Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Hydrogenases catalyze reversible reaction between hydrogen (H₂) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H₂production potential of [NiFe]-hydrogenase 1 of <it>Escherichia coli in vivo </it>and <it>in vitro</it>. The <it>hya</it>A and <it>hya</it>B genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an <it>E. coli </it>strain without H₂producing ability.</p> <p>Results</p> <p>Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H₂(12.5 mL H₂/(h·L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H₂even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified <it>E. coli </it>[NiFe]-hydrogenase 1 using his₆-tag displayed oxygen-tolerant activity of ~12 nmol H₂/(min·mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition.</p> <p>Conclusions</p> <p>This is the first report on physiological function of <it>E. coli </it>[NiFe]-hydrogenase 1 for H₂production. We found that [NiFe]-hydrogenase 1 has H₂production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H₂production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.</p

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.X119196sciescopu

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

<p>Abstract</p> <p>Background</p> <p>Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant <it>Escherichia coli </it>with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H₂) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H₂production yield under light conditions.</p> <p>Results</p> <p>Recombinant <it>E. coli </it>BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from <it>Hydrogenovibrio marinus </it>produced ~1.3-fold more H₂in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m²·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H₂production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m²·s) led to an increase (~1.8-fold) in H₂production from 287.3 to 525.7 mL H₂/L-culture in the culture of recombinant <it>E. coli </it>co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H₂achieved in this study was ~3.4%.</p> <p>Conclusion</p> <p>Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H₂production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant <it>E. coli </it>BL21(DE3) in a light intensity-dependent manner. These results demonstrate that <it>E. coli </it>can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.</p

Control of nacre biomineralization by Pif80 in pearl oyster

Molluscan nacre is a fascinating biomineral consisting of a highly organized calcium carbonate composite that provides unique fracture toughness and an iridescent color. Organisms elaborately control biomineralization using organic macromolecules. We propose the involvement of the matrix protein Pif80 from the pearl oyster Pinctada fucata in the development of the inorganic phase during nacre biomineralization, based on experiments using the recombinant form of Pif80. Through interactions with calcium ions, Pif80 participates in the formation of polymer-induced liquid precursor-like amorphous calcium carbonate granules and stabilizes these granules by forming calcium ion-induced coacervates. At the calcification site, the disruption of Pif80 coacervates destabilizes the amorphous mineral precursors, resulting in the growth of a crystalline structure. The redissolved Pif80 controls the growth of aragonite on the polysaccharide substrate, which contributes to the formation of polygonal tablet structure of nacre. Our findings provide insight into the use of organic macromolecules by living organisms in biomineralization.117Ysciescopu

A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase

Tyrosinase efficiently catalyzes the ortho-hydroxylation of monophenols and the oxidation of diphenols without any additional cofactors. Although it is of significant interest for the biosynthesis of catechol derivatives, the rapid catechol oxidase activity and inactivation of tyrosinase have hampered its practical utilization as a monophenol monooxygenase. Here, we prepared a functional tyrosinase that exhibited a distinguished monophenolase/diphenolase activity ratio (V max mono/ V max di = 3.83) and enhanced catalytic efficiency against L-tyrosine (k cat  = 3.33 ± 0.18 s−1, K m  = 2.12 ± 0.14 mM at 20 °C and pH 6.0). This enzyme was still highly active in ice water (>80%), and its activity was well conserved below 30 °C. In vitro DOPA modification, with a remarkably high yield as a monophenol monooxygenase, was achieved by the enzyme taking advantage of these biocatalytic properties. These results demonstrate the strong potential for this enzyme’s use as a monophenol monooxygenase in biomedical and industrial applications.113Nsciescopu

Site-specific immobilization of microbes using carbon nanotubes and dielectrophoretic force for microfluidic applications

We developed a microbial immobilization method for successful applications in microfluidic devices. Single-walled nanotubes and Escherichia coli were aligned between two cantilever electrodes by a positive dielectrophoretic force resulting in a film of single-walled nanotubes with attached Escherichia coli. Because this film has a suspended and porous structure, it has a larger reaction area and higher reactant transfer efficiency than film attached to the substrate surface. The cell density of film was easily controlled by varying the cell concentration of the suspension and varying the electric field. The film showed excellent stability of enzyme activity, as demonstrated by measuring continuous reaction and long-term storage times using recombinant Escherichia coli that expressed organophosphorus hydrolase.X1133sciescopu

Framework for online optimization of recombinant protein expression in highcell-density Escherichia coli cultures using GFP-fusion monitoring

Abstract: A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-celldensity cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-␤-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized

Framework for online optimization of recombinant protein expression in highcell-density Escherichia coli cultures using GFP-fusion monitoring

Abstract: A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-celldensity cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-␤-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized

Dual-Color-Emitting Carbon Nanodots for Multicolor Bioimaging and Optogenetic Control of Ion Channels

The development of intrinsically multicolor-emitting carbon nanodots (CNDs) has been one of the great challenges for their various fields of applications. Here, the controlled electronic structure engineering of CNDs is performed to emit two distinct colors via the facile surface modification with 4-octyloxyaniline. The so-called dual-color-emitting CNDs (DC-CNDs) can be stably encapsulated within poly(styrene-co-maleic anhydride) (PSMA). The prepared water-soluble DC-CNDs@PSMA can be successfully applied to in vitro and in vivo dual-color bioimaging and optogenetics. In vivo optical imaging can visualize the biodistribution of intravenously injected DC-CNDs@PSMA. In addition, the light-triggered activation of ion channel, channelrhodopsin-2, for optogenetic applications is demonstrated. As a new type of fluorophore, DC-CNDs offer a big insight into the design of charge-transfer complexes for various optical and biomedical applications.112Ysciescopu

The usefulness of serum delta neutrophil index for differentiating bacterial and viral meningitis in the emergency department

Objective When managing patients with acute meningitis in an emergency department (ED), early diagnosis of the type of infection (bacterial or viral) considerably affects the clinical course and treatment because of the high mortality and morbidity associated with bacterial meningitis (BM). The serum delta neutrophil index (DNI), a new inflammatory marker, reflects the fraction of circulating immature granulocytes and is elevated in cases of bacterial infection. The objective of this study was to evaluate whether serum DNI can be used to differentiate between BM and viral meningitis (VM) in the ED. Methods This retrospective, observational study included 104 consecutive patients (aged &gt;18 years) diagnosed with acute meningitis from January 2012 to November 2014 in a regional emergency center. White blood cell and neutrophil counts, C-reactive protein level, and DNI were evaluated regarding their usefulness for differentiating BM and VM. Results Serum DNI was not significantly higher in the BM group (n=12) than in the VM group (n=92) (0 [interquartile range, 0% to 2.73%] vs. 0 [interquartile range, 0 to 0%], P=0.057). However, the white blood cell count and C-reactive protein level were statistically higher in the BM group (P=0.034 and P=0.026, respectively). Serum DNI was not found to be a statistically useful differential diagnostic parameter (area under the curve, 0.628; 95% confidence interval, 0.438 to 0.818). Conclusion Currently, there is no evidence that the serum DNI aids in differentiating acute BM from acute VM in the ED. Keyword