Several studies suggest that petroleum biodegradation can be achieved by either aerobic or anaerobic microorganisms, depending on oxygen input or other electron acceptors and appropriate nutrients. Evidence from in vitro experiments with samples of petroleum formation water and oils from Pampo Field indicate that petroleum biodegradation is more likely to be a joint achievement of both aerobic and anaerobic bacterial consortium, refining our previous observations of aerobic degradation. The aerobic consortium depleted, in decreasing order, hydrocarbons > hopanes > steranes > tricyclic terpanes while the anaerobic consortium depleted hydrocarbons > steranes > hopanes > tricyclic terpanes. The oxygen content of the mixed consortia was measured from time to time revealing alternating periods of microaerobicity (O2 ~0.8 mg.L-1) and of aerobicity (O2~6.0 mg.L-1). In this experiment, the petroleum biodegradation changed from time to time, alternating periods of biodegradation similar to the aerobic process and periods of biodegradation similar to the anaerobic process. The consortia showed preferences for metabolizing hydrocarbons > hopanes > steranes > tricyclic terpanes during a 90-day period, after which this trend changed and steranes were more biodegraded than hopanes. The analysis of aerobic oil degrading microbiota by the 16S rRNA gene clone library detected the presence of Bacillus, Brevibacterium, Mesorhizobium and Achromobacter, and the analysis of the anaerobic oil degrading microbiota using the same technique detected the presence of Bacillus and Acinetobacter (facultative strains). In the mixed consortia Stenotrophomonas, Brevibacterium, Bacillus, Rhizobium, Achromobacter and 5% uncultured bacteria were detected. This is certainly a new contribution to the study of reservoir biodegradation processes, combining two of the more important accepted hypotheses.
Petroleum biodegradation; oxic environment; anoxic environment; 16S rRNA gene; petroleum biomarkers.
Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
TAIL-PCR; gene tagging; insertional mutagenesis
Systems Biology has taken advantage of computational tools and high-throughput experimental data to model several biological processes. These include signaling, gene regulatory, and metabolic networks. However, most of these models are specific to each kind of network. Their interconnection demands a whole-cell modeling framework for a complete understanding of cellular systems. We describe the features required by an integrated framework for modeling, analyzing and simulating biological processes, and review several modeling formalisms that have been used in Systems Biology including Boolean networks, Bayesian networks, Petri nets, process algebras, constraint-based models, differential equations, rule-based models, interacting state machines, cellular automata, and agent-based models. We compare the features provided by different formalisms, and discuss recent approaches in the integration of these formalisms, as well as possible directions for the future.
Systems Biology; Modeling Formalisms; Biological Networks
The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands.
Pseudozyma; Biodegradable plastic; Phyllosphere; Yeast
We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 220.127.116.11), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The Km values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible.
meso-Diaminopimelate dehydrogenase; Ureibacillus thermosphaericus; Thermostable amino acid dehydrogenase; Purification and characterization
Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.
NFAT-133; Streptomyces sp.; Antidiabetic; Actinomycetes
The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.
intestinal tract; biodiversity; carp; aquaculture; pyrosequencing; 16S rRNA
A major problem for manufacturers of cracked spores Ganoderma lucidum, a traditional functional food/Chinese medicine (TCM), is to ensure that raw materials are consistent as received from the producer. To address this, a feed-forward artificial neural network (ANN) method assisted by linear discriminant analysis (LDA) and principal component analysis (PCA) was developed for the spectroscopic discrimination of cracked spores of Ganoderma lucidum from uncracked spores. 120 samples comprising cracked spores, uncracked spores and concentrate of Ganoderma lucidum were analyzed. Differences in the absorption spectra located at ν1 (1143 - 1037 cm-1), ν2 (1660 - 1560 cm-1), ν3 (1745 - 1716 cm-1) and ν4 (2845 - 2798 cm-1) were identified by applying fourier transform infra-red (FTIR) spectroscopy and used as variables for discriminant analysis. The utilization of spectra frequencies offered maximum chemical information provided by the absorption spectra. Uncracked spores gave rise to characteristic spectrum that permitted discrimination from its cracked physical state. Parallel application of variables derived from unsupervised LDA/PCA provided useful (feed-forward) information to achieve 100% classification integrity objective in ANN. 100% model validation was obtained by utilizing 30 independent samples. ν1 was used to construct the matrix-matched calibration curve (n = 10) based on 4 levels of concentration (20%, 40%, 60% and 80% uncracked spores in cracked spores). A coefficient of correlation (r) of 0.97 was obtained. Relative standard deviation (RSD) of 11% was achieved using 100% uncracked spores (n = 30). These results demonstrate the feasibility of utilizing a combination of spectroscopy and prospective statistical tools to perform non destructive food quality assessment in a high throughput environment.
Adulteration; Artificial neural network; Cracked spores; Feed-forward; Ganoderma; Spectroscopy
With the emergence and spread of multidrug resistant bacteria, effective methods to eliminate both planktonic bacteria and those embedded in surface-attached biofilms are needed. Electric currents at μA-mA/cm2 range are known to reduce the viability of bacteria. However, the mechanism of such effects is still not well understood. In this study, Bacillus subtilis was used as the model Gram-positive species to systematically investigate the effects of electrochemical currents on bacteria including the morphology, viability, and gene expression of planktonic cells, and viability of biofilm cells. The data suggest that weak electrochemical currents can effectively eliminate B. subtilis both as planktonic cells and in biofilms. DNA microarray results indicate that the genes associated with oxidative stress response, nutrient starvation, and membrane functions were induced by electrochemical currents. These findings suggest that ions and oxidative species generated by electrochemical reactions might be important for the killing effects of these currents.
Bacillus subtilis; bioelectric effect; biofilm, gene expression; electrochemical current
Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.
Cyanophycinase; Cyanophycin degradation; Pseudomonas alcaligenes
In this work, Lactobacillus reuteri has been metabolically engineered for improving 1, 3-propanediol (1, 3-PD) production by the expression of an Escherichia coli alcohol dehydrogenase, yqhD, that is known to efficiently convert the precursor 3-hydroxypropionaldehyde (3-HPA) to 1, 3-PD. The engineered strain exhibited significantly altered formation rates for the product and other metabolites during the fermentation. An increase in the 1, 3-PD specific productivity of 34% and molar yield by 13% was achieved in the clone, relative to the native strain. A concomitant decrease in the levels of toxic intermediate, 3-HPA, was observed, with the specific productivity levels being 25% lesser than that of the native strain. Interestingly, the recombinant strain exhibited elevated rates of lactate and ethanol formation as well as reduced rate of acetate production, compared to the native strain. The preferential utilization of NADPH by YqhD with a possible decrease in the native 1, 3-PD oxidoreductase (NADH-dependent) activity, could have resulted in the diversion of surplus NADH towards increased lactate and ethanol productivities.
1, 3-propanediol oxidoreductase; YqhD; NADPH; 3-HPA; L. reuteri
In winemaking, nutrient supplementation is a common practice for optimising fermentation and producing quality wine. Nutritionally suboptimal grape juices are often enriched with nutrients in order to manipulate the production of yeast aroma compounds. Nutrients are also added to active dry yeast (ADY) rehydration media to enhance subsequent fermentation performance. In this study we demonstrate that nutrient supplementation at rehydration also has a significant effect on the formation of volatile sulfur compounds during wine fermentations. The concentration of the 'fruity' aroma compounds, the polyfunctional thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), was increased while the concentration of the 'rotten egg' aroma compound, hydrogen sulfide (H2S), was decreased. Nutrient supplementation of the rehydration media also changed the kinetics of H2S production during fermentation by advancing onset of H2S production. Microarray analysis revealed that this was not due to expression changes within the sulfate assimilation pathway, which is known to be a major contributor to H2S production. To gain insight into possible mechanisms responsible for this effect, a component of the rehydration nutrient mix, the tri-peptide glutathione (GSH) was added at rehydration and studied for its subsequent effects on H2S formation. GSH was found to be taken up during rehydration and to act as a source for H2S during the following fermentation. These findings represent a potential approach for managing sulfur aroma production through the use of rehydration nutrients.
Rehydration; yeast; nutrients; H2S; hydrogen-sulfide; GSH; glutathione
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.
Transcriptomic profiling; recombinant; fed-batch; Escherichia coli
Hydrophobicity is a very important surface property and there is a growing interest in the production and characterization of superhydrophobic surfaces. Accordingly, it was recently shown how to obtain a superhydrophobic surface using a simple and cost-effective method on a polymer named poly(L-lactic acid) (PLLA). To evaluate the ability of such material as a substrate for bacterial colonization, this work assessed the capability of different bacteria to colonize a biomimetic rough superhydrophobic (SH) PLLA surface and also a smooth hydrophobic (H) one. The interaction between these surfaces and bacteria with different morphologies and cell walls was studied using one strain of Staphylococcus aureus and one of Pseudomonas aeruginosa. Results showed that both bacterial strains colonized the surfaces tested, although significantly higher numbers of S. aureus cells were found on SH surfaces comparing to H ones. Moreover, scanning electron microscopy images showed an extracellular matrix produced by P. aeruginosa on SH PLLA surfaces, indicating that this bacterium is able to form a biofilm on such substratum. Bacterial removal through lotus leaf effect was also tested, being more efficient on H coupons than on SH PLLA ones. Overall, the results showed that SH PLLA surfaces can be used as a substrate for bacterial colonization and, thus, have an exceptional potential for biotechnology applications.
Poly(L-lactic acid); Superhydrophobicity; Biomimetic surfaces; Bacterial colonization substrate
The upper parts of oil field structures may leak gas which is supposed to be indirectly detected by the soil bacterial populations. Such microorganisms are capable of consuming this gas, supporting the Microbial Prospection of Oil and Gas (MPOG) methodology. The goal of the present work was to characterize microbial communities involved in short-chain alkane metabolism, namely methane, ethane and propane, in samples from a petroliferous (P) soil through clone libraries of the 16S rRNA gene of the Domains Bacteria and Archaea and the catabolic gene coding for the soluble di-iron monooxygenase (SDIMO) enzyme alpha subunit. The microbial community presented high abundance of the bacterial phylum Actinobacteria, which represented 53% of total clones, and the Crenarchaeota group I.1b from the Archaea Domain. The analysis of the catabolic genes revealed the occurrence of seven Operational Protein Families (OPF) and higher richness (Chao = 7; Ace = 7.5) and diversity (Shannon = 1.09) in P soil when compared with a non-petroliferous (Np) soil (Chao = 2; Ace = 0, Shannon = 0.44). Clones related to the ethene monooxygenase (EtnC) and methane monooxygenase (MmoX) coding genes occurred only in P soil, which also presented higher levels of methane and lower levels of ethane and propane, revealed by short-chain hydrocarbon measures. Real-time PCR results suggested that the SDIMO genes occur in very low abundance in the soil samples under study. Further investigations on SDIMOs genes in natural environments are necessary to unravel their still uncharted diversity and to provide reliable tools for the prospection of degrading populations.
Short-chain hydrocarbons; Microbial prospection; Community structure; Gene libraries; Soluble di-iron monooxygenases
Bovine viral diarrhea virus is a pestivirus in the family Flaviviridae that cause abortions and stillbirths in livestock and its traditional diagnosis is based on cell culture and virus neutralization test. In this study, for more sensitive, specific detection and determined the prevalence of virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses the antigen capture ELISA and RT-PCR were recommended. From the total of 2173 aborted fetuses, 347 (15.96%) and 402 (18.49%) were positive for presence of Bovine viral diarrhea virus by antigen capture ELISA and RT-PCR respectively. Statistical analysis of data showed significant differences between ELISA and RT-PCR for detection of virus in aborted fetuses.
These results indicate a high presence of this pathogen in Iran and that RT- PCR is considerably faster and more accurate than ELISA for identification of Bovine viral diarrhea virus.
To our knowledge the Camels and Bovine are the most resistant and sensitive to Bovine viral diarrhea's abortions respectively and the prevalence of virus in Caprine is more than Ovine aborted fetuses. This study is the first prevalence report of Bovine viral diarrhea virus in aborted Bovine, Ovine, Caprine, Buffalo and Camel fetuses by evaluation of ELISA and RT-PCR in Iran.
Prevalence study; Bovine viral diarrhea virus; antigen capture ELISA; RT-PCR; aborted fetuses; Iran
An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities.
Peroxidase; Basidiomycota; Cytochrome P450; Bioreactor
Methanogen populations and their domains are poorly understood; however, in recent years, research on this topic has emerged. The relevance of this field has also been enhanced by the growing economic interest in methanogen skills, particularly the production of methane from organic substrates. Management attention turned to anaerobic wastes digestion because the volume and environmental impact reductions. Methanogenesis is the biochemically limiting step of the process and the industrially interesting phase because it connects to the amount of biogas production. For this reason, several studies have evaluated the structure of methanogen communities during this process. Currently, it is clear that the methanogen load and diversity depend on the feeding characteristics and the process conditions, but not much data is available. In this study, we apply a Real-Time Polymerase Chain Reaction (RT-PCR) method based on mcrA target to evaluate, by specific probes, some subgroups of methanogens during the mesophilic anaerobic digestion process fed wastewater sludge and organic fraction of the municipal solid waste with two different pre-treatments. The obtained data showed the prevalence of Methanomicrobiales and significantly positive correlation between Methanosarcina and Methanosaetae and the biogas production rate (0.744 p < 0.01 and 0.641 p < 0.05). Methanosarcina detected levels are different during the process after the two pre-treatment of the input materials (T-test p < 0.05). Moreover, a role as diagnostic tool could be suggested in digestion optimisation.
methanogen; anaerobic digestion; biogas production; Methanosarcina; Archaea communities
Cellulose degradation is one of the major bottlenecks of a consolidated bioprocess that employs cellulolytic bacterial cells as catalysts to produce biofuels from cellulosic biomass. In this study, we investigated the spatial and temporal dynamics of cellulose degradation by Caldicellulosiruptfor obsidiansis, which does not produce cellulosomes, and Clostridium thermocellum, which does produce cellulosomes. Results showed that the degradation of either regenerated or natural cellulose was synchronized with biofilm formation, a process characterized by the formation and fusion of numerous crater-like depressions on the cellulose surface. In addition, the dynamics of biofilm formation were similar in both bacteria, regardless of cellulosome production. Only the areas of cellulose surface colonized by microbes were significantly degraded, highlighting the essential role of the cellulolytic biofilm in cellulose utilization. After initial attachment, the microbial biofilm structure remained thin, uniform and dense throughout the experiment. A cellular automaton model, constructed under the assumption that the attached cells divide and produce daughter cells that contribute to the hydrolysis of the adjacent cellulose, can largely simulate the observed process of biofilm formation and cellulose degradation. This study presents a model, based on direct observation, correlating cellulolytic biofilm formation with cellulose degradation.
biofilm; thermophile; cellulosome; cellulose
Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.
Bacteriophage; S. pneumoniae; Cpl-1; PEGylation; Endolysin; Enzybiotic
The black mould Alternaria alternata produces a wide diversity of mycotoxins which are of particular health concern. Since no maximum allowable limits are set for Alternaria toxins in food and feed, prevention of Alternaria infestations and mycotoxin spoilage is the only way to avoid health risks. Thus, the understanding of mycotoxin biosynthesis is essential. For that purpose, a reliable batch process in a 2 L bioreactor was established which enables the study of several parameters influencing the production of the mycotoxins alternariol (AOH), alternariol monomethylether (AME) and tenuazonic acid (TA) by A. alternata DSM 12633. Modified Czapek-Dox medium was used with glucose as carbon source and ammonium and nitrate as nitrogen sources. Consumption of carbon and nitrogen sources as well as formation of the three mycotoxins were monitored; the average data of five independent fermentations was plotted and fitted using a logistic equation with four parameters. Maximum mycotoxin concentrations of 3.49 ± 0.12 mg/L AOH, 1.62 ± 0.14 mg/L AME and 38.28 ± 0.1 mg/L TA were obtained.
In this system the effect of different aeration rates (0.53 vvm-0.013 vvm) was tested which exerted a great influence on mycotoxin production. The use of the semi-synthetic Czapek-Dox medium allowed the exchange of carbon and nitrogen sources for acetate and aspartic acid. The use of acetate instead of glucose resulted in the sole production of alternariol whereas the exchange of ammonium and nitrate for aspartate enhanced the production of both AOH and AME while TA production was not affected.
Alternaria alternata; Mycotoxin; Batch process; Aeration rate
Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.
chromatography; fibrinolysis; Perionyx excavatus; PMSF; serine protease; tandem MS analysis
Effective utilization of photosynthetic microorganisms as potential biocatalysts is favorable for the production of useful biomaterials and the reduction of atmospheric CO2. For example, biocatalytic transformations are used in the synthesis of optically active alcohols. We previously found that ketone reduction in cells of the cyanobacterium Synechococcus PCC 7942 is highly enantioselective and remarkably enhanced under light illumination. In this study, the mechanism of light-enhanced ketone reduction was investigated in detail using several inhibitors of photosynthetic electron transport and of enzymes of the Calvin cycle. It is demonstrated that light intensity and photosynthesis inhibitors significantly affect the ketone reduction activity in Synechococcus. This indicates that the reduction correlates well with photosynthetic activity. Moreover, ketone reduction in Synechococcus specifically depends upon NADPH and not NADH. These results also suggest that cyanobacteria have the potential to be utilized as biocatalytic systems for direct usage of light energy in various applications such as syntheses of useful compounds and remediation of environmental pollutants.
biocatalysis; cyanobacteria; ketone reduction; light energy; NADPH; photosynthesis
To determine the sensitivities to low electrical potential of human immunodeficiency virus type 1 (HIV-1) and its target cells, HIV-1 and MAGIC-5 cells were directly stimulated with a constant direct current potential of 1.0 V (vs. Ag/AgCl). HIV-1 was incubated for 3 h at 37°C on a poly-L-lysine-coated indium-tin oxide electrode, and then stimulated by an electrical potential. MAGIC-5 cells were seeded onto the electrically stimulated HIV-1 and cultured for 3 days at 37°C. HIV-1-infected cells were measured by multinuclear activation via a galactosidase indicator assay. MAGIC-5 cells were also stimulated by an electrical potential of 1.0 V; cell damage, proliferation and apoptosis were evaluated by trypan blue staining, cell counting and in situ apoptosis detection, respectively. HIV-1 was found to be damaged to a greater extent by electrical stimulation than the cells. In particular, after application of a 1.0-V potential for 3 min, HIV-1LAI and HIV-1KMT infection were inhibited by about 90%, but changes in cell damage, proliferation and apoptosis were virtually undetectable. These results suggested that HIV-1 is significantly more susceptible to low electrical potential than cells. This finding could form the basis of a novel therapeutic strategy against HIV-1 infection.
HIV-1 infectivity; electrical stimulation; indium-tin oxide; poly-L-lysine