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Characterization of NaCl-activated proteinases from moderate halophile isolated from fish sauce fermentation
New Biotechnology · Volume 25S · September 2009
Produced probiotic-loaded SLM revealed sizes below those reported in previous literature, for example that 10 m for Lactobacillus rhamnosus-loaded microparticles produced by (w1 /o/w2 ). The incorporation of probiotics in SLM is an extremely interesting approach for food product. Furthermore, because SLM are composed of lipid materials, these have the advantage of being easily digested in the intestine by lipases in the organism humans, causing microorganisms to be released in the vicinities of the site of action. doi:10.1016/j.nbt.2009.06.277
2.1.031 Fibrolytic enzymes produced in solid-state fermentation by two strains of white-rot fungi D.G. Gerardo 1,∗ , M. Marcos 2 , T.T. Maura 1 , L. Octavio 3 1
Universidad Autonoma de Tlaxcala, Ixtacuixtla, Mexico Colegio de Posgraduados, Montecillos, Mexico 3 Universidad Autónoma Metropolitana Iztapalapa, México, Mexico 2
Most commercial enzymatic complexes containing xylanases and cellulases are used to enhance fiber digestibility in ruminants; in this term, recent studies suggest that the enzymatic specificity depends on the fungal strain, substrate and culture conditions. Solid-state fermentation (SSF), using biodegradable substrates such as sugar cane bagasse (SCB), has proven higher enzymatic productivity than submerged fermentation. The aim of this research was to produce fibrolytic enzymes, including laccases, in SSF by two strains of white-rot fungi: Pleurotus ostreatus (ATCC-32783) and Trametes spp. (EUM1). SSF was carried out in Erlenmeyer flasks containing 2 g of dry SCB, the moisture was adjusted to 86% and then autoclaved. Each flask was inoculated with four mycelial plugs of either P. ostreatus or Trametes sp. SSF was incubated at 30◦ C for 17 days. The enzymatic extract was obtained by the lixiviation of solids with water and its separation by filtering. Xylanase and carboxymethylcellulase (CMCase) activities were evaluated by DNS method; laccases activity was evaluated using 2,2-azo-bis-(ethylbenzothiazoline-6-sulfonic acid) as substrate. The samples were analyzed daily and after a period of freezing of 30 days. Chemical composition of solids was determined. Both strains showed similar lacasses (2 U/gSCB) and CMCases activities (0.65 U/gSCB). The xylanases activity was fourfold higher in P. ostreatus (14 U/gSCB) than in Trametes spp. (3.5 U/gSCB). After the freezing period, enzymatic extracts lost 50 and 75% of laccase and CMCase activities, respectively for both strains; whereas xylanolytic activity was detected only in extracts from Trametes spp. (20% of initial value). Interestingly, SCB enhanced protein content and decreased lignin, hemicellulose and cellulose. In addition to the fact that both strains showed fibrolytic enzyme activity with different stability after freezing storage, this work shows that these enzymatic extracts could be used as alternatives to commercial products to improve the fiber digestibility in ruminants, with the advantage that laccases was also produced which could enhance fiber modification achieved by xylanases and CMCases. doi:10.1016/j.nbt.2009.06.278
ABSTRACTS
2.1.032 -Carotene production by means of fermentation from ındustrial waste M. Kıvan 1,∗ , M. Kahyaoglu 2 1
Anadolu University, Science of Faculty, Department of Biology, Eskisehir, Turkey 2 Dicle University, Education Faculty, Siirt, Turkey
Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The purpose of this study was to produce -carotene by microbial means from industrial wastes substrate. In this study, Blakesleea trispora, which synthesise -carotene structures and often used in biotechnology studies in fermentation. Molasses, vinasse of sugar factory and cheese whey, waste of milk factory were used as industrial waste and YPK and malt extract medium were used as synthetic medium in this research. The molasses, vinasse and cheese whey were pretreated before using. The absorbance values of the product obtained at the end of the fermentations were read by means of a spectrophotometer working with 455 and 483 nm and these values are converted to micrograms by utilizing a standard curve. Amount of -carotene were 1.8—5.3 mg/l YPK medium, 7.9—25 mg/l malt extract medium, 11.7—43.3 mg/l, molasses medium, 3.9—11.2 mg/l, vinasse medium and 4.9—12.5 mg/l cheese whey medium. Sunflower and soybean oil supported growth, but decreased -carotene production. Light stimulates the formation of -carotene in vinasse and molasses medium. As a result that vinasse, molasses and cheese whey waste could be effectively used for -carotene production through fermentation. doi:10.1016/j.nbt.2009.06.279
2.1.033 Characterization of NaCl-activated proteinases from moderate halophile isolated from fish sauce fermentation S. Sinsuwan 1,∗ , S. Rodtong 2 , J. Yongsawatdigul 1 1
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 2 School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
Fish sauce is popular condiment consumed in South East Asia. It relies on a natural fermentation process taking approximately 12—18 months for complete fermentation. Acceleration of natural fermentation process will reduce the capital cost and improve product quality. The use of starter cultures of proteinase-producing bacteria would overcome such limitations. Our objectives were to isolate halophiles producing NaCl-activated proteinases from fish sauce mash at various fermentation time (1—12-month-old) and to biochemically characterize the purified proteinases. Among 165 isolates cultivated in anchovy extract broth containing 25% www.elsevier.com/locate/nbt S57
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
NaCl, Virgibacilllus sp. SK33 obtained from 1-month-old fermentation mash showed the highest extracellular proteinase activity. Two dominant proteinases, 19- and 32-kDa proteinases, showed high activity in the presence of 25% NaCl (4.3 M) as determined by SDS-PAGE and activity staining. The purified 19- and 32-kDa proteinases showed subtilisin-like characteristics based on synthetic substrates tested. Activity of the purified enzyme was gradually increased with NaCl concentration up to 4 M, and stable at 4 M NaCl. Soluble anchovy protein used as a substrate was strongly hydrolyzed by the purified proteinases in the presence of 4 M NaCl. A 19-kDa proteinase showed broader substrate specificity toward oxidized insulin B than did 32-kDa proteinase. The common of cleavage sites of these two enzymes included Gln4 , Cys7 , Glu13 , Ala14 , Leu15,17 , Tyr16,26 , Arg22 , Phe24,25 and Lys29 . Moreover, a 19-kDa proteinase showed higher catalytic activity (Vmax ) and more binding ability (Km ) than did a 32-kDa proteinase. Molecular weight (MW) of a 19-kDa proteinase was 33.7 kDa when determined by MALDI-TOF. A 32-kDa proteinase was heterotrimer with three subunits with MW of 27,858, 33,918 and 35,368 Da, based on MALDI-TOF. Peptide mass fingerprint (PMF) and de novo peptide sequencing of both purified proteinases revealed that the purified proteinases seem to be a novel protein. doi:10.1016/j.nbt.2009.06.280
2.1.034 Decolourisation of textile waste waters — a prospective for anaerobic biotechnology A. Jain 1,∗ , A. Mandal 2 1
VIT University, Kanpur, India School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632014, India
2
Aromatic compounds are susceptible to biological degradation under both aerobic and anaerobic conditions. Under aerobic conditions, the enzymes mono- and di-oxygenase catalyse the incorporation of oxygen from O2 into the aromatic ring of organic compounds before ring fission. Under anaerobic conditions a low redox potential (≤50 mV) can be achieved, which is necessary for the effective decolourisation of dyes. Colour removal under anaerobic conditions is also referred as dye reduction in which literature mostly covers the biochemistry of azo dye reduction. The azo bond cleavage —N N— involves a transfer of four electrons (reducing equivalents), which proceeds through two stages at the azo linkage. In each stage two electrons are transferred to the azo dye, which acts as a final electron acceptor. The exact mechanism of azo dye reduction, whether occurring intracellularly or extracellularly, is still a subject of investigation. Reduced flavins can act as an electron shuttle from nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavoproteins to azo dye as electron acceptor. Intracellular azo dye reduction cannot be responsible for the conversion of all types of azo dyes, especially for sulfonated azo dyes, which have limited membrane permeability. The current hypothesis is that azo dye reduction mostly occurs by extracellular or membrane-bound S58
www.elsevier.com/locate/nbt
enzymes. Reduced cytoplasmatic cofactors such as reduced flavins do not contribute to the chemical dye reduction due to their inability to cross living cell membranes. doi:10.1016/j.nbt.2009.06.281
2.1.035 Biochemical evidence of a gene encoding an extracellular medium-chain-length polyhydroxyalkanoate depolymerase in the predator bacterium Bdellovibrio bacteriovorus HD100 V. Martínez ∗ , J.L. García, M.A. Prieto Department of Molecular Microbiology, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain
Bdellovibrio bacteriovorus are small highly motile deltaproteobacteria with an obligate predatory life cycle that utilizes other Gram-negative bacteria in the predation process. Bdellovibrio are ubiquitous in nature, having been discovered in a wide variety of environments and their prey includes plant, animal and human pathogens. The genome shows a large set of hydrolytic enzyme coding genes supporting the biotechnological interest on this bacterium. PHAs are biopolyesters that can be degraded by many microorganisms to soluble metabolites, which are reutilized by the cells as carbon and energy sources. PHAs can be degraded either intracellularly, by intracellular depolymerases of the accumulating strain or extracellularly by extracellular depolymerases. Intracellular PHA depolymerases catalyse the degradation of an endogenous storage reservoir of PHA being that of Pseudomonas putida KT2442 the prototype of this type of enzymes. By contrast, extracellular depolymerases catalyze the hydrolysis of exogenous PHA as carbon/energy source. The PHA granules are spread into the environment and hydrolyzed by secreted depolymerases into water-soluble oligomers and monomers. The ability to degrade extracellular-short-chain-length PHA (scl-PHA, three to five carbon atoms per monomer) is widespread among bacteria and many extracellular scl-PHA depolymerases have been characterized in depth. Extracellular mcl-PHA (six or more carbon atoms per monomer) degrading microorganisms are less frequently found in the environment and the molecular characteristics of the genes encoding these depolymerases are poorly understood. PhaZ of Pseudomonas fluorescens GK13 is the only mcl-PHA depolymerase which has been studied at molecular level. In this work, we report the in silico identification, cloning, extracellular production and functional characterization, of a new extracelullar mcl-PHA depolymerase from the predator bacterium B. bacteriovorus HD100 (PhaZBd). The primary structure of the protein has been identified. The high identity between the PhaZBd and the depolymerases of its Pseudomonad preys, suggests an ancient gene transfer between prey and predator. PhaZBd has been overproduced in Escherichia coli and P. putida strains. Subcellular localization of the enzyme showed that most of the active protein was associated to the membrane insoluble fraction, although no inclusion bodies were isolated. The depolymerase has successfully produced in tol-pal mutants of E. coli and P. putida, which favor the
Produced probiotic-loaded SLM revealed sizes below those reported in previous literature, for example that 10 m for Lactobacillus rhamnosus-loaded microparticles produced by (w1 /o/w2 ). The incorporation of probiotics in SLM is an extremely interesting approach for food product. Furthermore, because SLM are composed of lipid materials, these have the advantage of being easily digested in the intestine by lipases in the organism humans, causing microorganisms to be released in the vicinities of the site of action. doi:10.1016/j.nbt.2009.06.277
2.1.031 Fibrolytic enzymes produced in solid-state fermentation by two strains of white-rot fungi D.G. Gerardo 1,∗ , M. Marcos 2 , T.T. Maura 1 , L. Octavio 3 1
Universidad Autonoma de Tlaxcala, Ixtacuixtla, Mexico Colegio de Posgraduados, Montecillos, Mexico 3 Universidad Autónoma Metropolitana Iztapalapa, México, Mexico 2
Most commercial enzymatic complexes containing xylanases and cellulases are used to enhance fiber digestibility in ruminants; in this term, recent studies suggest that the enzymatic specificity depends on the fungal strain, substrate and culture conditions. Solid-state fermentation (SSF), using biodegradable substrates such as sugar cane bagasse (SCB), has proven higher enzymatic productivity than submerged fermentation. The aim of this research was to produce fibrolytic enzymes, including laccases, in SSF by two strains of white-rot fungi: Pleurotus ostreatus (ATCC-32783) and Trametes spp. (EUM1). SSF was carried out in Erlenmeyer flasks containing 2 g of dry SCB, the moisture was adjusted to 86% and then autoclaved. Each flask was inoculated with four mycelial plugs of either P. ostreatus or Trametes sp. SSF was incubated at 30◦ C for 17 days. The enzymatic extract was obtained by the lixiviation of solids with water and its separation by filtering. Xylanase and carboxymethylcellulase (CMCase) activities were evaluated by DNS method; laccases activity was evaluated using 2,2-azo-bis-(ethylbenzothiazoline-6-sulfonic acid) as substrate. The samples were analyzed daily and after a period of freezing of 30 days. Chemical composition of solids was determined. Both strains showed similar lacasses (2 U/gSCB) and CMCases activities (0.65 U/gSCB). The xylanases activity was fourfold higher in P. ostreatus (14 U/gSCB) than in Trametes spp. (3.5 U/gSCB). After the freezing period, enzymatic extracts lost 50 and 75% of laccase and CMCase activities, respectively for both strains; whereas xylanolytic activity was detected only in extracts from Trametes spp. (20% of initial value). Interestingly, SCB enhanced protein content and decreased lignin, hemicellulose and cellulose. In addition to the fact that both strains showed fibrolytic enzyme activity with different stability after freezing storage, this work shows that these enzymatic extracts could be used as alternatives to commercial products to improve the fiber digestibility in ruminants, with the advantage that laccases was also produced which could enhance fiber modification achieved by xylanases and CMCases. doi:10.1016/j.nbt.2009.06.278
ABSTRACTS
2.1.032 -Carotene production by means of fermentation from ındustrial waste M. Kıvan 1,∗ , M. Kahyaoglu 2 1
Anadolu University, Science of Faculty, Department of Biology, Eskisehir, Turkey 2 Dicle University, Education Faculty, Siirt, Turkey
Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The purpose of this study was to produce -carotene by microbial means from industrial wastes substrate. In this study, Blakesleea trispora, which synthesise -carotene structures and often used in biotechnology studies in fermentation. Molasses, vinasse of sugar factory and cheese whey, waste of milk factory were used as industrial waste and YPK and malt extract medium were used as synthetic medium in this research. The molasses, vinasse and cheese whey were pretreated before using. The absorbance values of the product obtained at the end of the fermentations were read by means of a spectrophotometer working with 455 and 483 nm and these values are converted to micrograms by utilizing a standard curve. Amount of -carotene were 1.8—5.3 mg/l YPK medium, 7.9—25 mg/l malt extract medium, 11.7—43.3 mg/l, molasses medium, 3.9—11.2 mg/l, vinasse medium and 4.9—12.5 mg/l cheese whey medium. Sunflower and soybean oil supported growth, but decreased -carotene production. Light stimulates the formation of -carotene in vinasse and molasses medium. As a result that vinasse, molasses and cheese whey waste could be effectively used for -carotene production through fermentation. doi:10.1016/j.nbt.2009.06.279
2.1.033 Characterization of NaCl-activated proteinases from moderate halophile isolated from fish sauce fermentation S. Sinsuwan 1,∗ , S. Rodtong 2 , J. Yongsawatdigul 1 1
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand 2 School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
Fish sauce is popular condiment consumed in South East Asia. It relies on a natural fermentation process taking approximately 12—18 months for complete fermentation. Acceleration of natural fermentation process will reduce the capital cost and improve product quality. The use of starter cultures of proteinase-producing bacteria would overcome such limitations. Our objectives were to isolate halophiles producing NaCl-activated proteinases from fish sauce mash at various fermentation time (1—12-month-old) and to biochemically characterize the purified proteinases. Among 165 isolates cultivated in anchovy extract broth containing 25% www.elsevier.com/locate/nbt S57
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
NaCl, Virgibacilllus sp. SK33 obtained from 1-month-old fermentation mash showed the highest extracellular proteinase activity. Two dominant proteinases, 19- and 32-kDa proteinases, showed high activity in the presence of 25% NaCl (4.3 M) as determined by SDS-PAGE and activity staining. The purified 19- and 32-kDa proteinases showed subtilisin-like characteristics based on synthetic substrates tested. Activity of the purified enzyme was gradually increased with NaCl concentration up to 4 M, and stable at 4 M NaCl. Soluble anchovy protein used as a substrate was strongly hydrolyzed by the purified proteinases in the presence of 4 M NaCl. A 19-kDa proteinase showed broader substrate specificity toward oxidized insulin B than did 32-kDa proteinase. The common of cleavage sites of these two enzymes included Gln4 , Cys7 , Glu13 , Ala14 , Leu15,17 , Tyr16,26 , Arg22 , Phe24,25 and Lys29 . Moreover, a 19-kDa proteinase showed higher catalytic activity (Vmax ) and more binding ability (Km ) than did a 32-kDa proteinase. Molecular weight (MW) of a 19-kDa proteinase was 33.7 kDa when determined by MALDI-TOF. A 32-kDa proteinase was heterotrimer with three subunits with MW of 27,858, 33,918 and 35,368 Da, based on MALDI-TOF. Peptide mass fingerprint (PMF) and de novo peptide sequencing of both purified proteinases revealed that the purified proteinases seem to be a novel protein. doi:10.1016/j.nbt.2009.06.280
2.1.034 Decolourisation of textile waste waters — a prospective for anaerobic biotechnology A. Jain 1,∗ , A. Mandal 2 1
VIT University, Kanpur, India School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632014, India
2
Aromatic compounds are susceptible to biological degradation under both aerobic and anaerobic conditions. Under aerobic conditions, the enzymes mono- and di-oxygenase catalyse the incorporation of oxygen from O2 into the aromatic ring of organic compounds before ring fission. Under anaerobic conditions a low redox potential (≤50 mV) can be achieved, which is necessary for the effective decolourisation of dyes. Colour removal under anaerobic conditions is also referred as dye reduction in which literature mostly covers the biochemistry of azo dye reduction. The azo bond cleavage —N N— involves a transfer of four electrons (reducing equivalents), which proceeds through two stages at the azo linkage. In each stage two electrons are transferred to the azo dye, which acts as a final electron acceptor. The exact mechanism of azo dye reduction, whether occurring intracellularly or extracellularly, is still a subject of investigation. Reduced flavins can act as an electron shuttle from nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavoproteins to azo dye as electron acceptor. Intracellular azo dye reduction cannot be responsible for the conversion of all types of azo dyes, especially for sulfonated azo dyes, which have limited membrane permeability. The current hypothesis is that azo dye reduction mostly occurs by extracellular or membrane-bound S58
www.elsevier.com/locate/nbt
enzymes. Reduced cytoplasmatic cofactors such as reduced flavins do not contribute to the chemical dye reduction due to their inability to cross living cell membranes. doi:10.1016/j.nbt.2009.06.281
2.1.035 Biochemical evidence of a gene encoding an extracellular medium-chain-length polyhydroxyalkanoate depolymerase in the predator bacterium Bdellovibrio bacteriovorus HD100 V. Martínez ∗ , J.L. García, M.A. Prieto Department of Molecular Microbiology, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain
Bdellovibrio bacteriovorus are small highly motile deltaproteobacteria with an obligate predatory life cycle that utilizes other Gram-negative bacteria in the predation process. Bdellovibrio are ubiquitous in nature, having been discovered in a wide variety of environments and their prey includes plant, animal and human pathogens. The genome shows a large set of hydrolytic enzyme coding genes supporting the biotechnological interest on this bacterium. PHAs are biopolyesters that can be degraded by many microorganisms to soluble metabolites, which are reutilized by the cells as carbon and energy sources. PHAs can be degraded either intracellularly, by intracellular depolymerases of the accumulating strain or extracellularly by extracellular depolymerases. Intracellular PHA depolymerases catalyse the degradation of an endogenous storage reservoir of PHA being that of Pseudomonas putida KT2442 the prototype of this type of enzymes. By contrast, extracellular depolymerases catalyze the hydrolysis of exogenous PHA as carbon/energy source. The PHA granules are spread into the environment and hydrolyzed by secreted depolymerases into water-soluble oligomers and monomers. The ability to degrade extracellular-short-chain-length PHA (scl-PHA, three to five carbon atoms per monomer) is widespread among bacteria and many extracellular scl-PHA depolymerases have been characterized in depth. Extracellular mcl-PHA (six or more carbon atoms per monomer) degrading microorganisms are less frequently found in the environment and the molecular characteristics of the genes encoding these depolymerases are poorly understood. PhaZ of Pseudomonas fluorescens GK13 is the only mcl-PHA depolymerase which has been studied at molecular level. In this work, we report the in silico identification, cloning, extracellular production and functional characterization, of a new extracelullar mcl-PHA depolymerase from the predator bacterium B. bacteriovorus HD100 (PhaZBd). The primary structure of the protein has been identified. The high identity between the PhaZBd and the depolymerases of its Pseudomonad preys, suggests an ancient gene transfer between prey and predator. PhaZBd has been overproduced in Escherichia coli and P. putida strains. Subcellular localization of the enzyme showed that most of the active protein was associated to the membrane insoluble fraction, although no inclusion bodies were isolated. The depolymerase has successfully produced in tol-pal mutants of E. coli and P. putida, which favor the