Engineering of carbohydrate oxidoreductases for sensors and bio-fuelcells

Pyranose dehydrogenase (PDH) and pyranose 2-oxidase (POx) are flavoproteins that catalyze the oxidation of free, non-phosphorylated sugars to the corresponding ketosugars. Pyranose dehydrogenase has a broad substrate specificity for monosaccharides (and few disaccharides), but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen, whereas pyranose 2-oxidase shows pronounced specificity for glucose and displays high oxidase as well as dehydrogenase activity. For bio-fuelcell and sensor applications, oxygen reactivity is undesirable as it leads to electron leakage and the formation of damaging hydrogen peroxide; for biocatalytic applications, oxygen reactivity is advantageous, as oxygen is freely available and obviates downstream removal of undesired electron acceptors. Site-saturation mutagenesis libraries of eleven (POx) and twelve (PDH) residues around the active sites were screened for oxidase and dehydrogenase activities. In POx, variants T166R, Q448H, L545C, L547R and N593C displayed reduced oxidase activities (between 40% and 0.2% of the wildtype) concomitant with unaffected or even increased dehydrogenase activity, dependent on the electron acceptor used (DCPIP, 1,4-benzoquinone or ferricenium ion). Kinetic characterization showed that both affinity and turnover numbers can be affected. The switch from oxidase to dehydrogenase activity was also observed electrochemically using screen-printed electrodes. In this miniaturized set-up with a reaction volume of only 50 µL the dehydrogenase activity of variant N593C was entirely preserved in the presence of a suitable mediator, shuttling electrons from the FAD cofactor to the electrode surface. The oxidase activity, utilizing molecular oxygen as acceptor, is abolished in this variant. Of all variants of PDH that were produced by saturation mutagenesis, only variants of one position displayed increased oxygen reactivity to a minor degree. Histidine 103, carrying the covalently attached FAD cofactor, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y displayed a five-fold increase of oxygen reactivity. Stopped flow analysis revealed that the mutation slowed down the reductive half-reaction whereas the oxidative half-reaction was affected to a minor degree. No alterations in the secondary structure were observed, but disruption of the FAD bond had negative effects on thermal and conformational stability. We also engineered PDH by systematically removing several N-glycosylation sites, in order to improve performance by reducing the distance of the active site to the electrode surface, improving accessibility for redox polymers and facilitate denser enzyme packing on the electrode. One glycosylation site, N319, was found to be indispensable for functional expression and folding of the enzyme, as no active variants could be obtained. A variant with two sites, N75 and N175 near the active site entrance, exchanged against G and Q, respectively, showed significantly improved properties when used on electrodes with Osmium-based redox polymers (Mediated Electron Transfer) and a low level of Direct Electron Transfer. The lack of two glycosylation sites results in minor negative effects on expression yield and stability. Removal of a third site, N252, on the opposite side of the active site entrance, does not bring further improvements in catalysis and electron transfer, but is detrimental to functional expression and stability. The bulk of hyperglycosylation of the recombinantly expressed enzyme (observed in both Pichia pastoris and Saccharomyces cerevisiae) is located only on this one glycosylation site. Please click Additional Files below to see the full abstract

Investigation of Physical Activity Levels of Eligible Students in Terms of Various Variables

This study aimed to examine the physical activity levels of the eligible students to study at the Faculty of Sports Sciences in the current academic year. A total of 200 students participated in the research as a result of the removal of the erroneous questionnaires from the questionnaires applied to 224 students who were eligible to attend the Faculty of Sports Sciences. The "International Physical Activity Questionnaire (IPAQ) short form was applied to the participants. While there was no significant difference according to the gender and body mass index variables values of the students who were entitled to study (p&gt;0.05), a significant difference was found according to the sports branches, sports experience, and the departments they attend (p&lt;0.05). As a result, a significant difference was found according to the departments of the students studying at the faculty of sports sciences according to their physical activity levels, sports experience, and the sports branches.</p

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

peer-reviewedHere for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 mu A and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.PUBLISHEDpeer-reviewe

Functional aspects of cellobiose dehydrogenase Applications for biosensor development

Electrochemcial investigations were conducted for elucidating and understanding the relation between the various electron transfer processes occurring in cellobiose dehydrogenase (CDH) bound to an electrode surface. Substrate inhibition caused by cellobiose was proven to act in competition with the electron transfer to a two-electron acceptor and under a partial inhibition mechanism for the internal electron transfer process. The implications of these findings have been discussed in relation to what has been stated previously but also exploited for analytical purposes by adjusting the sensitivity of the CDH based biosensor for catecholamines. Different thiols were used to form self-assembled monolayers (SAM) on gold electrodes and these have been used to electrochemically investigate and characterise the direct electron transfer reaction of CDH from two white-rot fungi (Phanerochaete sordida and Trametes villosa), and one soft-rot fungus (Myriococcum thermophilum) in the absence and presence of cellobiose. The high efficiency of the direct electron transfer between Phanerochaete sordida CDH and Trametes villosa CDH and SAM modified Au electrodes was emphasised, and a potential application of these enzymes was demonstrated by development of a third-generation biosensor for lactose determination. Toxic waste-water quinone type pollutants were also determined using a Phanerochaete chrysosporium CDH based biosensor, working under a mediated electron transfer mechanism

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 μM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e−/H+ acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH

Study regarding the use of gymnastics - related alternative methods during the physical education class

The improvement of motor qualities and skills represents the main concern of the Physical Education lesson. In the present paper we present a general motor exercise training routine, designed as an alternative to the traditional approach of the PE lessons. The routine was divided into three components, namely a cross fit exercise routine, an aerobics routine and an acrosport routine. This routine was implemented in the 5th grade syllabus. The test group was represented by 207 pupils, out of which 114 girls (55.07%) and 93 boys (44.93%). Most participants came from rural areas (66.18%) and 33.82% came from urban areas. The routine was implemented during the first semester of the current school year (2017-2018) in one of the two compulsory lessons. The topics from the traditional PE lesson were replaced by the proposed routines and used alternatively. The assessment consisted of 4 tests from the national assessment system. The results showed significant progress obtained in the final tests for all the 4 trials used (p < 0,001)

Study regarding the use of gymnastics - related alternative methods during the physical education class

The improvement of motor qualities and skills represents the main concern of the Physical Education lesson. In the present paper we present a general motor exercise training routine, designed as an alternative to the traditional approach of the PE lessons. The routine was divided into three components, namely a cross fit exercise routine, an aerobics routine and an acrosport routine. This routine was implemented in the 5th grade syllabus. The test group was represented by 207 pupils, out of which 114 girls (55.07%) and 93 boys (44.93%). Most participants came from rural areas (66.18%) and 33.82% came from urban areas. The routine was implemented during the first semester of the current school year (2017-2018) in one of the two compulsory lessons. The topics from the traditional PE lesson were replaced by the proposed routines and used alternatively. The assessment consisted of 4 tests from the national assessment system. The results showed significant progress obtained in the final tests for all the 4 trials used (p < 0,001)

Third-generation biosensor for lactose based on newly discovered cellobiose dehydrogenase

The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm(2)) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL(.)min(-1)). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 mu M, a sensitivity of 1100 mu A(.)mM(-1.)cm(-2), a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 mu M lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 mu M lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%)

Biosensor based on cellobiose dehydrogenase for detection of catecholamines

A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs AgAgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A(.)M(-1.)cm(2) 2 (1150 nA/muM) for noradrenaline, with a coverage of less than 2.5 mug of CDH adsorbed on the electrode surface (0.073 cm(2)). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones

Direct electron transfer - A favorite electron route for cellobiose dehydrogenase (CDH) from Trametes villosa. Comparison with CDH from Phanerochaete chrysosporium

This paper presents some functional differences as well as similarities observed when comparing the newly discovered cellobiose dehydrogenase (CDH) from Trametes Villosa (T.v) with the well-characterized one from Phanerochaete chrysosporium (P.c.). The enzymes were physically adsorbed on spectrographic graphite electrodes placed in an amperometric flow through cell connected to a flow system. In the case of T.v.-CDH-modified graphite electrodes, a high direct electron transfer (DET) current was registered at the polarized electrode in the presence of the enzyme substrate reflecting a very efficient internal electron transfer (IET) process between the reduced FAD-cofactor and the oxidized heme-cofactor. In the case of P.c.-CDH-modified graphite electrodes, the DET process is not as efficient, and the current will greatly increase in the presence of a mediator (mediated electron transfer, MET). As a consequence, when comparing the two types of enzyme-modified electrodes an inverted DET/MET ratio for T.v.-CDH is shown, in comparison with P.c.-CDH. The rates of the catalytic reaction were estimated to be comparable for both enzymes, by measuring the combined DET + MET currents. The inverted DET/MET ratio for T.v.-CDH-modified electrodes might suggest that probably there is a better docking between the two domains of this enzyme and that the linker region of P.c.-CDH might have an active role in modulating the rate of the IET (by changing the interdomain distance), with respect to pH. Based on the new properties of T.v.-CDH emphasized in the present study, an analytical application of a third-generation biosensor for lactose was recently published