Preparation and identification of carboxymethyl cellulose-degrading enzyme candidates for oilfield applications.

Carboxymethyl cellulose (CMC) is often used during hydraulic fracturing (fracking) operations as a fluid viscosifier to facilitate proppant delivery. However, the accumulation of residual CMC at fracture faces can result in formation damage, thereby impeding oil and gas recovery. Whereas harsh chemical oxidizers are typically added to disrupt these polymer accumulations, there is now industrial interest in developing clean, biological approaches for the degradation of CMC under fracking conditions. Using a methanogenic culture known to utilize CMC under conditions typically found in oil fields, we developed an efficient method to isolate and purify CMC-degrading enzymes. Initial purification and concentration of cellular components produced an increase in exo-ß-(1,4)-exoglucanase and ß-(1,4)-glucosidase activities by 9-fold and 26-fold, respectively. Partially purified extracts provided substantial degradation of CMC as monitored by viscosity reduction within three hours at 50 °C, an improvement over the untreated cell-free extract which required 48 h to achieve similar viscosity values, outperforming a commercially-available cellulase preparation. Putative cellulases were identified within the isolated enzyme population, with endo-ß-(1,4)-xylanase from Caldicoprobacter faecalis hypothesized to be an important contributor to CMC degradation. This study demonstrates that enzyme technology holds great promise as a viable approach to degrade CMC accumulations under field conditions.

Preserving Microbial Community Integrity in Oilfield Produced Water.

Determining a representative microbial signature from any given location is dependent on robust sample collection and handling. Different sampling locations and hence sample properties can vary widely; for example, soil would be collected and handled differently compared to liquid samples. In the event that sample material has a low concentration of biomass, large quantities need to be collected for microbial community analysis. This is certainly the case when investigating the microbiology of oilfield systems, wherein produced water (PW) is one of the most common sources for microbial sampling. As the detrimental effects of microbial metabolism within these industrial milieus are becoming increasingly well-established, the characterization of microbial community composition using molecular biological analyses is becoming more commonplace for accurate monitoring. As this field continues to develop, the importance for standardized operating protocols cannot be understated, so that industry can make the most informed operational decisions possible. Accurately identifying oilfield microbial communities is paramount, as improper preservation and storage following sample collection is known to lead to erroneous microbial identifications. Preserving oilfield PW can be challenging, as many locations are remote, requiring lengthy periods of time before samples can be processed and analyzed. While previous studies have characterized the effects of various preservatives on concentrated, filtered, or purified microbial samples, to the best of our knowledge, no such study has been undertaken on low biomass liquid samples. To this end, we investigated the effectiveness of nine different preservation conditions on PW collected from the same sampling location within a heavy-oil producing field, and monitored how the microbial community changed over the period of a month. Our results reveal that the choice of preservative drastically affects microbial community, and should be selected with careful consideration before sampling occurs.

Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, ß-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.

Transglutaminase-Catalyzed Bioconjugation Using One-Pot Metal-Free Bioorthogonal Chemistry.

General approaches for controlled protein modification are increasingly sought-after in the arena of chemical biology. Here, using bioorthogonal reactions, we present combinatorial chemoenzymatic strategies to effectuate protein labeling. A total of three metal-free conjugations were simultaneously or sequentially incorporated in a one-pot format with microbial transglutaminase (MTG) to effectuate protein labeling. MTG offers the particularity of conjugating residues within a protein sequence rather than at its extremities, providing a route to labeling the native protein. The reactions are rapid and circumvent the incompatibility posed by metal catalysts. We identify the tetrazine ligation as most-reactive for this purpose, as demonstrated by the fluorescent labeling of two proteins. The Staudinger ligation and strain-promoted azide-alkyne cycloaddition are alternatives. Owing to the breadth of labels that MTG can use as a substrate, our results demonstrate the versatility of this system, with the researcher being able to combine specific protein substrates with a variety of labels.

General C-H Arylation Strategy for the Synthesis of Tunable Visible Light-Emitting Benzo[a]imidazo[2,1,5-c,d]indolizine Fluorophores.

Herein we report the discovery of the benzo[a]imidazo[2,1,5-c,d]indolizine motif displaying tunable emission covering most of the visible spectrum. The polycyclic core is obtained from readily available amides via a chemoselective process involving Tf2O-mediated amide cyclodehydration, followed by intramolecular C-H arylation. Additionally, these fluorescent heterocycles are easily functionalized using electrophilic reagents, enabling divergent access to varied substitution. The effects of said substitution on the compounds' photophysical properties were rationalized by density functional theory calculations. For some compounds, emission wavelengths are directly correlated to the substituent's Hammett constants. Easily introduced nonconjugated reactive functional groups allow the labeling of biomolecules without modification of emissive properties. This work provides a straightforward platform for the synthesis of new moderately bright fluorescent dyes remarkable for their chemical stability, predictability, and unusually high excitation-emission differential.

Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency.

The clinical success of Escherichia colil-asparaginase II (EcAII) as a front line chemotherapeutic agent for acute lymphoblastic leukemia (ALL) is often compromised because of its silent inactivation by neutralizing antibodies. Timely detection of silent immune response can rely on immobilizing EcAII, to capture and detect anti-EcAII antibodies. Having recently reported the use of a portable surface plasmon resonance (SPR) sensing device to detect anti-EcAII antibodies in undiluted serum from children undergoing therapy for ALL (Aubé et al., ACS Sensors2016, 1 (11), 1358-1365), here we investigate the impact of the quaternary structure and the mode of immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity and reproducibility of immunosensing. We show that the native tetrameric structure of EcAII, while being essential for activity, is not required for antibody recognition because monomeric EcAII is equally antigenic. By modulating the mode of immobilization, we observed that low-density surface coverage obtained upon covalent immobilization allowed each tetrameric EcAII to bind up to two antibody molecules, whereas high-density surface coverage arising from metal chelation by N- or C-terminal histidine-tag reduced the sensing efficiency to less than one antibody molecule per tetramer. Nonetheless, immobilization of EcAII by metal chelation procured up to 10-fold greater surface coverage, thus resulting in increased SPR sensitivity and allowing reliable detection of lower analyte concentrations. Importantly, only metal chelation achieved highly reproducible immobilization of EcAII, providing the sensing reproducibility that is required for plasmonic sensing in clinical samples. This report sheds light on the impact of multiple factors that need to be considered to optimize the practical applications of plasmonic sensors.

Biotechnological applications of transglutaminases.

In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. In recent years, biotechnological applications of transglutaminases have come to light in areas ranging from material sciences to medicine. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as many of their characteristics that remain poorly understood. Those studies also work towards the goal of developing transglutaminases as more efficient catalysts. Progress in this area includes structural information and novel chemical and biological assays. Here, we review recent achievements in this area in order to illustrate the versatility of transglutaminases.