Showing papers in "Biotechnology Letters in 2011"
TL;DR: This study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses, and demonstrates howutive over-expression of a miR169 family member in tomato plant can efficiently down-regulate the transcripts of the target genes.
Abstract: Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.
294 citations
TL;DR: Screening of microbial extracts reveals the large structural diversity of natural compounds with broad biological activities, such as antimicrobial, antiviral, immunosuppressive, and antitumor activities, that enable the bacterium to survive in its natural environment.
Abstract: Bacillus species produce secondary metabolites that are the object of natural product chemistry studies. The wide structural variability of these compounds has attracted the curiosity of chemists and their biological activities have inspired the pharmaceutical industry to search for lead structures in microbial extracts. Screening of microbial extracts reveals the large structural diversity of natural compounds with broad biological activities, such as antimicrobial, antiviral, immunosuppressive, and antitumor activities, that enable the bacterium to survive in its natural environment. These findings widen the potential industrial importance of Bacillus spp., particularly of B. thuringiensis, beyond insecticidal usage and may help explain the role of Bacillus spp. in the soil ecosystem.
199 citations
TL;DR: A brief overview of the process of biodiesel production with microalgae as feedstock is provided, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification.
Abstract: Due to negative environmental influence and limited availability, petroleum-derived fuels need to be replaced by renewable biofuels. Biodiesel has attracted intensive attention as an important biofuel. Microalgae have numerous advantages for biodiesel production over many terrestrial plants. There are a series of consecutive processes for biodiesel production with microalgae as feedstock, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. To reduce the overall production cost, technology development and process optimization are necessary. Genetic engineering also plays an important role in manipulating lipid biosynthesis in microalgae. Many approaches, such as sequestering carbon dioxide from industrial plants for the carbon source, using wastewater for the nutrient supply, and maximizing the values of by-products, have shown a potential for cost reduction. This review provides a brief overview of the process of biodiesel production with microalgae as feedstock. The methods associated with this process (e.g. lipid determination, mass culture, oil extraction) are also compared and discussed.
195 citations
TL;DR: Research on this topic covers many decades, beginning with investigations of possible denaturation of enzymes during processing, whilst more recent concerns are how the quality of therapeutic proteins might be affected by shear or shear related effects.
Abstract: The effects of “shear” on proteins in solution are described and discussed. Research on this topic covers many decades, beginning with investigations of possible denaturation of enzymes during processing, whilst more recent concerns are how the quality of therapeutic proteins might be affected by shear or shear related effects. The paradigm that emerges from most studies is that shear in the fluid mechanical sense is unlikely by itself to damage most proteins and that interfacial phenomena are critically important. In particular, moving gas–liquid interfaces can be very deleterious. Aggregation of therapeutic proteins on nanoparticles shed from solid surfaces is a recent concern because of potential consequences on patient safety. It is clear that labeling such damage as “shear” is a mistake as this inhibits clear investigations of, and thinking about, the true causes of damage to proteins in solution during processing.
184 citations
TL;DR: This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microAlgal lipid production may be expected.
Abstract: Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.
178 citations
TL;DR: This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products.
Abstract: Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts.
157 citations
TL;DR: The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated and were interpreted using Kolmogorov’s theory of isotropic turbulence.
Abstract: The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, NJS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml−1 (ca. 1 g dry weight l−1), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at NJS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.
154 citations
TL;DR: In this paper, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow and adipose tissue (AT-MSC).
Abstract: To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.
153 citations
TL;DR: OsDREB2A confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier and reveals significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type.
Abstract: Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T0 rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T1-3 and T1-10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier.
141 citations
TL;DR: In this review, examples of successful flower color modification in floricultural plants focusing on recent advances in techniques are summarized.
Abstract: Recent advances in genetic transformation techniques enable the production of desirable and novel flower colors in some important floricultural plants. Genetic engineering of novel flower colors is now a practical technology as typified by commercialization of a transgenic blue rose and blue carnation. Many researchers exploit knowledge of flavonoid biosynthesis effectively to obtain unique flower colors. So far, the main pigments targeted for flower color modification are anthocyanins that contribute to a variety of colors such as red, pink and blue, but recent studies have also utilized colorless or faint-colored compounds. For example, chalcones and aurones have been successfully engineered to produce yellow flowers, and flavones and flavonols used to change flower color hues. In this review, we summarize examples of successful flower color modification in floricultural plants focusing on recent advances in techniques.
138 citations
TL;DR: In the present review an emphasis has been given on plant–microbe interactions and their mitigation under abiotic and biotic stresses.
Abstract: In subsistence agricultural systems, crop yields are directly dependent on the inherent soil fertility and on microbial processes that govern the mineralization and mobilization of nutrients required for plant growth. An impact of different crop species that are used in various combinations is likely to be an important factor in determining the structure of plant beneficial microbial communities that function in nutrient cycling, the production of plant growth hormones, and suppression of root diseases. In addition, studies are needed to elucidate the signal transduction pathways that result from treatment of plants with plant growth-promoting rhizobacteria under stress conditions. In the present review an emphasis has been given on plant–microbe interactions and their mitigation under abiotic and biotic stresses.
TL;DR: This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal under salt stress.
Abstract: Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme (d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content (l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness of this engineering approach for enhanced salt tolerance in transgenic potato (Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards developing salinity tolerant crop plants.
TL;DR: This paper reviews currently available self-cleaving fusion tags for recombinant protein production and describes how they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost.
Abstract: Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.
TL;DR: This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms, which is a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins.
Abstract: As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon. The silk gland is classified into two main regions: the posterior (PSG) and the middle silk gland (MSG). By controlling the expressed regions of the recombinant protein gene in the silk gland, we were able to control the localization of the synthesized protein in the silk thread. Expression in the PSG or MSG led to localization in the insoluble fibroin core or hydrophilic outer sericin layer, respectively. This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms. The recombinant protein secreted in the sericin layer is extractable from the cocoon with only a small amount of endogenous silk protein contamination by soaking the cocoon in mild aqueous solutions. The possibility of utilizing transgenic silkworms as a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins is discussed.
TL;DR: An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents and its mode of action is similar to end oxylanases of the family 10 glucoside hydrolases.
Abstract: An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T1/2 values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2–7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.
TL;DR: The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in hairy root cultures of Pueraria candollei.
Abstract: The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.
TL;DR: The science of yeast cell surface modification as well as current applications and future opportunities are reviewed.
Abstract: Cell surface engineering is a promising strategy for the molecular breeding of whole-cell biocatalysts. By using this strategy, yeasts can be constructed by the cell surface display of functional proteins; these yeasts are referred to as arming yeasts. Because reactions using arming yeasts as whole-cell biocatalysts occur on the cell surface, materials that cannot enter the cell can be used as reaction substrates. Numerous arming yeasts have therefore been constructed for a wide range of uses such as biofuel production, synthesis of valuable chemicals, adsorption or degradation of environmental pollutants, recovery of rare metal ions, and biosensors. Here, we review the science of yeast cell surface modification as well as current applications and future opportunities.
TL;DR: Re recombinant Corynebacterium glutamicum was genetically engineered to synthesize GABA using endogenous l-glutamate using exogenous glutamate decarboxylase genes by introducing Lbgad genes.
Abstract: Purpose of work
Purpose of this work is to synthesize γ-aminobutyric acid by glutamate-producing species expressing Lactobacillus brevis-derived glutamate decarboxylase genes, i.e. recombinant Corynebacterium glutamicum strains, which directly convert endogenous l-glutamate precursor into γ-aminobutyric acid (GABA) through single-step fermentation.
To express exogenous glutamate decarboxylase (GAD) in an l-glutamate-producing strain, Lactobacillus brevis Lb85, which can produce GABA, was used. Two Lb85 GAD genes, gadB1 and gadB2, and the ancillary genes, gadC-gadB2 and gadR-gadC-gadB2, were cloned separately into pDXW-8 and transformed into C. glutamicum. All four recombinant strains produced GABA whereas the wild-type strain did not. GABA produced by the recombinant strains continually increased after 36 h of fermentation. Although the mRNA levels of LbgadB2 and LbgadC were similar among the corresponding recombinants, GABA production of pDXW-8/gadRCB2 at 72 h (2.15 g/l) was higher than that of pDXW-8/gadCB2 (1.25 g/l) and pDXW-8/gadB2 (0.88 g/l). Thus, by introducing Lbgad genes, C. glutamicum was genetically engineered to synthesize GABA using endogenous l-glutamate.
TL;DR: An overview of the TAP method is given, with a focus on its key feature—the dual-affinity tag, and the application of this technology in various systems is briefly discussed.
Abstract: Tandem affinity purification (TAP) is a methodology for the isolation of protein complexes from endogenous sources. It involves incorporation of a dual-affinity tag into the protein of interest and introduction of the construct into desired cell lines or organisms. Using the two affinity handles, the protein complex assembled under physiological conditions, which contains the tagged target protein and its interacting partners, can be isolated by a sequential purification scheme. Compared with single-step purification, TAP greatly reduces non-specific background and isolates protein complexes with higher purity. TAP-based protein retrieval plus mass spectrometry-based analysis has become a standard approach for identification and characterization of multi-protein complexes. The present article gives an overview of the TAP method, with a focus on its key feature—the dual-affinity tag. In addition, the application of this technology in various systems is briefly discussed.
TL;DR: 16S rRNA gene analysis using PCR-DGGE indicated that the MFC operation with VFAs had enriched unique microbial species, which indicated that anodic microbes were competing for different substrates.
Abstract: Food wastes were used as feedstock for the direct production of electricity in a microbial fuel cell (MFC). MFC operations with volatile fatty acids (VFA) produced 533 mV with a maximum power density of 240 mW/m2. Short-chain VFAs, such as acetate, were degraded more rapidly and thus supported higher power generation than longer chain ones. In general, the co-existence of other, different VFAs slowed the removal of each VFA, which indicated that anodic microbes were competing for different substrates. 16S rRNA gene analysis using PCR-DGGE indicated that the MFC operation with VFAs had enriched unique microbial species.
TL;DR: The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.
Abstract: Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.
TL;DR: The lipase-catalyzed reaction is useful to obtain sugar esters with chemically defined structures and will contribute to the synthesis of sugar-based compounds by a chemo-enzymatic pathway.
Abstract: The lipase-catalyzed reaction is useful to obtain sugar esters with chemically defined structures and will contribute to the synthesis of sugar-based compounds by a chemo-enzymatic pathway. The synthesis of sugar esters in nonaqueous media has been attempted for a quarter century. To facilitate the reactions, they have been performed either in an organic solvent with/without a polar adjuvant or in an ionic liquid, or by using a hydrophobic sugar derivative. In this review, the following points are discussed: (1) various synthetic methods of sugar esters; (2) role of the solvents or adjuvants; and (3) improvement in the productivity.
TL;DR: All four microbial fuel cells inoculated with different bacterial species had low efficiencies in generating power from organic matter, which may have caused most of the protons produced in the anode chamber to leave the chamber with the effluent, which led to the low power generation performance of the MFCs.
Abstract: Four microbial fuel cells (MFCs) inoculated with different bacterial species were constructed. The species were Pseudomonas putida, Comamonas testosteroni, Corynebacterium gultamicum, and Arthrobacter polychromogenes. The MFCs were operated under identical continuous flow conditions. The factors affecting the capabilities of the MFCs for treating organic matter and generating power were evaluated and compared. The factors include microbial species type, organic loading, and substrate degradation rate. For all four MFCs, power output increased with the organic loading rate. Power density also increased with the substrate degradation rate. These findings implied that more organic matter was utilized for power generation at higher organic loading and substrate degradation rates. However, coulombic efficiency increased with decreased organic loading and substrate degradation rates. Apparently, all four MFCs had low efficiencies in generating power from organic matter. These low efficiencies are attributed to the long distance between the anode and the cathode, as well as to the small ratio of the proton exchange membrane surface area to the anode chamber surface area. These features may have caused most of the protons produced in the anode chamber to leave the chamber with the effluent, which led to the low power generation performance of the MFCs.
TL;DR: Th thin stillage has the potential to act as a substrate for the commercial production of food-grade malic acid by the A. niger strains.
Abstract: The ability of Aspergillus strains to utilize thin stillage to produce malic acid was compared. The highest malic acid was produced by Aspergillusniger ATCC 9142 at 17 g l−1. Biomass production from thin stillage was similar with all strains but ATCC 10577 was the highest at 19 g l−1. The highest malic acid yield (0.8 g g−1) was with A. niger ATCC 9142 and ATCC 10577 on the stillage. Thus, thin stillage has the potential to act as a substrate for the commercial production of food-grade malic acid by the A. niger strains.
TL;DR: This article reviews the use of three main techniques of whey fractionation together with an evaluation of their performance regarding the yield and purity of two major proteins in whey.
Abstract: Whey is a by-product of cheese manufacture that is normally treated as a waste. However, it contains a mixture of proteins with important nutritional and biological attributes. To extract these valuable proteins, whey fractionation has been developed using three main techniques; namely chromatographic (e.g., ion-exchange and hydrophobic adsorption), membrane (e.g., traditional pressure-driven and electro-separation)-, or combined methods. Recently, new promising techniques have been introduced such as aqueous two-phase separation (ATPS) and magnetic fishing. This article reviews the use of these techniques together with an evaluation of their performance regarding the yield and purity of two major proteins in whey.
TL;DR: This review of microRNAs provides a brief overview of their biogenesis, genomic organization and mode of action, followed by a description of the methods and approaches to studying their expression.
Abstract: The ability of microRNAs to influence gene expression is now recognized as a fundamental layer of regulation within the cell. MicroRNAs have a major impact on most biological processes and have generated considerable interest as potential biomarkers as well as therapeutic or engineering targets. In this review we provide a brief overview of their biogenesis, genomic organization and mode of action, followed by a description of the methods and approaches to studying their expression. We go on to consider some of the approaches to utilizing them as tools and their potential application in the bioprocessing area, with particular emphasis on Chinese hamster ovary cell engineering.
TL;DR: While chitosan solutions demonstrate strong bactericidal activity against a range of medically important bacteria, a loss of this beneficial property in thin films cast from the same solutions is reported.
Abstract: Chitosan is a promising biomaterial for biomedical applications and is currently applied as wound dressings. While chitosan solutions demonstrate strong bactericidal activity against a range of medically important bacteria, the study here reports a loss of this beneficial property in thin films cast from the same solutions. Chitosan films (20 μm) showed no inhibitory effects against Escherichia coli, Staphylococcus aureus or S. epidermidis species. In contrast, solutions used to prepare the films showed almost complete inhibition (~98 ± 2%) when tested on bacterial lawns and in liquid cultures. Increased acidity of the chitosan solutions (pH 5) was shown to promote the bactericidal effects of this biopolymer. The concept that devices fabricated from chitosan have an inherent antimicrobial activity is suggested as an important misconception.
TL;DR: The CM-α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi are biologically active and may therefore be used as diet or therapy supplements.
Abstract: To show biological activity of carboxymethylated α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi on human tumor and normal cells. Water-insoluble, alkali-soluble polysaccharides (WIP) were isolated from fruiting bodies of four macromycetes fungi: Lentinus edodes, Pleurotus ostreatus, Piptoporus betulinus and Laetiporus sulphureus. The structure of the polysaccharides was determined using composition analysis, methylation analysis, fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The chemical and spectroscopic investigations indicated that the polysaccharides were an α-(1 → 3)-d-glucans. A biological activity analysis of the carboxymethylated (CM) α-(1 → 3)-d-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects. The cytotoxic activity of the CM-glucans was concentration- and cell-type-dependent. The tested CM-glucans, generally, did not have a free radical scavenging effect. The CM-α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi are biologically active and may therefore be used as diet or therapy supplements.
TL;DR: Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges.
Abstract: Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.
TL;DR: Analysis of the genome of Streptomyces toxytricini NRRL 15443 revealed an ORF, stth, encoding a putative Trp halogenase within a non-ribosomal peptide synthetase gene cluster, which halogenated both l- and d-Trp to yield the corresponding 6-chlorinated derivatives.
Abstract: Tryptophan (Trp) halogenases are found in various bacteria and play an important role in natural product biosynthesis. Analysis of the genome of Streptomyces toxytricini NRRL 15443 revealed an ORF, stth, encoding a putative Trp halogenase within a non-ribosomal peptide synthetase gene cluster. This gene was cloned into pET28a and functionally overexpressed in Escherichia coli. The enzyme halogenated both l- and d-Trp to yield the corresponding 6-chlorinated derivatives. The optimum activity was at 40°C and pH 6 giving k
cat
/K
M value of STTH of 72,000 min−1 M−1. The enzyme also used bromide to yield 6-bromo-Trp.