Harnessing Redox Polymer Dynamics for Enhanced Glucose-Oxygen Coupling in Dual Biosensing and Therapeutic Applications.

The burgeoning field of continuous glucose monitoring (CGM) for diabetes management faces significant challenges, particularly in achieving precise and stable biosensor performance under changing environmental conditions such as varying glucose concentrations and O2 levels. To address this, we present a novel biosensor based on the electroless coupling of glucose oxidation catalyzed by flavin-dependent glucose dehydrogenase (FAD-GDH) and O2 reduction catalyzed by bilirubin oxidase (BOD) via a redox polymer, dimethylferrocene-modified linear poly(ethylenimine), FcMe2-LPEI. Initial cyclic voltammetry tests confirm the colocalization of both enzymatic reactions within the potential range of the polymer, indicating an effective electron shuttle mechanism. As a result, we created a hybrid biosensor that operates at open-circuit potential (OCP). It can detect glucose concentrations of up to 100 mM under various O2 conditions, including ambient air. This resulted from optimizing the enzyme ratio to 120 ± 10 mUBOD·UFAD-GDH-1·atmO2-1. This biosensor is highly sensitive, a crucial feature for CGM applications. This distinguishes it from FAD-GDH traditional biosensors, which require a potential to be applied to measure glucose concentrations up to 30 mM. In addition, this biosensor demonstrates the ability to function as a noninvasive, external device that can adapt to changing glucose levels, paving the way for its use in diabetes care and, potentially, personalized healthcare devices. Furthermore, by leveraging the altered metabolic pathways in tumor cells, this system architecture opened up new avenues for targeted glucose scavenging and O2 reduction in cancer therapy.

Wiring of bilirubin oxidases with redox polymers on gas diffusion electrodes for increased stability of self-powered biofuel cells-based glucose sensing.

A new redox polymer/bilirubin oxidase (BOD)-based gas diffusion electrode was designed to be implemented as the non-current and non-stability limiting biocathode in a glucose/O2 biofuel cell that acts as a self-powered glucose biosensor. For the proof-of-concept, a bioanode comprising the Os-complex modified redox polymer P(VI-co-AA)-[Os(bpy)2Cl]Cl and FAD-dependent glucose dehydrogenase to oxidize the analyte was used. In order to develop an optimal O2-reducing biocathode for the biofuel cell Mv-BOD as well as Bp-BOD and Mo-BOD have been tested in gas diffusion electrodes in direct electron transfer as well as in mediated electron transfer immobilized in the Os-complex modified redox polymer P(VI-co-AA)-[Os(diCl-bpy)2]Cl2. The resulting biofuel cell exhibits a glucose-dependent current and power output in the concentration region between 1 and 10 mM. To create a more realistic test environment, the performance and long-term stability of the biofuel cell-based self-powered glucose biosensor has been investigated in a flow-through cell design.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide.

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

Caged Activators of Artificial Allosteric Protein Biosensors.

The ability of proteins to interconvert unrelated biochemical inputs and outputs underlays most energy and information processing in biology. A common conversion mechanism involves a conformational change of a protein receptor in response to a ligand binding or a covalent modification, leading to allosteric activity modulation of the effector domain. Designing such systems rationally is a central goal of synthetic biology and protein engineering. A two-component sensory system based on the scaffolding of modules in the presence of an analyte is one of the most generalizable biosensor architectures. An inherent problem of such systems is dependence of the response on the absolute and relative concentrations of the components. Here we use the example of two-component sensory systems based on calmodulin-operated synthetic switches to analyze and address this issue. We constructed "caged" versions of the activating domain thereby creating a thermodynamic barrier for spontaneous activation of the system. We demonstrate that the caged biosensor architectures could operate at concentrations spanning 3 orders of magnitude and are applicable to electrochemical, luminescent, and fluorescent two-component biosensors. We analyzed the activation kinetics of the caged biosensors and determined that the core allosteric switch is likely to be the rate limiting component of the system. These findings provide guidance for predictable engineering of robust sensory systems with inputs and outputs of choice.

Synthesis of Formate from CO2 Gas Catalyzed by an O2-Tolerant NAD-Dependent Formate Dehydrogenase and Glucose Dehydrogenase.

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

Purification, characterization and gene identification of a membrane-bound glucose dehydrogenase from 2-keto-d-gluconic acid industrial producing strain Pseudomonas plecoglossicida JUIM01.

The membrane-bound glucose dehydrogenase (mGDH) is a rate-limiting enzyme for the industrial production of 2-keto-d-gluconic acid (2KGA) from glucose. In this study, mGDH was firstly purified from a 2KGA industrial producing strain Pseudomonas plecoglossicida JUIM01. The purified mGDH exhibited a specific activity of 16.85 U/mg and was identified as monomeric membrane-bound PQQ-dependent dehydrogenase with a molecular mass of ~87 kDa. The Km and Vmax value of d-glucose were 0.042 mM and 14.620 µM/min, and the optimal pH and temperature were of 6.0 and 35 °C with favorable acid resistance and poor heat tolerance. Ca2+/Mg2+ showed a significantly positive effect on mGDH activity with 20% increase, whereas EDTA/EGTA had a negative influence, and Ca2+ was essential for enzyme activity. Furthermore, a 2412 bp-length gcd was amplified by genome walking technique and heterologously expressed in Escherichia coli. Bioinformatics analysis and heterologous expression further confirmed it as a mGDH encoding gene. mGDH contained binding sites of Ca2+, cofactor PQQ and polypeptide binding sites concluded from alignment results of mGDHs from different genera. This study would lay the foundation for improving 2KGA productivity through further strain modification.

Inorganic iron-sulfur clusters enhance electron transport when used for wiring the NAD-glucose dehydrogenase based redox system.

Wiring the active site of an enzyme directly to an electrode is the key to ensuring efficient electron transfer for the proper performance of enzyme-based bioelectronic systems. Iron-sulfur complexes, the first link between proteins and mediating molecules in the biological electron transport chain(s), possess an intrinsic electron transport capability. The authors demonstrate the application of inorganic iron-sulfur clusters (Fe-S) viz. FeS, FeS2, Fe2S3, and Fe3S4, as molecular wires to mediate electron transport between a glucose-selective redox enzyme and the gold electrode. It is shown that Fe-S can emulate the functionality of the natural electron transport chain. Voltammetric studies indicate a significant improvement in electron transport, surface coverage, and resilience achieved by the Fe-S-based glucose anodes when compared to a conventional pyrroloquinoline quinone (PQQ)-based electrode. The Fe-S-based glucose anodes showed glucose oxidation at a potential of +0.5 V vs. Ag/AgCl with Tris-HCl buffer (pH 8) acting as a carrier. The current densities positively correlated with the concentrations of glucose in the range 0.1-100 mM displaying detection limits of 0.77 mM (FeS), 1.22 mM (FeS2), 2.95 mM (Fe2S3), and 14.57 mM (Fe3S4). The metal-anchorable sulfur atom, the strong π-coordinating iron atom, the favorable redox properties, low cost, and natural abundance make Fe-S an excellent electron-mediating relay capable of wiring redox active sites to electrode surfaces. Graphical abstract Schematic representation of inorganic iron-sulfur clusters used as molecular wires to facilitate direct electron transfer between NAD-glucose dehydrogenase and the gold electrode. The iron-sulfur based glucose anodes improve current response to selectively sense glucose concentrations in the range 0.1-100 mM.

Highly Efficient Deracemization of Racemic 2-Hydroxy Acids in a Three-Enzyme Co-Expression System Using a Novel Ketoacid Reductase.

Enantiopure 2-hydroxy acids (2-HAs) are important intermediates for the synthesis of pharmaceuticals and fine chemicals. Deracemization of racemic 2-HAs into the corresponding single enantiomers represents an economical and highly efficient approach for synthesizing chiral 2-HAs in industry. In this work, a novel ketoacid reductase from Leuconostoc lactis (LlKAR) with higher activity and substrate tolerance towards aromatic α-ketoacids was discovered by genome mining, and then its enzymatic properties were characterized. Accordingly, an engineered Escherichia coli (HADH-LlKAR-GDH) co-expressing 2-hydroxyacid dehydrogenase, LlKAR, and glucose dehydrogenase was constructed for efficient deracemization of racemic 2-HAs. Most of the racemic 2-HAs were deracemized to their (R)-isomers at high yields and enantiomeric purity. In the case of racemic 2-chloromandelic acid, as much as 300 mM of substrate was completely transformed into the optically pure (R)-2-chloromandelic acid (> 99% enantiomeric excess) with a high productivity of 83.8 g L-1 day-1 without addition of exogenous cofactor, which make this novel whole-cell biocatalyst more promising and competitive in practical application.

Enantioselective Biosynthesis of l-Phenyllactic Acid by Whole Cells of Recombinant Escherichia coli.

BACKGROUND: l-Phenyllactic acid (l-PLA)-a valuable building block in the pharmaceutical and chemical industry-has recently emerged as an important monomer in the composition of the novel degradable biocompatible material of polyphenyllactic acid. However, both normally chemically synthesized and naturally occurring phenyllactic acid are racemic, and the product yields of reported l-PLA synthesis processes remain unsatisfactory. METHODS: We developed a novel recombinant Escherichia coli strain, co-expressing l-lactate dehydrogenase (l-LDH) from Lactobacillus plantarum subsp. plantarum and glucose dehydrogenase (GDH) from Bacillus megaterium, to construct a recombinant oxidation/reduction cycle for whole-cell biotransformation of phenylpyruvic acid (PPA) into chiral l-PLA in an enantioselective and continuous manner. RESULTS: During fed-batch bioconversion with intermittent PPA feeding, l-PLA yield reached 103.8 mM, with an excellent enantiomeric excess of 99.7%. The productivity of l-PLA was as high as 5.2 mM·h-1 per OD600 (optical density at 600 nm) of whole cells. These results demonstrate the efficient production of l-PLA by the one-pot biotransformation system. Therefore, this stereoselective biocatalytic process might be a promising alternative for l-PLA production.

Efficient synthesis of (S)-N-Boc-3-hydroxypiperidine using an (R)-specific carbonyl reductase from Candida parapsilosis.

(S)-N-Boc-3-hydroxypiperidine (S-NBHP) is a critical chiral intermediate in the synthesis of pharmaceuticals, including ibrutinib, the active pharmaceutical ingredient of the new drug Imbruvica approved for the treatment of lymphoma. An (R)-specific carbonyl reductase from Candida parapsilosis (CprCR, also known as R-specific alcohol dehydrogenase) that catalyzes asymmetric reduction to produce (S)-N-Boc-3-hydroxypiperidine (S-NBHP) was identified for the first time. When co-expressed with a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3), recombinant crude enzyme exhibited an activity of 9 U/mg with N-Boc-3-piperidone as the substrate and 12 U/mg with glucose as the substrate. The biocatalysis of N-Boc-3-piperidone to S-NBHP using recombinant whole-cell biocatalysts was processed in a water/butyl acetate system as well as an aqueous monophasic system without extra NAD+/NADH. This process showed great commercial potential, with a 100 g/l substrate concentration and a whole cells loading (w/v) of 10%, with the conversion of 97.8% and an e.e. of 99.8% in an aqueous monophasic system.

An Enzyme-based 1:2 Demultiplexer Interfaced with an Electrochemical Actuator.

An enzyme-based 1:2 demultiplexer is designed in a flow system composed of three cells where each one is modified with a different enzyme: hexokinase, glucose dehydrogenase and glucose-6-phosphate dehydrogenase. The Input signal activating the biocatalytic cascade is represented by glucose, while the Address signal represented by ATP is responsible for directing the Input signal to one of the output channels, depending on the logic value of the Address. The biomolecular 1:2 demultiplexer is extended to include two electrochemical actuators releasing entrapped DNA molecules in the active output channel. The modular design of the system allows for easy exchange and extension of the functional elements. The present demultiplexer can be easily integrated in various biomolecular logic systems, including different logic gates based on the enzyme- or DNA-based reactions, as well as containing different chemical actuators, for example, with a biomolecular release function.

Reporter Immobilization Assay (REIA) for Bioconjugating Reactions.

Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 µg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour.

Visible-light driven biofuel cell based on hierarchically branched titanium dioxide nanorods photoanode for tumor marker detection.

In this work, a novel sensing platform based on visible light driven biofuel cell (BFC) has been facilely designed for sensitive detection of prostate-specific antigen (PSA) with the photo-response bioanode, realizing the dual route energy conversion of light energy and chemical energy to electricity. The hierarchical branched TiO2 nanorods (B-TiO2 NRs) decorated with CdS quantum dots (QDs) act as the substrate to confine glucose dehydrogenase (GDH) for the visible light driven glucose oxidation at the bioanode. Three dimensional flowers like hierarchical carbon/gold nanoparticles/bilirubin oxidase (3D FCM/AuNPs/BOD) bioconjugate served as biocatalyst for O2 reduction at the biocathode. With an increase in the concentration of PSA, the amount of BOD labels on biocathode increases, thus leading to the higher current output of the as-proposed visible light driven BFC. Based on this, this sensing platform provide great performance in sensitivity and specificity, increasing linear detection range from 0.3pgmL(-1) to 7µgmL(-1) with a detection limit of 0.1pgmL(-1). Most importantly, our new sensing strategy provided a simple and inexpensive sensing platform for tumor markers detection, suggesting its wide potential applications for clinical diagnostics.

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor.

Self-powered competitive immunosensor driven by biofuel cell based on hollow-channel paper analytical devices.

A mediator-less and compartment-less glucose/O2 enzymatic biofuel cell (BFC) was introduced into microfluidic paper-based analytical devices (µ-PADs) that relies on flow in hollow channels with silver nanoparticles/graphene modified paper electrode as the anodic and cathodic substrate, to implement self-powered sensitive carcinoembryonic antigen (CEA) detection. Glucose dehydrogenase (GDH)-gold nanoparticles bioconjugate modified with CEA acted as a biocatalyst for enhancing glucose oxidation in the bioanode, as well as the transducing enzyme for signaling magnification. Similarly, nanoporous PtNi/bilirubin oxidase (BOD) acted as a biocatalyst for enhancing O2 reduction in the biocathode. With an increase in the concentration of CEA, the amount of CEA-Au-GDH bioconjugate on bioanode decreases, thus leading to the lower output of the as-prepared BFC. This proposed BFC-based self-powered immunosensor for CEA possessed largely increased linear detection range from 1 pg mL(-1) to 0.5 µg mL(-)(1) with a detection limit of 0.7 pg mL(-)(1). The proposed BFC-based self-powered immunosensor shows high sensitivity, stability, and reproducibility and can become a promising platform for other protein detection.

Biofuel cell based self-powered sensing platform for L-cysteine detection.

L-cysteine (L-Cys) detection is of great importance because of its crucial roles in physiological and clinical diagnoses. In this study, a glucose/O2 biofuel cell (BFC) was assembled by using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH)-based bioanode and laccase-based biocathode. Interestingly, the open circuit potential (OCP) of the BFC could be inhibited by Cu(2+) and subsequently activated by L-Cys, by which a BFC-based self-powered sensing platform for the detection of L-Cys was proposed. The FAD-GDH activity can be inhibited by Cu(2+) and, in turn, subsequent reversible activation by L-Cys because of the binding preference of L-Cys toward Cu(2+) by forming the Cu-S bond. The preferential interaction between L-Cys and Cu(2+) facilitated Cu(2+) to remove from the surface of the bioanode, and thus, the OCP of the system could be turned on. Under optimized conditions, the OCP of the BFC was systematically increased upon the addition of the L-Cys. The OCP increment (ΔOCP) was linear with the concentration of L-Cys within 20 nM to 3 µM. The proposed sensor exhibited lower detection limit of 10 nM L-Cys (S/N = 3), which is significantly lower than those values for other methods reported so far. Other amino acids and glutathione did not affect L-Cys detection. Therefore, this developed approach is sensitive, facile, cost-effective, and environmental-friendly, and could be very promising for the reliable clinically detecting of L-Cys. This work would trigger the interest of developing BFCs based self-powered sensors for practical applications.

Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface.

Rational design was applied to glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10 to improve its thermal stability by introduction of disulfide bridges between subunits. One out of the eleven mutants, designated as DS255, displayed significantly enhanced thermal stability with considerable soluble expression and high specific activity. It was extremely stable at pH ranging from 4.5 to 10.5, as it retained nearly 100% activity after incubating at different buffers for 1h. Mutant DS255 also exhibited high thermostability, having a half-life of 9900min at 50°C, which was 1868-fold as that of its wild type. Moreover, both of the increased free energy of denaturation and decreased entropy of denaturation of DS255 suggested that the enzyme structure was stabilized by the engineered disulfide bonds. On account of its robust stability, mutant DS255 would be a competitive candidate in practical applications of chiral chemicals synthesis, biofuel cells and glucose biosensors.

Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins.

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.

Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal.

Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1U azoreductase:10U glucose 1-dehydrogenase, 250mM glucose, 1.0mM NAD(+) and 150µM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD(+) concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal.