Gemmobacter serpentinus sp. nov., isolated from conserved forages.

A Gram-stain-negative, long-rod-shaped and facultative aerobic bacterium, designated HB-1T, was isolated from a round hay bale at the Kansas State University Beef Stocker Unit. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that strain HB-1T clustered within the genus Gemmobacter and its closest relatives were Gemmobacter aquaticus A1-9T (98.0 %), Gemmobacter lutimaris YJ-T1-11T (98.0 %), Gemmobacter fontiphilus JS43T (97.8 %), Gemmobacter aquatilis DSM 3857T (97.5 %) and Gemmobacter lanyuensis Orc-4T (96.9 %). Additional phylogenomic analysis also indicated that strain HB-1T belongs to the genus Gemmobacter. The draft genome of strain HB-1T had a total length of 4.23 Mbp and contained 4071 protein-coding genes. The average nucleotide identity values between the genomes of strain HB-1T and the three most-related type strains ranged from 77.5 to 78.1 %. The DNA G+C content of strain HB-1T was 63.7 mol%. The novel strain grew at 10-37 °C, pH 5-10 and with 0-2 % NaCl. Oxidase and catalase activities were positive. Cells were 0.3-0.4 µm wide, 3.0-7.0 µm long and usually found in pairs or chains of cells. The major respiratory quinone of strain HB-1T was Q-10 (90 %), with a minor amount of Q-9 (10 %). The major fatty acids were C18 : 1 ω7c (54.6 %) and C16 : 0 (18.2 %). On the basis of phenotypic, phylogenetic and chemotaxonomic data, strain HB-1T (=DSM 109828T=ATCC TSD-211T) is considered to represent a novel species of the genus Gemmobacter, for which the name Gemmobacter serpentinus sp. nov. is proposed.

High-throughput analysis and protein engineering using microcapillary arrays.

We describe a multipurpose technology platform, termed µSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. µSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With µSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the µSCALE platform.

Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.

It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 µs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.

A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo.

Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe(III)-to-Fe(II) reduction potential and substantially improves NAD(P)H-driven activity.

A conformational ensemble derived using NMR methyl chemical shifts reveals a mechanical clamping transition that gates the binding of the HU protein to DNA.

Recent improvements in the accuracy of structure-based methods for the prediction of nuclear magnetic resonance chemical shifts have inspired numerous approaches for determining the secondary and tertiary structures of proteins. Such advances also suggest the possibility of using chemical shifts to characterize the conformational fluctuations of these molecules. Here we describe a method of using methyl chemical shifts as restraints in replica-averaged molecular dynamics (MD) simulations, which enables us to determine the conformational ensemble of the HU dimer and characterize the range of motions accessible to its flexible ß-arms. Our analysis suggests that the bending action of HU on DNA is mediated by a mechanical clamping mechanism, in which metastable structural intermediates sampled during the hinge motions of the ß-arms in the free state are presculpted to bind DNA. These results illustrate that using side-chain chemical shift data in conjunction with MD simulations can provide quantitative information about the free energy landscapes of proteins and yield detailed insights into their functional mechanisms.

Polymer Surface Dissection for Correlated Microscopic and Compositional Analysis of Bacterial Aggregates during Membrane Biofouling.

Multispecies biofilms are a common limitation in membrane bioreactors, causing membrane clogging, degradation, and failure. There is a poor understanding of biological fouling mechanisms in these systems due to the limited number of experimental techniques useful for probing microbial interactions at the membrane interface. Here, we develop a new experimental method, termed polymer surface dissection (PSD), to investigate multispecies assembly processes over membrane surfaces. The PSD method uses photodegradable polyethylene glycol hydrogels functionalized with bioaffinity ligands to bind and detach microscale, microbial aggregates from the membrane for microscopic observation. Subsequent exposure of the hydrogel to high resolution, patterned UV light allows for controlled release of any selected aggregate of desired size at high purity for DNA extraction. Follow-up 16S community analysis reveals aggregate composition, correlating microscopic images with the bacterial community structure. The optimized approach can isolate aggregates with microscale spatial precision and yields genomic DNA at sufficient quantity and quality for sequencing from aggregates with areas as low as 2000 µm2, without the need of culturing for sample enrichment. To demonstrate the value of the approach, PSD was used to reveal the composition of microscale aggregates of different sizes during early-stage biofouling of aerobic wastewater communities over PVDF membranes. Larger aggregates exhibited lower diversity of bacterial communities, and a shift in the community structure was found as aggregate size increased to areas between 25,000 and 45,000 µm2, below which aggregates were more enriched in Bacteroidetes and above which aggregates were more enriched with Proteobacteria. The findings demonstrate that community succession can be observed within microscale aggregates and that the PSD method is useful for identification and characterization of early colonizing bacteria that drive biofouling on membrane surfaces.

Shrimp shell peptide hydrolysates inhibit human cancer cell proliferation.

BACKGROUND: Shrimp wastes contain high-quality protein that is underutilized, and particularly peptides derived from shrimp wastes (normally used as animal feed) have not been utilized for bioactive properties. Hence the objective was to utilize shrimp waste proteins in generating peptides and to investigate these for cancer antiproliferative activities. The objectives involved hydrolyzing shrimp proteins (intact in shell) using a food-grade Cryotin enzyme, obtaining gastrointestinal resistant peptides, fractionation to generate < 10, 10-30 and > 30 kDa fractions, and evaluating for colon and liver cancer cell growth inhibitory effects. Three shrimp shells--whole langostino lobster shells from El Salvador (South America), shrimp shells from St Petersburg, FL (USA), and shrimp shell whites from the Gulf of Mexico, LA (USA)--were evaluated for the study. RESULTS: Peptide fractions (<10 and 10-30 kDa) obtained from shrimp shell whites (Gulf of Mexico) as well as from langostino shells (El Salvador) significantly inhibited the growth of both colon and liver cancer cells by 60%, while < 10 kDa fraction from shrimp shells (FL) inhibited growth of liver cancer cells alone by 55%, compared to controls. CONCLUSION: The promising anticancer peptide fractions from shrimp waste proteins has the potential for novel nutraceutical ingredient applications.

SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.

A quantitative linear model accurately (R(2) = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 degrees C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 degrees C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.

Long-term microbial community dynamics in a pilot-scale gas sparged anaerobic membrane bioreactor treating municipal wastewater under seasonal variations.

This study evaluates the microbial community development in the suspended sludge within a pilot-scale gas sparged Anaerobic membrane bioreactor (AnMBR) under ambient conditions, as well as understand the influence of microbial signatures in the influent municipal wastewater on the bioreactor using amplicon sequence analysis. The predominant bacterial phyla comprised of Bacteroidetes, Proteobacteria, Firmicutes, and Chloroflexi demonstrated resiliency with ambient temperature operation over a period of 472 days. Acetoclastic Methanosaeta were predominant during most of the AnMBR operation. Beta diversity analysis indicated that the microbial communities present in the influent wastewater did not affect the AnMBR core microbiome. Syntrophic microbial interactions were evidenced by the presence of the members from Synergistales, Anaerolineales, Clostridiales, and Syntrophobacterales. The proliferation of sulfate reducing bacteria (SRB) along with sulfate reduction underscored the competition of SRB in the AnMBR. Operational and environmental variables did not greatly alter the core bacterial population based on canonical correspondence analysis.

Phosphorous in the environment: characteristics with distribution and effects, removal mechanisms, treatment technologies, and factors affecting recovery as minerals in natural and engineered systems.

Phosphorus (P), an essential element for living cells, is present in different soluble and adsorbed chemical forms found in soil, sediment, and water. Most species are generally immobile and easily adsorbed onto soil particles. However, P is a major concern owing to its serious environmental effects (e.g., eutrophication, scale formation) when found in excess in natural or engineered environments. Commercial chemicals, fertilizers, sewage effluent, animal manure, and agricultural waste are the major sources of P pollution. But there is limited P resources worldwide. Therefore, the fate, effects, and transport of P in association with its removal, treatment, and recycling in natural and engineered systems are important. P removal and recycling technologies utilize different types of physical, biological, and chemical processes. Moreover, P minerals (struvite, vivianite, etc.) can precipitate and form scales in drinking water and wastewater systems. Hence, P minerals (e.g., struvite, vivianite etc.) are problems when left uncontrolled and unmonitored although their recovery is beneficial (e.g., slow release fertilizers, sustainable P sources, soil enhancers). Sources like wastewater, human waste, waste nutrient solution, etc. can be used for P recycling. This review paper extensively summarizes the importance and distribution of P in different environmental compartments, the effects of P in natural and engineered systems, P removal mechanisms through treatment, and recycling technologies specially focusing on various types of phosphate mineral precipitation. In particular, the factors controlling mineral (e.g., struvite and vivianite) precipitation in natural and engineered systems are also discussed.

Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy-whey proteins and fractions.

Soy proteins when prepared to high purity can confer good functional properties and the whey by-product is a potential source for bioactivity. In this study, we determined the protein, moisture, fiber, solubility, foaming, emulsion properties, as well as Angiotensin-I converting enzyme (ACE-I) inhibitory activity of prepared soy-whey proteins and its fractions. The soy-whey proteins were fractionated into <5, >5, >10, and >50kDa using ultrafiltration. The expanded AACC methods were used to determine protein, moisture, and fiber analyses of the whey and its fractions. Solubility method was conducted to determine the protein solubility profile of the soy-whey and its fractions at varying pHs. Turbidimetric method was used to evaluate emulsifying activity (EA) and emulsion stability (ES). There were significant differences observed in moisture, protein and salt contents between unfractionated, >50kDa and smaller sized fractions. No significant differences were observed with phytic acid and total dietary fiber contents among all samples. The unfractionated whey protein and >50kDa fraction showed better solubility than other fractions. Unfractionated whey protein had the highest foam capacity (42.7mL) while the fraction >5kDa showed the greatest foaming stability (46min). The highest emulsion activity (0.33±0.1) and stability (825.1±45.1) was obtained with the >50kDa fraction while the unfractionated whey protein had the highest ACE-I inhibition activity. The findings indicate that soy-whey protein fraction (>50kDa) had good solubility, emulsion activity and stability, while the unfractionated whey protein exhibited the strongest ACE-I inhibition percentage (19%).

Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes.

Transition metal-catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C-H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C-C bond-forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450(BM3) that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.

Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran.

Food-derived bioactive peptides promote functional activity against diseases and present as nutraceutical agents. The purpose of our research was to isolate and fully characterize peptide(s) derived from rice bran having anti-cancer properties. Gastrointestinal juices resistant peptide fractions were initially generated from heat stabilized de-fatted rice bran from which <5 kDa fraction was shown to inhibit proliferation of cancer cells. Based on these published findings the <5 kDa peptide fraction was selected for further characterization to obtain single pure peptide(s) with enhanced anti-cancer properties. Purification and characterization from the fraction was done employing chromatography and mass spectrometric techniques. Cancer cell viability was measured using a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; (MTS)] and the electron coupling reagent, phenazine methosulfate. Ion-exchange chromatography elutes that showed anti-cancer properties were further purified to liberate pure peptide. The pure peptide at 600-700 microg/mL dose caused 84% inhibition to colon cancer cells (Caco-2, HCT-116) growth, 80% to breast cancer cells (MCF-7, MDA-MB-231) growth and 84% to liver cancer cells (HepG-2) growth. Mass spectrometry analysis and de novo sequencing revealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptide with a molecular mass of 685.378 Da. A novel pentapeptide was isolated from rice bran to possess cancer growth inhibitory properties on colon, breast, lung and liver cancer cells. This peptide could serve as a nutraceutical agent against cancer.

Human colon and liver cancer cell proliferation inhibition by peptide hydrolysates derived from heat-stabilized defatted rice bran.

Rice bran, an economical, underutilized coproduct of rough rice milling, was used to produce peptide hydrolysates, which were investigated for anticancer activity. Protein hydrolysates prepared by Alcalase hydrolysis under optimized conditions were treated further to obtain gastrointestinal (GI)-resistant peptide hydrolysates. They were fractionated into >50, 10-50, 5-10, and <5 kDa sizes and evaluated for inhibitory activity on proliferation of human colon (Caco-2) and liver (HepG2) cancer cell lines by Trypan blue dye exclusion assay. GI-resistant <5 and 5-10 kDa sized peptide fractions inhibited growth of Caco-2 cells by 80%, and the <5 kDa fraction inhibited growth of HepG2 cells by approximately 50% compared to controls and nonresistant fractions. An MTS cell titer assay confirmed antiproliferative effects of the peptide fractions. The results demonstrated that 5-10 and <5 kDa sized GI-resistant fractions promoted significant (p < 0.05) inhibitory activities on both cancer cell lines compared to controls. More investigations are needed to show such value-added effects on the technofunctional and sensorial properties of the food protein and peptide matrices.