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Applied Microbiology and Biotechnology (v.69, #4)
Aptamers—basic research, drug development, and clinical applications by Daniela Proske; Michael Blank; Raymund Buhmann; Ansgar Resch (pp. 367-374).
Since its discovery in the early 1990s, aptamer technology has progressed tremendously. Automated selection procedures now allow rapid identification of DNA and RNA sequences that can target a broad range of extra- and intracellular proteins with nanomolar affinities and high specificities. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for different areas of biotechnology. Moreover, the approval of an aptamer for vascular endothelial growth factor by the US Food and Drug Administration highlights the potential of aptamers for therapeutic applications. This review summarizes recent developments and demonstrates that aptamers are valuable tools for diagnostics, purification processes, target validation, drug discovery, and even therapeutic approaches.
Morphology and productivity of filamentous fungi by L. H. Grimm; S. Kelly; R. Krull; D. C. Hempel (pp. 375-384).
Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.
Expression of porcine lactoferrin by using recombinant baculovirus in silkworm, Bombyx mori L., and its purification and characterization by Yizhen Wang; Xiaofeng Wu; Guangfu Liu; Cuiping Cao; Haiqing Huang; Zirong Xu; Jianxin Liu (pp. 385-389).
Lactoferrin is a multifunctional glycoprotein that is present in several mucosal secretions. In this study, we exploited the silkworm, Bombyx mori, as host for the recombinant baculovirus harboring the porcine lactoferrin (PLF) gene to produce the recombinant PLF (rPLF). Around 205 μg of rPLF was purified from a single silkworm pupa infected by the virus and the rPLF was proved to be biologically active. This method established in our study will pave the way for efficient industrial production of rPLF on a large scale for further utilization of this protein as a feed additive in the future.
Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate by Chen Xiong; Chen Shouwen; Sun Ming; Yu Ziniu (pp. 390-396).
Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett–Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on γ-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box–Behnken design. The average γ-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.
d-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli by Björn Kaup; Stephanie Bringer-Meyer; Hermann Sahm (pp. 397-403).
Recently, we reported on the construction of a whole-cell biotransformation system in Escherichia coli for the production of d-mannitol from d-fructose (Kaup B, Bringer-Meyer S, Sahm H (2004) Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation. Appl Microbiol Biotechnol 64:333–339). Supplementation of this strain with extracellular glucose isomerase resulted in the formation of 800 mM d-mannitol from 1,000 mM d-glucose. Co-expression of the xylA gene of E. coli in the biotransformation strain resulted in a d-mannitol concentration of 420 mM from 1,000 mM d-glucose. This is the first example of conversion of d-glucose to d-mannitol with direct coupling of a glucose isomerase to the biotransformation system.
Utilization of keratin-containing biowaste to produce biohydrogen by Balázs Bálint; Zoltán Bagi; András Tóth; Gábor Rákhely; Katalin Perei; Kornél L. Kovács (pp. 404-410).
A two-stage fermentation system was constructed to test and demonstrate the feasibility of biohydrogen generation from keratin-rich biowaste. We isolated a novel aerobic Bacillus strain (Bacillus licheniformis KK1) that displays outstanding keratinolytic activity. The isolated strain was employed to convert keratin-containing biowaste into a fermentation product that is rich in amino acids and peptides. The process was optimized for the second fermentation step, in which the product of keratin fermentation—supplemented with essential minerals—was metabolized by Thermococcus litoralis, an anaerobic hyperthermophilic archaeon. T. litoralis grew on the keratin hydrolysate and produced hydrogen gas as a physiological fermentation byproduct. Hyperthermophilic cells utilized the keratin hydrolysate in a similar way as their standard nutrient, i.e., bacto-peptone. The generalization of the findings to protein-rich waste treatment and production of biohydrogen is discussed and possible means of further improvements are listed.
Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana by Tak-Hyun Park; Ki-Won Choi; Cheon-Seok Park; Soo-Bok Lee; Ho-Young Kang; Kwang-Jae Shon; Jang-Su Park; Jaeho Cha (pp. 411-422).
The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.
Construction of a selective cleavage system for a protein displayed on the cell surface of yeast by Michiko Kato; Haruko Maeda; Masayuki Kawakami; Seizaburo Shiraga; Mitsuyoshi Ueda (pp. 423-427).
We constructed a novel protein-purification system in which Saccharomyces cerevisiae with a protein displayed on the cell surface is harvested and the displayed protein is then cleaved from the cell surface. GFPuv was used as a model protein in this cell surface engineering experiment. In this system, the C-terminal 320 amino acids of α-agglutinin were bound to the C-terminal of GFPuv for display on the cell surface. In this novel system, the insertion of the recognition sequence-encoding gene of protease factor Xa between GFPuv and α-agglutinin was successfully carried out. The GFPuv, displayed by the insertion, was successfully cleaved from yeast cell surface by treatment with factor Xa, and could be easily recovered. By removing such a protease with well-known properties, the displayed protein could be isolated and purified with relative ease.
Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae by Alessandra Piscitelli; Paola Giardina; Cristina Mazzoni; Giovanni Sannia (pp. 428-439).
Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.
The yeast split-ubiquitin system to study chloroplast membrane protein interactions by Jan Christoph Pasch; Jörg Nickelsen; Danja Schünemann (pp. 440-447).
Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF0III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.
Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2 by Bharat Bhushan; Annamaria Halasz; Jalal Hawari (pp. 448-455).
In a previous study, a marine isolate Clostridium sp. EDB2 degraded 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) under anaerobic conditions (Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004c) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821); however, the enzyme responsible for CL-20 degradation was not known. In the present study, we isolated and purified an enzyme, from strain EDB2, responsible for CL-20 degradation. The enzyme was membrane-associated and NADH-dependent and had a molecular weight of 56 kDa (with SDS-PAGE). N-terminal amino acid sequence of enzyme revealed that it belonged to dehydrogenase class of enzymes. The purified enzyme degraded CL-20 at a rate of 18.5 nmol/h mg protein under anaerobic conditions. Carbon and nitrogen mass balance of the products were 100 and 64%, respectively. In LC–MS–MS studies, we detected three different initial metabolites from CL-20, i.e., mono-nitroso derivative, denitrohydrogenated product, and double-denitrated isomers with molecular weight of 422, 393, and 346 Da, corresponding to presumed empirical formulas of C6H6N12O11, C6H7N11O10, and C6H6N10O8, respectively. Identity of all the three metabolites were confirmed by using ring-labeled [15N]CL-20 and the nitro-group-labeled [15NO2]CL-20. Taken together, the above data suggested that the enzyme degraded CL-20 via three different routes: Route A, via two single electron transfers necessary to release two nitro-groups from CL-20 to produce two double-denitrated isomers; Route B, via a hydride transfer necessary to produce a denitrohydrogenated product; and Route C, via transfer of two redox equivalents to CL-20 necessary to produce a mono-nitroso derivative of CL-20. This is the first biochemical study which showed that CL-20 degradation can be initiated via more than one pathway.
Modeling of growth kinetics for Pseudomonas spp. during benzene degradation by D.-J. Kim; J.-W. Choi; N.-C. Choi; B. Mahendran; C.-E. Lee (pp. 456-462).
A modeling study was conducted on growth kinetics of three different strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) during benzene degradation to determine optimum substrate concentrations for most efficient biodegradation. Batch tests were performed for eight different initial substrate concentrations to observe cell growth and associated substrate degradation using benzene-adapted cells. Kinetic parameters of both inhibitory (Haldane–Andrews, Aiba–Edwards) and noninhibitory (Monod) models were fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that half-saturation constant of P. fluorescens was the highest among the three strains, indicating that this strain could grow well at high concentration, while P. putida could grow best at low concentration. The inhibition constant of P. aeruginosa was the highest, implying that it could tolerate high benzene concentration and therefore could grow at a wider concentration range. Estimated specific growth rate of P. putida was lower, but half-saturation constant was higher than those from literature study due to high substrate concentration range used in this study. These two kinetic parameters resulted in substantial difference between Monod- and Haldane-type models, indicating that distinction should be made in applying those models.
Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2 by Ching T. Hou (pp. 463-468).
We identified [Hou CT (2003) New uses of vegetable oils: novel oxygenated fatty acids by biotransformation. SIM News 53:56–61] many novel oxygenated fatty acids produced from linoleic acid by Bacillus megaterium ALA2: 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. 12,13,17-THOA, the main product, has antiplant pathogenic fungal activity. To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products were studied. Dextrose at 5 g/l was the optimum amount for the carbon source. A combination of 15 g yeast extract and 10 g tryptone showed good results as nitrogen sources. Among the metal ions tested, the optimum concentrations for the reaction for the different ions were as follows (in mM): magnesium 2.0, iron 0.5, zinc 0.1, nickel 0.01, and cobalt 0.05. Copper ions did not affect the production of oxygenated products; however, manganese ions inhibited the reaction. Addition of these metal ions did not alter the distribution of products. The optimum temperature and pH for the production of THOAs were 30°C and pH 6.5. Time course studies showed 40–48 h is the optimum for the production of both THOAs. These data provide the basis for engineering scale-up production of these new products.
Distribution of EPS and cell surface hydrophobicity in aerobic granules by Zhi-Wu Wang; Yu Liu; Joo-Hwa Tay (pp. 469-473).
This study described the distribution of extracellular polysaccharides (EPS) and hydrophobicity in aerobic granule as well as the essential role of EPS in maintaining the stable structure of aerobic granules. Aerobic granules showed a heterogeneous structure, which had an outer shell with high biomass density and an inner core having a relatively low biomass density. Results showed that the outer shell of aerobic granule was composed of poorly soluble and noneasily biodegradable EPS, whereas its core part was filled with readily soluble and biodegradable EPS. It was further found that the shell of aerobic granule exhibited a higher hydrophobicity than the core of granule. The insoluble EPS present in the granule shell would play a protective role with respect to the structure stability and integrity of aerobic granules.
Kinetic analysis of the transformation of phthalate esters in a series of stoichiometric reactions in anaerobic wastes by Vasily A. Vavilin; Susanne Jonsson; Bo H. Svensson (pp. 474-484).
Phthalates such as dimethyl phthalate, dimethyl terephthalate (DMT), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate (MEHP) are degraded to varying degrees under anaerobic conditions in waste treatment systems. Here we kinetically analyse the enzymatic hydrolyses involved and the subsequent stoichiometric reactions. The resulting model indicates that the degradation of the alcohols released and the transformation of the phthalic acid (PA) result in biphasic kinetics for the methane formation during transformation of DMT, DEP and MEHP. The ester hydrolysis and the PA transformation to methane appear to be the two rate-limiting steps. The PA-fermenting bacteria, which have biomass-specific growth rates between 0.04 and 0.085 day−1, grow more slowly than the other bacteria involved. Anaerobic microorganisms that remove intermediate products during phthalic acid ester conversion appear to be important for the efficiency of the ultimate phthalate degradation and to be inhibited by elevated hydrogen partial pressures. The model was based on (and the simulations corresponded well with) data obtained from experimental waste treatment systems.