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.66, #4)
Microbial hyaluronic acid production by Barrie Fong Chong; Lars M. Blank; Richard Mclaughlin; Lars K. Nielsen (pp. 341-351).
Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.
Towards electronic paper displays made from microbial cellulose by Jay Shah; R. Malcolm Brown Jr. (pp. 352-355).
Cellulose (in the form of printed paper) has always been the prime medium for displaying information in our society and is far better than the various existing display technologies. This is because of its high reflectivity, contrast, low cost and flexibility. There is a major initiative to push for a dynamic display technology that emulates paper (popularly known as “electronic paper”). We have successfully demonstrated the proof of the concept of developing a dynamic display on cellulose. To the best of our knowledge, this is the first significant effort to achieve an electronic display using bacterial cellulose. First, bacterial cellulose is synthesized in a culture of Acetobacter xylinum in standard glucose-rich medium. The bacterial cellulose membrane thus formed (not pulp) is dimensionally stable, has a paper-like appearance and has a unique microfibrillar nanostructure. The technique then involves first making the cellulose an electrically conducting (or semi-conducting) sheet by depositing ions around the microfibrils to provide conducting pathways and then immobilizing electrochromic dyes within the microstructure. The whole system is then cased between transparent electrodes, and upon application of switching potentials (2–5 V) a reversible color change can be demonstrated down to a standard pixel-sized area (ca. 100 μm2). Using a standard back-plane or in-plane drive circuit, a high-resolution dynamic display device using cellulose as substrate can be constructed. The major advantages of such a device are its high paper-like reflectivity, flexibility, contrast and biodegradability. The device has the potential to be extended to various applications, such as e-book tablets, e-newspapers, dynamic wall papers, rewritable maps and learning tools.
Biotechnology of desulfurization of diesel: prospects and challenges by Nidhi Gupta; P. K. Roychoudhury; J. K. Deb (pp. 356-366).
To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to make a huge investment to bring down the sulfur content in diesel to the desired level, using conventional hydrodesulfurization (HDS) technology, by which sulfur is catalytically converted to hydrogen sulfide in the presence of hydrogen. These reactions proceed rapidly only at high temperature and pressure and therefore the capital cost as well as the operating cost associated with HDS very high. Biological desulfurization has the potential of being developed as a viable technology downstream of classical HDS. Various attempts have been made to develop biotechnological processes based on microbiological desulfurization employing aerobic and anaerobic bacteria. However, there are several bottlenecks limiting commercialization of the process. This review discusses various aspects of microbial desulfurization and the progress made towards its commercialization.
Mycotoxins as harmful indoor air contaminants by Bruce B. Jarvis; J. David Miller (pp. 367-372).
Fungal metabolites (mycotoxins) that pose a health hazard to humans and animals have long been known to be associated with mold-contaminated food and feed. In recent times, concerns have been raised about exposures to mycotoxin-producing fungi in indoor environments, e.g., damp homes and buildings. The principal mycotoxins that contaminate food and feed (alfatoxins, fumonisins, ochratoxin A, deoxynivalenol, zearalenone) are rarely if ever found in indoor environments, but their toxicological properties provide an insight into the difficulties of assessing the health effects of related mycotoxins produced by indoor molds. Although the Penicillium and Aspergillus genera of fungi are major contaminants of both food and feed products and damp buildings, the particular species and hence the array of mycotoxins are quite different in these environments. The mycotoxins of these indoor species and less common mycotoxins from Stachybotrys and Chaetomium fungi are discussed in terms of their health effects and the need for relevant biomarkers and long-term chronic exposure studies.
HBMMD: an enhanced database of the microorganisms associated with deeper water marine invertebrates by Aravinda S. Gunasekera; Karen S. Sfanos; Dedra K. Harmody; Shirley A. Pomponi; Peter J. McCarthy; Jose V. Lopez (pp. 373-376).
The Harbor Branch Marine Microbial Database (HBMMD) provides preliminary taxonomic identifications and features of microorganisms maintained in the Harbor Branch Oceanographic Institution Marine Microbial Culture Collection. The microbes are primarily derived from marine invertebrates such as sponges (phylum Porifera) and soft corals (phylum Cnidaria) found in deep water environments [>120 feet (>35 m) seawater]. The microbes isolated from within marine invertebrates represent some unique taxa and phylogenetic signatures. The database provides a user-friendly method to systemically search or sort a desired input. The site allows a powerful search for multiple parameters of any entry. Images of the microbes are contained within the database and can be accessed from the website. The HBMMD homepage is located at http://www.hboi.edu/dbmr/dbmr_hbmmd.html.
A screen-printed biosensor using pyruvate oxidase for rapid determination of phosphate in synthetic wastewater by Roger C. H. Kwan; H. F. Leung; Phoebe Y. T. Hon; J. P. Barford; R. Renneberg (pp. 377-383).
A screen-printed phosphate biosensor based on immobilized pyruvate oxidase (PyOD, E.C. 1.2.3.3) has been developed for monitoring phosphate concentrations in a sequencing batch reactor (SBR) system. The enzyme was immobilized by a nafion matrix and covered a poly(carbamoyl) sulfonate (PCS) hydrogel on a screen-printed electrode. PyOD consumes phosphate in the presence of pyruvate and oxygen and generates hydrogen peroxide (H2O2), carbon dioxide and acetylphosphate. The electroactive H2O2, monitored at +420 mV vs Ag/AgCl, is generated in proportion to the concentration of phosphate. The sensor has a fast response time (2 s) and a short recovery period (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 μM to 625 μM phosphate with a detection limit of 3.6 μM. There was good agreement (R2=0.9848) between the commercial phosphate testing kit and the phosphate sensor in measurements of synthetic wastewater in a SBR system. This sensor maintained a high working stability (>85%) after 12 h of operation and involved a simple operation procedure. It therefore serves as a useful tool for rapid and accurate phosphate measurements in the SBR system and probably for process control.
Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori by Vivi Joosten; Robin J. Gouka; Cees A. M. J. J. van den Hondel; C. Theo Verrips; B. Christien Lokman (pp. 384-392).
We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional VHHs were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted VHHs were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.
Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining by Marco W. Fraaije; Jin Wu; Dominic P. H. M. Heuts; Erik W. van Hellemond; Jeffrey H. Lutje Spelberg; Dick B. Janssen (pp. 393-400).
Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcat = 1.9 s−1, KM = 59 μM). The enzyme exhibits moderate enantioselectivity with α-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.
Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization by Pauliina Lankinen; Kristiina Hildén; Nina Aro; Mirja Salkinoja-Salonen; Annele Hatakka (pp. 401-407).
The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.
Identification of Pyrococcus furiosus amylopullulanase catalytic residues by S. Kang; C. Vieille; J. G. Zeikus (pp. 408-413).
Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (kcat/Km) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.
Quantification by real-time PCR of Lactococcus lactis subsp. cremoris in milk fermented by a mixed culture by F. Grattepanche; C. Lacroix; P. Audet; G. Lapointe (pp. 414-421).
During cheese making, interactions between different strains of lactic acid bacteria play an important role. However, few methods are available to specifically determine each bacterial population in mixed cultures, in particular for strains of the same species. The aim of this study was to develop a real-time PCR quantification method to monitor the population of Lactococcus cremoris ATCC 19257 in mixed culture with Lactobacillus rhamnosus RW-9595M and the bacteriocin-producing microorganism Lc. diacetylactis UL719. The specificity of the two primers 68FCa33 and 16SR308 used to amplify a 240-bp fragment of DNA from Lc. cremoris was demonstrated by conventional PCR. Using these primers for real-time PCR, the detection limit was 2 cfu/reaction or 200 cfu of Lc. cremoris ATCC 19257 per millilitre of mixed culture in milk. In pure culture batch fermentation, good correlation was obtained between real-time PCR and the conventional plating method for monitoring Lc. cremoris growth. In mixed culture batch fermentation, Lb. rhamnosus and Lc. cremoris decreased due to nisin Z production by Lc. diacetylactis. The decrease of the Lc. cremoris cell population detected by real-time PCR was not possible to observe by the plate count method in the presence of a Lc. diacetylactis population that was 1 log higher.
Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds by Lingyun Rui; Kenneth F. Reardon; Thomas K. Wood (pp. 422-429).
Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase α-subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium. Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V. To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106. Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography–mass spectroscopy. Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G). The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound. Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring. Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed. Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions. Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.
Enzymatic synthesis of a catechin conjugate of polyhedral oligomeric silsesquioxane and evaluation of its antioxidant activity by N. Ihara; M. Kurisawa; J. E. Chung; H. Uyama; S. Kobayashi (pp. 430-433).
The antioxidant activity of catechin was amplified by conjugation with amine-terminated polyhedral oligomeric silsesquioxane (POSS) using horseradish peroxidase as catalyst. Compared to intact catechin, the scavenging activity of the POSS-catechin conjugate against superoxide anion was greatly improved. In addition, the conjugate strongly inhibited xanthine oxidase activity.
Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain by Frank Hoster; Jessica E. Schmitz; Rolf Daniel (pp. 434-442).
Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples. Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi. Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces. The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B. ehimensis (MG1), and Streptomyces griseus (MG3). The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45°C and 50°C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5–7 (MG3). The crude chitinase preparations of all five strains possessed antifungal activity. The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested. The chiIS gene was cloned and the gene product was purified. The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da. Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases. Purified ChiIS showed remarkable antifungal activity and stability.
Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus by Beata Kluczek-Turpeinen; Kari T. Steffen; Marja Tuomela; Annele Hatakka; Martin Hofrichter (pp. 443-449).
The soil mold Paecilomyces inflatus is capable of modifying and partially mineralizing synthetic and natural humic acids (HAs) in compost environments. HA degradation studies using a synthetic HA (14C-HA) in autoclaved compost microcosms showed that, after 12 weeks of cultivation, P. inflatus mineralized approximately 5% of the 14C-labeled HA to14CO2, while 6% of the 14C-HA was converted into 14C-labeled water-soluble fragments (fulvic-acid-like fraction). About 40% was still present as NaOH-soluble HA representing unmodified or only slightly modified humic material (compared with 60% in the controls). Modification of natural HAs extracted from compost was followed by their partial decolorization (30%) in liquid cultures of P. inflatus. Bleaching of the medium was accompanied by moderate changes in the molecular mass distribution of both the HA and fulvic-acid fractions, which were analyzed with high-performance size exclusion chromatography. HA modification was most pronounced during the primary growth phase of the fungus and was associated with increased laccase activity.
Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications by Claude Jolivalt; Catherine Madzak; Agathe Brault; Eliane Caminade; Christian Malosse; Christian Mougin (pp. 450-456).
Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.
Quantifying bacterial population dynamics in compost using 16S rRNA gene probes by Patrick D. Schloss; Anthony G. Hay; David B. Wilson; James M. Gossett; Larry P. Walker (pp. 457-463).
Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17–33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).
Quantification by real-time PCR of Lactococcus lactis subsp. cremoris in milk fermented by a mixed culture
by F. Grattepanche; C. Lacroix; P. Audet; G. Lapointe (pp. 464-464).