Maria E.
Yakovleva‡
a,
Christoph
Gonaus‡
b,
Katharina
Schropp
ab,
Peter
ÓConghaile
c,
Dónal
Leech
c,
Clemens K.
Peterbauer
b and
Lo
Gorton
*a
aDepartment of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, PO Box 124, SE-221 00 Lund, Sweden. E-mail: lo.gorton@biochemistry.lu.se
bDepartment of Food Sciences and Technology, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Wien, Austria
cSchool of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland
First published on 27th February 2015
In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 μA cm−2 at room temperature and when “wired” to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.
An obvious example of such a device would be an EBFC powering a cardiac pacemaker – medical devices currently powered by lithium-iodine batteries invented by Wilson Greatbatch and his team in 1972, with a life time of about 10 years.10 EBFCs can also be applied as implantable self-powered sensors, for instance in monitoring of the glucose concentration in blood for diabetes management.11
One of the reasons why EBFCs still did not yet reach the market is the low power output, which is directly related to the current generated in the anodic part, where sugar is oxidised at the anode, onto which the biocatalyst is immobilised, and the cathodic part, where oxygen is reduced. In order to produce an EBFC with a sufficient power capacity it is important to maximise the current density generated in both parts of the cell. This can be done by a variety of methods.
One of the most efficient ways is to improve the electrical communication between the redox enzyme and the electrode, which to a large degree determines the current output. This can be done by “wiring” the enzyme to osmium redox polymers, which will facilitate the electron transfer between the enzyme's active site and electrode surface.12–16 Such an approach was demonstrated in a number of recent publications, where various oxidising enzymes were cross-linked with redox polymers based on polyvinylimidazole-bound osmium complexes in order to improve the current density of both bioanodes17–19 and biocathodes.20,21
Another way to increase the current output of the bioanode and the biocathode is to downsize the dimensions of the enzyme.22 This can be achieved by employing whole deglycosylated enzymes or by utilising separate enzyme domains responsible for the catalytic activity. By removing the carbohydrate shell from glycosylated redox enzymes it is possible to physically bring the enzyme's active site closer to the electrode surface or mediator matrix, which will increase the electron transfer rate according to the Marcus theory.23 This has been previously demonstrated for horseradish peroxidase (HRP),24,25 glucose oxidase (GOx),26,27 cellobiose dehydrogenase (CDH)28,29 and pyranose dehydrogenase (PDH).30–32 Shao et al. proposed a bioanode based on the separately expressed deglycosylated dehydrogenase domain of CDH (DHCDH).33,34 When using only DHCDH instead of the whole enzyme for construction of EBFCs, a greater load of the biocatalyst can be packed onto the electrode surface due to the smaller size of DHCDH resulting in higher currents. Downsizing the redox enzyme for improved current density was also demonstrated for deglycosylated PDH, which spontaneously loses a part of its C-terminus and forms a fragmented product with excellent electrocatalytic performance.35
PDH (EC 1.1.99.29) is a glycosylated extracellular enzyme produced by a group of litter-degrading basidiomycete Agaricales. It is a monomeric enzyme, which carries one flavin adenine dinucleotide (FAD) prosthetic group covalently bound to the polypeptide chain of the protein36 in contrast to other glucose oxidising enzymes carrying their cofactor only through non-covalent linkages. PDH belongs to the glucose–methanol–choline oxidoreductase superfamily,37 which also includes GOx (EC 1.1.3.4), pyranose oxidase (POx, EC 1.1.3.10), and CDH (EC 1.1.99.18).38 In contrast to other members of the same family, which can only monooxidise sugars at the C-1 position, PDH (as well as POx) is able to mono- and dioxidise a variety of substrates at the C-2 and/or C-3 to their corresponding aldonolactones, or (di)dehydrosugars (aldos(di)uloses) and it is thus anomerically insensitive.36,39,40 Unlike many sugar oxidising oxidoreductases PDH does not show reactivity towards molecular oxygen but makes use of other electron acceptors/mediators instead.41 Together with a broad substrate tolerance and regioselectivity, this lack of electron donation to oxygen can be explained by a unique structure of the flavin pocket specific for the members of VAO [6-S-cysteinyl-8α-(N1-histidyl)-FAD] family.42 These three properties make the enzyme attractive for construction of EBFCs, where a broad substrate tolerance is not considered a disadvantage, the covalently bound cofactor increases the long-term stability of the enzyme in its immobilised state and the inability to utilise oxygen prevents competition with O2 as electron acceptor as well as any damaging effects of hydrogen peroxide, which is otherwise formed when oxygen-dependent oxidoreductases are employed (e.g., GOx or POx).
The suitability of PDH for bioanode fabrication was first demonstrated for naturally occurring glycosylated Agaricus meleagris enzyme when “wired” to osmium redox polymers on the surface of graphite electrodes.43 The electrodes modified with PDH showed a higher current response to glucose in flow-injection amperometry compared to those modified with GOx. It was possible to oxidise the substrate at potentials close to the formal potential of the enzyme in near-physiological conditions.18 However, low production levels of PDH purified from the natural source hindered widespread application of the enzyme for fabrication of EBFCs and other biotechnological purposes. The problem was solved by heterologous expression of the enzyme in several host organisms, of which Pichia pastoris was shown to be most suitable, resulting in production of active recombinant enzyme (recAmPDH) with 30% glycosylation.44 A further step in engineering of recAmPDH was achieved through enzymatic deglycosylation of the overglycosylated enzyme, which resulted in improved electrocatalytical performance of deglycosylated PDH compared to its glycosylated analogue.30–32 Time-consuming and generally expensive production of enzymatically deglycosylated enzyme suggested that other approaches for engineering of PDH should be implemented. In the present study we utilise a novel approach for engineering PDH, based on a recent report,35 as follows: asparagine residues (N) carrying the glycan moieties in the enzyme were first assigned using MALDI-MS measurements in combination with endoglycosidase treatment and tryptic digestion.35 Three of the five possible glycosylation positions: N75, N175 and N252 were confirmed to carry carbohydrate moieties. Using site-directed mutagenesis it was then possible to exchange glycosylated asparagine (N) residues and express an enzyme in Pichia pastoris that lacks part of the glycosyl moiety. Two single mutant PDHs (mPDHs) (SM) with asparagine residues exchanged to glycine (G), N75G, and glutamine (Q), N175Q, one double mutant with N exchanged to G in N75 and to Q in N175 position (DM; N75G N175Q) and one triple mutant with N exchanged to G, Q and Q in positions N75, N175 and N252, respectively (TM; N75G N175Q N252Q) were produced. In the work described herein all four mutants were electrochemically “wired” to three polyvinylimidazole-bound osmium complexes with different formal potentials (E°′ = 140 mV, 320 mV and 420 mV vs. NHE) and the electrocatalytic properties of the enzyme electrodes studied using flow-injection amperometry and cyclic voltammetry. The mPDHs were also directly adsorbed onto the working electrodes in order to study their ability to directly communicate with the electrode surface in a mediator-less mode.
Oligonucleotides were synthesised by VBC Biotech (Vienna, Austria) and polymerase chain reactions were conducted in a BioRad (Hercules, CA, USA) C1000 Thermal Cycler. Molecular biology reagents were obtained from Thermo Scientific (Waltham, MA, USA) and used according to the manufacturer's recommendations. Chemically competent E. coli NEB5α from New England Biolabs (Ipswich, MA, USA) was used for vector constructions and P. pastoris X33 from Invitrogen (Carlsbad, CA, USA) for expression. Chromatography resins were purchased from GE Healthcare (Little Chalfont, UK). Endo Hf (1000
000 U mL−1; 232
000 U mg−1) was supplied by New England Biolabs (Ipswich, MA, USA).
Overlap extension PCR was used for site directed mutagenesis. Two amplicons were generated using Phusion DNA polymerase (Thermo Scientific) and one mutating oligonucleotide (Table S2, ESI†) with the complementary AOX oligonucleotide each (3′AOX: 5′-GCAAATGGCATTCTGACATCC-3′, 5′AOX: 5′-GACTGGTTCCAATTGACAAG-3′). In a subsequent polymerase chain reaction with 5′AOX and 3′AOX the two amplicons were fused. The resulting DNA fragment was ligated into the XbaI and KpnI restriction sites of pPICZ-B. The vector was amplified in E. coli NEB5α, recovered and electroporated into P. pastoris according to the manufacturer's recommendations.
AmPDH1 [GeneBank: AY753307.1] glycosylation sites were previously predicted from the sequence in silico. Five consensus sites NXS/T are found, one (N313) is ruled out by the presence of proline at the middle position, another one (N319) was judged to have a low probability of glycosylation by the prediction software NetNGlyc 1.0. Positions N75, N175 and N252 were since confirmed by mass spectrometry analysis of tryptic fragments.35 The locations of these sites, and of the additional site at N319, are shown in Fig. 1a, based on the AmPDH1 crystal structure.42 N319 is located in a surface cavity but the other sites are well exposed. N75 is located next to the largest active site access and close to the covalently bound catalytic co-factor FAD. N175 is situated on the other side of the active site access. The fragmentation site into the ∼46 kDa and ∼20 kDa fragments is between residues 416 and 417, positioned between N75 (∼34 Å away) and N175 (∼24 Å away) (Fig. 1b). N252 is on the far side of the protein. N75 and N175 were selected for elimination due to the expected effect of reducing the distance from the active site and the FAD co-factor to the electrode surface when the enzyme is immobilised on the electrode. The third site, N252, was eliminated to additionally reduce the overall size of the enzyme. The AmPDH1 N75X site saturation mutagenesis library was created from Ampdh1 in pPICZ-B (Invitrogen, Life Technologies) with the primers fwAmPDHN75 and rvAmPDHN75X. 10% of the screened clones expressed active PDH and were rescreened in triplicates, and 14 mutants with expression of active enzyme were sequenced. Half of the clones were wild type, while the others were mutated to N75G, Q, K or H. These four mutants were expressed in shake flasks with AmPDH1 wild type (wt) as positive control and purified in a simplified 2-step chromatography scheme (Table S1, ESI†). AmPDH1 N75G showed the best expression at almost wild type levels and was chosen for further study. The other N-glycosylation site mutants were constructed by site directed mutagenesis (Table S2, ESI†). AmPDH1 N75G/N175Q could be expressed in shake flasks to twice the volumetric activity of AmPDH1 N75G/N175G. Gln was therefore chosen as replacement for Asn at N175 and N252.
The enzyme was purified from the harvested supernatant by salt precipitation ((NH4)2SO4) followed by hydrophobic interaction chromatography at room temperature (750 mL of Phenyl-Sepharose fast flow, 15 mL min−1). The eluted fractions of the enzyme were subjected to buffer exchange with Bis-Tris buffer (50 mM, pH 6.5) and subsequent anion exchange chromatography (60 mL of DEAE-Sepharose fast flow, 10 mL min−1) was conducted. The fractions with high enzyme activity were pooled and subjected to a second purification step, which included salt precipitation and hydrophobic interaction chromatography. The resulting fractions were pooled for highest specific activity, washed with sodium phosphate buffer (50 mM, pH 6.5) and concentrated by ultracentrifugation (10 kDa Amicon Ultra-15 Centrifugal Filter Units, Millipore Corp.). If necessary, the resulting enzyme solution was further purified by gel filtration (180 mL of Superose12, 1 mL min−1). Purified and concentrated enzyme solutions were stored at −20 °C.
The recombinant expression of N-glycosylation site mutants and the wt enzyme were compared in simultaneous expression in an Infors HT Multifors 6 × 0.5 L bioreactor system, according to the Invitrogen “Pichia Fermentation Process Guidelines”. Wet biomass, protein concentration and enzyme activity at standard conditions were determined (see ESI†).
The not optimised expression yielded 0.1–0.4 U mL−1 (0.3–3.7 U mg−1) in the supernatant. Purification was done by a 3-step chromatography scheme (Phenyl-Sepharose, DEAE-Sepharose, Phenyl-Source), whereas AmPDH1 DM was polished in an additional gel filtration step with Superose12 resin (Table S3, ESI†). An eight- to 115-fold increase in specific activity could be achieved with a purification yield of 11–30%, due to strict pooling. In parallel production experiments of mutant enzymes with wild type enzyme, the single mutation N75G had no significant impact on expression efficiency. The mutation at N175 in AmPDH1 DM, however, decreased the volumetric and specific yield by half. An additional mutation at N252 in AmPDH1 TM did not lead to a further decrease in yield. The single mutation N175Q showed an ambiguous picture with expression levels between wt/N75G and AmPDH1 DM (data not shown). This suggests a different importance of the particular glycosylation sites, in this case namely that position N175 requires intact N-glycosylation for proper folding and/or secretion of the enzyme. AmPDH1 N175Q, DM and TM showed sufficient expression levels in 5 L stirred-tank bioreactors and AmPDH1 N75G in shake flasks (0.1–0.4 U mL−1) for subsequent characterisation of the purified enzyme.
The electrocatalytic response of the films containing the different Os-polymer/mPDHs couples to glucose were measured in the flow injection system in 50 mM phosphate buffer pH 7.4 (137 mM NaCl) (Fig. S2, ESI†). The current densities obtained for the oxidation of glucose by the mPDHs-containing films increase with a shift in E°′-values of the Os-polymer towards more positive values as a function of the ligand used: Os(dmobpy)PVI < Os(dmbpy)PVI < Os(bpy)PVI (Table 1).
mPDH | Type of ETa | MET | DET | ||
---|---|---|---|---|---|
Kin. param.b | Os(dmobpy)-PVI | Os(dmbpy)-PVI | Os(bpy)-PVI | ||
a ET – electron transfer. b Kin. param. – kinetic parameter. | |||||
N75G | J max/μA cm−2 | 0.5 ± 0.02 | 2.5 ± 0.09 | 22.2 ± 1.8 | 1 ± 0.06 |
N175Q | 6.2 ± 0.2 | 20.9 ± 0.7 | 152.7 ± 2.8 | 1.8 ± 0.05 | |
DM | 9.8 ± 0.3 | 290.1 ± 15.9 | 802.4 ± 101.07 | 7.8 ± 0.7 | |
TM | 35.2 ± 0.5 | 51.7 ± 0.6 | 215 ± 2.5 | 6.6 ± 0.2 | |
N75G | K appM/mM | 1.9 ± 0.1 | 5.9 ± 0.2 | 6.7 ± 0.1 | 4.5 ± 0.4 |
N175Q | 0.7 ± 0.1 | 3.8 ± 0.4 | 7.6 ± 1.3 | 2.1 ± 0.4 | |
DM | 0.2 ± 0.06 | 2.1 ± 0.2 | 1.7 ± 0.1 | 5.2 ± 0.4 | |
TM | 0.7 ± 0.1 | 4.4 ± 0.6 | 10.2 ± 2.4 | 3.9 ± 0.9 |
This result is expected considering that the driving force for electron transfer is increasing with the increase in potential difference between the bound FAD of mPDH and the Os-polymer. Maximum response is obtained for each of the mPDHs electrochemically “wired” with the Os(bpy)PVI-based polymer. The KappM-values obtained from the curve fitting of the results are displayed in Table 1 and the smallest KappM-values are obtained for the electrodes containing mPDH “wired” to the Os(dmobpy)PVI polymer exhibiting the lowest E°′.
The CVs recorded in the absence of substrate (Fig. 3) indicate that films of the DM and TM enzyme contain approximately half the amount of redox-active polymer compared to films of the N75G and N175Q enzyme, whilst providing higher maximum current densities (Table 1). Osmium coverage alone therefore does not determine glucose oxidation current response. The cyclic voltammetry results presented in Fig. 3 are in a good agreement with the FIA data (Fig. S2, ESI†).
When comparing the various mPDHs a clear correlation between the electrocatalytic response and the degree of deglycosylation can be observed for the SMs and the DM (Fig. 3 and Fig. S2, ESI†). The DM co-immobilised with the different Os-polymers generates current densities greater than those obtained for either of the SMs covalently bound to the corresponding Os-polymers. The concentration in terms of mg mL−1 is higher for the SMs (N75 g: 26.3 mg mL−1; N175Q: 24.1 mg mL−1) than for the DM (17.0 mg mL−1) and the TM (16.1 mg mL−1). Even though the specific activity of the enzyme solutions (N75G: 1440 U mL−1; N175Q: 1103 U mL−1; DM: 624 U mL−1; TM: 820 U mL−1) is around twice as high for the SMs, the current densities obtained for the oxidation of glucose increase in the following order: N75 < N175 < DM (Table 1). The explanation may be that the electron transfer rate between the two electroactive species is increased by bringing them closer to each other.23 The glycan shell of DM is smaller than that of the SMs. Therefore, the active site of the DM is more accessible to be “wired” to the Os-polymer compared to mPDHs with a higher glycosylation degree and the electron transfer will hence become faster.
Taking these observations into consideration, it is expected that the TM should outperform both the DM and the SMs when “wired” with the various Os-polymers, because the TM has less carbohydrates attached to its polypeptide chain. Surprisingly, the opposite effect is observed. Analysis of the biological functions of carbohydrates indicates that the latter play an important role in the physical maintenance and the catalytic performance of the whole glycoprotein. Moreover, it is difficult to predict a priori an effect of elimination of the glycan on the properties of the glycoprotein.56 This may explain why the DM shows much better catalytic response to glucose compared to the TM in both flow injection and cyclic voltammetric measurements, when the same mass of enzymes is co-immobilised with the mediator on the surface of the working electrodes. Apparently, glycosylation in N252 is needed for retaining a high activity of mPDH using the osmium polymers as mediators, and exchanging N to Q in the protein polypeptide chain does not further improve the catalytic efficiency of mPDH.
The onset of the oxidation current is observed for all of the enzyme electrodes starting at a potential close to the E°′ of each redox polymer. The films modified with cross-linked Os(bpy)PVI/DM display the highest current densities (Table 1) compared to the other mPDHs prepared using the same redox polymer. With an increase in E°′ of the mediator the catalytic response of the Os-polymer/mPDH-modified electrodes becomes more clearly observed in the cyclic voltammograms, as expected considering the increase in the thermodynamic driving force of the electron transfer reaction.
The performance of the Os(dmbpy)PVI/DM electrode exhibiting the highest response to glucose in the FI and the CV studies was compared with the previously reported data on films prepared by co-immobilisation of an Os-polymer with enzymatically deglycosylated recombinantly expressed AmPDH (dgPDH)31,32 or the more active fragmented form of the same enzyme (fdgPDH).35 Equal mass amounts of the enzyme, mediator and cross-linker were utilised for drop-coating onto the electrodes and the obtained films were screened under similar experimental conditions (buffer composition, applied potential, glucose as a substrate). The Os(dmbpy)PVI/DM-based films reported here produce a higher catalytic response (Jmax = 290 μA cm−2) compared to the Jmax of 146 μA cm−2 and 248 μA cm−2 for the Os(dmbpy)PVI/dgPDH- and the Os(dmbpy)PVI/fdgPDH-based films, respectively.32,35
Comparison with results of other sugar-oxidising enzyme electrodes is difficult due not only to considerable differences in the testing conditions such as glucose concentration, temperature and pH selected, but also to the effect of the electrode preparation methodologies, variability in experimental approaches and the method used to measure current density. For example, Murata et al.57 recently reported that deglycosylated FAD-dependent glucose dehydrogenase from Aspergillus terreus, co-immobilised, through diepoxide crosslinking with an Os-polymer on glassy carbon electrodes, rotated at 5000 rpm, yields glucose oxidation current densities of 0.95 mA cm−2 and 4 mA cm−2 in 5 mM and 200 mM glucose, respectively, at 0.5 V vs. Ag|AgCl in pH 7.0 buffered solution at 25 °C.
Due to the drawbacks associated with general use of mediators,2,3,9 DET approach for construction of bioanodes and cathodes is quite appealing. In the present study current output from mPDH-modified electrodes was studied using CV and FI (see ESI†). Although all of the films containing mPDHs possess an ability to directly communicate with the electrode surface, DET takes place at high overpotentials and current densities produced are much lower in comparison with the MET mode (Fig. S3 and S4, ESI†). Therefore, application of mPDH-modified films in fabrication of bioanodes is still limited.
All forms of the mutant enzyme possess an ability to directly communicate with the electrode surface with DM having the highest and the most stable current response to glucose (Jmax = 7.8 μA cm−2).
Footnotes |
† Electronic supplementary information (ESI) available: Information concerning purification of mutant PDHs. See DOI: 10.1039/c5cp00430f |
‡ These authors contributed equally. |
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