Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Applied Microbiology and Biotechnology (v.69, #2)
Chip devices for miniaturized biotechnology by J. Michael Köhler; Thomas Henkel (pp. 113-125).
Chip devices were introduced in chemistry and molecular biology to improve the read-out of information from molecular systems by efficient analytical procedures and to organize automated experiments. Biochips and chip reactor systems are of interest for cellular processes, too, and can be regarded as components in interfaces for the information exchange between living nature and digital electronic systems. In this minireview, different types of chip reactors for biotechnological applications like nanotiterplates, chip thermocyclers and devices for segmented flow operations are discussed. Finally, an outlook is given on the application of chip reactor systems, which are promising tools for automated experiments with highly parallelized screening procedures, for artificial microcompartmentation, cell analogue systems, micro-ecological studies, investigations on modulated morphogenesis, and for a bioanalogue molecular nanotechnology.
Keywords: Chip reactors; Microsystems; Microreaction technology; μ–TAS; Flow–through devices; Cell manipulation; Microcultivation; Microsegmented flow
Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo by Yong Peng; Xiaojuan Yang; Yizheng Zhang (pp. 126-132).
Accumulation of fibrin in the blood vessels usually results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. For thrombolytic therapy, microbial fibrinolytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesirable side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus from traditional fermented foods. The physiochemical properties of these enzymes have been characterized, and their effectiveness in thrombolysis in vivo has been further identified. Therefore, microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases.
Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor by Hannes Hofer; Walter Steiner (pp. 133-140).
The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K M of oxygen must be known. The determination of this constant was done using progress curve analysis (K M=260 μmol l−1). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35°C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5°C to 9 min at 35°C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.
Characterization of Sphingomonas aldehyde dehydrogenase catalyzing the conversion of various aromatic aldehydes to their carboxylic acids by Xue Peng; Kazutoshi Shindo; Kaneo Kanoh; Yukie Inomata; Seon-Kang Choi; Norihiko Misawa (pp. 141-150).
An aldehyde dehydrogenase gene, designated phnN, was isolated from a genome library of the 1,4-dimethylnaphthalene-utilizing soil bacterium, Sphingomonas sp. 14DN61. Escherichia coli expressing the phnN gene converted 1,4-dihydroxymethylnaphthalene to 1-hydroxymethyl-4-naphthoic acid. The putative amino acid sequence of the phnN gene product had 31–42% identity with those of NAD+-dependent short-chain aliphatic aldehyde dehydrogenases and a secondary alcohol dehydrogenase. The NAD(P)+-binding site and two consensus sequences involved in the active site for aldehyde dehydrogenase are conserved among these proteins. The PhnN enzyme purified from recombinant E. coli showed broad substrate specificity towards various aromatic aldehydes, i.e., 1- and 2-naphaldehydes, cinnamaldehyde, vanillin, syringaldehyde, benzaldehyde and benzaldehydes substituted with a hydroxyl, methyl, methoxy, chloro, fluoro, or nitro group were converted to their corresponding carboxylic acids. Interestingly, E. coli expressing phnN was able to biotransform a variety of not only aromatic aldehydes, but also aromatic alcohols to carboxylic acids.
Multiple large segment deletion method for Corynebacterium glutamicum by Nobuaki Suzuki; Hiroshi Nonaka; Yota Tsuge; Satoshi Okayama; Masayuki Inui; Hideaki Yukawa (pp. 151-161).
A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.
Selection strategies for the establishment of recombinant Chinese hamster ovary cell line with dihydrofolate reductase-mediated gene amplification by Seung Chul Jun; Min Soo Kim; Jong Youn Baik; Sun Ok Hwang; Gyun Min Lee (pp. 162-169).
To evaluate the efficacy of selection strategies for recombinant Chinese hamster ovary (rCHO) clones undergone with dihydrofolate reductase-mediated gene amplification, rCHO cell lines producing a chimeric antibody were established using two strategies, one based on individual clones and the other based on cell pools. In a selection based on individual clones, cell cloning by limiting dilution method was performed twice, once after a round of selection of parental cell clones and once after obtaining high-producer clones. Thirty parental clones selected from 300 parental clones were cultivated independently throughout the gene amplification procedure. Using this labor-intensive strategy, it took approximately 17 weeks to obtain high-producing clones such as CS11-8 and CS18-3 clones. A selection based on cell pools, in which cell cloning was performed once at the final selection stage, required less effort and time to amplify large numbers of individual parental clones within the pool. However, high-producing clones were lost during the amplification procedure. The antibody expression level of high-producing clones such as PS7-2 and PS7-32 chosen on the basis of cell pools was less than one third of that of CS11-8 and CS18-3 clones. Taken together, a selection strategy based on individual clones is favored for establishment of high-producing rCHO clones because it is more efficient to perform cell cloning at the initial selection stage of parental cell clones.
The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose by Karin Nijkamp; Nicole van Luijk; Jan A. M. de Bont; Jan Wery (pp. 170-177).
A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.
Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica by Mei Guo; Fuping Lu; Jun Pu; Dongqing Bai; Lianxiang Du (pp. 178-183).
A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79 U ml−1. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.
Functional GNA expressed in Escherichia coli with high efficiency and its effect on Ceratovacuna lanigera Zehntner by Sulan Luo; Dongting Zhangsun; Kexuan Tang (pp. 184-191).
The mannose-specific GNA (Galanthus nivalis agglutinin, snowdrop lectin) are the resistant proteins with many bioactivities. Snowdrop lectin is different with plant organs and development periods in lectin species, content, and bioactivities. It is an effective and cheap way to obtain much active GNA through overexpression of GNA gene in Escherichia coli. Constructs encoding mature GNA fused with an N-terminal pelB signal sequence protein (PelB) were expressed in E. coli with high efficiency. Recombinant protein productivity was higher than values published before. The insecticidal activity of purified recombinant proteins was assayed on feeding sugarcane wooly aphid (Ceratovacuna lanigera Zehntner), as well as spraying on sugarcane plants infected by aphids. The insecticidal activity was found to be comparable to native GNA. Oral delivery has obvious positive implications for crop protection against insect pests since peptides can be present in, or sprayed on, plant tissues susceptible to damage. A highly efficient expression of functional recombinant GNA would decrease the cost of GNA and promote its wide use, especially to give crop protection in the field.
Characterization of biochemical properties and biological activities of biosurfactants produced by Pseudomonas aeruginosa mucoid and non-mucoid strains isolated from hydrocarbon-contaminated soil samples by Kishore Das; Ashis K. Mukherjee (pp. 192-199).
Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseudomonas aeruginosa mucoid (M) and non-mucoid (NM) strains, isolated from hydrocarbon-contaminated soil samples, were characterized. Both the strains secreted appreciable amount of biosurfactants (5.0–6.5 g/l), responsible for the reduction of surface tension of the medium from 68 to 29±0.5 mN/m post 96 h of growth. Maximum yield of biosurfactants was observed following the supplementation of NH4Cl and glycerol as nitrogenous source and carbon source, respectively. These thermostable biosurfactants exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand-pack column. Pharmacological characterization of these biosurfactants revealed that they induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were non-detrimental to chicken lung, liver, heart and kidney tissues. Our study has documented that biosurfactants from P. aeruginosa M and NM strains could be exploited for use in petroleum sectors as well in pharmaceutical industries.
Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius by Silvia Guzmán; Itzel Ramos; Elizabeth Moreno; Beatriz Ruiz; Romina Rodríguez-Sanoja; Laura Escalante; Elizabeth Langley; Sergio Sanchez (pp. 200-206).
Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.
Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production by M. Reverberi; A. A. Fabbri; S. Zjalic; A. Ricelli; F. Punelli; C. Fanelli (pp. 207-215).
Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and β-glucan content. Mushroom β-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with β-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, β-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.
Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances by Guo-Ping Sheng; Han-Qing Yu; Zheng-Bo Yue (pp. 216-222).
A hydrogen-producing photosynthetic bacteria strain, Rhodopseudomonas acidophila, was used to investigate the production of extracellular polymeric substances (EPS) in the presence of toxic substances and the effect of toxicants on bacterial surface characteristics. Addition of the toxic substances including Cu(II), Cr(VI), Cd(II) and 2,4-dichlorophenol (2,4-DCP) stimulated the production of EPS but reduced the cell dry weight. At concentrations of 30 mg l−1 Cu(II), 40 mg l−1 Cr(VI), 5 mg l−1 Cd(II) and 100 mg l−1 2,4-DCP, the EPS content increased by 5.5, 2.5, 4.0 and 1.4 times, respectively, than the control. These toxic substances also greatly influenced the proteins/carbohydrates ratio of EPS. The ratios in the presence of toxic substances were always higher than that of control. Furthermore, under toxic conditions, the increase in the protein content far exceeded than that of others in EPS, suggesting that extracellular proteins could protect cells against toxic substances. The toxic substances significantly changed the surface characteristics and flocculation ability of R. acidophila, such as surface energy, relative hydrophobicity and free energy of adhesion.
Enhancement of the Gibberella zeae growth inhibitory lipopeptides from a Bacillus subtilis mutant by ion beam implantation by J. Liu; M. Liu; J. Wang; J. M. Yao; R. R. Pan; Z. L. Yu (pp. 223-228).
Bacillus subtilis JA antagonized the growth of Gibberella zeae. In order to reduce growth of this fungi pathogen to a greater extent, low-energy ion beam implantation was applied in mutant breeding. We studied the effects of different energies and different doses of nitrogen ion implantation. The mutant strain designated as JA026 was obtained showing higher inhibition activity in the screening plate. Its inhibition zone against indicator organism increased by 14.3% compared to the original strain. The electrospray ionization mass spectrometry (ESI/MS) analysis indicated that the antifungal lipopeptides produced by the mutant were identical to those produced by the wild-type strain. The mutant strain exhibited favorable properties including the high yield of antifungal lipopeptides production and faster growth over the parent strain, which suggested that this strain would be a promising biocontrol candidate in agriculture.
A novel membrane bioreactor enhanced by effective microorganisms for the treatment of domestic wastewater by Min Jin; Xin-Wei Wang; Tai-Shi Gong; Chang-Qin Gu; Bin Zhang; Zhi-Qiang Shen; Jun-Wen Li (pp. 229-235).
The activated sludge membrane bioreactor (MBR) has been shown to have some advantages for the processing and reclamation of domestic wastewater. We hypothesized that certain microorganisms, chosen for their abilities to decompose the chemical components of raw sewage, would, when coupled with the MBR, significantly improve the stability and efficiency of this system. We selected environmental bacterial strains which oxidize ammonia and nitrites and produce protease, amylase, and cellulase for the development and testing of a novel biologically enhanced MBR (eMBR). We compared the eMBR with the activated sludge MBR. With the eMBR, the average values of effluent quality were: chemical oxygen demand (COD), 40 mg/l(average efficiency of removal 90.0%); and NH4 +–N, 0.66 mg/l(average efficiency of removal 99.4%). Effluent qualities met the standard and were stable during the entire 90 days of this study. For the activated sludge MBR, the COD removal rate was 91.7%, and the NH4 +–N removal (94.8%) was less than that of the eMBR. Start-up time for the eMBR was only 24–48 h, much shorter than the 7–8 days required to initiate function of the standard MBR. The biomass concentrations of total heterotrophic bacteria and autotrophic bacteria in the eMBR did not fluctuate significantly during the course of the study. Various kinds of microorganisms will establish an ecological balance in the reactor. Compared with the activated sludge MBR, the eMBR not only produced an excellent and stable quality of effluent but also resulted in a shorter time to start-up and significantly improved the efficiency of NH4 +–N removal.