Showing papers in "Applied Microbiology and Biotechnology in 2002"
TL;DR: The present review analyzes the pecularities of polysaccharides derived from fruiting bodies and cultured mycelium in selected examples of medicinal mushrooms and concludes that high molecular weight glucans appear to be more effective than those of low molecular weight.
Abstract: The number of mushrooms on Earth is estimated at 140,000, yet maybe only 10% (approximately 14,000 named species) are known. Mushrooms comprise a vast and yet largely untapped source of powerful new pharmaceutical products. In particular, and most importantly for modern medicine, they represent an unlimited source of polysaccharides with antitumor and immunostimulating properties. Many, if not all, Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, culture broth. Data on mushroom polysaccharides have been collected from 651 species and 7 infraspecific taxa from 182 genera of higher Hetero- and Homobasidiomycetes. These polysaccharides are of different chemical composition, with most belonging to the group of β-glucans; these have β-(1→3) linkages in the main chain of the glucan and additional β-(1→6) branch points that are needed for their antitumor action. High molecular weight glucans appear to be more effective than those of low molecular weight. Chemical modification is often carried out to improve the antitumor activity of polysaccharides and their clinical qualities (mostly water solubility). The main procedures used for chemical improvement are: Smith degradation (oxydo-reducto-hydrolysis), formolysis, and carboxymethylation. Most of the clinical evidence for antitumor activity comes from the commercial polysaccharides lentinan, PSK (krestin), and schizophyllan, but polysaccharides of some other promising medicinal mushroom species also show good results. Their activity is especially beneficial in clinics when used in conjunction with chemotherapy. Mushroom polysaccharides prevent oncogenesis, show direct antitumor activity against various allogeneic and syngeneic tumors, and prevent tumor metastasis. Polysaccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antitumor action of polysaccharides requires an intact T-cell component; their activity is mediated through a thymus-dependent immune mechanism. Practical application is dependent not only on biological properties, but also on biotechnological availability. The present review analyzes the pecularities of polysaccharides derived from fruiting bodies and cultured mycelium (the two main methods of biotechnological production today) in selected examples of medicinal mushrooms.
1,962 citations
TL;DR: The types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors are discussed, with an overview on the use in the detergent industry.
Abstract: Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN′ and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.
1,573 citations
TL;DR: The current status of the technology for ethanol production from softwood is reviewed, with focus on hemicellulose and cellulose hydrolysis, which is the major problem in the overall process.
Abstract: Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline One of the major resources in the Northern hemisphere is softwood This paper reviews the current status of the technology for ethanol production from softwood, with focus on hemicellulose and cellulose hydrolysis, which is the major problem in the overall process Other issues of importance, eg overall process configurations and process economics are also considered
1,153 citations
TL;DR: An integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.
Abstract: Biofouling is referred to as the unwanted deposition and growth of biofilms. This phenomenon can occur in an extremely wide range of situations, from the colonisation of medical devices to the production of ultra-pure, drinking and process water and the fouling of ship hulls, pipelines and reservoirs. Although biofouling occurs in such different areas, it has a common cause, which is the biofilm. Biofilms are the most successful form of life on Earth and tolerate high amounts of biocides. For a sustainable anti-fouling strategy, an integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.
833 citations
TL;DR: It is concluded that A. niger is a safe production organism and new and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms.
Abstract: Aspergillus niger is one of the most important microorganisms used in biotechnology. It has been in use already for many decades to produce extracellular (food) enzymes and citric acid. In fact, citric acid and many A. niger enzymes are considered GRAS by the United States Food and Drug Administration. In addition, A. niger is used for biotransformations and waste treatment. In the last two decades, A. niger has been developed as an important transformation host to over-express food enzymes. Being pre-dated by older names, the name A. niger has been conserved for economical and information retrieval reasons and there is a taxonomical consensus based on molecular data that the only other common species closely related to A. niger in the Aspergillus series Nigri is A. tubingensis. A. niger, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. However, compared with other filamentous fungi, it does not stand out as a particular problem concerning allergy or mycopathology. A few medical cases, e.g. lung infections, have been reported, but always in severely immunocompromised patients. In tropical areas, ear infections (otomycosis) do occur due to A. niger invasion of the outer ear canal but this may be caused by mechanical damage of the skin barrier. A. niger strains produce a series of secondary metabolites, but it is only ochratoxin A that can be regarded as a mycotoxin in the strict sense of the word. Only 3-10% of the strains examined for ochratoxin A production have tested positive under favourable conditions. New and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms. It is concluded, with these restrictions, that A. niger is a safe production organism.
713 citations
TL;DR: The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described, and current strategies for the improvement ofitamin B12 production using modern biotechnological techniques are outlined.
Abstract: One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species. The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.
576 citations
TL;DR: TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals and the principal function of bacterial TAG seems to be as a reserve compound.
Abstract: Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.
572 citations
TL;DR: Molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the Bryozoan Bugula neritina.
Abstract: The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.
552 citations
TL;DR: Recent advances in the molecular genetics of biodegradation and studies on enzyme-tailoring and DNA-shuffling are discussed in this paper.
Abstract: With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds, industrial solvents, pesticides and metals. A number of bioremediation strategies have been developed to treat contaminated wastes and sites. Selecting the most appropriate strategy to treat a specific site can be guided by considering three basic principles: the amenability of the pollutant to biological transformation to less toxic products (biochemistry), the accessibility of the contaminant to microorganisms (bioavailability) and the opportunity for optimization of biological activity (bioactivity). Recent advances in the molecular genetics of biodegradation and studies on enzyme-tailoring and DNA-shuffling are discussed in this paper.
499 citations
TL;DR: This Mini-Review describes the properties of Pleurotus spp in relation to their biotechnological applications and potential and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants.
Abstract: The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and important biotechnological and environmental applications. The cultivation of Pleurotus spp is an economically important food industry worldwide which has expanded in the past few years. P. ostreatus is the third most important cultivated mushroom for food purposes. Nutritionally, it has unique flavor and aromatic properties; and it is considered to be rich in protein, fiber, carbohydrates, vitamins and minerals. Pleurotus spp are promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic and immunomodulation activities. The bioactive molecules isolated from the different fungi are polysaccharides. One of the most important aspects of Pleurotus spp is related to the use of their ligninolytic system for a variety of applications, such as the bioconversion of agricultural wastes into valuable products for animal feed and other food products and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants. In this Mini-Review, we describe the properties of Pleurotus spp in relation to their biotechnological applications and potential.
494 citations
TL;DR: In this review, advances made in using renewable substrates for biosurfactant production and their newer applications are reported.
Abstract: Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes. Microbes synthesize them, especially during growth on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. Because of their structural diversity (i.e., glycolipids, lipopeptides, fatty acids, etc.), low toxicity, and biodegradability, these molecules could be widely used in cosmetic, pharmaceutical, and food processes as emulsifiers, humectants, preservatives, and detergents. Moreover, they are ecologically safe and can be applied in bioremediation and waste treatments. They can be produced from various substrates, mainly renewable resources such as vegetable oils, distillery and dairy wastes, which are economical but have not been reported in detail. In this review, we report advances made in using renewable substrates for biosurfactant production and their newer applications.
TL;DR: The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pECTin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment to explore the potential of these enzymes and to encourage new alkalinepectinase-based industrial technology.
Abstract: The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes. Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations. The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment. It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential. The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology.
TL;DR: This review attempts to describe in detail the three major classes of nitrile-converting enzymes, namely nitrilases,Nitrile hydratases and amidases, including their occurrence, mechanism of action, characteristics and applicability in different sectors.
Abstract: Nitrile-converting enzymes are becoming commonplace in the synthesis of pharmaceuticals and commodity chemicals. These versatile biocatalysts have potential applications in different fields including synthetic biocatalysis and bioremediation. This review attempts to describe in detail the three major classes of nitrile-converting enzymes, namely nitrilases, nitrile hydratases and amidases. Various aspects of these enzymes including their occurrence, mechanism of action, characteristics and applicability in different sectors have been elaborately elucidated. Cloning of genes related to nitrile-converting enzymes is also discussed.
TL;DR: Alkaline proteases useful for detergent applications are mostly active in the pH range 8–12 and at temperatures between 50 and 70°C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80–90°C.
Abstract: Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8–12 and at temperatures between 50 and 70°C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80–90°C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8–11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.
TL;DR: The use of statins can lead to a reduction in coronary events related to hypercholesterolemia, but the relationship between benefit and risk, and any possible interaction with other drugs, must be taken into account.
Abstract: Hypercholesterolemia is considered an important risk factor in coronary artery disease. Thus the possibility of controlling de novo synthesis of endogenous cholesterol, which is nearly two-thirds of total body cholesterol, represents an effective way of lowering plasma cholesterol levels. Statins, fungal secondary metabolites, selectively inhibit hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductase, the first enzyme in cholesterol biosynthesis. The mechanism involved in controlling plasma cholesterol levels is the reversible inhibition of HMG-CoA reductase by statins, related to the structural similarity of the acid form of the statins to HMG-CoA, the natural substrate of the enzymatic reaction. Currently there are five statins in clinical use. Lovastatin and pravastatin (mevastatin derived) are natural statins of fungal origin, while symvastatin is a semi-synthetic lovastatin derivative. Atorvastatin and fluvastatin are fully synthetic statins, derived from mevalonate and pyridine, respectively. In addition to the principal natural statins, several related compounds, monacolins and dihydromonacolins, isolated fungal intermediate metabolites, have also been characterized. All natural statins possess a common polyketide portion, a hydroxy-hexahydro naphthalene ring system, to which different side chains are linked. The biosynthetic pathway involved in statin production, starting from acetate units linked to each other in head-to-tail fashion to form polyketide chains, has been elucidated by both early biogenetic investigations and recent advances in gene studies. Natural statins can be obtained from different genera and species of filamentous fungi. Lovastatin is mainly produced by Aspergillus terreus strains, and mevastatin by Penicillium citrinum. Pravastatin can be obtained by the biotransformation of mevastatin by Streptomyces carbophilus and simvastatin by a semi-synthetic process, involving the chemical modification of the lovastatin side chain. The hypocholesterolemic effect of statins lies in the reduction of the very low-density lipoproteins (VLDL) and LDL involved in the translocation of cholesterol, and in the increase in the high-density lipoproteins (HDL), with a subsequent reduction of the LDL- to HDL-cholesterol ratio, the best predictor of atherogenic risk. The use of statins can lead to a reduction in coronary events related to hypercholesterolemia, but the relationship between benefit and risk, and any possible interaction with other drugs, must be taken into account.
TL;DR: A nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30°C by implementing exponential feeding of vitamins throughout the fermentation process.
Abstract: Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30°C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l–1 to 135 g l–1 and 147 g l–1, respectively. A maximum instantaneous productivity of 9.5 g l–1 h–1 was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.
TL;DR: This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.
Abstract: 2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.
TL;DR: Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the recently reclassified genus Mannheimia as a novel species, and has been named MannheimIA succiniciproducens MBEL55E.
Abstract: A novel succinic acid-producing bacterium was isolated from bovine rumen. The bacterium is a non-motile, non-spore-forming, mesophilic and capnophilic gram-negative rod or coccobacillus. Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the recently reclassified genus Mannheimia as a novel species, and has been named Mannheimia succiniciproducens MBEL55E. Under 100% CO2 conditions, it grows well in the pH range of 6.0–7.5 and produces succinic acid, acetic acid and formic acid at a constant ratio of 2:1:1. When M. succiniciproducens MBEL55E was cultured anaerobically in medium containing 20 g l–1 glucose as carbon source, 13.5 g l–1 of succinic acid was produced.
TL;DR: An initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background.
Abstract: Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59→Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311→Ile mutation in the aspartokinase gene (lysC), and a Pro-458→Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l–1 h–1) so far reported for microbes producing L-lysine in fed-batch fermentation.
TL;DR: The data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.
Abstract: Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes. Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete. The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h. The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated. Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads. Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.
TL;DR: Recombinant Escherichia coli strains expressing atrazine chlorohydrolase have been constructed and chemically cross-linked to generate catalytic particles used forAtrazine remediation in soil, used for cleaning up a spill of 1,000 pounds of atrazin to attain a level of herbicide acceptable to regulatory agencies.
Abstract: s-Triazine ring compounds are common industrial chemicals: pesticides, resin intermediates, dyes, and explosives. The fate of these compounds in the environment is directly correlated with the ability of microbes to metabolize them. Microbes metabolize melamine and the triazine herbicides such as atrazine via enzyme-catalyzed hydrolysis reactions. Hydrolytic removal of substituents on the s-triazine ring is catalyzed by enzymes from the amidohydrolase superfamily and yields cyanuric acid as an intermediate. Cyanuric acid is hydrolytically processed to yield 3 mol each of ammonia and carbon dioxide. In those cases studied, the genes underlying the hydrolytic reactions are localized to large catabolic plasmids. One such plasmid, pADP-1 from Pseudomonas sp. ADP, has been completely sequenced and contains the genes for atrazine catabolism. Insertion sequence elements play a role in constructing different atrazine catabolic plasmids in different bacteria. Atrazine chlorohydrolase has been purified to homogeneity from two sources. Recombinant Escherichia coli strains expressing atrazine chlorohydrolase have been constructed and chemically cross-linked to generate catalytic particles used for atrazine remediation in soil. The method was used for cleaning up a spill of 1,000 pounds of atrazine to attain a level of herbicide acceptable to regulatory agencies.
TL;DR: A novel and quick enrichment technology that can be used as a preliminary method to obtain a hydrogen-producing species from the biological sludge produced by wastewater treatment and hydrogen accumulation from sludge with acid or base enrichment is higher than that of the control.
Abstract: This study offers a novel and quick enrichment technology that can be used as a preliminary method to obtain a hydrogen-producing species from the biological sludge produced by wastewater treatment. The influences of acid–base enrichment (by sludge pH adjustment) on the anaerobic hydrogen-producing micro-organisms were investigated using serum bottle assays. The enrichment pH values were controlled at 3, 4, 5, 7, 10, 11 and 12 with 1 N hydrochloric acid and 1 N sodium hydroxide. For each enrichment pH, the cultivation pH values were controlled at 5, 6 and 7. Based on the experimental results, hydrogen accumulation from sludge with acid or base enrichment is higher than that of the control. The hydrogen-production potential of the sludge with acid or base enrichment is 200 and 333 times enhanced, compared with the control, when the enrichment pH is 10 and 3, respectively. The enhancement is due to a shortening of the micro-organisms' lag-time which occurs at a proper cultivation-pH level.
TL;DR: This review will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity and discuss the application of combinatorial and evolutionary strategies to carOTenoid pathway engineering to broaden the diversity of carotanoid structures synthesized in recombinant hosts.
Abstract: Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.
TL;DR: A tabular overview of α-CD production processes is presented and the implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction.
Abstract: Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly α-amylases. Due to its low solubility in water, separation and purification of β-CD is relatively easy compared to α- and γ-CD. In recent years more economic processes for γ-CD and especially α-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of α-CD production processes is presented.
TL;DR: The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions, which makes them ideal organisms for microbial process development.
Abstract: The genus Gluconobacter belongs to the group of acetic acid bacteria, which are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are excreted almost completely into the medium. In most cases, the reactions are catalyzed by dehydrogenases connected to the respiratory chain. Since the reactive centers of the enzymes are oriented towards the periplasmic space, transport of substrates and products into, and out of, the cell is not necessary. Thus, rapid accumulation of incompletely oxidized products in the medium is facilitated. These organisms are able to grow in highly concentrated sugar solutions and at low pH-values. High oxidation rates correlate with low biomass production, which makes Gluconobacter strains interesting organisms for industrial applications. Modern fermentation processes, such as the production of L-sorbose (vitamin C synthesis) and 6-amino-L-sorbose (synthesis of the antidiabetic drug miglitol) are carried out with members of this genus. Other important products are dihydroxyacetone, gluconate and ketogluconates. The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions. This uniqueness makes them ideal organisms for microbial process development.
TL;DR: The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium, and Y. lipolytica presented a strong fatty acid specificity.
Abstract: The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.
TL;DR: Current production by S. putrefaciens was enhanced 10-fold when an electron mediator (i.e., Mn4+ or neutral red) was incorporated into the graphite anode.
Abstract: The production of electricity by Shewanella putrefaciens in the absence of exogenous electron acceptors was examined in a single compartment fuel cell with different types of electrodes and varying physiological conditions. Electricity production was dependent on anode composition, electron donor type and cell concentration. A maximum current of 2.5 mA and a current density of 10.2 mW/m2 electrode was obtained with a Mn4+ graphite anode, 200 mM sodium lactate and a cell concentration of 3.9 g cell protein/ml. Current production by S. putrefaciens was enhanced 10-fold when an electron mediator (i.e., Mn4+ or neutral red) was incorporated into the graphite anode.
TL;DR: Enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.
Abstract: Cysteine desulfurase is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. Increased evidence for the functions of cysteine desulfurases has revealed their important roles in the biosyntheses of Fe-S clusters, thiamine, thionucleosides in tRNA, biotin, lipoic acid, molybdopterin, and NAD. The enzymes are also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. The mechanisms for sulfur mobilization mediated by cysteine desulfurases are as yet unknown, but enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.
TL;DR: The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively, possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis.
Abstract: Sorbitol, a polyol found in many fruits, is of increasing industrial interest as a sweetener, humectant, texturizer and softener. At present, it is produced chemically. The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis. The possibilities for the industrial production of sorbitol by Z. mobilis are discussed, and compared with the current chemical production method as well as other microbiological processes.
TL;DR: The N-terminal amino acid sequence of Lcc 1 showed close homology to the N- terminal sequences determined for laccases from Phlebia radiata, Trametes villosa, and Tramsetes versicolor, but only low similarity was observed to a previously reported laccase from L. edodes.
Abstract: A laccase (EC 1.10.3.2) was isolated from the culture filtrate of Lentinula edodes. The enzyme was purified to a homogeneous preparation using hydrophobic, anion-exchange, and size-exclusion chromatographies. SDS-PAGE analysis showed the purified laccase, Lcc 1, to be a monomeric protein of 72.2 kDa. The enzyme had an isoelectric point of around pH 3.0. The optimum pH for enzyme activity was around 4.0, and it was most active at 40°C and stable up to 35°C. The enzyme contained 23.8% carbohydrate and some copper atoms. The enzyme oxidized 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, p-phenylendiamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol, and ferulic acid, but not veratryl alcohol, tyrosine, and β-(3,4-dihydroxyphenyl) alanine. The N-terminal amino acid sequence of Lcc 1 showed close homology to the N-terminal sequences determined for laccases from Phlebia radiata, Trametes villosa, and Trametes versicolor, but only low similarity was observed to a previously reported laccase from L. edodes. Lcc 1 was effective in the decolorization of chemically different dyes – Remazole Brilliant Blue R, Bromophenol Blue, methyl red, and Naphtol Blue Black – without any mediators, but the decolorization of two dyes – red poly(vinylamine)sulfonate-anthrapyridone dye and Reactive Orange 16 – did require some redox mediators.