- Email: [email protected]
Isolation and screening of R-(+)-limonene-resistant microorganisms
Abstracts / Journal of Biotechnology 131S (2007) S211–S241
S231
43. New aldoxime-dehydrating enzymes from filamentous fungi
Kato, Y., Yoshida, S., Xie, S.-X., Asano, Y., 2004. Bioengineering 97, 250–259. Kato, Y., Asano, Y., 2005. Biosci. Biotechnol. Biochem. 69, 2254–2257.
David Kubac ∗ , Vojtech Vejvoda, Michal Himl, Ondrej Kaplan, Vladimir Kren, Ludmila Martinkova
doi:10.1016/j.jbiotec.2007.07.420
Institute of Microbiology, Videnska 1083, 14220 Prague, Czech Republic The enzymes able to synthesize nitriles from aldoximes have been only reported in the late 1990s. However, such enzymes (aldoxime dehydratases) appear to be widely distributed in microorganisms (Kato et al., 2000). Aldoxime dehydratases became acknowledged as useful tools for the synthesis of nitriles from aldoximes under mild conditions. Moreover they can be used in conjunction with nitrile hydratases or nitrilases, which, in general, coexist with aldoxime dehydratases in the same microorganisms. Thus, amides or carboxylic acids can be obtained via a bienzymatic process from aldoximes, which, in turn, are easily accessible from aldehydes and hydroxylamine. Several enzymes of this type have been purified from bacteria and characterized (Kato et al., 2004). On the other hand, aldoxime dehydratases from filamentous fungi are much less explored though they have been detected in many species of these microorganisms (Kato et al., 2000). Very recently, a note on the purification and characterization of a heterologous aldoxime dehydratase from Fusarium graminearum appeared (Kato and Asano, 2005). We aimed at isolating a non-recombinant aldoxime dehydratase from a fungal strain based on induction of the aldoxime dehydratase-nitrilase pathway. We reported on the induction of high nitrilase activites in various filamentous fungi (Kaplan et al., 2007) and became interested also in the aldoxime dehydrating activities of these microorganisms. Aldoxime dehydratases were detected in all nitrile-hydrolyzing fungi examined, i.e. in Aspergillus niger, Fusarium oxysporum, Fusarium solani and Penicillium multicolor. These enzyme activities were induced by either pyridine-2-aldoxime, pyridine-3-aldoxime or nitriles (2cyanopyridine, 3-cyanopyridine). The latter inducers were more suitable as they did not inhibit the fungal growth so strongly as the aldoximes. Penicillium multicolor CCF 2244 exhibited the highest activity of the fungal strains examined (150 U/L of culture. . .). While intact cells exhibited a good catalytic activity for phenylacetaldoxime in buffer (5.5 U/g of wet mycelia), the crude extract and the semi-purified enzyme required addition of Na2 S2 O4. Acknowledgments Financial support through projects 203/05/2267 (Czech Science Foundation), OC 171, D25 (Ministry of Education, Czech Republic) and the institutional research concept AV0Z50200510 (Institute of Microbiology). References Kaplan, O., Vejvoda, V., Charv´atov´a-Piˇsvejcov´a, A., Mart´ınkov´a, L., 2007. J. Ind. Microbiol. Biotechnol. 33, 891–896. Kato, Y., Ooi, R., Asano, Y., 2000. Appl. Environ. Microbiol. 66, 2290–2296.
44. Isolation and screening of R-(+)-limonene-resistant microorganisms Juliano Lemos Bicas ∗ , Cedenir Pereira Quadros, Rosˆangela dos Santos, Francisco F´abio Cavalcante Barros, Ana Paula Dion´ısio, Mariana Uenojo, Iramaia Ang´elica Neri, Gl´aucia Maria Pastore Universidade Estadual de Campinas, Laborat´orio de Bioaromas, Depto Ciencia de alimentos-FEA, Rua Monteiro Lobato 80, CP 6121, 13083 862 Campinas, Sao Paulo, Brazil R-(+)-limonene is one of the most abundant monocyclic monoterpene in nature and it represents more than 90% of orange peel oil (Bauer et al., 2001). Its chemical structure is similar to many oxygenated monoterpenoids with pleasant fragrance and, therefore, R-(+)-limonene may be used in flavor industry as a precursor in the synthesis of those compounds. Beyond their desirable fragrances, some of these R-(+)-limonene oxigenated derivates, e.g. perillyl alcohol, carveol, carvone, geraniol and menthol, have shown biological activity against certain types of tumors in in vivo studies, not only preventing the formation or progression of cancer, but also regressing the existing malignant tumors (Crowell, 1999). These characteristics greatly enhances industrial interest in such compounds. In this context, biotransformation processes emerges as an attractive alternative for the d-limonene transformation since, comparing to the traditional chemical methods, they proceed under mild conditions, has a elevated regio and enantioselectivety, do not generate toxic wastes and the products obtained might be labeled as “natural” (Giri et al., 2001; Janssens et al., 1992). In the scientific literature the biotransformation of limonene by microorganisms has been well documented (Duetz et al., 2003). However, a commercial process which allies practice and high productivity is yet to be developed. In this study, microorganisms were isolated from strategic places of a citrus processing plant, some citrus fruit and mint. The objective was to recover strains more adapted to limonene-containing environment. The samples were incubated in rotary shaker at 30 ◦ C/150 rpm for 48 h or 7 days in YM medium containing 0.1% limonene. Great part of the 112 stains recovered after 48 h and the 126 after 7 days were identified as Gram-positive bacillus, followed by a smaller amount of Gram-negative bacillus, yeasts and Gram-positive cocci, beyond five fungi. About one half of the Gram-positive bacillus, Gramnegative bacillus, yeasts and some Gram-positive cocci were selected based on their ability to resist limonene concentrations up to 2%. Amongst these resistant strains, seventy of them were able to grow in mineral medium containing 1% limonene as sole carbon source. This characteristic make of the last microorganisms potentially attractive to flavor compounds production via biotransformation of limonene, a non-expensive by-product of citrus industry.
S232
Abstracts / Journal of Biotechnology 131S (2007) S211–S241
References Bauer, K., Garbe, D., Surburg, H., 2001. Common Fragrance and Flavor Materials. Preparation, Properties and Uses, 4th ed. Wiley-VCH, Weinheim, p. 293. Crowell, P.L., 1999. J. Nutr. 129, 775S–778S. Duetz, W.A., Bouwmeester, H., Van Beilen, J.B., Witholt, B., 2003. Appl. Microbiol. Biotechnol. 61, 269–277. Giri, A., Dhingra, V., Giri, C.C., Singh, A., Ward, O.P., Narasu, M.L., 2001. Biotechnol. Adv. 19, 175–199. Janssens, L., De Pooter, H.L., Schamp, N.M., Vandamme, E.J., 1992. Process Biochem. 27, 195–215.
doi:10.1016/j.jbiotec.2007.07.421 45. Partially purified and characterization of the ␣-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation Ana Fl´avia Azevedo Carvalho ∗ , Rodrigo Sim˜oes Ribeiro Leite, Eduardo Martins, Nat´alia Martin, Roberto Silva, Eleni Gomes Universidade Estadual Paulista, Rua Cristov˜ao Colombo no 2310, 15054-000 S˜ao Jos´e do Rio Preto, S˜ao Paulo, Brazil The ␣-glucosidase (EC 3.2.1.20) is an amylase that catalyses the successive liberation of ␣-d-glucose from the non-reducing end of short saccharides contains ␣-1 → 4 and ␣-1 → 6 linkage. Its applications are proposed for industrial starch processing as well as a guide to structure-based design of anti-HIV inhibitors. Many other papers reported that the inhibition of ␣-glucosidase is a potential therapy for the treatment of diseases such as cancer and diabetes. The objective of this work was the partial purification and characterize ␣-glucosidase from Thermoascus aurantiacus CBMAI 756. The ␣-glucosidase was partially purified by concentration of the crude enzyme solution and gel filtration chromatographies such as sephadex G-100 and sephacryl S-100. After purification steps, the ␣-glucosidase activity increased 16.5-fold with a yield of 10%. Analysis by PAGE, reveled two band indicating the partial purification of ␣-glucosidase. Using gel filtration column Sephadex G-100 and PAGE, the molecular mass of the enzyme was estimated to be between 150 and 200 kDa. The temperature and pH optimum of partially purified ␣-glucosidase were 70 ◦ C and 4.5, respectively. The ␣-glucosidase enzyme showed lost 10% of activity during incubation for 1 h at 50 ◦ C, in absence of substrate. The enzyme maintained 100% activity at pH 4.0–6.5. It is important to establish molecular nature, action mechanisms and functions of this enzyme from several eukaryotic organisms with a view to industrial application and to provide a feasible patter for structural study of these enzymes and their action mechanism. In view the importance of this enzyme is justified the interest for new microorganism producer as well as their characteristics. doi:10.1016/j.jbiotec.2007.07.422
46. Beta-fructofuranosidase from Bifidobacterium longum KN29,1 Marzena Jedrzejczak-Krzepkowska ∗ , Boguslawa Korona, Dagmara Korona, Karolina L. Tkaczuk, Stanislaw Bielecki Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland In recent years the significant increase in commercial interest in bifidobacteria has been observed. Bifidobacteria are a predominant bacterial genus among microorganisms populating human and animal gastrointestinal tracts. The bifidobacteria play an important role in normalization of the composition of intestinal microflora and inhibition of many pathogens, improvement of lactose tolerance, reduction of serum cholesterol and blood ammonia concentration, synthesis of vitamins and stimulation of gastrointestinal immunity. For these reasons, bifidobacterial strains are often added as probiotic components to many foods, mainly dairy products. The growth and/or activity of the bifidobacteria are stimulated by prebiotics such as fructooligosacharides. These carbohydrates are not digested in the upper gastrointestinal tract of the host and thus are able to reach the colon where they are fermented by bifidobacteria. These bacteria can use fructooligosacharides as substrates because they produce intracellular beta-fructofuranosidase which cleaves the beta (2-1) glycosidic bond. Our research deals with identification of the Bifidobacterium longum KN29,1 beta-fructofuranosidase encoding gene, and characterization of the enzyme expressed in Pichia pastoris. Chromosomal DNA from this microorganism was isolated using TriReagent according to the procedure described by Chomczynski. The gene enconding beta-fructofuranosidase from B. longum KN29,1 was amplified by PCR technique. The alignment of the B. longum KN29,1 betafructofuranosidase gene sequence with that of B. longum NCC2705, Bifidobacterium breve strain UCC2003 and Bifidobacterium adolescentis ATCC 15703 genes recorded in data bases, revealed that 99, 96 and 85% of these genes were identical, respectively. The alignment of the deduced amino acid sequences of B. longum KN29,1 -fructofuranosidase with amino acid sequences of beta-fructofuranosidases from B. longum NCC2705, B. breve strain UCC2003, B. longum DJO10A B. adolescentis ATCC 15703, Escherichia coli E24377A and Zymomonas mobilis showed 99, 97, 96, 84, 39 and 37% sequence identity, respectively. On the basis of the deduced amino acid sequence of 518 residues, the molecular mass of 58.09 kDa and isoelectric point of 4.87 were calculated. These values confirmed our earlier results. The two-dimensional electrophoresis (2D-PAGE) of the purified -fructofuranosidase from B. longum KN29,1 revealed a single spot with an apparent molecular mass of about 65 kDa and pI of 4.5. The protein was analyzed by MS and it was found to show 74% identity with the deduced amino acid sequence.
S231
43. New aldoxime-dehydrating enzymes from filamentous fungi
Kato, Y., Yoshida, S., Xie, S.-X., Asano, Y., 2004. Bioengineering 97, 250–259. Kato, Y., Asano, Y., 2005. Biosci. Biotechnol. Biochem. 69, 2254–2257.
David Kubac ∗ , Vojtech Vejvoda, Michal Himl, Ondrej Kaplan, Vladimir Kren, Ludmila Martinkova
doi:10.1016/j.jbiotec.2007.07.420
Institute of Microbiology, Videnska 1083, 14220 Prague, Czech Republic The enzymes able to synthesize nitriles from aldoximes have been only reported in the late 1990s. However, such enzymes (aldoxime dehydratases) appear to be widely distributed in microorganisms (Kato et al., 2000). Aldoxime dehydratases became acknowledged as useful tools for the synthesis of nitriles from aldoximes under mild conditions. Moreover they can be used in conjunction with nitrile hydratases or nitrilases, which, in general, coexist with aldoxime dehydratases in the same microorganisms. Thus, amides or carboxylic acids can be obtained via a bienzymatic process from aldoximes, which, in turn, are easily accessible from aldehydes and hydroxylamine. Several enzymes of this type have been purified from bacteria and characterized (Kato et al., 2004). On the other hand, aldoxime dehydratases from filamentous fungi are much less explored though they have been detected in many species of these microorganisms (Kato et al., 2000). Very recently, a note on the purification and characterization of a heterologous aldoxime dehydratase from Fusarium graminearum appeared (Kato and Asano, 2005). We aimed at isolating a non-recombinant aldoxime dehydratase from a fungal strain based on induction of the aldoxime dehydratase-nitrilase pathway. We reported on the induction of high nitrilase activites in various filamentous fungi (Kaplan et al., 2007) and became interested also in the aldoxime dehydrating activities of these microorganisms. Aldoxime dehydratases were detected in all nitrile-hydrolyzing fungi examined, i.e. in Aspergillus niger, Fusarium oxysporum, Fusarium solani and Penicillium multicolor. These enzyme activities were induced by either pyridine-2-aldoxime, pyridine-3-aldoxime or nitriles (2cyanopyridine, 3-cyanopyridine). The latter inducers were more suitable as they did not inhibit the fungal growth so strongly as the aldoximes. Penicillium multicolor CCF 2244 exhibited the highest activity of the fungal strains examined (150 U/L of culture. . .). While intact cells exhibited a good catalytic activity for phenylacetaldoxime in buffer (5.5 U/g of wet mycelia), the crude extract and the semi-purified enzyme required addition of Na2 S2 O4. Acknowledgments Financial support through projects 203/05/2267 (Czech Science Foundation), OC 171, D25 (Ministry of Education, Czech Republic) and the institutional research concept AV0Z50200510 (Institute of Microbiology). References Kaplan, O., Vejvoda, V., Charv´atov´a-Piˇsvejcov´a, A., Mart´ınkov´a, L., 2007. J. Ind. Microbiol. Biotechnol. 33, 891–896. Kato, Y., Ooi, R., Asano, Y., 2000. Appl. Environ. Microbiol. 66, 2290–2296.
44. Isolation and screening of R-(+)-limonene-resistant microorganisms Juliano Lemos Bicas ∗ , Cedenir Pereira Quadros, Rosˆangela dos Santos, Francisco F´abio Cavalcante Barros, Ana Paula Dion´ısio, Mariana Uenojo, Iramaia Ang´elica Neri, Gl´aucia Maria Pastore Universidade Estadual de Campinas, Laborat´orio de Bioaromas, Depto Ciencia de alimentos-FEA, Rua Monteiro Lobato 80, CP 6121, 13083 862 Campinas, Sao Paulo, Brazil R-(+)-limonene is one of the most abundant monocyclic monoterpene in nature and it represents more than 90% of orange peel oil (Bauer et al., 2001). Its chemical structure is similar to many oxygenated monoterpenoids with pleasant fragrance and, therefore, R-(+)-limonene may be used in flavor industry as a precursor in the synthesis of those compounds. Beyond their desirable fragrances, some of these R-(+)-limonene oxigenated derivates, e.g. perillyl alcohol, carveol, carvone, geraniol and menthol, have shown biological activity against certain types of tumors in in vivo studies, not only preventing the formation or progression of cancer, but also regressing the existing malignant tumors (Crowell, 1999). These characteristics greatly enhances industrial interest in such compounds. In this context, biotransformation processes emerges as an attractive alternative for the d-limonene transformation since, comparing to the traditional chemical methods, they proceed under mild conditions, has a elevated regio and enantioselectivety, do not generate toxic wastes and the products obtained might be labeled as “natural” (Giri et al., 2001; Janssens et al., 1992). In the scientific literature the biotransformation of limonene by microorganisms has been well documented (Duetz et al., 2003). However, a commercial process which allies practice and high productivity is yet to be developed. In this study, microorganisms were isolated from strategic places of a citrus processing plant, some citrus fruit and mint. The objective was to recover strains more adapted to limonene-containing environment. The samples were incubated in rotary shaker at 30 ◦ C/150 rpm for 48 h or 7 days in YM medium containing 0.1% limonene. Great part of the 112 stains recovered after 48 h and the 126 after 7 days were identified as Gram-positive bacillus, followed by a smaller amount of Gram-negative bacillus, yeasts and Gram-positive cocci, beyond five fungi. About one half of the Gram-positive bacillus, Gramnegative bacillus, yeasts and some Gram-positive cocci were selected based on their ability to resist limonene concentrations up to 2%. Amongst these resistant strains, seventy of them were able to grow in mineral medium containing 1% limonene as sole carbon source. This characteristic make of the last microorganisms potentially attractive to flavor compounds production via biotransformation of limonene, a non-expensive by-product of citrus industry.
S232
Abstracts / Journal of Biotechnology 131S (2007) S211–S241
References Bauer, K., Garbe, D., Surburg, H., 2001. Common Fragrance and Flavor Materials. Preparation, Properties and Uses, 4th ed. Wiley-VCH, Weinheim, p. 293. Crowell, P.L., 1999. J. Nutr. 129, 775S–778S. Duetz, W.A., Bouwmeester, H., Van Beilen, J.B., Witholt, B., 2003. Appl. Microbiol. Biotechnol. 61, 269–277. Giri, A., Dhingra, V., Giri, C.C., Singh, A., Ward, O.P., Narasu, M.L., 2001. Biotechnol. Adv. 19, 175–199. Janssens, L., De Pooter, H.L., Schamp, N.M., Vandamme, E.J., 1992. Process Biochem. 27, 195–215.
doi:10.1016/j.jbiotec.2007.07.421 45. Partially purified and characterization of the ␣-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation Ana Fl´avia Azevedo Carvalho ∗ , Rodrigo Sim˜oes Ribeiro Leite, Eduardo Martins, Nat´alia Martin, Roberto Silva, Eleni Gomes Universidade Estadual Paulista, Rua Cristov˜ao Colombo no 2310, 15054-000 S˜ao Jos´e do Rio Preto, S˜ao Paulo, Brazil The ␣-glucosidase (EC 3.2.1.20) is an amylase that catalyses the successive liberation of ␣-d-glucose from the non-reducing end of short saccharides contains ␣-1 → 4 and ␣-1 → 6 linkage. Its applications are proposed for industrial starch processing as well as a guide to structure-based design of anti-HIV inhibitors. Many other papers reported that the inhibition of ␣-glucosidase is a potential therapy for the treatment of diseases such as cancer and diabetes. The objective of this work was the partial purification and characterize ␣-glucosidase from Thermoascus aurantiacus CBMAI 756. The ␣-glucosidase was partially purified by concentration of the crude enzyme solution and gel filtration chromatographies such as sephadex G-100 and sephacryl S-100. After purification steps, the ␣-glucosidase activity increased 16.5-fold with a yield of 10%. Analysis by PAGE, reveled two band indicating the partial purification of ␣-glucosidase. Using gel filtration column Sephadex G-100 and PAGE, the molecular mass of the enzyme was estimated to be between 150 and 200 kDa. The temperature and pH optimum of partially purified ␣-glucosidase were 70 ◦ C and 4.5, respectively. The ␣-glucosidase enzyme showed lost 10% of activity during incubation for 1 h at 50 ◦ C, in absence of substrate. The enzyme maintained 100% activity at pH 4.0–6.5. It is important to establish molecular nature, action mechanisms and functions of this enzyme from several eukaryotic organisms with a view to industrial application and to provide a feasible patter for structural study of these enzymes and their action mechanism. In view the importance of this enzyme is justified the interest for new microorganism producer as well as their characteristics. doi:10.1016/j.jbiotec.2007.07.422
46. Beta-fructofuranosidase from Bifidobacterium longum KN29,1 Marzena Jedrzejczak-Krzepkowska ∗ , Boguslawa Korona, Dagmara Korona, Karolina L. Tkaczuk, Stanislaw Bielecki Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland In recent years the significant increase in commercial interest in bifidobacteria has been observed. Bifidobacteria are a predominant bacterial genus among microorganisms populating human and animal gastrointestinal tracts. The bifidobacteria play an important role in normalization of the composition of intestinal microflora and inhibition of many pathogens, improvement of lactose tolerance, reduction of serum cholesterol and blood ammonia concentration, synthesis of vitamins and stimulation of gastrointestinal immunity. For these reasons, bifidobacterial strains are often added as probiotic components to many foods, mainly dairy products. The growth and/or activity of the bifidobacteria are stimulated by prebiotics such as fructooligosacharides. These carbohydrates are not digested in the upper gastrointestinal tract of the host and thus are able to reach the colon where they are fermented by bifidobacteria. These bacteria can use fructooligosacharides as substrates because they produce intracellular beta-fructofuranosidase which cleaves the beta (2-1) glycosidic bond. Our research deals with identification of the Bifidobacterium longum KN29,1 beta-fructofuranosidase encoding gene, and characterization of the enzyme expressed in Pichia pastoris. Chromosomal DNA from this microorganism was isolated using TriReagent according to the procedure described by Chomczynski. The gene enconding beta-fructofuranosidase from B. longum KN29,1 was amplified by PCR technique. The alignment of the B. longum KN29,1 betafructofuranosidase gene sequence with that of B. longum NCC2705, Bifidobacterium breve strain UCC2003 and Bifidobacterium adolescentis ATCC 15703 genes recorded in data bases, revealed that 99, 96 and 85% of these genes were identical, respectively. The alignment of the deduced amino acid sequences of B. longum KN29,1 -fructofuranosidase with amino acid sequences of beta-fructofuranosidases from B. longum NCC2705, B. breve strain UCC2003, B. longum DJO10A B. adolescentis ATCC 15703, Escherichia coli E24377A and Zymomonas mobilis showed 99, 97, 96, 84, 39 and 37% sequence identity, respectively. On the basis of the deduced amino acid sequence of 518 residues, the molecular mass of 58.09 kDa and isoelectric point of 4.87 were calculated. These values confirmed our earlier results. The two-dimensional electrophoresis (2D-PAGE) of the purified -fructofuranosidase from B. longum KN29,1 revealed a single spot with an apparent molecular mass of about 65 kDa and pI of 4.5. The protein was analyzed by MS and it was found to show 74% identity with the deduced amino acid sequence.