An automatic immuno-microfluidic system integrating electrospun polystyrene microfibrous reactors for rapid detection of salivary cortisol.

Conventional competitive enzyme-linked immunosorbent assay (ELISA) to measure the cortisol level in body fluid consumes a large amount of time, owing to complicated operations involved and requirement for precise control of reagent addition. We developed an automatic microfluidic system to detect salivary cortisol rapidly, and an electrospun polystyrene (PS) microfiber-based reactor providing considerable binding sites for antibody immobilization, thus resolving the time limitations of competitive ELISA. Cortisol sample, horseradish peroxidase (HRP)-conjugated cortisol, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate were delivered to the PS reactor from containers in sequence by pumps automatically. The color variation due to oxidized TMB complex reflects the cortisol concentration level measured using an RGB phototransistor. In addition, the entire procedure from sample introduction to obtaining the photocurrent took only 15 min. This system can be implemented to quantify cortisol from 0.37 ng/mL to 30 ng/mL, and the limit of detection was estimated at 0.37 ng/mL.

Fluidic Patch Device to Sample Sweat for Accurate Measurement of Sweat Rate and Chemical Composition: A Proof-of-Concept Study.

Sweat sensors that can continuously sample sweat are critical for determining the time-dependent physiological responses occurring in normal daily life. Here, a new device, termed fluidic patch, for collecting human sweat samples at defined time intervals is developed, and the proof-of-concept is demonstrated. The device comprises micropumps and a disposable microfluidic patch attached to the human skin. The fluidic patch continuously collects aliquots of freshly secreted sweat accumulated in the fluidic pathway at accurately defined time windows (typically 5 min). By measuring the weight of the collected samples, the local sweat rate is calculated. The sweat sample collected can be directly subjected to a wide range of chemical analyses. For the proof-of-concept, we compared the sweat rates during passive heating in human trials using the fluidic patch and the conventional ventilated sweat capsule system. Although the sweat rate obtained using the fluidic patch highly correlated with that of the ventilated sweat capsule (R2 = 0.96, y = 1.4x - 0.05), the fluidic patch overestimated the sweat rate compared with the ventilated capsule system when the sweat rate exceeded 0.5 mg/(cm2·min). The sampled sweat was analyzed for sodium, potassium, chloride, lactate, pyruvate, and cortisol. The device could obtain the time courses of the concentrations of the abovementioned three ions; the concentrations of sodium and chloride increased linearly with the sweat rate during passive heating (R2 = 0.76 and 0.66, respectively). The device can reliably measure the sweat rate and collect sweat samples for chemical analysis. It can be utilized for real-time physiological investigations toward wider applications.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

Feasibility study of on-site solid-state enzyme production by Aspergillus oryzae.

BACKGROUND: The development of biorefinery systems that use lignocellulosic biomass as a renewable carbon source to produce fuels and chemicals is attracting increasing attention. The process cost of enzymatic saccharification of biomass is a major challenge for commercialization. To decrease this cost, researchers have proposed on-site solid-state fermentation (SSF). This study investigated the feasibility of using Aspergillus oryzae as a host microorganism for SSF recombinant enzyme production with ammonia-treated rice straw as model biomass. Eight A. oryzae strains were tested, all of which are used in the food industry. We evaluated the effects of acetic acid, a fermentation inhibitor. We also developed a platform strain for targeted recombinant enzyme production by gene engineering technologies. RESULTS: The SSF validation test showed variation in the visibility of mycelium growth and secreted protein in all eight A. oryzae strains. The strains used to produce shoyu and miso grew better under test conditions. The ammonia-treated rice straw contained noticeable amounts of acetic acid. This acetic acid enhanced the protein production by A. oryzae in a liquid-state fermentation test. The newly developed platform strain successfully secreted three foreign saccharifying enzymes. CONCLUSIONS: A. oryzae is a promising candidate as a host microorganism for on-site SSF recombinant enzyme production, which bodes well for the future development of a more cost-efficient saccharifying enzyme production system.

Alanine substitution in cellobiohydrolase provides new insights into substrate threading.

The glycoside hydrolase family 7 (GH7) member cellobiohydrolase (CBH) is a key enzyme that degrades crystalline cellulose, an important structural component of plant cell walls. As GH7 CBH is a major component in the enzyme mixture used to degrade biomass into fermentable glucose in biorefineries, enhancing its catalytic activity will significantly impact development in this field. GH7 CBH possesses a catalytic tunnel through which cellulose substrates are threaded and hydrolysed. Despite numerous studies dissecting this processive mechanism, the role of amino acid residues in the tunnel remains not fully understood. Herein, we examined the respective contributions of nine amino acid residues in the catalytic tunnel of GH7 CBH from Talaromyces cellulolyticus by substitution with alanine. As a result, N62A and K203A mutants were found to possess significantly higher cellulase activities than wild type. Molecular dynamics simulations showed that the N62 residue interacted strongly with the cellulose substrate, impeding threading, while the N62A mutant allowed cellulose to proceed more smoothly. Furthermore, the W63 residue was observed to facilitate twisting of the cellulose substrate in our simulations. This study helps elucidate cellulose threading and provides insight into biomass hydrolysis.

The rosettazyme: a synthetic cellulosome.

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds. Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg(2+) assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin-rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.

Cell structure degradation in Escherichia coli and Thermococcus sp. strain Tc-1-95 associated with thermal death resulting from brief heat treatment.

The thermal death mechanism of microorganisms when heated at lethally high temperatures is still not fully understood. In this study, we examined the relationship between thermal death and degradation of the cell structure in the mesophilic bacterium Escherichia coli strain W3110 and the hyperthermophilic archaeon Thermococcus sp. strain Tc-1-95. By heating the microorganisms at lethally high temperatures only briefly (1.5 s duration) in a flow-type apparatus, we studied the microbial cells at very early and critical stages of the thermal death process. For E. coli, it was found that the loss of viability was not associated with thermal damage to the cell envelope. Deformation of the nucleoid was observed. These results suggest that the thermal death of E. coli is attributed to thermal denaturation or degradation of cytoplasmic molecules. On the other hand, the thermal death of Thermococcus sp. strain Tc-1-95 was strongly associated with rupture of the cell envelope. Furthermore, massive deformation of the S-layer with lethal thermal stress was observed. These results demonstrate that the thermal deaths of the two microorganisms investigated proceed via very different mechanisms. The contrast can be attributed to the difference in their cell envelope structures.

'Reverse chemical evolution': a new method to search for thermally stable biopolymers.

The primitive sea on Earth may have had high-temperature and high-pressure conditions similar to those in present-day hydrothermal environments. If life originated in the hot sea, thermal stability of the constituent molecules would have been necessary. Thus far, however, it has been reported that biopolymers hydrolyze too rapidly to support life at temperatures of more than 200 degrees C. We herein propose a novel approach, called reverse chemical evolution, to search for biopolymers notably more stable against thermal decomposition than previously reported. The essence of the approach is that hydrolysis of a protein or functional RNA (m-, t-, r-RNA) at high temperature and high pressure simulating the ancient sea environment may yield thermally stable peptides or RNAs at higher concentrations than other peptides or RNAs. An experimental test hydrolyzing bovine ribonuclease A in aqueous solution at 205 degrees C and 25 MPa yielded three prominently stable molecules weighing 859, 1030 and 695 Da. They are thermally some tens or hundreds times more stable than a polyglycine of comparable mass. Sequence analyses of the 859- and 1030-Da molecules revealed that they are a heptapeptide and its homologue, respectively, elongated by two amino acids at the N-terminal region, originally embedded as residues 112-120 in the protein. They consist mainly of hydrophobic amino acids.