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Somatic embryogenesis is a promising method of propagation of conifers, but it requires optimized protocols according to the different stages of the process and the model species. Pinus halepensis Mill. (Aleppo pine) is a species widely used in reforestation and the somatic embryogenesis procedure was successfully developed, but even so, there is low germination and conversion of somatic embryos into plants. In this sense, promoting changes in the chemical environment in the germination stage is an alternative to increase germination rates and the consequent obtaining of somatic plants. Taking this into account, the objective of this work was to evaluate the influence of different carbohydrate sources applied during the germination stage of the somatic embryos of P. halepensis, on the success of this process and the morphology of the somatic plants obtained. A statistically significant increase in germination rates, total length of somatic plants, as well as principal root length was observed when somatic embryos were cultured in maltose-supplemented germination medium., La embriogénesis somática es un método prometedor de propagación de coníferas, pero necesita de protocolos optimizados de acuerdo con las diferentes etapas del proceso y la especie modelo. Pinus halepensis Mill. (pino carrasco) es una especie utilizada ampliamente en la reforestación y se logró desarrollar el procedimiento de embriogénesis somática satisfactoriamente pero aun así, existe baja germinación y conversión de embriones somáticos en plantas. En este sentido, promover cambios en el ambiente químico en la etapa de germinación es una alternativa para aumentar las tasas de germinación y la consecuente obtención de plantas somáticas. Teniendo esto en cuenta, el objetivo de este trabajo fue evaluar la influencia de diferentes fuentes de carbohidratos aplicadas durante la etapa de germinación de los embriones somáticos de P. halepensis, sobre el éxito de este proceso y la morfología de las plantas somáticas obtenidas. Se observó un aumento estadísticamente significativo en las tasas de germinación, en la longitud total de las plantas somáticas, así como en la longitud de la raíz principal cuando los embriones somáticos fueron cultivados en el medio de germinación suplementado con maltosa.

Microorganisms often live in complex habitats, where changes in the environment are predictable, providing an opportunity for microorganisms to learn, anticipate the upcoming environmental changes and prepare in advance for better survival and growth. One such environment is the mammalian intestine, where the abundance of different carbon sources is spatially distributed. In this study, we identified seven spatially distributed carbon sources in the mammalian intestine and tested whether Escherichia coli exhibits phenotypes that are consistent with an anticipatory response given their spatial order and abundance within the mammalian intestine. Through RNA-Seq and RT-PCR validation measurements, we found that there was a 67% match in the expression patterns between the measured phenotypes and what would otherwise be expected in the case of anticipatory behavior, while 83% and 0% were in agreement with the homeostatic and random response, respectively. To understand the genetic and phenotypic basis of the discrepancies between the expected and measured anticipatory responses, we thoroughly investigated the discrepancy in D-galactose treatment and the expression of maltose operon in E. coli. Here, the expected anticipatory response, based on the spatial distribution of D-galactose and D-maltose, was that D-galactose should upregulate the maltose operon, but it was the opposite in experimental validation. We performed whole genome random mutagenesis and screening and identified E. coli strains with positive expression of maltose operon in D-galactose. Targeted Sanger sequencing and mutation repair identified that the mutations in the promoter region of malT and in the coding region of the crp gene were the factors responsible for the reversion in the association. Further, to identify why positive association in the D-galactose treatment and the expression of the maltose operon did not evolve naturally, fitness measurements were performed. Fitness experiments demonstrate

Microorganisms often live in complex habitats, where changes in the environment are predictable, providing an opportunity for microorganisms to learn, anticipate the upcoming environmental changes and prepare in advance for better survival and growth. One such environment is the mammalian intestine, where the abundance of different carbon sources is spatially distributed. In this study, we identified seven spatially distributed carbon sources in the mammalian intestine and tested whether Escherichia coli exhibits phenotypes that are consistent with an anticipatory response given their spatial order and abundance within the mammalian intestine. Through RNA-Seq and RT-PCR validation measurements, we found that there was a 67% match in the expression patterns between the measured phenotypes and what would otherwise be expected in the case of anticipatory behavior, while 83% and 0% were in agreement with the homeostatic and random response, respectively. To understand the genetic and phenotypic basis of the discrepancies between the expected and measured anticipatory responses, we thoroughly investigated the discrepancy in D-galactose treatment and the expression of maltose operon in E. coli. Here, the expected anticipatory response, based on the spatial distribution of D-galactose and D-maltose, was that D-galactose should upregulate the maltose operon, but it was the opposite in experimental validation. We performed whole genome random mutagenesis and screening and identified E. coli strains with positive expression of maltose operon in D-galactose. Targeted Sanger sequencing and mutation repair identified that the mutations in the promoter region of malT and in the coding region of the crp gene were the factors responsible for the reversion in the association. Further, to identify why positive association in the D-galactose treatment and the expression of the maltose operon did not evolve naturally, fitness measurements were performed. Fitness experiments demonstrate

Phosphorus has an essential role in cellular and extracellular metabolism; maintenance of normal phosphorus homeostasis is critical. Phosphorus homeostasis can be affected by diet and certain medications; some intravenous iron formulations can induce renal phosphate excretion and hypophosphatemia, likely through increasing serum concentrations of intact fibroblast growth factor 23. Case studies provide insights into two types of hypophosphatemia: acute symptomatic and chronic hypophosphatemia, while considering the role of pre-existing conditions and comorbidities, medications, and intravenous iron. This review examines phosphorus homeostasis and hypophosphatemia, with emphasis on effects of iron deficiency and iron replacement using intravenous iron formulations.

Phosphorus has an essential role in cellular and extracellular metabolism; maintenance of normal phosphorus homeostasis is critical. Phosphorus homeostasis can be affected by diet and certain medications; some intravenous iron formulations can induce renal phosphate excretion and hypophosphatemia, likely through increasing serum concentrations of intact fibroblast growth factor 23. Case studies provide insights into two types of hypophosphatemia: acute symptomatic and chronic hypophosphatemia, while considering the role of pre-existing conditions and comorbidities, medications, and intravenous iron. This review examines phosphorus homeostasis and hypophosphatemia, with emphasis on effects of iron deficiency and iron replacement using intravenous iron formulations.

Iron(III)-polymaltose pharmaceutical ferritin analogue Ferrifol® was investigated by high resolution transmission electron microscopy (HRTEM), X-ray diffraction, thermogravimetry, electron magnetic resonance (EMR) spectroscopy, direct current magnetization measurements and 57Fe Mössbauer spectroscopy to get novel information about the structural arrangement of the iron core. The Ferrifol® Mössbauer spectra measured in the range from 295 K to 90 K demonstrated non-Lorentzian two-peak pattern. These spectra were better fitted using a superposition of 5 quadrupole doublets with the same line width. The obtained Mössbauer parameters were different and an unusual line broadening with temperature decrease was observed. Measurements of the Ferrifol® Mössbauer spectra from 60 K to 20 K demonstrated a slow decrease of magnetic relaxation in the iron core. Zero-field-cooled and field-cooled magnetization measurements revealed a blocking temperature at ~33 K and a paramagnetic state of the Ferrifol® iron core at higher temperatures. Isothermal magnetization measurements at 5 K show that the saturation magnetic moment is ~0.31 emu/g. X-band EMR spectroscopy measurements revealed the presence of different magnetic species in the sample. Transmission electron microscopy demonstrated that the size of the iron cores in Ferrifol® is in the range 2–6 nm. The lattice periodicity in these iron cores, measured on the HRTEM images, vary in the range 2.2–2.7 Å. This can be best understood as sets of close packed O(OH) layers in ferrihydrite cores without long range correlation. © 2020 Elsevier Inc.

[ES] La obtención de líneas puras es un proceso de gran relevancia para la industria agronómica, pues a partir de su cruce, se pueden obtener híbridos comerciales de mayor rendimiento y homogeneidad en la producción. La mejora genética convencional permite obtener líneas puras tras siete u ocho generaciones de autofecundación, proceso demasiado largo y costoso. Por consiguiente, la obtención de dichas líneas conlleva la utilización de técnicas biotecnológicas si se desea ahorrar tiempo y costes de producción, con el fin de obtener individuos haploides y dobles haploides. Una de estas técnicas, denominada androgénesis, consiste en la regeneración de individuos haploides que han sido inducidos a partir del precursor del gametófito masculino, la microspora. En determinadas condiciones de estrés abiótico dichas células haploides se pueden inducir a proliferar y después duplicar su genoma, generando individuos dobles haploides, los cuales son interesantes porque son homocigotos para todos sus loci, por lo que pueden utilizarse como líneas puras. En el caso del pimiento destacan dos técnicas de obtención de dobles haploides: el cultivo de microsporas en medio líquido y el cultivo in vitro de anteras. La ventaja del cultivo de anteras frente al de microsporas es que las células están protegidas por la pared de la antera durante el cultivo, proporcionándole nutrientes y un ambiente más propicio para la inducción de haploides. No obstante, ambas técnicas no están aún optimizadas y presentan aún problemas de rendimiento en la obtención de embriones viables haploides o doble haploides en algunas variedades de pimiento. En este trabajo se han probado distintos medios de cultivo con el objetivo de optimizar el protocolo de obtención de dobles haploides a partir de anteras. Se trata, por lo tanto, de modificar las condiciones de estrés y posterior cultivo a las que se ven sometidas las microsporas para que tenga lugar la división celular y la proliferación que precede a la obtenc, [EN] Obtaining pure lines is a highly relevant process for the agronomic industry, since from their crossing, commercial hybrids of high yield and homogeneity in production can be obtained. Conventional plant breeding allows for the production of pure lines after seven or eight generations of self-fertilization, which is remarkably expensive and timeconsuming. Alternatively these lines can be obtained by using biotechnological approaches based on the production of haploids and doubled haploids, which save time and production costs. One of these approaches, known as induction of androgenesis consists in the regeneration of haploid individuals that have been induced from the precursor of the male gametophyte, the microspore. Under certain conditions of abiotic stress, haploid cells can be induced to proliferate and then duplicate their genome, generating doubled haploid individuals, which are interesting because they are homozygous for all of their loci, so they can be used as pure lines. In the case of pepper, two techniques for obtaining doubled haploids stand out: culture of microspores in liquid medium and in vitro culture of anthers. The advantage of anther culture compared to microspore culture is that the cells are protected by the anther wall during culture, providing them with nutrients and an environment more favorable to haploid induction. However, both techniques are not yet optimized and still present performance problems when applied to obtain viable haploid or doubled haploid embryos in some pepper varieties. In this work, different culture media have been tested in order to optimize the protocol for obtaining doubled haploids from anthers. We tried to modify the stress conditions and subsequent in vitro culture to which the microspores are subjected, in order to promote the initial cell division and proliferation that takes place as a consequence of induction. The factors analyzed include the addition of maltose or silver nitrate to the culture medium

[ES] La obtención de líneas puras es un proceso de gran relevancia para la industria agronómica, pues a partir de su cruce, se pueden obtener híbridos comerciales de mayor rendimiento y homogeneidad en la producción. La mejora genética convencional permite obtener líneas puras tras siete u ocho generaciones de autofecundación, proceso demasiado largo y costoso. Por consiguiente, la obtención de dichas líneas conlleva la utilización de técnicas biotecnológicas si se desea ahorrar tiempo y costes de producción, con el fin de obtener individuos haploides y dobles haploides. Una de estas técnicas, denominada androgénesis, consiste en la regeneración de individuos haploides que han sido inducidos a partir del precursor del gametófito masculino, la microspora. En determinadas condiciones de estrés abiótico dichas células haploides se pueden inducir a proliferar y después duplicar su genoma, generando individuos dobles haploides, los cuales son interesantes porque son homocigotos para todos sus loci, por lo que pueden utilizarse como líneas puras. En el caso del pimiento destacan dos técnicas de obtención de dobles haploides: el cultivo de microsporas en medio líquido y el cultivo in vitro de anteras. La ventaja del cultivo de anteras frente al de microsporas es que las células están protegidas por la pared de la antera durante el cultivo, proporcionándole nutrientes y un ambiente más propicio para la inducción de haploides. No obstante, ambas técnicas no están aún optimizadas y presentan aún problemas de rendimiento en la obtención de embriones viables haploides o doble haploides en algunas variedades de pimiento. En este trabajo se han probado distintos medios de cultivo con el objetivo de optimizar el protocolo de obtención de dobles haploides a partir de anteras. Se trata, por lo tanto, de modificar las condiciones de estrés y posterior cultivo a las que se ven sometidas las microsporas para que tenga lugar la división celular y la proliferación que precede a la obtenc, [EN] Obtaining pure lines is a highly relevant process for the agronomic industry, since from their crossing, commercial hybrids of high yield and homogeneity in production can be obtained. Conventional plant breeding allows for the production of pure lines after seven or eight generations of self-fertilization, which is remarkably expensive and timeconsuming. Alternatively these lines can be obtained by using biotechnological approaches based on the production of haploids and doubled haploids, which save time and production costs. One of these approaches, known as induction of androgenesis consists in the regeneration of haploid individuals that have been induced from the precursor of the male gametophyte, the microspore. Under certain conditions of abiotic stress, haploid cells can be induced to proliferate and then duplicate their genome, generating doubled haploid individuals, which are interesting because they are homozygous for all of their loci, so they can be used as pure lines. In the case of pepper, two techniques for obtaining doubled haploids stand out: culture of microspores in liquid medium and in vitro culture of anthers. The advantage of anther culture compared to microspore culture is that the cells are protected by the anther wall during culture, providing them with nutrients and an environment more favorable to haploid induction. However, both techniques are not yet optimized and still present performance problems when applied to obtain viable haploid or doubled haploid embryos in some pepper varieties. In this work, different culture media have been tested in order to optimize the protocol for obtaining doubled haploids from anthers. We tried to modify the stress conditions and subsequent in vitro culture to which the microspores are subjected, in order to promote the initial cell division and proliferation that takes place as a consequence of induction. The factors analyzed include the addition of maltose or silver nitrate to the culture medium

[ES] La obtención de líneas puras es un proceso de gran relevancia para la industria agronómica, pues a partir de su cruce, se pueden obtener híbridos comerciales de mayor rendimiento y homogeneidad en la producción. La mejora genética convencional permite obtener líneas puras tras siete u ocho generaciones de autofecundación, proceso demasiado largo y costoso. Por consiguiente, la obtención de dichas líneas conlleva la utilización de técnicas biotecnológicas si se desea ahorrar tiempo y costes de producción, con el fin de obtener individuos haploides y dobles haploides. Una de estas técnicas, denominada androgénesis, consiste en la regeneración de individuos haploides que han sido inducidos a partir del precursor del gametófito masculino, la microspora. En determinadas condiciones de estrés abiótico dichas células haploides se pueden inducir a proliferar y después duplicar su genoma, generando individuos dobles haploides, los cuales son interesantes porque son homocigotos para todos sus loci, por lo que pueden utilizarse como líneas puras. En el caso del pimiento destacan dos técnicas de obtención de dobles haploides: el cultivo de microsporas en medio líquido y el cultivo in vitro de anteras. La ventaja del cultivo de anteras frente al de microsporas es que las células están protegidas por la pared de la antera durante el cultivo, proporcionándole nutrientes y un ambiente más propicio para la inducción de haploides. No obstante, ambas técnicas no están aún optimizadas y presentan aún problemas de rendimiento en la obtención de embriones viables haploides o doble haploides en algunas variedades de pimiento. En este trabajo se han probado distintos medios de cultivo con el objetivo de optimizar el protocolo de obtención de dobles haploides a partir de anteras. Se trata, por lo tanto, de modificar las condiciones de estrés y posterior cultivo a las que se ven sometidas las microsporas para que tenga lugar la división celular y la proliferación que precede a la obtenc, [EN] Obtaining pure lines is a highly relevant process for the agronomic industry, since from their crossing, commercial hybrids of high yield and homogeneity in production can be obtained. Conventional plant breeding allows for the production of pure lines after seven or eight generations of self-fertilization, which is remarkably expensive and timeconsuming. Alternatively these lines can be obtained by using biotechnological approaches based on the production of haploids and doubled haploids, which save time and production costs. One of these approaches, known as induction of androgenesis consists in the regeneration of haploid individuals that have been induced from the precursor of the male gametophyte, the microspore. Under certain conditions of abiotic stress, haploid cells can be induced to proliferate and then duplicate their genome, generating doubled haploid individuals, which are interesting because they are homozygous for all of their loci, so they can be used as pure lines. In the case of pepper, two techniques for obtaining doubled haploids stand out: culture of microspores in liquid medium and in vitro culture of anthers. The advantage of anther culture compared to microspore culture is that the cells are protected by the anther wall during culture, providing them with nutrients and an environment more favorable to haploid induction. However, both techniques are not yet optimized and still present performance problems when applied to obtain viable haploid or doubled haploid embryos in some pepper varieties. In this work, different culture media have been tested in order to optimize the protocol for obtaining doubled haploids from anthers. We tried to modify the stress conditions and subsequent in vitro culture to which the microspores are subjected, in order to promote the initial cell division and proliferation that takes place as a consequence of induction. The factors analyzed include the addition of maltose or silver nitrate to the culture medium

Xanthophyllomyces dendrorhous is a carotenogenic yeast with a singular metabolic capacity to produce astaxanthin, a valuable antioxidant pigment. This yeast can assimilate several carbon sources and sustain fermentation even under aerobic conditions. Since astaxanthin biosynthesis is affected by the carbon source, the study of carotenogenesis regulatory mechanisms is key for improving astaxanthin yield in X. dendrorhous This study aimed to elucidate the regulation of the metabolism of different carbon sources and the phenomenon of catabolic repression in this yeast. To this end, protein and transcript levels were quantified by iTRAQ (isobaric tags for relative and absolute quantification) and transcriptomic sequencing (RNA-seq) in the wild-type strain under conditions of glucose, maltose, or succinate treatment and in the mutant strains for genes MIG1, CYC8, and TUP1 under conditions of glucose treatment. Alternative carbon sources such as maltose and succinate affected the relative abundances of 14% of the wild-type proteins, which were mainly grouped into the carbohydrate metabolism category, with the glycolysis/gluconeogenesis and citrate cycle pathways being the most highly represented pathways. Each mutant strain showed significant proteomic profile changes, affecting approximately 2% of the total proteins identified, compared to the wild-type strain under glucose treatment conditions. Similarly to the results seen with the alternative carbon sources, the changes in the mutant strains mainly affected carbohydrate metabolism, with glycolysis/gluconeogenesis and the pentose phosphate and citrate cycle pathways being the most highly represented pathways. Our results showed convergence between carbon assimilation and catabolic repression in the strains studied. Interestingly, indications of cooperative, opposing, and overlapping processes during catabolic regulation were found. We also identified target proteins of the regulatory processes, reinforcing the likeliho

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains., BT/Industrial Microbiology

Comparative studies of taste perception have found that primates may differ markedly in their sensitivity for substances perceived as sweet by humans. These findings raise questions about the reason that may underlie these differences in sweet-taste sensitivity between species. The aim of the present study was to assess the taste responsiveness of chimpanzees (Pan troglodytes verus) to seven substances tasting sweet to humans and to compare the results with those of other primate species. Using a two-bottle preference test (1 min) I found that the taste preference thresholds of the chimpanzees for five food-associated carbohydrates ranged between 20-30 mM for sucrose, 20-50 mM for fructose, 60-80 mM for glucose, 50-80 mM for maltose, and 30-80 mM for lactose. Taste preference thresholds for two steviol glycosides ranged from 0.04-0.05 mM for stevioside, and 0.03-0.05 mM for rebaudioside A. The chimpanzees displayed clear preferences for all sweet-tasting substances presented. In line with data obtained in other primates, the taste preference threshold of the chimpanzees for sucrose was lower compared to the other carbohydrates presented and the taste preference thresholds for stevioside and rebaudioside A were lower compared to sucrose. In general, the taste sensitivity of the chimpanzees fell into the range of data reported in other nonhuman primate species. Interestingly, the taste preference thresholds of the chimpanzees reported here are similar to the taste detection thresholds obtained in humans, despite the fact that the former are only a conservative approximation of an animal’s taste sensitivity. This suggests that chimpanzees may be as sweet-taste sensitive as humans.

Comparative studies of taste perception have found that primates may differ markedly in their sensitivity for substances perceived as sweet by humans. These findings raise questions about the reason that may underlie these differences in sweet-taste sensitivity between species. The aim of the present study was to assess the taste responsiveness of chimpanzees (Pan troglodytes verus) to seven substances tasting sweet to humans and to compare the results with those of other primate species. Using a two-bottle preference test (1 min) I found that the taste preference thresholds of the chimpanzees for five food-associated carbohydrates ranged between 20-30 mM for sucrose, 20-50 mM for fructose, 60-80 mM for glucose, 50-80 mM for maltose, and 30-80 mM for lactose. Taste preference thresholds for two steviol glycosides ranged from 0.04-0.05 mM for stevioside, and 0.03-0.05 mM for rebaudioside A. The chimpanzees displayed clear preferences for all sweet-tasting substances presented. In line with data obtained in other primates, the taste preference threshold of the chimpanzees for sucrose was lower compared to the other carbohydrates presented and the taste preference thresholds for stevioside and rebaudioside A were lower compared to sucrose. In general, the taste sensitivity of the chimpanzees fell into the range of data reported in other nonhuman primate species. Interestingly, the taste preference thresholds of the chimpanzees reported here are similar to the taste detection thresholds obtained in humans, despite the fact that the former are only a conservative approximation of an animal’s taste sensitivity. This suggests that chimpanzees may be as sweet-taste sensitive as humans.

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains., BT/Industrial Microbiology

In this issue of Structure, McCoy et al. (2016) describe the 2.55-Å X-ray structure of the outward-facing occluded conformation of the Bacillus cereus maltose transporter MalT. This structure represents the penultimate piece needed to complete the picture of the transport cycle of the glucose superfamily of membrane-spanning EIIC components.

Molasses and sugar beet pulp, by-products in sugar industry, are produced in large amounts annually which create disposal problems. High sugar content in sugar beet molasses enables its use for fermentations while sugar beet pulp represents an interesting cheap raw material source for enzyme production. beta-amylase production by a new strain Paenibacillus chitinolyticus CKS1 was performed using a molasses and sugar beet pulp. Two different pretreatments of SBP were investigated while scanning electron microscopy (SEM) showed immobilization of P. chitinolyticus CKS1 cells onto alkali treated SBP. The statistical model was design via Central Composite Design (CCD) under the Response Surface Methodology (RSM) using three variables (inoculum concentration, time of incubation and sugar beet pulp concentration) for obtaining maximum beta-amylase activity. An approximately 3.66-fold increase in beta-amylase production was achieved in the optimized medium as compared with the nonoptimized medium. A maximum beta-amylase production 2.237 U/ml was achieved under optimal conditions inoculum concentration 10%, incubation time 83.07 h and SBP concentration 3%. A high performance liquid chromatography showed that P. chitinolyticus CKS1 hydrolyzed starch to form maltose as a major product. These results indicate that beta-amylase production could be improved using these kind of processes. Also, taking into consideration cheap substrates, economic side of the process is justified.

Molasses and sugar beet pulp, by-products in sugar industry, are produced in large amounts annually which create disposal problems. High sugar content in sugar beet molasses enables its use for fermentations while sugar beet pulp represents an interesting cheap raw material source for enzyme production. beta-amylase production by a new strain Paenibacillus chitinolyticus CKS1 was performed using a molasses and sugar beet pulp. Two different pretreatments of SBP were investigated while scanning electron microscopy (SEM) showed immobilization of P. chitinolyticus CKS1 cells onto alkali treated SBP. The statistical model was design via Central Composite Design (CCD) under the Response Surface Methodology (RSM) using three variables (inoculum concentration, time of incubation and sugar beet pulp concentration) for obtaining maximum beta-amylase activity. An approximately 3.66-fold increase in beta-amylase production was achieved in the optimized medium as compared with the nonoptimized medium. A maximum beta-amylase production 2.237 U/ml was achieved under optimal conditions inoculum concentration 10%, incubation time 83.07 h and SBP concentration 3%. A high performance liquid chromatography showed that P. chitinolyticus CKS1 hydrolyzed starch to form maltose as a major product. These results indicate that beta-amylase production could be improved using these kind of processes. Also, taking into consideration cheap substrates, economic side of the process is justified.

Molasses and sugar beet pulp, by-products in sugar industry, are produced in large amounts annually which create disposal problems. High sugar content in sugar beet molasses enables its use for fermentations while sugar beet pulp represents an interesting cheap raw material source for enzyme production. beta-amylase production by a new strain Paenibacillus chitinolyticus CKS1 was performed using a molasses and sugar beet pulp. Two different pretreatments of SBP were investigated while scanning electron microscopy (SEM) showed immobilization of P. chitinolyticus CKS1 cells onto alkali treated SBP. The statistical model was design via Central Composite Design (CCD) under the Response Surface Methodology (RSM) using three variables (inoculum concentration, time of incubation and sugar beet pulp concentration) for obtaining maximum beta-amylase activity. An approximately 3.66-fold increase in beta-amylase production was achieved in the optimized medium as compared with the nonoptimized medium. A maximum beta-amylase production 2.237 U/ml was achieved under optimal conditions inoculum concentration 10%, incubation time 83.07 h and SBP concentration 3%. A high performance liquid chromatography showed that P. chitinolyticus CKS1 hydrolyzed starch to form maltose as a major product. These results indicate that beta-amylase production could be improved using these kind of processes. Also, taking into consideration cheap substrates, economic side of the process is justified.

The density, viscosity and hydrogen solubility of selected sugars (l-arabinose, d-galactose, d-maltose and l-rhamnose) were determined at different temperatures (generally 60, 90 and 130 °C). The role of internal diffusion resistance in porous catalyst layers for sugar hydrogenation was confirmed by numerical simulations based on kinetic data and physical properties. The simulations suggested the use of small catalyst particles or structured catalysts in continuous hydrogenation of the sugars to sugar alcohols. Continuous hydrogenation of l-arabinose was carried out in a laboratory-scale fixed bed reactor with ruthenium catalysts on three different supports (active carbon clothes, carbon nanotubes on sponge-like metallic structures, conventional active carbon catalyst particles). It was proved that continuous hydrogenation is a feasible alternative to batch technology for sugar hydrogenation over conventional catalyst particles and structured catalysts: l-arabinose was converted to arabitol with a very high selectivity., Bio4Energy

In this issue of Structure, McCoy et al. (2016) describe the 2.55-Å X-ray structure of the outward-facing occluded conformation of the Bacillus cereus maltose transporter MalT. This structure represents the penultimate piece needed to complete the picture of the transport cycle of the glucose superfamily of membrane-spanning EIIC components.

Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.

With the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (alpha-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto-hydrolysis of starch from triticale (95.88 U mL(-1)). Carbohydrate mixtures from triticale induced the production of alpha-amylase in A. oryzae. More alpha-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL(-1)). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

With the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (alpha-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto-hydrolysis of starch from triticale (95.88 U mL(-1)). Carbohydrate mixtures from triticale induced the production of alpha-amylase in A. oryzae. More alpha-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL(-1)). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

With the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (alpha-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto-hydrolysis of starch from triticale (95.88 U mL(-1)). Carbohydrate mixtures from triticale induced the production of alpha-amylase in A. oryzae. More alpha-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL(-1)). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

With the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (alpha-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto-hydrolysis of starch from triticale (95.88 U mL(-1)). Carbohydrate mixtures from triticale induced the production of alpha-amylase in A. oryzae. More alpha-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL(-1)). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide- forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism., QC 20131104

Plasma membrane H+-ATPase isoforms with increased H+/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H+/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H+/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly., BT/Biotechnology, Applied Sciences

Plasma membrane H+-ATPase isoforms with increased H+/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H+/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H+/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly., BT/Biotechnology, Applied Sciences

The paper reports the in vitro cultivation of two commercial lines and 23 wild populations (with 10, 20 and 30 chromosomes) of Brachypodium distachyon. Callus induction was assayed on Murashige and Skoog medium containing 1 mg dm -3 2,4-dichlorophenoxyacetic acid (2,4-D) with 30 g dm -3 of sucrose (MSs) or maltose (MSm). No significant differences were seen between the two media with respect to callus induction. Calli were transferred to MSm medium without 2,4-D but containing 0. 1 mg dm -3 of 6-benzylaminopurine for plant regeneration. The plant regeneration response was very variable depending on the original induction medium, although no overall preference for one or the other medium was seen. The three main culture stages (callus induction, plant regeneration, and green plantlets formation) are probably differently controlled in the plants with different chromosome numbers. This supports the idea that the three cytotypes of Brachypodium cultured actually belong to different species. © 2011 Springer Science+Business Media B.V.

[EN] Thermal processing of vegetables has pronounced effects on the cell structure, often negatively affecting the final textural properties of the product. In order to study the effect of thermal processing and the protective effect of sugars on the tissue, sliced carrots were subjected to blanching treatments under different time and temperature combinations both in water and in 4% sugar solutions made of trehalose or maltose. The influence of these process conditions on mass transfer, texture, and microstructure (Cryo-scanning electron microscopy) was thus investigated. The total mass loss of all the samples blanched in water was associated to their cook value (C-100(18)) except for the overprocessed one (90 degrees C, 10 min) that showed a total mass change significantly lower due to water uptake. The use of trehalose and maltose in the blanching solution reduced the solute loss while increasing the water loss. Microstructural analysis of the differently blanched carrots showed detachments between adjacent cell walls as well as plasmolysis phenomena as the time and temperature of the thermal treatment were increased. A protective effect of both sugars on cell structures was observed mostly in the sample treated at 90 degrees C. At macroscopic level, textural changes upon blanching were observed by a penetration test. As blanching time was increased, samples processed at 75 degrees C showed a hardness increase, while those processed at 90 degrees C showed a hardness decrease. However, both trehalose and maltose did not exert significant effects on the textural properties of blanched carrots when compared with those blanched in water.

Powder X-ray diffraction (PXRD) measurements on rapid freezing samples of disaccharide (trehalose, sucrose, and maltose) solutions indicated that the crystalline phases in the sample were both hexagonal and cubic ice. The cubic ice existed at a higher ratio in the higher disaccharide concentration samples. The temperature ramping experiments revealed that the cubic ice was stable below 233 K, which was obviously higher than the temperature expected for a pure water system. The diffraction peak width of the hexagonal ice crystal was independent in the disaccharide concentrations. This indicated that the crystallite size of the hexagonal ice was more than several hundreds of nanometer, which coincided with the ice particle size previously observed in the freeze-fractured replica samples. The comparison of the present PXRD data with the replica observations by transmission electron microscope in an earlier study allows us to conclude that the cubic ice was formed at the grain boundary between the hexagonal ice and the coexisted non-crystalline disaccharide phase.

Powder X-ray diffraction (PXRD) measurements on rapid freezing samples of disaccharide (trehalose, sucrose, and maltose) solutions indicated that the crystalline phases in the sample were both hexagonal and cubic ice. The cubic ice existed at a higher ratio in the higher disaccharide concentration samples. The temperature ramping experiments revealed that the cubic ice was stable below 233 K, which was obviously higher than the temperature expected for a pure water system. The diffraction peak width of the hexagonal ice crystal was independent in the disaccharide concentrations. This indicated that the crystallite size of the hexagonal ice was more than several hundreds of nanometer, which coincided with the ice particle size previously observed in the freeze-fractured replica samples. The comparison of the present PXRD data with the replica observations by transmission electron microscope in an earlier study allows us to conclude that the cubic ice was formed at the grain boundary between the hexagonal ice and the coexisted non-crystalline disaccharide phase.

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters vs. sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesize different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalyzed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process

The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of α-amylase, glucoamylase and α-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative α-glucosidase (AgdB) and an α-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative α-glucan modifying processes are discussed. © 2008 The Author(s).

While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-beta-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.

While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-beta-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.

In this report we have evaluated the potential of using fluorescence/Förster resonance energy transfer (FRET) in a competitive immunosensor for continuous monitoring of the carbohydrate hapten maltose. The cyanine dyes Cy5 and Cy5.5 were used as a donor–acceptor pair by conjugation to maltose-labeled bovine serum albumin (BSA) and the monoclonal antibody IgG 39.5, giving Cy5–BSA–maltotriitol (3.1/1/18) and Cy5.5–mAb39.5 (2.2/1), respectively. This antibody with weak affinity towards maltose showed full reversibility to both the free maltose and the maltose-labeled conjugate. It allowed us to measure continuously the maltose content by monitoring the FRET signal change over time due to displacement of Cy5–BSA–maltotriitol from Cy5.5–mAb39.5 inside a semipermeable capsule. A near 22% total increase was seen in the fluorescence intensity ratio I670/I700 in the presence of maltose, with a calculated EC50 = 1.87 ± 0.13 mM (R2 = 0.9984) from the sigmoidal dose–response curve at 25 °C. Specificity of the immunosensor was shown with the structural analog to maltose, cellobiose, and it generated no detectable response. A minor drift in the sensor baseline was seen with 0.4% per 24 h, which was in the same magnitude as the signal-to-noise ratio, during the 4 weeks of measurements. The immunosensor was applied to crude samples of oat drinks for direct quantification of the maltose content. Overall, this work demonstrates the potential to use an immunosensor based on weakly binding antibodies and FRET technology for remote and non-invasive carbohydrate monitoring.

Our group previously reported the preparation of water-soluble Au-Fe(x)O(y) nanoparticles functionalized with a maltose neoglycoconjugate. A fraction soluble in methanol was also separated and originated a new supramolecular polymeric aggregate. We report here the full characterization of this novel material by transmission electron microscopy (TEM), fluorescence emission, and atomic force microscopy. By means of noncontact dynamic atomic force microscopy, we have been able to obtain information about the organization of the organic components of the polymers, which eluded TEM analysis. We have observed that polymers packed in units about 65 nm in length and 40 nm in width on Au surfaces. The nanoparticles seem to be encapsulated by the organic material. We propose interactions between the sugar residues and the amphiphilic character of the maltose neoglycoconjugate (with a lipophilic undecane spacer) as responsible for the origin of these amazing supramolecular arrangements.

Interactions between water and saccharide in food are very important. The water activity coefficient in saccharide-water systems was measured by freezing point depression. Glucose-water systems were used, being ideal solutions. Freezing point depression in these systems was proportional to mole fraction and the accuracy of the experimental data was confirmed. Maltose-water systems behaved as ideal solutions. For two systems, water activity coefficient decreased from 1.0 at mole fractions above 0.02 in sucrose-water systems., 凝固点降下法を用いて糖水溶液の活量係数を測定した。 グルコース水溶液は理想溶液と考えられるので用い，実 験結果より凝固点降下はモル分率と比例し，この実験精 度が確かめられた。マルトース水溶液も理想溶液の挙動 を示した。これら2種類の系に対し，ショ糖水溶液では モル分挙が0.02以上になると，活量係数は1から漸減し た。また活量係数を用いて水分収着現象を理論的に考察 することが可能であることがわかった。

Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail., Att känna igen en motståndare är viktigt i många sammanhang, inte minst i kroppens immunförsvar som är utvecklat för att angripa främmande ämnen i kroppen. Antikroppen spelar en central roll i immunförsvaret där den lär sig att känna igen sin motståndare (antigen) och därmed binda sitt antigen. De antikroppsproducerande cellerna kan användas i laboratoriet för att producera antikroppar som härstammar från försöksdjur. I denna avhandling har antikroppar använts som binder betydligt svagare till antigenet än vad man i de flesta analyser använder sig av för att t.ex. detektera sjukdomar. Antikroppar som binder till olika typer av socker, däribland maltsocker (maltos) och blodsocker (glukos) har studerats. Dessa antikroppar har använts för att undersöka hur hårt de binder till sitt antigen beroende på temperatur och om antikropparna kan känna igen liknande motståndare (korsreaktivitet). Fördelen med dessa svaga bindningar är att antikroppen snabbt kan binda in och släppa sitt antigen istället för att nästan permanent sitta på sitt antigen, som vid starka bindningar. Bindningens styrka (affinitet) har i avhandlingen studerats med hjälp av fluorescensteknik och affinitets-separation. Den maltosbindande antikroppen har använts tillsammans med fluorescensteknik för att designa två olika biosensorer (immunosensorer). Immunosensorerna kan mäta förändringen av maltoskoncentration över tid, vilket är attraktivt i t.ex. livsmedelsindustrin när man vill mäta maltoshalten kontinuerligt under tillverkningen. Den glukosbindande antikroppen har använts i affinitets-separation för att bestämma dess affinitet mot glukos och olika polymerer av glukos. En glukosbindande antikropp är åtråvärt för att t.ex. kontinuerligt mäta koncentrationen av blodsocker genom huden hos diabetiker och därmed minska antalet blodprover man idag behöver ta.

Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail., Att känna igen en motståndare är viktigt i många sammanhang, inte minst i kroppens immunförsvar som är utvecklat för att angripa främmande ämnen i kroppen. Antikroppen spelar en central roll i immunförsvaret där den lär sig att känna igen sin motståndare (antigen) och därmed binda sitt antigen. De antikroppsproducerande cellerna kan användas i laboratoriet för att producera antikroppar som härstammar från försöksdjur. I denna avhandling har antikroppar använts som binder betydligt svagare till antigenet än vad man i de flesta analyser använder sig av för att t.ex. detektera sjukdomar. Antikroppar som binder till olika typer av socker, däribland maltsocker (maltos) och blodsocker (glukos) har studerats. Dessa antikroppar har använts för att undersöka hur hårt de binder till sitt antigen beroende på temperatur och om antikropparna kan känna igen liknande motståndare (korsreaktivitet). Fördelen med dessa svaga bindningar är att antikroppen snabbt kan binda in och släppa sitt antigen istället för att nästan permanent sitta på sitt antigen, som vid starka bindningar. Bindningens styrka (affinitet) har i avhandlingen studerats med hjälp av fluorescensteknik och affinitets-separation. Den maltosbindande antikroppen har använts tillsammans med fluorescensteknik för att designa två olika biosensorer (immunosensorer). Immunosensorerna kan mäta förändringen av maltoskoncentration över tid, vilket är attraktivt i t.ex. livsmedelsindustrin när man vill mäta maltoshalten kontinuerligt under tillverkningen. Den glukosbindande antikroppen har använts i affinitets-separation för att bestämma dess affinitet mot glukos och olika polymerer av glukos. En glukosbindande antikropp är åtråvärt för att t.ex. kontinuerligt mäta koncentrationen av blodsocker genom huden hos diabetiker och därmed minska antalet blodprover man idag behöver ta.

Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail., Att känna igen en motståndare är viktigt i många sammanhang, inte minst i kroppens immunförsvar som är utvecklat för att angripa främmande ämnen i kroppen. Antikroppen spelar en central roll i immunförsvaret där den lär sig att känna igen sin motståndare (antigen) och därmed binda sitt antigen. De antikroppsproducerande cellerna kan användas i laboratoriet för att producera antikroppar som härstammar från försöksdjur. I denna avhandling har antikroppar använts som binder betydligt svagare till antigenet än vad man i de flesta analyser använder sig av för att t.ex. detektera sjukdomar. Antikroppar som binder till olika typer av socker, däribland maltsocker (maltos) och blodsocker (glukos) har studerats. Dessa antikroppar har använts för att undersöka hur hårt de binder till sitt antigen beroende på temperatur och om antikropparna kan känna igen liknande motståndare (korsreaktivitet). Fördelen med dessa svaga bindningar är att antikroppen snabbt kan binda in och släppa sitt antigen istället för att nästan permanent sitta på sitt antigen, som vid starka bindningar. Bindningens styrka (affinitet) har i avhandlingen studerats med hjälp av fluorescensteknik och affinitets-separation. Den maltosbindande antikroppen har använts tillsammans med fluorescensteknik för att designa två olika biosensorer (immunosensorer). Immunosensorerna kan mäta förändringen av maltoskoncentration över tid, vilket är attraktivt i t.ex. livsmedelsindustrin när man vill mäta maltoshalten kontinuerligt under tillverkningen. Den glukosbindande antikroppen har använts i affinitets-separation för att bestämma dess affinitet mot glukos och olika polymerer av glukos. En glukosbindande antikropp är åtråvärt för att t.ex. kontinuerligt mäta koncentrationen av blodsocker genom huden hos diabetiker och därmed minska antalet blodprover man idag behöver ta.

Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail., Att känna igen en motståndare är viktigt i många sammanhang, inte minst i kroppens immunförsvar som är utvecklat för att angripa främmande ämnen i kroppen. Antikroppen spelar en central roll i immunförsvaret där den lär sig att känna igen sin motståndare (antigen) och därmed binda sitt antigen. De antikroppsproducerande cellerna kan användas i laboratoriet för att producera antikroppar som härstammar från försöksdjur. I denna avhandling har antikroppar använts som binder betydligt svagare till antigenet än vad man i de flesta analyser använder sig av för att t.ex. detektera sjukdomar. Antikroppar som binder till olika typer av socker, däribland maltsocker (maltos) och blodsocker (glukos) har studerats. Dessa antikroppar har använts för att undersöka hur hårt de binder till sitt antigen beroende på temperatur och om antikropparna kan känna igen liknande motståndare (korsreaktivitet). Fördelen med dessa svaga bindningar är att antikroppen snabbt kan binda in och släppa sitt antigen istället för att nästan permanent sitta på sitt antigen, som vid starka bindningar. Bindningens styrka (affinitet) har i avhandlingen studerats med hjälp av fluorescensteknik och affinitets-separation. Den maltosbindande antikroppen har använts tillsammans med fluorescensteknik för att designa två olika biosensorer (immunosensorer). Immunosensorerna kan mäta förändringen av maltoskoncentration över tid, vilket är attraktivt i t.ex. livsmedelsindustrin när man vill mäta maltoshalten kontinuerligt under tillverkningen. Den glukosbindande antikroppen har använts i affinitets-separation för att bestämma dess affinitet mot glukos och olika polymerer av glukos. En glukosbindande antikropp är åtråvärt för att t.ex. kontinuerligt mäta koncentrationen av blodsocker genom huden hos diabetiker och därmed minska antalet blodprover man idag behöver ta.

The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized. Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter. poxB mRNA expression was strongly induced by aeration and was repressed by glucose. Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted. Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region. The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production. Constitutive overproduction of PoxB in L. plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions. The DeltapoxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose). In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production. In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.

Amylosucrase from Neisseria polysaccharea (AS) is a remarkable transglycosidase of family 13 of the glycoside hydrolases that catalyses the synthesis of an amylose-like polymer from sucrose and is always described as a sucrose-specific enzyme. Here, we demonstrate for the first time the ability of pure AS to catalyse the disproportionation of maltooligosaccharides by cleaving the alpha-1,4 linkage at the non-reducing end of a maltooligosaccharide donor and transferring the glucosyl unit to the non-reducing end of another maltooligosaccharide acceptor. Surprisingly, maltose, maltotriose and maltotetraose are very poor glucosyl donors whereas longer maltooligosaccharides are even more efficient glucosyl donors than sucrose. At least five glucose units are required for efficient transglucosylation, suggesting the existence of strong binding subsites, far from the sucrose binding site, at position +4 and above.

Amylosucrase from Neisseria polysaccharea (AS) is a remarkable transglycosidase of family 13 of the glycoside hydrolases that catalyses the synthesis of an amylose-like polymer from sucrose and is always described as a sucrose-specific enzyme. Here, we demonstrate for the first time the ability of pure AS to catalyse the disproportionation of maltooligosaccharides by cleaving the alpha-1,4 linkage at the non-reducing end of a maltooligosaccharide donor and transferring the glucosyl unit to the non-reducing end of another maltooligosaccharide acceptor. Surprisingly, maltose, maltotriose and maltotetraose are very poor glucosyl donors whereas longer maltooligosaccharides are even more efficient glucosyl donors than sucrose. At least five glucose units are required for efficient transglucosylation, suggesting the existence of strong binding subsites, far from the sucrose binding site, at position +4 and above.

A taxonomic study was carried out for isolates of Saccharomyces spp. identified as contaminants ("wild yeast") in 24 different lager breweries. With reference to the current taxonomy all isolates were found to belong to the Saccharomyces sensu stricto complex and 58% of the isolates were further identified as S. cerevisiae, 26% as S. pastorianus and 3% as S. bayanus. The remaining isolates (13%) could not be identified to the species level based on their phenotypic characteristics. However, some of these isolates were identified as S. cerevisiae by HaeIII restriction digest of PCR-amplified intergenic transcribed spacer (ITS) regions. Chromosome length polymorphism (CLP) was evident among the Saccharomyces brewing contaminants with chromosome profiles typical of Saccharomyces sensu stricto. Based upon cluster analysis of their chromosome profiles the majority of the brewing contaminants could be grouped as either S. cerevisiae or S. pastorianus/S. bayanus. Further, the technique was able to differentiate between almost all brewing contaminants and to separate them from any specific lager brewing yeast. The diversity of the Saccharomyces brewing contaminants clearly demonstrated by their CLP was further reflected by MAL genotyping. For the majority of the isolates more than two MAL loci were found with MAL1, MAL2 MAL3, MAL4 and MAL11, MAL31, MAL41 as the dominant genotypes. For all isolates MAL11 and MAL31 were found whereas MAL61 only was found for one isolate. The high number of MAL loci found in the SaccharomYces brewing contaminants indicate their adaptation to a maltose-enriched environment.

A taxonomic study was carried out for isolates of Saccharomyces spp. identified as contaminants ("wild yeast") in 24 different lager breweries. With reference to the current taxonomy all isolates were found to belong to the Saccharomyces sensu stricto complex and 58% of the isolates were further identified as S. cerevisiae, 26% as S. pastorianus and 3% as S. bayanus. The remaining isolates (13%) could not be identified to the species level based on their phenotypic characteristics. However, some of these isolates were identified as S. cerevisiae by HaeIII restriction digest of PCR-amplified intergenic transcribed spacer (ITS) regions. Chromosome length polymorphism (CLP) was evident among the Saccharomyces brewing contaminants with chromosome profiles typical of Saccharomyces sensu stricto. Based upon cluster analysis of their chromosome profiles the majority of the brewing contaminants could be grouped as either S. cerevisiae or S. pastorianus/S. bayanus. Further, the technique was able to differentiate between almost all brewing contaminants and to separate them from any specific lager brewing yeast. The diversity of the Saccharomyces brewing contaminants clearly demonstrated by their CLP was further reflected by MAL genotyping. For the majority of the isolates more than two MAL loci were found with MAL1, MAL2 MAL3, MAL4 and MAL11, MAL31, MAL41 as the dominant genotypes. For all isolates MAL11 and MAL31 were found whereas MAL61 only was found for one isolate. The high number of MAL loci found in the SaccharomYces brewing contaminants indicate their adaptation to a maltose-enriched environment.

An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PglaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations, PglaA-driven GFP expression by the glucoamylase promoter could be demonstrated in xylose-limited continuous cultures. In these cultivations, the xylose concentration was therefore too low to cause repression. Transient experiments initiated with a maltose pulse did not further induce red-shifted GFP production in xylose-limited continuous cultures. Maltose induction under conditions of xylose repression was microscopically observed and quantified in a flow-through chamber. Red-shifted GFP was first produced after 5 h induction. Finally the strain was characterized in glucose-limited continuous cultures, and here the area of the mycelium stained with cytoplasmic GFP increased with increasing specific growth rate, indicating that GFP can be used as a marker of cellular activity in this type of cultivation.