imagination at work

Application note 29-0211-99 AA Chromatography systems

Purification and immobilization of a transaminase for the preparation of an enzyme bioreactorA histidine-tagged transaminase was overexpressed in E. coli and purified using immobilized metal ion affinity chromatography (IMAC). Purification was achieved in a single step using HisTrap™ HP columns on ÄKTA™ pure chromatography system. Using the predefined NHS coupling method in UNICORN™ v6.3 software for ÄKTA pure system, the purified enzyme was immobilized on HiTrap™ NHS-activated HP columns for the preparation of an enzyme bioreactor to enable catalysis of chiral amination reactions. Columns with immobilized transaminase were used to investigate their efficiency in catalyzing conversion of a model substrate applied to the column. The study, which included use of the UNICORN Design of Experiment (DoE) software, showed the proof-of-principle for the application of a Sepharose™ based enzyme bioreactor for industrial-scale synthesis of chemical precursors.

IntroductionBiological effects differ depending on the enantiomeric form of a substance. In a racemate (mixture of two enantiomers), one enantiomer may be fully active whereas the other enantiomer is inactive. This could mean that only a fraction of a chemically synthesized material is functional, increasing the cost of production. Chemical synthesis of only the desired enantiomer may be extremely complicated but can be simplified by use of enzymes catalyzing chiral reactions.

Transaminases are a group of enzymes that catalyze the transfer of an amino group from an amine to an acceptor, creating a ketone and an amino acid with a single chirality. There is great interest in the use of these enzymes for chiral production of chemical precursors. Currently, chiral production is usually performed by overexpressing the enzyme in a suitable host and the crude extract of the material is used for mixing

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with the precursor to be modified. The method is relatively straightforward but has the drawback that purification of the product becomes more complex due to the addition of large amounts of biomass without catalytic function. The use of purified enzyme reduces the purification efforts required to some extent. An even more interesting approach is to use enzyme immobilized on a solid support for easy separation of product substance from the enzyme (1). Immobilization can give increased stability of the enzyme, allowing the use of the bioreactor for an extended period of time.

A proof-of-principle for the use of NHS-activated Sepharose High Performance medium (resin) for immobilization of a purified transaminase for chiral synthesis of chemical precursors was investigated (Fig 1). The aim for the industrial application was to prepare a transaminase column that can be used for an extended period for chiral biosynthesis (Fig 2).

The histidine-tagged transaminase purified and immobilized in the study has a broad substrate range and gives an enantiomeric excess of > 99%. This dimeric protein (2 × relative molecular mass [Mr] 57 000) requires pyridoxal-5’-phosphate (PLP) as cofactor (2). The transaminase was purified from freeze-dried E. coli in a single step using nickel-charged IMAC (Ni-IMAC). After buffer exchange the transaminase was immobilized on NHS-activated media in suspension or in prepacked columns. The on-column catalytic function of the immobilized transaminase was evaluated in continuous-flow experiments where a substrate was passed through the column; the concentration (yield) of product was monitored by reversed-phase chromatography (RPC) analysis of collected fractions.

The use of immobilized transaminase in prepacked columns could potentially replace an existing process for the manufacture of chemical precursors based on crude E. coli extract (Fig 3).

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Purification

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Fig 3. Schematic views of (A) the current process based on crude E. coli extract that includes enrichment of the product from biological material and (B) the proposed new process based on immobilized pure transaminase. The latter process could potentially replace the existing process to yield a product mixture without biological material.

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Fig 1. Overall workflow for preparation and use of the enzyme bioreactor.

Materials and methodsSample preparationHistidine-tagged (S)-aminotransferase (also called histidine-tagged omega-transaminase or transaminase) derived from Arthobacter citreus was overexpressed in E. coli MG1655 (3) at Cambrex, Karlskoga, Sweden and the cells were freeze dried. The E. coli cells were suspended (1 g cells/10 ml buffer) in Homogenization buffer (50 mM sodium phosphate with 0.5 mM pyridoxal-5’-phosphate [PLP, required enzyme cofactor], 10 mM Triton™ X-100, 5% [v/v] glycerol, and 1 mM DTT, pH 7.65) and rehydrated for 1 h at 4̊ C. The cells were homogenized using EmulsiFlex™-C3 high-pressure homogenizer by four passes at 40 psi. The homogenate was clarified by centrifugation at 26 000 × g for 20 min.

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Fig 2. Overall plan for the bioreactor project.

Immobilized metal ion affinity chromatography (IMAC)The columns (HisTrap HP 1 ml or HisTrap HP 5 ml) were equilibrated with five column volumes (CV) of Binding buffer (20 mM sodium phosphate, 20 mM imidazole, 0.5 mM PLP and 500 mM NaCl, pH 7.65). Up to 70 ml of sample was applied per milliliter packed bed, and the column was washed with 5 CV of Binding buffer. Step elution (10 CV) was performed using Elution buffer (20 mM sodium phosphate, 500 mM imidazole, 0.5 mM PLP, and 500 mM NaCl, pH 7.65). The purification was performed at room temperature.

Buffer exchangeTo prepare the purified transaminase for NHS coupling it was subjected to buffer exchange at room temperature using HiPrep™ 26/10 Desalting column. The column was equilibrated with 4 CV of Coupling buffer (200 mM sodium carbonate with 500 mM NaCl and 0.5 mM PLP; pH 8.0, pH 8.5, or pH 9.0). Aliquots of 4.5 ml of sample were applied to the column. Proteins were eluted isocratically for 2 CV and collected using peak fractionation via the outlet valve of ÄKTA pure, with 50 mAU (A280) as start and end settings.

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Activity determination was performed in the wells used for coupling in a bench shaker placed in a temperature controlled oven (45°C). The plate was incubated for 5 min before addition of 200 µl of Reaction mixture (100 mM sodium phosphate buffer, pH 7.8 with 0.5 mM PLP, 20 mM α-Methylbenzylamine, [α-MBA, substrate] and 20 mM sodium pyruvate [co-substrate]). The reaction mixture was added to three sets of wells after 5, 10, and 20 min. The reaction was then immediately stopped by addition of 400 µl of 200 mM HCl and 0.5 mM PLP, and the absorbance was measured at 290 nm using a SpectraMax Plus384 absorbance microplate reader. A graph was plotted for the absorbance values at the different reaction times for determination of activity in each well.

Immobilization of transaminase in HiTrap NHS-activated HP 1 ml columnImmobilization of proteins on HiTrap NHS-activated HP 1 ml prepacked columns works well above pH 8. The robustness of coupling of purified transaminase was controlled by a DoE setup with the following factors at their high and low settings: pH 8 to 9, incubation time for coupling reaction of 30 to 120 min, and transaminase concentration of 3 to 5 mg/ml. Three center points were included in the design. The coupling efficiency was selected as response. The coupling was performed at room temperature on two ÄKTA pure systems in parallel. The systems were equipped with Column valve (V9-C) allowing up to five columns to be used per system, and Loop valve (V9-L) that allows up to five defined volumes of different samples to be applied. The predefined NHS coupling method in UNICORN software was used. A full factorial robustness test experiment was set up with 11 runs. The protocol for each column was in brief: protein samples were loaded into 1-ml sample loops connected to the loop valve using the Manual loop fill method in UNICORN. The NHS coupling method was then used for coupling using the DoE and scouting function of UNICORN. Column activation was performed using a wash with 5 CV of ice-cold 1 mM HCl. Protein solution in the loop valve was applied on the column (0.7 CV = 0.7 ml) and the system entered an “Incubation” phase for 30 to 120 min. Deactivation of remaining active sites on the medium was performed with 3 × 2 ml of 100 mM Tris-HCl buffer containing 0.5 mM PLP, pH 8.5. The medium was washed with 3 × 2 ml of 100 mM acetate buffer containing 500 mM NaCl, 0.5 mM PLP, pH 4. The deactivation and wash steps were applied in an alternating fashion. The prepared columns were stored in 20% ethanol with 0.5 mM PLP at 4̊ C.

On-column enzyme catalysisContinuous enzyme reactions were investigated using the model reaction (Fig 4) on ÄKTA pure. A HiTrap NHS-activated HP 1 ml column with immobilized transaminase was placed in a column oven at 40°C. A metal capillary loop was attached upstream of the column inside the oven for thermal equilibration of the reaction mixture entering the column.

Fig 4. Model reaction used in the transaminase activity assay and for the on-column reactions. Activity was determined as rate of formation of acetophenone.

SDS-PAGEPurified transaminase was analyzed using SDS-PAGE in precast ExcelGel™ SDS Gradient 8–18 gel with ExcelGel SDS Buffer Strips on a Multiphor™ II electrophoresis system. Electrophoresis was run at limiting settings of 50 mA, 600 V, 30 W for 1.5 h. The gel was stained using Deep Purple™ Total Protein Stain and scanned with Ettan™ DIGE Imager fluorescence scanner. The image was analyzed using ImageQuant™ TL software.

Activity assayTransaminase activity was measured by detecting the amount of acetophenone formed. The reaction (Fig 4) was performed in a 96-well microplate. Aliquots of 200 µl reaction mix containing 100 mM sodium phosphate buffer, 0.5 mM PLP, 20 mM sodium pyruvate, and 20 mM (S)-(-)-α-Methylbenzylamine, pH 7.65 were incubated for 5 min at 45˚C in a SpectraMax™ Plus384 absorbance microplate reader. Samples of 40 µl of purified enzyme were added into the mix and the absorbance increase at 290 nm was followed by measurement every 10 s for 40 min. Transaminase activity in microplate immobilization experiments was determined as described in Results.

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Immobilization of transaminase on NHS-activated Sepharose Fast Flow medium in 96-well filterplateA 96-well filterplate was prepared by dispensing 110 µl of 50% gel slurry in 20% ethanol with a 12-channel multi-pipette. The gel was activated by washing with ice-cold 1 mM HCl (3 × 200 µl) for 1 min each on a microplate bench shaker at 1100 rpm with draining of the wells using a vacuum manifold at 0.5 bar. In the Design of Experiments (DoE) setup, purified transaminase was applied (in each well, 5 to 20 mg/ml sedimented medium) as varying volumes of 2.5 mg/ml transaminase in 100 mM sodium phosphate buffer with 0.5 mM PLP, pH 7.5 to 9.5.

The plate was incubated at room temperature for 4 h. Remaining reactive groups were deactivated with 100 mM Tris-HCl containing 0.5 mM PLP, pH 8.5 (2 × 200 µl) for 30 min with shaking at 1100 rpm. Washing was performed by first shaking with 100 mM acetate buffer, 0.5 mM PLP, 500 mM NaCl, pH 4.0 (3 × 200 µl), followed by 100 mM Tris-HCl containing 0.5 mM PLP, pH 8.5 (3 × 200 µl) for 1 min each. This step was repeated five times.

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The reaction mixture was placed at an inlet of ÄKTA pure and the system pump was used for application of the mixture on the column. The outlet of the column was attached to the UV detector of the chromatography system for detection of product formed, and was also connected to Fraction collector F9-R. Measurements were performed by first equilibrating with 5 CV of Reaction buffer (100 mM sodium phosphate, 0.5 mM PLP, pH 7) and then applying 10 ml of substrate solution using a 10-ml sample loop at various flow rates (0.1 to 10 ml/min). The substrate solutions were: 0.1 to 100 mM α-MBA, 183 mM sodium pyruvate (co-substrate) dissolved in Reaction buffer, and adjusted to a final pH of 7.65.

Fractions of 2 ml were collected. After passage of the substrate solution, the column was emptied of substrate and product by washing with 10 CV of Reaction buffer. Collected fractions were analyzed using RPC.

To check effects of column length/residence time, five 1-ml HiTrap columns with immobilized transaminase were connected in series and substrate solution composed of 10 mM α-MBA and 17 mM sodium pyruvate in Reaction buffer was passed through the columns at 0.1 ml/min.

Analysis of acetophenone (reaction product) by RPCRPC analyses of reaction mixtures passed through immobilized-transaminase columns were performed on ÄKTAmicro™ chromatography system. The RPC column (µRPC C2/C18 ST 4.6/100) was equilibrated at 1 ml/min with 5 CV of an eluent composed of 20% acetonitrile and 0.1% trifluoroacetic acid. Aliquots of 10 µl were applied using Autosampler A-905 and followed by isocratic elution with 15 CV of the same eluent as for equilibration. A standard curve for subsequent analysis was prepared by sequential application of 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 10 mM acetophenone in 100 mM sodium phosphate buffer with 0.5 mM PLP, pH 7. Acetophenone is the product of the model reaction used for evaluation of transaminase columns. In a separate run, a mixture of α-MBA (18 mM) and sodium pyruvate (30 mM) in 0.1 M Tris-HCl, 0.5 mM PLP, pH 7.65, was applied to determine the elution positions of these substances that are substrate and co-substrate, respectively, in the model reaction. Acetophenone shows a strong absorbance at 245 nm (4) and the UV detector was therefore set to 245 nm, together with 254 nm and 280 nm.

ResultsPurificationTransaminase was purified from freeze-dried E. coli cells using Ni-IMAC with step elution (Fig 5 and 6) followed by desalting using HiPrep 16/10 Desalting column (Fig 7). A preparation from 7 g of cells yielded 35 mg transaminase. Gel filtration (GF) analyses (not shown) indicated that the enzyme (Mr 57 000) behaves as an elongated dimer or possibly a trimer (apparent Mr of 160 000). The purity of the preparation was 86% as judged from UV peak integration, which was confirmed by SDS-PAGE (data not shown). The preparation did not contain significant amounts of aggregates as determined by GF analyses. The specific activity was determined using a model reaction (α-MBA and pyruvate as substrates) to be 56 U/mg. Aliquots of the purified transaminase were subjected to buffer exchange in solution suitable for coupling of transaminase on HiTrap NHS-activated HP columns.

Fig 5. Workflow for the purification.

Cell harvest

Cell disruption

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Preparation of enzyme

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Fig 6. Single-step purification of histidine-tagged transaminase using HisTrap HP 1 ml.

Fig 7. Buffer exchange of purified transaminase on HiPrep 26/10 Desalting column in buffer suitable for NHS coupling on chromatography medium.

Column: HisTrap HP 1 mlSample: Clarified homogenate from recombinant E. coli

expressing transaminaseSample volume: 70 mlInjection method: System pump via Mixer valve V9-MBinding buffer: 20 mM sodium phosphate buffer, 0.5 mM pyridoxal phosphate (PLP), 500 mM NaCl, 20 mM imidazole, pH 7.65Elution buffer: 20 mM sodium phosphate buffer, 0.5 mM PLP,

500 mM NaCl, 500 mM imidazole, pH 7.65Flow rate: 1 ml/minUV cell: 2 mmSystem: ÄKTA pure equipped with Column valve V9-C,

UV-monitor U9-M, pH valve V9-pH,Outlet valve V9-O

Columns: HiPrep 26/10 DesaltingInjection method: Superloop™Sample volume: 4.5 mlSample: Eluate from IMAC purificationCoupling buffer: 200 mM NaHCO3, 0.5 mM PLP, 500 mM NaCl, pH 8Flow: 5 ml/minUV cell: 2 mmSystem: ÄKTA pure equipped with Column valve V9-C, UV-monitor U9-M, pH/restrictor valve V9-pH,

Outlet valve V9-O

Immobilization on NHS-activated Sepharose Fast Flow: Condition screeningTransaminase was coupled in a 96-well filterplate for screening of coupling conditions. A full factorial DoE set up was used with pH and amount of protein as factors, and activity as response (Fig 8). Different amounts of protein (5 to 20 mg/ml medium) at pH 7.5 to 9.5 were coupled in wells containing the equivalent of 55 µl of NHS-activated Sepharose Fast Flow. Activity in each well was determined using the model reaction for transaminase based on α-MBA as substrate. Three series of wells were set up to allow three measurements (at different times) for each activity determination (each experimental point).

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Fig 8. Screening model of the NHS coupling. A full factorial design with two factors was applied. (A) pH and protein loading amount. Four conditions and a center point analyzed as triplicate were included. The corner colors represent the activity response level. (B) Protein load is given as mg protein/ml medium (illustration generated from MODDE 9.0 evaluation software).

Investigation: DoE immobilizationDesign: Full Fac (2 levels)Response: Activity

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Evaluation of DoE results was performed using software MODDE™ 9.0 (Fig 9). The Summary-of-fit plot shows that the design model is good (A), the Coefficient plot (B) shows that the protein load is a valid factor. A higher transaminase activity was observed with high protein load during coupling. The results also suggest that there was no significant effect of pH on immobilized activity (results not shown).

Fig 9. Evaluation of NHS coupling in 96-well filter plate. (A) Summary-of-fit plot, (B) Coefficient plot.

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Fig 10. Workflow for coupling of transaminase on HiTrap NHS-activated HP 1 ml.

Fig 11. The predefined NHS coupling method in UNICORN was used for the coupling of transaminase. Phases in the UNICORN method are shown on the right.

Method outline

Immobilization on HiTrap NHS-activated HP: Robustness testing

A protocol for coupling of transaminase on HiTrap NHS-activated Sepharose HP 1 ml was designed (Fig 10) based on the findings from the condition screening experiment and using the UNICORN predefined method, NHS coupling (Fig 11). The experiment was run on ÄKTA pure equipped with Loop valve (V9-L) and Column valve (V9-C) to allow automatic runs of five experimental points at a time. A robustness test based on DoE was performed with three factors; coupling pH (8 to 9), load concentration (3 to 5 mg/ml medium) and incubation time (30 to 120 min), and with coupling efficiency as response.

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Fig 12. Example of NHS-coupling of transaminase on HiTrap NHS-activated HP 1 ml.

Fig 13. Evaluation of DoE results for the robustness test of the NHS coupling of transaminase on HiTrap NHS-activated HP 1 ml. (A) Summary-of-fit plot and (B) Coefficient plot.

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Columns: HiTrap NHS-activated HP 1 mlSample: Purified transminase in 200 mM sodium carbonate

buffer, pH 8.0, pH 8.5, pH 9.0Sample volume: up to 0.7 mlInjection method: Loop valve, V9-L, with 1-ml loopsBuffer A: 1 mM HClBuffer B: 100 mM Tris-HCl, 0.5 mM PLP, pH 8.5

(Deactivation and wash)Buffer C: 100 mM sodium acetate buffer with 0.5 mM PLP,

500 mM NaCl, pH 4 (Wash)Flow: 1 ml/minUV cell: 2 mmSystem: ÄKTA pure equipped with Column valve V9-C,

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Coupling of transaminase on HiTrap NHS-activated HP 1 ml (Fig 12) was initiated by applying dilute ice-cold HCl to remove isopropanol used to stabilize the preactivated columns before use. Transaminase solutions were applied on the column and left to react for different periods of time. After the incubation, deactivation using Tris buffer (pH 8.5), and wash with acetate buffer (pH 4) was performed three times in sequence. The high peak early in the chromatogram (flowthrough) is caused by N-hydroxysuccinimide (NHS) leaving the activated groups in the medium upon reacting with amine groups on the protein surface. The flowthrough peaks were analyzed as described below. Additional minor squared peaks correspond to the deactivation and washing steps.

The coupling efficiency was determined by the desalting method as described in the instruction for the columns (article code number 71-7006-00). In brief, the flowthrough (upon restart of flow after incubation) was applied on a HiTrap Desalting column to separate NHS from uncoupled protein, using absorbance at 280 nm for quantitation. The coupling efficiencies obtained were in the range of 87.5% to 98.3% for the robustness test experiment. The R2 and Q2 parameters in the Summary-of-fit and the Coefficient plot showed weak or no relationship between the factors and the response, which indicates that the method was robust (Fig 13). Columns prepared by the method were used for investigation of continuous on-column reactions.

Interpreting DoE data

The Summary-of-fit plot shows four parameters describing the quality of the model obtained from the DoE results. For each parameter, the value 1 means perfect fit of model to experimental data.

R2 (Goodness-of-fit) is a measure of how well the model fits the experimental data. If R2 is around 0.5, the model is of rather low significance.

Q2 (Goodness of prediction) shows how well the model predicts tentative new data. This parameter is often considered a more realistic and useful performance indicator than R2, since it can better measure how the model can accurately predict the outcome of data points. Q2 should be greater than 0.1 for a model to be significant and greater than 0.5 for a good model. Q2 is negative in this model. In robustness testing the optimal result for Q2 is close to zero. A negative Q2 indicates weak or no relationship between the factors and the response.

Model validity is a test of diverse model problems. It reflects whether the model is appropriate in a general sense. The higher the value the more valid the model is. A value above 0.25 suggests a valid model. A value lower than 0.25 indicates statistically significant model problems, such as the presence of outliers, an incorrect model, or a transformation problem. The model validity in the present study was around 0.5.

Reproducibility is the variation of the response under the same conditions (pure error), often at the center points, compared to the total variation of the response. A reproducibility value of 1 represents perfect reproducibility.

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Fig 14. Principle of on-column (HiTrap NHS-activated HP) transaminase activity studies.

Fig 15. System setup for on-column reaction studies with columns containing immobilized transaminase. The column was kept in a column oven.

Fig 16. Chromatogram from on-column reaction studies with transaminase on HiTrap NHS-activated HP 1 ml column. Absorbance at 210 nm (purple), 245 nm (green), and 280 nm (dark blue), and conductivity (%, light blue).

On-column enzyme-catalyzed reactionsThe model reaction based on α-MBA and pyruvate as substrates (Fig 4) was used for evaluation of the activity of the immobilized transaminase in the 1-ml HiTrap NHS-activated HP column (4 mg immobilized transaminase/ml medium, Fig 14). The experiments were performed using ÄKTA pure system combined with a column oven heated to 40°C (Fig 15). The column was equilibrated in buffer with the cofactor PLP, and reaction mixtures containing various concentrations of α-MBA and pyruvate in excess were applied on the column. Figure 16 shows a resulting chromatogram. Effluent was collected using a fraction collector and analyzed for content of acetophenone (the product) by RPC on ÄKTAmicro chromatography system (Fig 17).

Apply substrate and cofactor (PLP)

Factors tested:Substrate concentration Flow rate (residence time)TemperaturepHIonic strengthStabilizers

Collect and analyze flowthrough

Column: HiTrap NHS-activated HP 1 ml with immobilized transaminase

Sample: 110 mM MBA, 183 mM pyruvate (co-substrate) in eluentSample volume: 10 mlInjection method: Sample loopEluent: 100 mM sodium phosphate, 0.5 mM pyridoxal

phosphate, pH 7.0Flow rate: 0.5 ml/minUV cell: 0.5 mm (short path length to increase linear range)System: ÄKTA pure and external column oven (40°C)

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A standard curve was prepared using pure acetophenone (Fig 17D). The two outlying points (15 mM and 20 mM) were excluded from the linear regression analysis to obtain an equation for concentration calculations. The concentration of product in the effluent represents the yield in the reaction. The conversion factor was defined and calculated as conc. acetophenone/conc. α-MBA, where α-MBA concentration was the start concentration.

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Fig 17. RPC analysis of substrate and product standards in 100 mM sodium phosphate buffer, pH 7 and 0.5 mM PLP. (A) 18 mM α-MBA and 100 mM sodium pyruvate. Sodium pyruvate (first peak) eluted at 1.07 ml and α-MBA (second peak) eluted at 1.49 ml. (B) 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, and 20 mM acetophenone applied in a series of runs for the preparation of standard curve. (C) Enlargement of acetophenone peaks in B. (D) Acetophenone standard curve.

Column: µRPC C2/C18 ST 4.6/100Sample: Standard or fraction from on-column reaction experiment on HiTrap NHS-activated HP column with immobilized transaminaseSample volume: 10 mlInjection method: Sample loopEluent: 30% acetonitrile with 0.1% TFA (isocratic separation, 10 CV)Flow rate: 1 ml/minUV cell: 10 mm; 245 nm for specific detection of acetophenoneSystem: ÄKTAmicro

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245 nm254 nm280 nm

245 nm254 nm280 nm

(A) Substrate and co-substrate (B) Product (acetophenone)

(C) Product peaks from a series of analyses with different concentrations of product

(D) Acetophenone standard curve

A condition screening was performed with concentration of α-MBA and flow rate as factors with yield (concentration of the product, acetophenone) and conversion factor as responses (Fig 18). Center points were not included. For visualization of the screening results yield and substrate conversion factor was subjected to multiple linear regression analysis and preparation of contour plots using SigmaPlot™ software (Fig 19). The highest yield (2.0 mM acetophenone) was unexpectedly obtained using the low level of substrate concentration combined with low flow rate. This may indicate product inhibition effects, which can be a drawback with asymmetric synthesis (5). The conversion factor was also highest for the same data point. As expected the conversion factor plot indicated that a high substrate concentration reduces the conversion factor of the reaction. The highest

conversion factor obtained was 20%. It was hypothesized that a longer (larger) column will increase the yield and conversion factor since this corresponds to an increased residence time on the column. This was tested by connecting five transaminase columns in series and applying a reaction mixture with 10 mM α-MBA and 17 mM sodium pyruvate at 0.1 ml/min. The yield was 3.9 mM acetophenone and the substrate conversion factor was 40%. The dramatic increase in yield upon increasing residence time (from 10 to 50 min) indicates that the reaction rate or mass transport of substrate in the chromatographic medium is limiting the yield. Further work will be performed to investigate this.

10 29-0211-99 AA

Fig 19. Contour plots of on-column reaction data obtained in the condition screening experiment. (A) Yield (mM) and (B) conversion factor (%). The plots serve as simplified visualization of the few data points, and should only be viewed as preliminary evaluation. More data is needed for a valid model of the reactions.

600

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Investigation: Transaminase reaction on column study (MLR)Design: Full Fac (2 levels)Response: Product formation

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Fig 18. Screening model for the on-column reactions study. (A) The factors included were concentration of substrate (10 and 110 mM) and flow rate (0.1 and 0.5 ml/min). The corner colors represent the product yield. (B) Example of RPC analysis of column effluent in experiment with 10 mM α-MBA at 0.1 ml/min.

ConclusionsHistidine-tagged transaminase could be purified in a single step on ÄKTA pure system using Ni-IMAC giving a yield of 5 mg transaminase/g dried E. coli cells with a purity of 86%. The specific activity was 56 U/mg in solution as determined using a model enzyme reaction. The transaminase enzyme (Mr 57 000) behaved as a dimer or possibly a trimer as determined from GF analyses (apparent Mr of 160 000) under the conditions used. The preparation did not contain significant amounts of aggregates. By buffer exchange on a HiPrep 26/10 Desalting column, purified enzyme could be quickly transferred into buffers suitable for NHS coupling to Sepharose medium. Conditions that gave a robust method for NHS coupling on HiTrap NHS-activated Sepharose HP 1 ml were determined, which allowed the immobilization of 4 mg transaminase/ml packed medium bed. Investigation of on-column reactions based on catalysis by immobilized transaminase showed that passage through the column of 10 mM α-MBA (the substrate) at 40˚C with a residence time of 10 min gave a yield of 2 mM acetophenone (the product). This yield corresponds to a substrate conversion factor of 20%. The conversion factor was doubled to 40% by using five HiTrap NHS-activated HP 1 ml columns in series (residence time 50 min) under the same conditions.

References1. Tischer, W. and Wedekind, F. Immobilized Enzymes: Methods

and Applications. In Biocatalysis. From Discovery to Application, (Fessner, W-D., ed.), Springer-Verlag, Berlin (1999).

2. Percudani, R. and Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Reports 4, 850–854 (2003).

3. Martin, A. R., et al.. Characterization of free and immobilized (S)-aminotransferase for acetophenone production. Appl. Microbiol. Biotechnol. 76, 843–851 (2007).

4. Schätzle, S., et al. Rapid and Sensitive Kinetic Assay for Characterization of omega-transaminases. Anal. Chem. 81, 8244–8248 (2009).

5. Shin J.-S. and Kim B.-G. (1999). Asymmetric synthesis of chiral amines with omega-Transaminase. Biotechnol. Bioeng. 65, 206–211.

AcknowledgementsCloning and expression were performed at Cambrex, Karlskoga, Sweden. Purification, immobilization, and on-column reaction studies were performed at GE Healthcare by D. Wei, S. Jouda, J. Senewiratne, and T. Frigård.

29-0211-99 AA 11

Ordering informationProduct Code number

ÄKTA pure M2 29-0182-27

Loop valve V9-L 29-0113-58

pH valve kit V9-pH 29-0113-59

Fraction collector F9-R 29-0113-62

Autosampler A-905 18-5050-65

ÄKTAmicro 28-9483-03

Deep Purple Total Protein Stain RPN6305

Dithiothreitol (DTT) 17-1318-02

ExcelGel SDS Buffer Strips 17-1342-01

ExcelGel SDS Gradient 8-18 80-1255-53

Glycerol 17-1325-01

HiTrap NHS-activated HP (5 × 1 ml) 17-0716-01

HisTrap HP (5 × 1 ml) 17-5247-01

HisTrap HP (5 × 5 ml) 17-5248-01

HiTrap Desalting (5 × 5 ml) 17-1408-01

HiPrep Desalting 26/10 (1 × 53 ml) 17-5087-01

NHS-activated Sepharose 4 Fast Flow (25 ml) 17-0906-01

IEF Sample application pieces 18-1129-46

ImageQuant TL 28-9380-94

µRPC C2/C18 ST 4.6/100 17-5057-01

LMW-SDS Marker Kit 17-0446-01

Sodium dodecyl sulfate (SDS) 17-1313-01

Tris 17-1321-01

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