fine chemicals and pharmaceuticals as well as.
References Kruegener, S., Krings, U., Zorn, H., Berger, R.G., 2010. A dioxygenase of Pleurotus sapidus transforms (+)-valencene regio-specifically to (+)-nootkatone via a stereo-specific allylic hydroperoxidation. Bioresour. Technol. 101 (2), 457–462. Linke, D.; Krings, U.; Zorn, H.; Rabe, S.; Ulmer, H. Production of gluten-specific peptidases from basidiomycetes and their use in hydrolysis of proteins for food industry applications. WO 2008131938 A1. Linke, D., Nimtz, M., Berger, R.G., Zorn, H., 2009. Separation of extracellular esterases from pellet cultures of the basidiomycete Pleurotus sapidus by foam fractionation. J. Am. Oil Chem. Soc. 86 (5), 437–444.
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Linke, D., Zorn, H., Gerken, B., Parlar, H., Berger, R.G., 2005. Foam fractionation of exo-lipases from a growing fungus (Pleurotus sapidus). Lipids 40 (3), 323–327. Scheibner, M., Huelsdau, B., Zelena, K., Nimtz, M., de Boer, L., Berger, R.G., Zorn, H., 2008. Novel peroxidases of Marasmius scorodonius degrade -carotene. Appl. Microbiol. Biotechnol. 77 (6), 1241–1250. Schrader, J., 2007. In: Berger, R.G. (Ed.), Microbial flavour production. In Flavours and Fragrances. Springer, Berlin Heidelberg, pp. 507–574.
doi:10.1016/j.jbiotec.2010.08.164 [F.12] Insights into the reaction mechanism of the coagulation of soy proteins induced by subtilisin Carlsberg K. Inouye ∗ , M. Nakano, K. Asaoka, K. Yasukawa Kyoto University, Japan Keywords: Coagulation; Protease; Soy protein; Thermal treatment It is known that coagula are formed when soy proteins are treated by proteases such as subtilisin Carlsberg (SC) and thermolysin (Asaoka et al., 2009; Inouye et al., 2009; Nagai and Inouye, 2004). In the present study, we examined the effects of thermal treatment on the SC-induced coagulation of soy protein isolates (SPI) and soy proteins in 7S (-conglycinin) and 11S (glycinin) fractions in order to elucidate the coagulation mechanism. The soy proteins were treated at 37–96 ◦ C for 30 min, followed by hydrolysis by SC at pH 8.0 and 37 ◦ C. Time-course of the coagulation was monitored by measuring the turbidity (OD660 ) of the reaction solution. With the treatment at 37–60 ◦ C, the turbidity did not increase at all by the proteolysis while with that at 70–96 ◦ C, drastically increased. The degree of the coagulation was highest for the treatment at 80 ◦ C and most remarkable for 11S. Changes in the SDS-PAGE pattern of the digests during the proteolysis were in good agreement with those in the turbidity for SPI, 7S, 11S proteins. CD analysis showed that the amounts of nonstructured form in these proteins were initially 40-50%, increased to 55-60% by the treatment at 80 ◦ C, and further increased to 65-75% by the proteolysis. The fluorescence intensities of the proteins increased with increasing the treatment temperature up to 80 ◦ C. These findings suggest that the thermal treatment at 80 ◦ C most effectively changes the secondary structure of soy proteins and renders them coagulate when hydrolyzed by SC. The thermal treatment is crucial in the coagulation of soy proteins induced by SC. Elucidation of the mechanism of the coagulation might be useful to establish a method for controlling the coagulation. References Asaoka, K., et al., 2009. Enz. Microb. Technol. 44, 229–234. Inouye, K., et al., 2009. J. Agric. Food Chem. 57, 717–723. Nagai, K., Inouye, K., 2004. J. Agric. Food Chem. 52, 4921–4927.
doi:10.1016/j.jbiotec.2010.08.165 [F.13] Golden rice in a mission P. Beyer ∗ University of Freiburg, Germany Keywords: Rice; Carotenoids; Provitamin A; Product development Golden rice (GR) is the name given to a genetically modified rice (Oryza sativa) that produces -carotene (provitamin A) in the
The various aspects of nutrigenomics can and should help in all of the above issues. Examples will be given of approaches related to target discovery, mechanism of action, inter-individual differences and health claim substantiation. doi:10.1016/j.jbiotec.2010.08.163 [F.11] Performance of a Novel dioxygenase of pleurotus sapidus to generate terpene-derived flavours U. Krings ∗ , R.G. Berger Institut für Lebensmittelchemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167 Hannover, Germany Keywords: Pleurotus sapidus; Dioxygenase; Biotransformation; Flavours Due to their unique capability to degrade all major components of wood and other recalcitrant organic wastes basidomycetes express a broad diversity of highly efficient degrading enzymes. Hydrolases (esterases, lipases, and peptidases) as well as carotene bleaching oxidoreductases of basisomycetes has been recently described for application in the food and feed industry (Scheibner et al., 2008; Linke et al., 2008; Linke et al., 2009; Linke et al., 2005). One of the most challenging, but also potentially most rewarding fields of current biotechnology of flavours is the transformation of abundant terpene hydrocarbons to oxyfunctionalised terpenoid derivatives. Because of their large genome cells of basidiomycetes often generated a complex mixture of products including terpenoids with distinctive flavour attributes (Schrader, 2007). Recently, a labelling study revealed a dioxygenase of Pleurotus sapidus as the active principle for the allylic oxidation of the sesquiterpene valencene to yield the valuable flavour compound nootkatone (Kruegener et al., 2010). Within this study further terpene precursors were applied to characterise the substrate specificity and to reveal the catalytic mechanism of the novel dioxygenase of P. sapidus. Either cell homogenates or freeze dried mycelium were re-suspended in a buffer and incubated agitated with pre-selected mono- and sequiterpenes. After 16 h of incubation resulting products of the dioxygenase were analyzed by means of HRGC–MS amongst them attractive flavour compounds such as carvone and verbenone. Deduced from the results obtained so far it can be concluded that the dioxygenase preferably accepted mono- and sesquiterpenes with a cyclic isoprenyl moiety as an indispensable structure element. The catalytic reaction starts with the abstraction of an hydrogen radical which might occur at all allylic positions but products indicated a distinct preference towards cyclic methylene groups. The novel fungal dioxygenase of P. sapidus offers a powerful tool for the selective oxidation of cyclic terpenes at moderate conditions and thereby open access to natural flavour compounds or fine chemicals and pharmaceuticals as well as.
References Kruegener, S., Krings, U., Zorn, H., Berger, R.G., 2010. A dioxygenase of Pleurotus sapidus transforms (+)-valencene regio-specifically to (+)-nootkatone via a stereo-specific allylic hydroperoxidation. Bioresour. Technol. 101 (2), 457–462. Linke, D.; Krings, U.; Zorn, H.; Rabe, S.; Ulmer, H. Production of gluten-specific peptidases from basidiomycetes and their use in hydrolysis of proteins for food industry applications. WO 2008131938 A1. Linke, D., Nimtz, M., Berger, R.G., Zorn, H., 2009. Separation of extracellular esterases from pellet cultures of the basidiomycete Pleurotus sapidus by foam fractionation. J. Am. Oil Chem. Soc. 86 (5), 437–444.
S63
Linke, D., Zorn, H., Gerken, B., Parlar, H., Berger, R.G., 2005. Foam fractionation of exo-lipases from a growing fungus (Pleurotus sapidus). Lipids 40 (3), 323–327. Scheibner, M., Huelsdau, B., Zelena, K., Nimtz, M., de Boer, L., Berger, R.G., Zorn, H., 2008. Novel peroxidases of Marasmius scorodonius degrade -carotene. Appl. Microbiol. Biotechnol. 77 (6), 1241–1250. Schrader, J., 2007. In: Berger, R.G. (Ed.), Microbial flavour production. In Flavours and Fragrances. Springer, Berlin Heidelberg, pp. 507–574.
doi:10.1016/j.jbiotec.2010.08.164 [F.12] Insights into the reaction mechanism of the coagulation of soy proteins induced by subtilisin Carlsberg K. Inouye ∗ , M. Nakano, K. Asaoka, K. Yasukawa Kyoto University, Japan Keywords: Coagulation; Protease; Soy protein; Thermal treatment It is known that coagula are formed when soy proteins are treated by proteases such as subtilisin Carlsberg (SC) and thermolysin (Asaoka et al., 2009; Inouye et al., 2009; Nagai and Inouye, 2004). In the present study, we examined the effects of thermal treatment on the SC-induced coagulation of soy protein isolates (SPI) and soy proteins in 7S (-conglycinin) and 11S (glycinin) fractions in order to elucidate the coagulation mechanism. The soy proteins were treated at 37–96 ◦ C for 30 min, followed by hydrolysis by SC at pH 8.0 and 37 ◦ C. Time-course of the coagulation was monitored by measuring the turbidity (OD660 ) of the reaction solution. With the treatment at 37–60 ◦ C, the turbidity did not increase at all by the proteolysis while with that at 70–96 ◦ C, drastically increased. The degree of the coagulation was highest for the treatment at 80 ◦ C and most remarkable for 11S. Changes in the SDS-PAGE pattern of the digests during the proteolysis were in good agreement with those in the turbidity for SPI, 7S, 11S proteins. CD analysis showed that the amounts of nonstructured form in these proteins were initially 40-50%, increased to 55-60% by the treatment at 80 ◦ C, and further increased to 65-75% by the proteolysis. The fluorescence intensities of the proteins increased with increasing the treatment temperature up to 80 ◦ C. These findings suggest that the thermal treatment at 80 ◦ C most effectively changes the secondary structure of soy proteins and renders them coagulate when hydrolyzed by SC. The thermal treatment is crucial in the coagulation of soy proteins induced by SC. Elucidation of the mechanism of the coagulation might be useful to establish a method for controlling the coagulation. References Asaoka, K., et al., 2009. Enz. Microb. Technol. 44, 229–234. Inouye, K., et al., 2009. J. Agric. Food Chem. 57, 717–723. Nagai, K., Inouye, K., 2004. J. Agric. Food Chem. 52, 4921–4927.
doi:10.1016/j.jbiotec.2010.08.165 [F.13] Golden rice in a mission P. Beyer ∗ University of Freiburg, Germany Keywords: Rice; Carotenoids; Provitamin A; Product development Golden rice (GR) is the name given to a genetically modified rice (Oryza sativa) that produces -carotene (provitamin A) in the