This article was submitted to Biocatalysis, a section of the journal Frontiers in Catalysis
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
A novel fusion protein has been rationally designed, combining the hexameric glutamate dehydrogenase from
A fusion protein is a molecule consisting of two or more protein domains incorporated into one single complex. Naturally occurring fusion proteins are widespread, and their modular organization has been identified as an important evolutionary phenomenon (
Artificial fusion proteins may be designed to achieve improved properties or new functionality (
Furthermore, the very close proximity of the two protein domains yields a higher catalytic efficiency of the two sequential reactions. In fact, the molecule being produced from one enzyme could directly migrate to the active site of the second enzyme without first diffusing to the bulk environment, through substrate channeling (
Fusion enzyme engineering is one of the promising approaches for the efficient
Fraaije and co-workers described the construction of three different ADHs fused with an NADPH oxidase (NOX). Each enzyme retained its catalytic properties and the coupling of the two proteins offered a rapid qualitative screening system for the detection of ADH/NOX oxidase activity to make large mutant libraries (
It is worthy to mention a further example, where enzymes were engineered to both retain and recycle their cofactor (NAD+ and ATP) with the final purpose of producing a drug precursor in a three-step continuous-flow reactor system. In this case, the fusion of three enzymes had little effect on the kinetic performance or thermal stability of each domain. Moreover, the enzyme fusions have been successfully immobilized with 86–98% yield and high recovered activity (
Glutamate dehydrogenase (GluDH) from bovine liver is the enzyme of choice for the quantification of ammonia in solution. It catalyzes the reductive amination of α-ketoglutaric acid to glutamic acid using NADPH and ammonia, which is measured by monitoring the decrease in absorbance at 340 nm caused by the oxidation of the cofactor (
Ammonia commonly enters the environment through municipal, industrial, agricultural, and natural processes (
The methods to quantify ammonia are based normally on biosensors for concentrations up to 500 µmol/L, or assay kits (up to 800 µmol/L) (
On the other hand, there are biotransformations, for instance, where ammonia can be steadily produced on a millimolar scale as a by-product, so theoretically its continuous removal by a different enzyme can shift the reaction equilibrium towards the formation of the product of interest, increasing the overall biocatalytic efficiency of the desired reaction. Similarly, if the reagents and products of those biotransformations cannot be easily monitored and quantified, the accurate determination of ammonia
Formate dehydrogenase (FDH) catalyzes the oxidation of formic acid to CO2 using NAD+ as cofactor and is often used in the recycling of NADH due to its highly favorable thermodynamic equilibrium, and the inertness of the substrate and reaction product (
The reaction of interest in this work is shown in
Coupled reaction where GluDH and FDH are involved.
Here, the NAD(H)-dependent glutamate dehydrogenase from bacteria
The new complex was fully characterized and later immobilized to enhance the efficiency of the bifunctional system, which was additionally proven by performing biotransformations.
Chemicals, reagents, and medium component, unless stated otherwise, were obtained as analytical grade from Sigma-Aldrich and Fisher Scientific. All the materials and kits employed for the cloning were acquired from New England Biolabs (NEB). Primers were synthetized by Microsynth AG; NADH and NAD+ were purchased from Apollo Scientific Ltd., while immobilization supports were kindly provided by Resindion S.r.l and Purolite Ltd.
The pET28b(+) vector harboring the formate dehydrogenase gene was used as template for PCR amplification (Fwd: 5′-CTCAGGGCATTGCTTGGTAA
The genes were amplified by PCR using Q5 High-Fidelity DNA polymerase and the obtained fragments (insert and vector) were used for cloning the fusion construct with the Gibson Assembly Cloning kit. To transform host cells with the assembled product, 2 µl of the Gibson Assembly reaction mixture were added to the chemically competent
Through the Gibson assembly method, the linker and the glutamate dehydrogenase sequence have been joined with the sequence of the formate dehydrogenase to successfully assemble a double-stranded fully sealed DNA sequence of the recombinant fusion protein.
The transformed
Cells were first resuspended in loading buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM NaCl, 30 mM imidazole) and the resulting suspension subjected to sonication to disrupt cells (5 s pulse on and 10 s off for 8 min). Cell debris was removed by centrifugation (14,500 rpm for 45 min at 4°C) to yield a cell-free extract which was filtered (0.45 µm pore size). The affinity chromatography was performed using an AKTA Start system, equilibrated with the loading buffer. The crude extract was loaded (0.5 ml/min flow rate) onto a 5 ml His-trap Ni-affinity column (GE Healthcare) collecting the flow-through and washed with the loading buffer until the non-specific proteins were completely eluted. The collected solution was then applied to the column a second time to ensure that all the proteins were effectively bound, and the column was washed again. After that, an isocratic wash step with only 10% of elution buffer (50 mM potassium phosphate buffer pH 7.5, 300 mM NaCl, 300 mM imidazole) was performed to remove any non-specific protein still bound onto the column. Finally, recombinant proteins were eluted with 100% of elution buffer and dialyzed at 4°C under mild stirring, against storage buffer (50 mM potassium phosphate buffer pH 7.5). The dialysis was performed for an overnight replacing the buffer at least 2 times.
The purity of the obtained proteins was tested by 12% (w/v) SDS-PAGE and the concentration was quantified by Bradford assay.
One unit of activity was determined as the amount of enzyme needed to produce or consume 1 µmol of NADH per minute at 25°C. The activity assay was performed in a 96-well plate and detected by UV absorbance at 340 nm (ɛ340 = 6.22 mM−1 cm−1). Each protein domain was evaluated singularly (GluDH: 10 mM α-ketoglutaric acid disodium salt and 200 mM ammonium chloride for reductive amination, 40 mM L-glutamic acid monosodium salt for oxidative deamination, in potassium phosphate buffer 50 mM pH 7.5; FDH: 100 mM sodium formate in potassium phosphate 50 mM pH 7.5). The assay was started by the addition of the cofactor in the reaction environment (0.2 mM of NADH or 1.0 mM of NAD+ as final concentration). The specific activity was calculated in U/mg considering the overall concentration of the fusion protein.
The kinetic constants were found in triplicate by measuring the dependence of initial rate on substrate concentration at 25°C in potassium phosphate buffer 50 mM (pH 8.0 for GluDH and pH 7.5 for FDH). For oxidative deamination reaction of GluDH, glutamic acid concentration was varied from 0.1 to 100 mM in the presence of 1 mM NAD+. For the reverse reaction, α-ketoglutaric acid concentration was ranging between 0.05 and 20 mM at a fixed concentration of 200 mM ammonium chloride; ammonium chloride was varied from 1 to 200 mM at 10 mM α-ketoglutaric acid. In both cases, the concentration of NADH was 0.35 mM. The kinetic constants for formic acid in the reaction of FDH were found by using a range between 1 and 100 mM formate concentration at 1 mM NAD+. The cofactor affinity was also evaluated by changing the concentration of NADH from 0.01 to 0.4 mM (10 mM α-ketoglutaric acid, 200 mM ammonium chloride) and 0.1–1.0 mM for NAD+ (40 mM glutamic acid for GluDH and 100 mM formic acid for FDH). Reactions were made by adding an appropriate amount of enzyme to the substrate solution and incubating the mixture for at least 10 min to allow the enzyme to adjust to the environment. The measurement started after mixing the cofactor and following the change in absorbance at 340 nm.
The stability and activity assays at different temperatures and pH were executed in triplicate by incubating the biocatalyst solution at different temperatures (pH 7.5) or at chosen pH values (temperature 25°C) and withdrawing samples at regular times for proceeding with specific activity assays of the fusion protein in parallel with the original GluDH and FDH. The activity of the enzyme was also checked using the previous protocol at several pH and temperature conditions. The reagents were prepared with the chosen pH or incubated at the chosen temperature before proceeding with the activity tests. The results were fitted in a graph showing the trend of the stability/activity of both glutamate and formate DH at chosen temperature or pH values.
The purified fusion protein was applied to a gel filtration column (Superdex 200 10/300 GL, GE Healthcare), which was equilibrated with buffer (50 mM Tris/HCl, 150 mM NaCl, pH 7.5) and mounted onto an ÄKTA Pure instrument (GE Healthcare). The flow rate was set at 0.5 ml/min. A calibration curve was made by plotting the elution times of the following protein standards: carbonic anhydrase (29 KDa), albumin (66 KDa), alcohol dehydrogenase from yeast (150 KDa), ß-amylase from sweet potato (200 KDa), apoferritin from horse spleen (443 KDa), thyroglobulin bovine (669 KDa). Blue dextran (2,000 KDa) was employed to determine the void volume and to check column packing. The elution of the standard proteins and the sample was followed by absorbance readings at 280 nm. The molecular weight of the fusion protein was calculated through the elution time fitted in the equation obtained from the calibration curve.
The CsGluDH-pRSETb and the CbFDH-pET28b were separately used to transform
The chosen support was left in incubation with the enzyme solubilized in the appropriate buffer. Typically, 1 g of support was added to 1 ml of a solution containing the desired amount of protein and incubated at room temperature under mild agitation. The protein load is defined as the amount of protein that has been immobilized per Gram of carrier (mg/g).
Small amounts of supernatant were taken over time to detect enzymatic activity, that was compared to the activity of the initial enzyme solution that was offered to the carrier (control sample). To better confirm the full immobilization, the protein concentration was then checked by Bradford assay and SDS-PAGE. When the maximum achievable yield was obtained, the support was washed.
The activity assay was performed by adding 5–10 mg of immobilized enzyme to a 2 ml reaction mixture containing substrates and cofactor and monitoring it using the same procedure as for the activity assay of the free enzyme. The aim is to determine the expressed activity in U/g (units of enzyme catalyzing the formation of 1 µmol of product per minute per Gram of support), the specific activity in U/mg (expressed activity divided by the amount of protein loaded to the carrier) and the recovered activity, defined as a percentage calculated from the ratio between the specific activity of the immobilized enzyme and the specific activity of the free enzyme.
All the procedures are done at room temperature, if not otherwise stated, under mild agitation. The washing steps are performed with deionized water. All the resins are stored at 4°C after preparation or immobilization.
For the activation of epoxy resin to aldehyde groups, 1 g of support was mixed with 10 ml of 100 mM H2SO4 and incubated overnight. The support was washed, incubated with 10 ml of 30 mM NaIO4 for 2 h and washed again. For the immobilization,
For the covalent immobilization on epoxy resin by first interaction with a metal, 1 g of resin was incubated with 2 ml of modification buffer (100 mM sodium borate, 2 M iminodiacetic acid, 50 mM phosphate buffer pH 8) for 2 h. After washing, the support was incubated with 5 ml of metal buffer (1 M NaCl, 5 mg/ml CoCl2 in 50 mM phosphate buffer pH 6) for 2 h. The support was then washed and incubated with the protein solution in phosphate buffer 50 mM pH 8.5. The support was then washed with 3 ml of desorption buffer (50 mM EDTA, 500 mM NaCl in 50 mM phosphate buffer pH 7) to remove the metal and rinsed with deionized water. The support was left in 4 ml blocking buffer (3 M glycine in 20 mM phosphate buffer pH 8.5) for an overnight and then washed (
For the covalent immobilization on epoxy groups by interaction with ethylenediamine (EDA), 1 g of the epoxy resin was incubated with 6 ml of 2% (v/v) EDA in 100 mM NaHCO3 buffer pH 8.5 for 2 h. The support was then washed (water, 1 M NaCl and again with water). The protein solution was prepared in 5 mM phosphate buffer pH 7. After being washed, the immobilize enzyme was incubated with 1 ml of 5 mM NaHCO3 buffer pH 9 for at least 2 h. Afterwards, the support was washed and left in 4 ml blocking buffer (prepared as previously described) for an overnight (
For the coating of epoxy and glyoxyl resin with polyethyleneimine (PEI), a solution of 10 mg/ml of PEI 60 kDa was prepared in 100 mM NaHCO3 buffer pH 10 and 10 ml were incubated with 1 g of support for an overnight. After that, the epoxy resin was washed and stored, while the glyoxyl resin was both washed and incubated with 1 mg of NaBH4 in 10 ml of 100 mM NaHCO3 buffer at pH 10 for 30 min at 4°C. The protein solution was prepared in 5 mM phosphate buffer pH 7. After immobilization, the resin was washed and stored (
For the qualitative assay with picrylsulfonic acid, an adapted version of a previously reported protocol was used (
The EP400/SS resin was firstly activated with glyoxyl groups and then coated with PEI 60 KDa. Several protein loadings were tested: 1, 5 and 10 mg per Gram of resin.
Different strategies of immobilization were chosen, using the resins ReliSorb EP400/SS (Resindion S.R.L.) and Lifetech ECR8204F, ECR8304F, ECR8285 (Purolite Ltd.). The resins EP400/SS and ECR8204F were activated to obtain aldehyde groups. A part of them was employed for the covalent immobilization on glyoxyl groups, while the other part was functionalized with PEI, 60 KDa for the respective immobilization. The same epoxy resins were also modified with iminodiacetic acid and cobalt, with EDA or with PEI for the relative immobilizations. The biocatalysts were also immobilized directly to Lifetech ECR8304F and ECR8285 (protein eluted in 5 mM phosphate buffer pH 7). All the carriers presenting amino groups were qualitatively analyzed using picrylsulfonic acid as reagent.
The immobilization yield and the recovered activity were calculated, by following the same conditions as for the activity assays (oxidative deamination direction for GluDH).
For the co-immobilization, ReliSorb EP400/SS was firstly treated to get glyoxyl groups and after that, it was coated with PEI 60 KDa. The two enzymes were incubated with the carrier both simultaneously and stepwise adding firstly GluDH and then FDH, and vice versa, to compare any difference in recovered activity. Different amounts of enzymes as well as different ratios were loaded.
The immobilized protein was tested in terms of operational stability by performing consecutive biotransformations and checking the decrease of relative rate of conversion for each cycle of reaction.
The biotransformations were prepared in 1 ml total volume containing 50 mM α-ketoglutaric acid disodium salt, 200 mM ammonium chloride, 200 mM sodium formate and 1 mM NADH in potassium phosphate buffer 50 mM pH 7.5, adding 10–40 mg of immobilized biocatalyst. The reaction was left in incubation at 37°C under agitation for 30 min, then the supernatant was taken for sample preparation and subsequent GC-FID analysis.
Enough free or immobilized biocatalyst was added to a 2 ml tube containing 1 ml of reaction mixture (50/300/400 mM α-ketoglutaric acid disodium salt, 200/400/300 mM ammonium chloride, 200/400 mM sodium formate and 1 mM NADH in 50 mM potassium phosphate buffer pH 7.5). A control without enzyme was run in parallel. The mixture was then incubated at 37°C under agitation (150 RPM) and aliquots were taken over time.
Samples were prepared and derivatized following the available protocol (
The production of glutamic acid and the consumption of α-ketoglutaric acid were monitored and compared between free and immobilized biocatalytic reaction to assess the efficiency of the system.
The genetic construction of the recombinant fusion protein has been developed using the original plasmid encoding the FDH from
Genetic construction of the recombinant fusion protein.
A 9 aa flexible linker was inserted between the two protein domains to facilitate folding (
The linker has been designed to include two flanking restriction sites to facilitate direct modification of the genetic sequence (length of the linker, flexibility, amino acids involved, etc.) and versatility of the construct (see
Computational studies have been performed to probe the rationale behind the gene design, before proceeding with the experimental development of the fusion protein. The PDB file resulting from the
Modelling of the hexamer fusion protein construct created with molecular modelling and minimized with openMM (
The FDH-GluDH fused gene was successfully created via Gibson assembly and confirmed by sequence analysis.
Several conditions to maximize overexpression have been tested (data not shown). LB media induced with 0.1 mM of IPTG at 20°C for an overnight yielded the best results. The correct, soluble expression of the large fusion protein (93 KDa per monomer with a total of 558 KDa if hexamer), was facilitated by a lower concentration of inducer together with a lower incubation and expression temperature. At 20°C, the undesired formation of inclusion bodies was not observed. Following IMAC purification and dialysis, a yield of 40–60 mg of protein per liter of culture were obtained (see ESI paragraph 1 for further details).
The fusion protein yield was comparable to the results obtained with the individual enzymes. Indeed, similar expression levels between fused and free enzymes were also reported in the literature for other fusion proteins (
The specific activity of the fusion protein was determined for both catalytic domains (GluDH and FDH) and compared with the activity of the single enzymes. The monomeric molecular mass of the free enzymes (GluDH 53 and FDH 43 KDa) is about half than the fusion protein (93 KDa), therefore the results are reported in
Specific activity of the two protein domains compared to the normalized specific activity of the wild-type FDH and GluDH (shown only as reference values without the standard deviations).
Formate dehydrogenase | Glutamate dehydrogenase | ||
---|---|---|---|
Amination | Deamination | ||
Fusion protein | 1.2 ± 0.2 [U/mg] | 135 ± 6 [U/mg] | 17.0 ± 0.5 [U/mg] |
Wild-type enzyme | 1.5 | 156 | 19.7 |
The two domains exhibited very similar activity to the wild-type free proteins with an average of 86.4% of GluDH and an 80% of FDH activity. This outcome is consistent with a previous report where the FDH moiety retained less parental activity than partner enzyme (
The predicted assembly suggested by the molecular modelling has been therefore validated experimentally. The subunits of the two domains were clearly assembled without any major distortion, meaning that the GluDH possessed a hexameric quaternary structure as the wild-type, while the FDH presented a dimeric configuration. The size exclusion chromatography further reinforced the accuracy of the molecular model, showing a predominant hexameric and octameric form with traces of assembled 2 and 12 monomers (see paragraph 3 in the
Kinetic constants for the fusion protein, the glutamate- and the formate dehydrogenase.
Fusion protein–GluDH | Wild-type CsGluDH | |||
---|---|---|---|---|
KM [mM] | Vmax [mmol min−1 mg−1] | KM [mM] | Vmax [mmol min−1 mg−1] | |
L-glutamate | 2.9 ± 0.8 | 0.04 ± 0.01 | 3.0 ± 1.1 | 0.08 ± 0.01 |
α-ketoglutarate | 0.41 ± 0.22 | 0.07 ± 0.05 | 0.48 ± 0.31 | 0.13 ± 0.1 |
Ammonia | 32.5 ± 10.0 | 0.18 ± 0.06 | 32.3 ± 10.0 | 0.36 ± 0.15 |
NADH | 0.20 ± 0.08 | 0.06 ± 0.05 | 0.10 ± 0.06 | 0.12 ± 0.1 |
NAD
|
0.17 ± 0.04 | 0.02 ± 0.01 | 0.17 ± 0.02 | 0.05 ± 0.02 |
|
|
|||
|
Vmax [mmol min−1 mg−1] | KM [mM] | Vmax [mmol min−1 mg−1] | |
Formate | 6.0 ± 1.0 | 0.0010 ± 0.0003 | 5.0 ± 1.0 | 0.003 ± 0.001 |
NAD
|
0.088 ± 0.014 | 0.0010 ± 0.0004 | 0.168 ± 0.030 | 0.005 ± 0.001 |
The resulting KM values were comparable between the fusion protein and the wild-type enzymes; the affinity to the substrates has not been affected where the fusion of the two proteins could have destabilized the quaternary structure. The FDH domain showed a lower KM value towards NAD+ compared with that of the free FDH, while the GluDH had a decreased affinity for the NADH after the fusion. The same alteration of the cofactor kinetic parameters was found in the LeuDH-FDH fusion protein as well as in the KR fused with the mutant formate dehydrogenase MycFDH (
The maximum velocities (Vmaxs) of the fusion protein were consistently half of those of GluDH, and 3 to 5 times lower than FDH, but this simply reflects the fact that the weight of the fusion protein is approximately twice that of the wild-type enzymes, therefore also the rate of conversion is conserved. The bifunctional protein retained the overall catalytic properties of the individual enzymes, as seen for some fused enzymes. In other cases, however, such as for some ADH-CHMO fusion proteins, the catalytic efficiency was dramatically diminished, possibly due to the orientation of the two domains or the fusion itself that hindered the association between the subunits to form the oligomers. It is in fact possible that one protein domain covers the oligomerization interface of its fusion partner, preventing the multimeric assembly, essential for the activity (
The fusion protein presented the same profiles as the wild-type enzymes for both pH and temperature activity/stability (details in paragraph two of ESI). Furthermore, the FDH moiety acquired a cooperative behavior that is typical of the GluDH but does not occur in the WT FDH. The fusion, therefore, led to a tight connection between the two enzymes where one domain is dependent to the other for structural modifications induced by temperature shift, for example.
To further probe the applicability of this new assembly, the fusion protein was immobilized on a solid support. Preliminary immobilization tests on the individual WT enzymes, based on a trial-and-error approach, showed that all the immobilization strategies based on covalent bonds were exhibiting almost no recovered activity (
Recently, a python-based GUI-application called CapiPy has been developed to assist in protein immobilization, which supplies information to rationalize the choice of immobilization strategy (
CapiPy offered an initial evaluation of the amino acid residues available on the surface of the enzyme through a detailed analysis of the hexameric structure, whose model was given by minimizing a manually assembled hexameric structure followed by minimization with openMM (
3D structure of the assembled fusion protein after minimization with outputs retrieved from CapiPy tool after the analysis of the structure (
The polymethacrylate epoxy resin ReliSorb EP400/SS has been selected as a support, and it provides a highly porous structure. The material of the resin offers a robust support to the enzyme, while the porosity gives many points of anchoring. Polyethyleneimine (PEI) has been used to coat the carrier, to enhance the enzyme binding through the high number of amino groups introduced and create a more hydrophilic environment.
The enzyme was efficiently bound through ionic interactions between the amino groups of PEI and the exposed carboxyl groups of the enzyme (aspartate and the glutamate residues). This favors the retention of enzyme flexibility which is essential for its activity since it undergoes a conformational change from an “active” to an “inactive” form, where the six subunits have the capacity for cooperative interaction (
A complete immobilization yield was achieved, with no remaining protein in the supernatant. As indicated in
Recovered activity (in bars) and expressed activity (in lines) after the immobilization of the fused protein onto ReliSorb EP400/SS, loading an increasing amount of protein.
The enzyme was trialed first for its potential application in the synthesis of glutamic acid at different concentrations of starting material (α-ketoglutarate) and sub-stoichiometric amounts of NADH (1:10, 1:50, 1:300). In this case, the ammonia and formate substrates are kept in excess with respect to the α-ketoglutaric acid (see
At a 50 mM scale of α-ketoglutaric acid (1 mM NADH), the free protein and the immobilized form appeared to biocatalyse similarly the reaction, showing analogous conversion rates (
Percentage of conversion of 20 mg of immobilized fusion protein (5 mg/g) versus 0.1 mg/ml of the soluble form with 50 mM α-ketoglutarate, 200 mM ammonium, 200 mM formate, 1 mM NADH at 37°C.
Then, a large-scale biotransformation of 300 mM of α-ketoglutaric acid (
Percentage of conversion of 15 mg of immobilized fusion protein (10 mg/g) versus 0.15 mg/ml of the soluble form with 300 mM α-ketoglutarate, 400 mM ammonium, 400 mM formate, 1 mM NADH at 37°C.
The GluDH-FDH fused system could be envisaged as a tool to remove ammonia, for example in contaminated waters following spillages. To better evaluate the efficiency of the system in such potential application, different experiments where the ammonia must be fully depleted have been designed. The removal of ammonia was determined by measuring the consumption of the α-ketoglutarate (substrate provided in excess) combined with the rate of production of the glutamate, since the ammonia employed for the amination is stoichiometric with the two consumed/formed components.
Firstly, to compare the performance of GluDH alone and the GluDH in the fused assembly, with or without the assistance of the FDH, a low amount of ammonia (15 mM) was supplemented with an excess of α-ketoglutarate (30 mM) and the catalytic efficiency tested in the absence or presence of the formate substrate, with either stoichiometric or catalytic amounts of NADH. The reactions were monitored at 1, 2, and 24 h but, in all cases, after 1 h no further change in the ammonia elimination was observed. The fusion protein emerged to be more efficient (
Percentage of conversion of 15 mM ammonia without (first line) and with (second line) NADH recycling.
Entry | Concentration [mM] | Conversion [%] | ||||
---|---|---|---|---|---|---|
Ammonia | α-ketoglutarate | NADH | Formate | Wild-type CsGluDH | GluDH domain | |
1 | 15 | 30 | 15 | / | 80% | 80% |
2 | 15 | 30 | 1 | 100 | / | 100% |
While the fusion protein faired very well with low concentration of ammonia, the robustness of the system was tested under significantly harsher conditions. A second set of reactions was set up with 300 mM ammonia together with a moderate excess of α-ketoglutarate (400 mM) and formate (400 mM), and 1 mM NADH. Surprisingly, only the immobilized fusion protein showed a >99% conversion in 48 h (
Percentage of conversion of the fusion protein and the individual enzymes in both soluble and immobilized form with 300 mM ammonium, 400 mM α-ketoglutarate, 400 mM formate, 1 mM NADH. The final concentration of the fusion protein in both forms was 0.1 mg/ml, the final concentration of the free and co-immobilized individual enzymes was 0.057 mg/ml for GluDH and 0.046 mg/ml for FDH.
Despite the general improved stability attained with the immobilization method, the conversion rates achieved in 48 h with the immobilized fusion protein and the co-immobilized WT enzymes are clearly divergent (>99 versus 65%, respectively) with 1.5-fold higher rate for the bifunctional protein. The performance in working conditions provides a suitable method of comparison of the catalytic efficiency of the two systems (with and without the fusion) where the new enzyme is more effective timewise.
Looking at the literature, also the PheDH-FDH fusion protein showed a 1.5-fold higher molecular conversion rate than the individual enzymes, due to the improved cofactor regeneration process, where the product of the first reaction can be quickly transferred to the second enzyme, which leads to a lower time of diffusion (
Consequently, the bifunctional enzyme gained remarkable enhancements, especially in the performance, reducing the reaction duration and intensifying the overall conversion degree. The fusion protein results a superior biocatalyst than the soluble or co-immobilized wild-type enzymes under several points, from the time- and cost-effectiveness production (including transformation, expression, purification, immobilization steps) to the enhanced biocatalytic reaction.
The GluDH from
The original contributions presented in the study are included in the article/
FP conceptualized the idea and supervised the project, VM performed the experiments and the characterization studies, and wrote the initial draft; DRP performed the MD analysis, AIB-M designed and supervised the genetic experiments and the immobilization studies. All the authors discussed the results and reviewed the manuscript.
SNSF grant number 200021_192274/1.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The authors thank SNSF (200021_192274) for funding this research.
The Supplementary Material for this article can be found online at: