Search

Brettanomyces yeasts have gained popularity in many sectors of the biotechnological industry, specifically in the field of beer production, but also in wine and ethanol production. Their unique properties enable Brettanomyces to outcompete conventional brewer’s yeast in industrially relevant traits such as production of ethanol and pleasant flavors. Recent advances in next-generation sequencing (NGS) and high-throughput screening techniques have facilitated large population studies allowing the selection of appropriate yeast strains with improved traits. In order to get a better understanding of Brettanomyces species and its potential for beer production, we sequenced the whole genome of 84 strains, which we make available to the scientific community and carried out several in vitro assays for brewing-relevant properties. The collection includes isolates from different substrates and geographical origin. Additionally, we have included two of the oldest Carlsberg Research Laboratory isolates. In this study, we reveal the phylogenetic pattern of Brettanomyces species by comparing the predicted proteomes of each strain. Furthermore, we show that the Brettanomyces collection is well described using similarity in genomic organization, and that there is a direct correlation between genomic background and phenotypic characteristics. Particularly, genomic patterns affecting flavor production, maltose assimilation, beta-glucosidase activity, and phenolic off-flavor (POF) production are reported. This knowledge yields new insights into Brettanomyces population survival strategies, artificial selection pressure, and loss of carbon assimilation traits. On a species-specific level, we have identified for the first time a POF negative Brettanomyces anomalus strain, without the main spoilage character of Brettanomyces species. This strain (CRL-90) has lost DaPAD1, making it incapable of converting ferulic acid to 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP). This loss of fun

Many factors strongly influence the enzymatic hydrolysis of biomass to fermentable sugars (feedstock composition, pretreatment, enzymes and enzyme loading). In order to optimize the reaction conditions for the hydrolysis of biomass, an accurate high-throughput bioprocess development tool is mandatory, which enables a parallelization and an easy scale-up. New S-shaped impellers were developed for magnetically inductive driven stirred-tank bioreactors at a 10 mL-scale. An efficient and reproducible homogenization was shown at 20% w/w solids loading of microcrystalline cellulose and at, 4-10% with wheat straw in 48 parallel operated stirred-tank bioreactors. The scale-up was successfully validated for the enzymatic hydrolysis of wheat straw suspensions and microcrystalline cellulose mixtures by application of a cellulase complex at a milliliter- and liter-scale. As an example, the parallel stirred-tank bioreactor system was applied for the evaluation of enzymatic batch hydrolyses of plant materials with varying pretreatments.

Many factors strongly influence the enzymatic hydrolysis of biomass to fermentable sugars (feedstock composition, pretreatment, enzymes and enzyme loading). In order to optimize the reaction conditions for the hydrolysis of biomass, an accurate high-throughput bioprocess development tool is mandatory, which enables a parallelization and an easy scale-up. New S-shaped impellers were developed for magnetically inductive driven stirred-tank bioreactors at a 10 mL-scale. An efficient and reproducible homogenization was shown at 20% w/w solids loading of microcrystalline cellulose and at, 4-10% with wheat straw in 48 parallel operated stirred-tank bioreactors. The scale-up was successfully validated for the enzymatic hydrolysis of wheat straw suspensions and microcrystalline cellulose mixtures by application of a cellulase complex at a milliliter- and liter-scale. As an example, the parallel stirred-tank bioreactor system was applied for the evaluation of enzymatic batch hydrolyses of plant materials with varying pretreatments.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest. The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes. The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsuta ⁄ Trametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose. T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugar

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs., QC 20160808

Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest. The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes. The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsuta ⁄ Trametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose. T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugar

Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat ) and enzymatic efficiency (kcat /Km ). Four enzymes displayed an average seven-fold acceleration, with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.

Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat ) and enzymatic efficiency (kcat /Km ). Four enzymes displayed an average seven-fold acceleration, with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest. The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes. The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsuta ⁄ Trametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose. T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugar

Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest. The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes. The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsuta ⁄ Trametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose. T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugar

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. beta-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the beta-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the beta-glucosidase from H. jecorina (HjCel3A) with the beta-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 beta-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest. The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes. The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsuta ⁄ Trametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose. T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugar

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated., QC 20150331

© 2014 Elsevier Masson SAS. Pyranoid-type glycomimetics having a cis-1,2-fused glucopyranose-2-alkylsulfanyl-1,3-oxazoline (Glc-PSO) structure exhibit an unprecedented specificity as inhibitors of mammalian β-glucosidase. Notably, their inhibitory potency against human β-glucocerebrosidase (GCase) was found to be strongly dependent on the nature of aglycone-type moieties attached at the sulfur atom. In the particular case of υ-substituted hexadecyl chains, an amazing influence of the terminal group was observed. A comparative study on a series of Glc-PSO derivatives suggests that hydrogen bond acceptor functionalities, e.g. fluoro or methyloxycarbonyl, significantly stabilize the Glc-PSO:GCase complex. The S-(16-fluorohexadecyl)-PSO glycomimetic turned out to be a more potent GCase competitive inhibitor than ambroxol, a non glycomimetic drug currently in pilot trials as a pharmacological chaperone for Gaucher disease. Moreover, the inhibition constant increased by one order of magnitude when shifting from neutral (pH 7) to acidic (pH 5) media, a favorable characteristic for a chaperone candidate. Indeed, the fluoro-PSO derivative also proved superior to ambroxol in mutant GCase activity enhancement assays in N370S/N370S Gaucher fibroblasts. The results presented here represent a proof of concept of the potential of exploiting long-range non-glycone interactions for the optimization of glycosidase inhibitors with chaperone activity.

Heterobasidion annosum sensu lato is a serious pathogen causing root and stem rot to conifers in the northern hemisphere and rendering the timber defective for sawing and pulping. In this study we applied next-generation sequencing to i) identify transcriptional responses unique to Heterobasidion-inoculated Norway spruce and ii) investigate the H. annosum transcripts to identify putative virulence factors. To address these objectives we wounded or inoculated 30-year-old Norway spruce clones with H. annosumand 454-sequenced the transcriptome of the interaction at 0, 5 and 15 days post inoculation. The 491860 high-quality reads were de novo assembled and the relative expression was analysed. Overall, very few H. annosum transcripts were represented in our dataset. Three delta-12 fatty acid desaturase transcripts and one Clavaminate synthase-like transcript, both associated with virulence in other pathosystems, were found among the significantly induced transcripts. The analysis of the Norway spruce transcriptional responses produced a handful of differentially expressed transcripts. Most of these transcripts originated from genes known to respond to H. annosum. However, three genes that had not previously been reported to respond to H. annosum showed specific induction to inoculation: an oxophytodienoic acid-reductase (OPR), a beta-glucosidaseand a germin-like protein (GLP2) gene. Even in a small data set like ours, five novel highly expressed Norway spruce transcripts without significant alignment to any previously annotated protein in Genbank but present in the P. abies (v1.0) gene catalogue were identified. Their expression pattern suggests a role in defence. Therefore a more complete survey of the transcriptional responses in the interactions between Norway spruce and its major pathogen H. annosumwould probably provide a better understanding of gymnosperm defence than accumulated until now.

Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.

Endo-beta-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 degrees C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-beta-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences. (C) 2013 Elsevier Inc. All rights reserved.

Endo-beta-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 degrees C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-beta-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences. (C) 2013 Elsevier Inc. All rights reserved.

Endo-beta-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 degrees C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-beta-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences. (C) 2013 Elsevier Inc. All rights reserved.

ScopeThis study compares conversion of three major soy isoflavone glucosides and their aglycones in a series of in vitro intestinal models. Methods and resultsIn an in vitro human digestion model isoflavone glucosides were not deconjugated, whereas studies in a Caco-2 transwell model confirmed that deconjugation is essential to facilitate transport across the intestinal barrier. Deconjugation was shown upon incubation of the isoflavone glucosides with rat as well as human intestinal S9. In incubations with rat intestinal S9 lactase phlorizin hydrolase, glucocerebrosidase, and cytosolic broad-specific -glucosidase all contribute significantly to deconjugation, whereas in incubations with human intestinal S9 deconjugation appeared to occur mainly through the activity of broad-specific -glucosidase. Species differences in glucuronidation and sulfation were limited and generally within an order of magnitude with 7-O-glucuronides being the major metabolites for all three isoflavone aglycones and the glucuronidation during first pass metabolism being more efficient in rats than in humans. Comparison of the catalytic efficiencies reveals that deconjugation is less efficient than conjugation confirming that aglycones are unlikely to enter the systemic circulation. ConclusionAltogether, the data point at possible differences in the characteristics for intestinal conversion of the major soy isoflavones between rat and human, especially with respect to their deconjugation.

Background: The commercially important glycoside hydrolase family 3 (GH3) beta-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined. Methods: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 beta-glucosidase from A. niger ASKU28. Results: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K-m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on add catalysis, although changes in the rate-determining catalytic step were revealed for both, pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl beta-glucosides and yielded beta-glucosyl azide as a product. Conclusions and general significance: These results strongly support the assignment of E490 as the acid/base catalyst in a beta-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members., QC 20130325

This study is a first attempt to investigate the impact of urban contamination on metal tolerance of heterotrophic river biofilms using a short-term test based on beta-glucosidase activity. Tolerance levels to Cu, Cd, Zn, Ni and Pb were evaluated for biofilms collected at three sites along an urban gradient in the Seine river (France). Metallic pollution increased along the river, but concentrations remained low compared to environmental quality standards. Biofilm metal tolerance increased downstream from the urban area. Multivariate analysis confirmed the correlation between tolerance and contamination and between multi-metallic and physico-chemical gradients. Therefore, tolerance levels have to be interpreted in relation to the whole chemical and physical characteristics and not solely metal exposure. We conclude that community tolerance is a sensitive biological response to urban pressure and that mixtures of contaminants at levels lower than quality standards might have a significant impact on periphytic communities. (C) 2011 Elsevier Ltd. All rights reserved.

This study is a first attempt to investigate the impact of urban contamination on metal tolerance of heterotrophic river biofilms using a short-term test based on beta-glucosidase activity. Tolerance levels to Cu, Cd, Zn, Ni and Pb were evaluated for biofilms collected at three sites along an urban gradient in the Seine river (France). Metallic pollution increased along the river, but concentrations remained low compared to environmental quality standards. Biofilm metal tolerance increased downstream from the urban area. Multivariate analysis confirmed the correlation between tolerance and contamination and between multi-metallic and physico-chemical gradients. Therefore, tolerance levels have to be interpreted in relation to the whole chemical and physical characteristics and not solely metal exposure. We conclude that community tolerance is a sensitive biological response to urban pressure and that mixtures of contaminants at levels lower than quality standards might have a significant impact on periphytic communities. (C) 2011 Elsevier Ltd. All rights reserved.

This study is a first attempt to investigate the impact of urban contamination on metal tolerance of heterotrophic river biofilms using a short-term test based on beta-glucosidase activity. Tolerance levels to Cu, Cd, Zn, Ni and Pb were evaluated for biofilms collected at three sites along an urban gradient in the Seine river (France). Metallic pollution increased along the river, but concentrations remained low compared to environmental quality standards. Biofilm metal tolerance increased downstream from the urban area. Multivariate analysis confirmed the correlation between tolerance and contamination and between multi-metallic and physico-chemical gradients. Therefore, tolerance levels have to be interpreted in relation to the whole chemical and physical characteristics and not solely metal exposure. We conclude that community tolerance is a sensitive biological response to urban pressure and that mixtures of contaminants at levels lower than quality standards might have a significant impact on periphytic communities. (C) 2011 Elsevier Ltd. All rights reserved.

The word sustainability is more and more frequently used within the field of chemistry and in many other fields. Green or sustainable chemistry was introduced during the 90 ́s as a field of chemistry where the environmental and health impacts of processes and products are evaluated and improved. Within analytical chemistry the awareness of sustainability has also increased, which has resulted in optimisation and development of new techniques and methods. The easiest way to improve the sustainability of an analytical method is to decrease and/or replace the use of hazardous chemicals with ones known to have less negative impact on the environment. This thesis reports on a study of the extraction and hydrolysis of quercetin glucosides from onion using sustainable solvents and catalysts. Water at elevated temperatures has been used as a green extraction solvent and, to catalyse the hydrolysis reaction, a thermostable beta-glucosidase expressed from Thermotoga neapolitana, TnBgl1A has been used. It is shown that the use of water as extraction solvent and enzyme as catalyst have less environmental impact than the conventional method where aqueous methanol and a high concentration of HCl are used as extraction solvent and catalyst, respectively. In studying the rate of hydrolysis, it was found that glucose bound at the 3-position of quercetin was slower to hydrolyse than glucose bound at 4 ́-position when TnBgl1A was used as catalyst. Different amino acids in the active site of TnBgl1A were mutated and one mutant, TnBgl1A_N221S/P342L, showed an increased hydrolysis rate of glucose at 4 ́-position, but also of glucose at 3-position, compared to the wild type enzyme. TnBgl1A_N221S/P342L was then immobilised to support materials to allow reuse and also to be used in a continuous flow system. The combination of extraction and hydrolysis of quercetin glucosides from onion in a continuous flow system was not as trivial as originally expected. The total time was quite long, 2 ho

Byla provedena literární rešerše týkající se detekce beta-glukosidáz (EC 3.2.1.21) s ohledem na využití v diagnostice nebezpečných patogenů. Kromě detekce bakterie Listeria monocytogenes v potravinách lze uvažovat i o využití popsaných metod k diagnostice ischemizace tenkého střeva u lidských pacientů. V experimentální části práce byly prověřeny tři metody syntézy 2-amino-5-brombenzoové kyseliny s výtěžky 50-64%. Reakcí s kyselinou chloroctovou byla 2-amino-5-brombenzoová kyselina převedena na 5-brom-2-(karboxymethylamino)benzoovou kyselinu (51%) z které byl následně připraven 3-acetoxy-1-acetyl-5-bromindol (89%)., A research related to methods for detection of beta-glucosidase in order to possible use in diagnosis of dangerous patogens has been done. Besides the detection of bacterium Listeria monocytogenes in food, described methods can be potentially used in diagnosis of small intestine ischemic injury of human patients. In experimental part were verified three ways of synthesis 2-amino-5-bromobenzoic acid with yields 50-64%. In next step, 2-amino-5-bromobenzoic acid reacted with chloroacetic acid obtaining 5-bromo-2-(carboxymethylamino)benzoic acid (51%), which was consequently converted into 3-acetoxy-1-acetyl-5-bromoindole (89%)., Ústav organické chemie a technologie, Student přednesl cca 10-ti minutovou obhajobu své bakalářské práce s názvem Diagnostické systémy založené na detekci beta-glukosidáz. Poté bylo přečteno hodnocení školitele. K obhajobě byly vzneseny následující dotazy a připomínky: prof. Ing. Petr Kalenda, CSc. Jaké je praktické využití Vašich výsledků? Zeptal se na typ polymerního materiálu pro imobilizaci detekčního systému. doc. Ing. Petr Šimůnek, Ph.D. Zeptal se na typ oxidačního činidla pro oxidaci indolu.

Recent sequencing of a number of fungal genomes has revealed the presence of multiple putative beta-glucosidases. Here, we report the cloning of two beta-glucosidase genes (bg1 and aven1), which have very different biological functions and represent two of a number of beta-glucosidases from Talaromyces emersonii. The bg1 gene, encoding a putative intracellular beta-glucosidase, shows significant similarity to other fungal glucosidases from glycosyl hydrolase family 1, known to be involved in cellulose degradation. Solka floc, methyl-xylose, gentiobiose, beech wood xylan, and lactose induced expression of bg1, whereas glucose repressed expression. A second beta-glucosidase gene isolated from T. emersonii, aueni, encodes a putative avenacinase, an enzyme that deglucosylates the anti-fungal saponin, avenacin, rendering it less toxic to the fungus. This gene displays high homology with other fungal saponin-hydrolysing enzymes and beta-glucosidases within GH3. A putative secretory signal peptide of 21 amino acids was identified at the N-terminus of the predicted aven1 protein sequence suggesting that this enzyme is extracellular. Furthermore, T. emersonii cultivated on oat plant biomass was shown to deglucosylate avenacin. The presence of the avenacinase transcript was confirmed by RT-PCR on RNA extracted from mycelia grown in the presence of avenacin. The expression pattern of aven1 on various carbon sources was distinctly different from that of bg1. Only methyl-xylose and gentiobiose induced transcription of aven1. Gentiobiose induces synthesis of a number of cellulase genes by T. emersonii and it may be a possible candidate for the natural cellulase inducer observed in Penicillium purpurogenum. This work represents the first report of an avenacinase gene from a thermophilic, saprophytic fungal source, and suggests that this gene is not exclusive to plant pathogens., QC 20100525

Glycosidehydrolasesform a class of enzymes that play an important role in sugar-converting processes. They are applied as biocatalyst in both the hydrolysis of natural polymers to mono- andoligo-saccharides, and the reverse hydrolysis ortransglycosylation, by which oligosaccharides are synthesized from mono- and disaccharides. The latter synthesis process requires high substrate concentrations; to avoid technical problems (e.g. poor solubility, low diffusion rate, microbial contamination) this process is best performed at higher temperatures. Specific oligosaccharides (prebiotics) can stimulate the growth of so-called beneficialmicroorganismsin the gastro-intestinal tract. The complexity of this ecological system requires a specific combination of oligosaccharides which appears not to be present in currently available commercial preparations. Therefore, the production of highly specific oligosaccharides is desired. Biocatalysts for such a synthesis preferably need to have high catalytic specificity as well as high thermo-activity, and -stability. In this context, the work described in this thesis is aimed to gain knowledge on (i) catalytic mechanisms as well as stability strategies ofthermostableglycosidehydrolases, (ii) state-of-the-art engineering methods to optimize these features, and (iii) novel molecular strategies to enhance protein production.Three classes of glycosidehydrolaseshave been selected for detailed analysis:a-galactosidase,b-glucosidaseandb-glucanase. Thea-galactosidasefrom thehyperthermophilicarchaeonPyrococcusfuriosus has been cloned, functionally produced and characterized. Successful identification of its catalyticnucleophileallows for the future application of asynthase. Theb-glucanasefrom P.furiosus (laminarinase,LamA) has been converted into aglycosynthaseby a similarnucleophilemutation:glycosylationwas observed with yields of up to 30% of oligosaccharides. In addition, analysis of the molecular basis for the extremely high chemical and the

The distribution of substituents along the polymer backbone will have a strong influence on the properties of modified cellulose. Endoglucanases were used to degrade a series of hydroxypropyl cellulose (HPC) derivatives with a high degree of substitution. The HPCs were characterized with cloud-point analysis prior to degradation. The extent of enzymatic degradation was determined with size-exclusion chromatography with online multi-angle light scattering and refractive index detection and also with high-pH anion exchange chromatography with pulsed amperometric detection. To further characterize the formed products, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was employed for analysis of short-chained oligosaccharides. The different endoglucanases showed varying degradation capability depending on structure of the active site. The highly substituted HPCs had different susceptibility to degradation by the endoglucanases. The results show a difference in substituent distribution between HPCs, which would explain the differing cloud-point behaviors. Increased number of regions with low substitution could be, correlated with lower polymer cloud point. The study shows the usefulness of enzymatic degradation to study the distribution of substituents in soluble biopolymer derivates., QC 20100525

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-laminarin oligosaccharides of degree of polymerisation 2-8 were synthesized enzymatically. Catalytic efficiency values, k (cat)/K (m), of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-beta-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside., QC 20100525

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth., QC 20100525

Two strategies to increase the productivity of secreted Thai Rosewood beta-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K(L)a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g(-1) g(-1)) and high-pressure processes (0.035 g g(-1) g(-1)), compared to 0.022 g g(-1) g(-1) in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total beta-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the beta-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins., QC 20100525

The purpose of this study was to measure the effects of plant growth on soil microbial biomass C (C-mic) and soil enzyme activities. In a pot experiment using spring barley and sugar beet, we investigated the response of C-mic, hot water extractable C and N fractions (C-hwe, N-hwe), and enzyme activities involved in C, N and P cycling in a loess-derived Chernozem from Bad Lauchstadt (Central Germany). The study site has been receiving the same fertilisation treatments for 100 years. The soil originated from plots fertilised with 15 t ha(-1) farmyard manure (FYM) year(-1) + mineral fertiliser (NPK), or 15 t ha(-1) FYM year(-1), or NPK or from an unfertilised control. Pots were sampled monthly, and alkaline phosphatase- (AP), beta-glucosidase- and protease-activities were analysed. At the beginning and the end of study, levels of C-mic, C-hwe and N-hwe were also measured. All three enzyme activities and C-mic were significantly and positively correlated with C-org and C-hwe. Results suggest that the enzyme activities measured originated mostly from microorganisms and that C-hwe is an important C source for soil microorganisms. beta-Glucosidase and AP activities were higher in summer months than at other times. In contrast, protease activity changed only slightly during the growing period.

The economic crisis at the beginning of the eighties impacted 20th-century Costa Rica inmany different ways. Government programs were successful in reducing the proportionof poor people from 35% in 1985 to 23% in 2000. This article utilizes official HouseholdSurveys corresponding to the period 1981-2002 and Population Censuses to performan estimation of age, cohort, and period effects, in order to show that poverty amongthe elderly can be understood as a cohort effect. Their poverty conditions areassociated with their low schooling, mainly among men. The probability of having theright to a retirement pension and the fact that individuals with lower educational levelsearned low income during their later working years, intervene in the relationshipbetween schooling and poverty as a cohort effect.Soon after the end of the civil war in 1948, Costa Rica underwent political and economicchanges that aided the expansion of the social benefits initialized in the 40’s, such asuniversalizing Social Security, promoting health and educational policy, and providinginfrastructure and services to both rural and urban communities. Nevertheless, at theend of the 70’s, increasing oil prices had a strong negative impact on many countriesaround the world. Costa Rica was no exception, and during the 1980 – 1982 period itwent through an economic crisis characterized by hyperinflation, increasedunemployment and underemployment rates, and the declaration of a moratorium on foreign debt payments (Barahona Montero 1999a). The governments after 1982 wererelatively successful in promoting economic recovery by changing the developmentmodel based on import substitution to one promoting non-traditional product exportsand tourism (Barahona Montero 1999a, González Mejía 1999). In spite of the recoveryand public policies designed to combat poverty, since 1991, it has not been possible toreduce the proportion of poor households below 18%, maintaining an annual average ofaround 20%.2 In addition, econo

The economic crisis at the beginning of the eighties impacted 20th-century Costa Rica inmany different ways. Government programs were successful in reducing the proportionof poor people from 35% in 1985 to 23% in 2000. This article utilizes official HouseholdSurveys corresponding to the period 1981-2002 and Population Censuses to performan estimation of age, cohort, and period effects, in order to show that poverty amongthe elderly can be understood as a cohort effect. Their poverty conditions areassociated with their low schooling, mainly among men. The probability of having theright to a retirement pension and the fact that individuals with lower educational levelsearned low income during their later working years, intervene in the relationshipbetween schooling and poverty as a cohort effect.Soon after the end of the civil war in 1948, Costa Rica underwent political and economicchanges that aided the expansion of the social benefits initialized in the 40’s, such asuniversalizing Social Security, promoting health and educational policy, and providinginfrastructure and services to both rural and urban communities. Nevertheless, at theend of the 70’s, increasing oil prices had a strong negative impact on many countriesaround the world. Costa Rica was no exception, and during the 1980 – 1982 period itwent through an economic crisis characterized by hyperinflation, increasedunemployment and underemployment rates, and the declaration of a moratorium on foreign debt payments (Barahona Montero 1999a). The governments after 1982 wererelatively successful in promoting economic recovery by changing the developmentmodel based on import substitution to one promoting non-traditional product exportsand tourism (Barahona Montero 1999a, González Mejía 1999). In spite of the recoveryand public policies designed to combat poverty, since 1991, it has not been possible toreduce the proportion of poor households below 18%, maintaining an annual average ofaround 20%.2 In addition, econo

Pre-plant fumigation of agricultural soils with a combination of methyl bromide (MeBr) and chloropicrin (CP) to control nematodes, soil-borne pathogens and weeds has been a common practice in strawberry (Fragaria X ananassa Duchesne) production since the 1960s. MeBr will be phased out by 2005, but little is known about the impacts of alternative fumigants on soil microbial processes. We investigated the response of microbial biomass and enzyme activities in soils fumigated over two years with MeBr+CP and the alternatives propargyl bromide (PrBr), InLine, Midas and CP. Results were compared to control soils, which were not fumigated for the last 4-5 years for Watsonville and Oxnard, respectively, but had a 10 year history of MeBr+CP fumigation (history soils). Soil samples (0-15 cm) were taken from two sites in the coastal areas of California, USA, in Watsonville and Oxnard, at peak strawberry production after two years of repeated application. In addition to the soil enzymes, the activities of purified reference enzymes of beta-glucosidase, acid phosphatase and arylsulfatase were assayed before and after fumigation with MeBr+CP and alternative biocides. At the Oxnard site, microbial respiration significantly decreased in soils fumigated with MeBr+ CP (P=0.036), while microbial biomass C and N showed no response to fumigation at both sites. These results may indicate that fumigation promotes the growth of resistant species or that soil microorganisms had recovered at the time of sampling. Repeated soil fumigation with MeBr+CP significantly decreased the activities of P-glucosidase and acid phosphatase at the Watsonville site, and dehydrogenase activity at the Oxnard site. Although, enzyme activities in soils fumigated with PrBr, InLine, Midas and CP were lower compared to the control soil, effects were, in general, not significant. Fumigation with MeBr+ CP and alternatives reduced the activities of purified reference enzymes by 13, 76 and 28% for acid phosphatase, be

Pre-plant fumigation of agricultural soils with a combination of methyl bromide (MeBr) and chloropicrin (CP) to control nematodes, soil-borne pathogens and weeds has been a common practice in strawberry (Fragaria X ananassa Duchesne) production since the 1960s. MeBr will be phased out by 2005, but little is known about the impacts of alternative fumigants on soil microbial processes. We investigated the response of microbial biomass and enzyme activities in soils fumigated over two years with MeBr+CP and the alternatives propargyl bromide (PrBr), InLine, Midas and CP. Results were compared to control soils, which were not fumigated for the last 4-5 years for Watsonville and Oxnard, respectively, but had a 10 year history of MeBr+CP fumigation (history soils). Soil samples (0-15 cm) were taken from two sites in the coastal areas of California, USA, in Watsonville and Oxnard, at peak strawberry production after two years of repeated application. In addition to the soil enzymes, the activities of purified reference enzymes of beta-glucosidase, acid phosphatase and arylsulfatase were assayed before and after fumigation with MeBr+CP and alternative biocides. At the Oxnard site, microbial respiration significantly decreased in soils fumigated with MeBr+ CP (P=0.036), while microbial biomass C and N showed no response to fumigation at both sites. These results may indicate that fumigation promotes the growth of resistant species or that soil microorganisms had recovered at the time of sampling. Repeated soil fumigation with MeBr+CP significantly decreased the activities of P-glucosidase and acid phosphatase at the Watsonville site, and dehydrogenase activity at the Oxnard site. Although, enzyme activities in soils fumigated with PrBr, InLine, Midas and CP were lower compared to the control soil, effects were, in general, not significant. Fumigation with MeBr+ CP and alternatives reduced the activities of purified reference enzymes by 13, 76 and 28% for acid phosphatase, be

A range of new C-1 modified derivatives of the powerful glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol has been synthesised and their biological activities probed with the beta-glucosidase from Agrobacterium sp. Ki values are compared with those of previously prepared close relatives. Findings suggest dramatic effects exerted by the aglycon binding site on substrate/inhibitor binding.

A range of new C-1 modified derivatives of the powerful glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol has been synthesised and their biological activities probed with the beta-glucosidase from Agrobacterium sp. Ki values are compared with those of previously prepared close relatives. Findings suggest dramatic effects exerted by the aglycon binding site on substrate/inhibitor binding.

β-Glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety such as cellobiose and other beta-linked oligosaccharides. In maize (Zea mays L.), β-glucosidase exists as 120 kD homodimers, but also forms high-molecular-weight (HMW) aggregates in certain maize inbreds (nulls). In this study we show that the null β-glucosidase phenotype is caused by the formation of HMW enzyme aggregates (>1.5 X 10⁶ Daltons), caused by a β-glucosidase aggregating factor (BGAF). BGAF is a 32 kD protein that binds specifically to β-glucosidase and renders it insoluble during extraction. The data unequivocally demonstrate that BGAF is solely responsible for β-glucosidase aggregation and insolubility, and thus, the apparent null phenotype. Additionally, I have isolated the cDNA encoding BGAF and have identified BGAF as a member of the small heat-shock protein (sHsp) family. Interestingly, BGAF binds to both maize β-glucosidase isozymes (Glu1 and Glu2), but does not bind to their sorghum homolog Dhurrinase-1 (Dhr1; Sorghum beta-glucosidase), that shares 70% sequence identity with Glu1 and Glu2. Therefore, these proteins provide an excellent system to study functional differences at nonconserved residues and elucidate the mechanism of enzyme aggregation and insolubility. By examining the behavior of β-glucosidase chimeras in binding assays, I demonstrate that BGAF binding is conformation dependent, highly specific, and reminiscent of antigen-antibody interactions. Additionally, I have identified two disparate polypeptide segments in the primary structure of the maize beta-glucosidase isozyme Glu1 that form a BGAF binding site in the tertiary structure of the enzyme.

Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase dan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D- lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2,2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2,6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases., QC 20100525

Objective: To study the effect of the intake of 15 g nondigestible oligosaccharides per day on various parameters of large-bowel function, as well as on blood lipid concentrations and glucose absorption in man. Design: Latin square, randomized, double-blind, diet-controlled. Setting: Metabolic research unit. Subjects: Twelve apparently healthy men (mean age 23 years), recruited from the Institute's pool of volunteers, no drop-outs. Interventions: Four treatment periods of 3 weeks: inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and control; analyses of stool weight, intestinal transit, faecal pH, short-chain fatty acids, bile acids, faecal enzymes, blood lipids and glucose absorption. Results: As compared to the control treatment: higher concentration of faecal acetate (inulin and GOS, P < 0.05) and valerate (inulin, P < 0.05), significantly lower concentration of faecal deoxycholic acid (inulin and FOS, P < 0.05 and P < 0.02, respectively) and β-glucuronidase activity (inulin and GOS, P < 0.05 and P < 0.02 respectively). Other changes of faecal parameters and those of blood lipids and glucose absorption were statistically not significant. Conclusions: Results indicate that nondigestible oligosaccharides are (partly) fermented in the human colon, but in healthy young men the effects are limited. Also the consumption of 15 g nondigestible oligosaccharides does not seem to alter blood lipid concentrations and glucose absorption in our young healthy adults.

A 4.7-kb DNA insert encoding a secretory cellobiase (Cba) was cloned from Cellulomonas biazotea in Escherichia coli using an excretion vector, pM. Host cells transformed with the recombinant construct, designated pBZ4.7, were able to utilize cellobiose as the sole carbon source. Part of the Cba activity encoded by pBZ4.7 could be detected in the periplasm and even in the culture supernatant. The Cba protein was purified from the culture supernatant and analysed by SDS-PAGE to have an apparent M-r of 86 000. The insert consisted of two PstI fragments with lengths of 0.75 and 3.95 kb, both of which were found to be crucial for expressing the Cba activity. Sequencing of the first 3.95 kb of the insert revealed that the coding sequence for Cba, designated the cba gene, was 2484 bp long. Comparison of the deduced Cba sequence with those of published beta-glucosidases revealed a potential active site located at the N-terminal portion of the former. The cba gene has a high G+C content of 76.4\% and is flanked by a putative ribosome-binding site and potential transcriptional termination signals upstream and downstream from its coding sequence, respectively. (C) 1998 Elsevier Science B.V.

A 4.7-kb DNA insert encoding a secretory cellobiase (Cba) was cloned from Cellulomonas biazotea in Escherichia coli using an excretion vector, pM. Host cells transformed with the recombinant construct, designated pBZ4.7, were able to utilize cellobiose as the sole carbon source. Part of the Cba activity encoded by pBZ4.7 could be detected in the periplasm and even in the culture supernatant. The Cba protein was purified from the culture supernatant and analysed by SDS-PAGE to have an apparent M-r of 86 000. The insert consisted of two PstI fragments with lengths of 0.75 and 3.95 kb, both of which were found to be crucial for expressing the Cba activity. Sequencing of the first 3.95 kb of the insert revealed that the coding sequence for Cba, designated the cba gene, was 2484 bp long. Comparison of the deduced Cba sequence with those of published beta-glucosidases revealed a potential active site located at the N-terminal portion of the former. The cba gene has a high G+C content of 76.4\% and is flanked by a putative ribosome-binding site and potential transcriptional termination signals upstream and downstream from its coding sequence, respectively. (C) 1998 Elsevier Science B.V.