29021199aa purification and immobilization of a transaminase

7/21/2019 29021199AA Purification and Immobilization of a Transaminase

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Application note 29-0211-99 AA Chromatography systems

Purification and immobilization of atransaminase for the preparation ofan enzyme bioreactorA histidine-tagged transaminase was overexpressed inE. coli and purified using immobilized metal ion affinitychromatography (IMAC). Purification was achieved in asingle step using HisTrap HP columns on KTA pure

chromatography system. Using the predefined NHS couplingmethod in UNICORN v6.3 software for KTA puresystem, the purified enzyme was immobilized on HiTrapNHS-activated HP columns for the preparation of anenzyme bioreactor to enable catalysis of chiral aminationreactions. Columns with immobilized transaminase wereused to investigate their efficiency in catalyzing conversionof 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 theapplication of a Sepharose based enzyme bioreactor forindustrial-scale synthesis of chemical precursors.

IntroductionBiological effects differ depending on the enantiomeric formof a substance. In a racemate (mixture of two enantiomers),one enantiomer may be fully active whereas the otherenantiomer is inactive. This could mean that only a fractionof a chemically synthesized material is functional, increasingthe cost of production. Chemical synthesis of only the desiredenantiomer may be extremely complicated but can besimplified 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 chiralproduction of chemical precursors. Currently, chiral productionis usually performed by overexpressing the enzyme in a suitablehost and the crude extract of the material is used for mixing

GE HealthcareLife Sciences

with the precursor to be modified. The method is relativelystraightforward but has the drawback that purification ofthe product becomes more complex due to the addition oflarge amounts of biomass without catalytic function. The use

of purified enzyme reduces the purification efforts requiredto some extent. An even more interesting approach is to useenzyme immobilized on a solid support for easy separation ofproduct substance from the enzyme (1). Immobilization cangive increased stability of the enzyme, allowing the use of thebioreactor for an extended period of time.

A proof-of-principle for the use of NHS-activated SepharoseHigh Performance medium (resin) for immobilization of a purifiedtransaminase for chiral synthesis of chemical precursors wasinvestigated (Fig 1). The aim for the industrial application wasto prepare a transaminase column that can be used for anextended period for chiral biosynthesis (Fig 2).

The histidine-tagged transaminase purified and immobilizedin the study has a broad substrate range and gives anenantiomeric excess of > 99%. This dimeric protein (2 relativemolecular mass [M

r] 57 000) requires pyridoxal-5-phosphate

(PLP) as cofactor (2). The transaminase was purified fromfreeze-dried E. coliin a single step using nickel-chargedIMAC (Ni-IMAC). After buffer exchange the transaminasewas immobilized on NHS-activated media in suspension orin prepacked columns. The on-column catalytic function ofthe immobilized transaminase was evaluated in continuous-flow experiments where a substrate was passed through the

column; the concentration (yield) of product was monitoredby reversed-phase chromatography (RPC) analysis ofcollected fractions.

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


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Purification

E. colifeed

Pure enzyme

Immobilization

Asymmetric

synthesis

~100% amine

Ketone + cofactor(s)

Fig 3. Schematic views of (A) the current process based on crude E. coliextract that includes enrichment of the product from biological material and

(B) the proposed new process based on immobilized pure transaminase. Thelatter process could potentially replace the existing process to yield a productmixture without biological material.

Present process

New process

Homogenized E. coli expressingS-transaminase, ketone

(substrate) and cofactors

Product for further purification

Acid andorganic solvent

Enzyme reaction Floatation

Incubation

Chiral amine(productmixture)

pH adjustmentand distillation

Two-phaseextraction

m3-scale

Organic solvent

Ketone + cofactor(s)

Immobilized enzymeS-transaminase

Chiral amine

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

Materials and methodsSample preparation

Histidine-tagged (S)-aminotransferase (also called histidine-tagged omega-transaminase or transaminase) derived from

Arthobacter citreuswas overexpressed in E. coliMG1655 (3) atCambrex, Karlskoga, Sweden and the cells were freeze dried.

The E. colicells were suspended (1 g cells/10 ml buffer)in Homogenization buffer (50 mM sodium phosphate with0.5 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 4 C. The cells were homogenizedusing EmulsiFlex-C3 high-pressure homogenizer byfour passes at 40 psi. The homogenate was clarified bycentrifugation at 26 000 g for 20 min.

(A) Current process

(B) New process

Pilot/production bioreactor setup

Proof-of-concept using model reaction

Scale-up

Enzyme purificationCoupling on

chromatographymedium

Enzyme reaction

Enzyme purificationCoupling on

chromatographymedium

Bioreactor process

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) wereequilibrated with five column volumes (CV) of Binding buffer(20 mM sodium phosphate, 20 mM imidazole, 0.5 mM PLP and500 mM NaCl, pH 7.65). Up to 70 ml of sample was applied permilliliter packed bed, and the column was washed with 5 CVof 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 wasperformed at room temperature.

Buffer exchange

To prepare the purified transaminase for NHS coupling itwas subjected to buffer exchange at room temperatureusing HiPrep 26/10 Desalting column. The column wasequilibrated with 4 CV of Coupling buffer (200 mM sodiumcarbonate 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 thecolumn. Proteins were eluted isocratically for 2 CV and

collected using peak fractionation via the outlet valve ofKTA pure, with 50 mAU (A

280) as start and end settings.


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Activity determination was performed in the wells used forcoupling in a bench shaker placed in a temperature controlledoven (45C). The plate was incubated for 5 min before additionof 200 l of Reaction mixture (100 mM sodium phosphatebuffer, pH 7.8 with 0.5 mM PLP, 20 mM -Methylbenzylamine,[-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 stoppedby addition of 400 l of 200 mM HCl and 0.5 mM PLP, and the

absorbance was measured at 290 nm using a SpectraMaxPlus384absorbance microplate reader. A graph was plottedfor the absorbance values at the different reaction times fordetermination of activity in each well.

Immobilization of transaminase inHiTrap NHS-activated HP 1 ml column

Immobilization of proteins on HiTrap NHS-activated HP 1 mlprepacked columns works well above pH 8. The robustness ofcoupling of purified transaminase was controlled by a DoEsetup with the following factors at their high and low settings:

pH 8 to 9, incubation time for coupling reaction of 30 to120 min, and transaminase concentration of 3 to 5 mg/ml.Three center points were included in the design. The couplingefficiency was selected as response. The coupling wasperformed at room temperature on two KTA pure systemsin parallel. The systems were equipped with Column valve(V9-C) allowing up to five columns to be used per system, andLoop valve (V9-L) that allows up to five defined volumes ofdifferent samples to be applied. The predefined NHS couplingmethod in UNICORN software was used. A full factorialrobustness test experiment was set up with 11 runs. Theprotocol for each column was in brief: protein samples were

loaded into 1-ml sample loops connected to the loop valve usingthe Manual loop fill method in UNICORN. The NHS couplingmethod was then used for coupling using the DoE and scoutingfunction of UNICORN. Column activation was performedusing a wash with 5 CV of ice-cold 1 mM HCl. Protein solutionin 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 wasperformed with 3 2 ml of 100 mM Tris-HCl buffer containing0.5 mM PLP, pH 8.5. The medium was washed with 3 2 mlof 100 mM acetate buffer containing 500 mM NaCl, 0.5 mM PLP,

pH 4. The deactivation and wash steps were applied in analternating fashion. The prepared columns were stored in20% ethanol with 0.5 mM PLP at 4 C.

On-column enzyme catalysis

Continuous enzyme reactions were investigated using the modelreaction (Fig 4) on KTA pure. A HiTrap NHS-activated HP 1 mlcolumn with immobilized transaminase was placed in acolumn oven at 40C. A metal capillary loop was attachedupstream of the column inside the oven for thermalequilibration of the reaction mixture entering the column.

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

SDS-PAGE

Purified transaminase was analyzed using SDS-PAGE in precastExcelGel SDS Gradient 818 gel with ExcelGel SDS Buffer Stripson a Multiphor II electrophoresis system. Electrophoresiswas run at limiting settings of 50 mA, 600 V, 30 W for 1.5 h.The gel was stained using Deep Purple Total Protein Stainand scanned with Ettan DIGE Imager fluorescence scanner.The image was analyzed using ImageQuant TL software.

Activity assayTransaminase activity was measured by detecting the amountof acetophenone formed. The reaction (Fig 4) was performed ina 96-well microplate. Aliquots of 200 l reaction mix containing100 mM sodium phosphate buffer, 0.5 mM PLP, 20 mM sodiumpyruvate, and 20 mM (S)-(-)--Methylbenzylamine, pH 7.65 wereincubated for 5 min at 45 C in a SpectraMax Plus384absorbancemicroplate reader. Samples of 40 l of purified enzyme wereadded into the mix and the absorbance increase at 290 nmwas followed by measurement every 10 s for 40 min.Transaminase activity in microplate immobilization experiments

was determined as described in Results.

NH2 NH2O

()-MBA Sodium pyruvate Acetophenone Sodium alanine

O

ONa

O

O

ONa

+ +

Immobilization of transaminase on NHS-activatedSepharose Fast Flow medium in 96-well filterplate

A 96-well filterplate was prepared by dispensing 110 l of50% 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 l) for 1 min each on a microplate bench shaker at1100 rpm with draining of the wells using a vacuum manifoldat 0.5 bar. In the Design of Experiments (DoE) setup, purifiedtransaminase was applied (in each well, 5 to 20 mg/mlsedimented medium) as varying volumes of 2.5 mg/mltransaminase 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 with100 mM Tris-HCl containing 0.5 mM PLP, pH 8.5 (2 200 l) for30 min with shaking at 1100 rpm. Washing was performedby first shaking with 100 mM acetate buffer, 0.5 mM PLP,500 mM NaCl, pH 4.0 (3 200 l), followed by 100 mM Tris-HClcontaining 0.5 mM PLP, pH 8.5 (3 200 l) for 1 min each.This step was repeated five times.


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The reaction mixture was placed at an inlet of KTA pure andthe system pump was used for application of the mixture onthe column. The outlet of the column was attached to theUV detector of the chromatography system for detectionof product formed, and was also connected to Fractioncollector F9-R. Measurements were performed by firstequilibrating with 5 CV of Reaction buffer (100 mM sodiumphosphate, 0.5 mM PLP, pH 7) and then applying 10 ml ofsubstrate solution using a 10-ml sample loop at various flow

rates (0.1 to 10 ml/min). The substrate solutions were: 0.1to 100 mM -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 substratesolution, the column was emptied of substrate and productby washing with 10 CV of Reaction buffer. Collected fractionswere analyzed using RPC.

To check effects of column length/residence time, five1-ml HiTrap columns with immobilized transaminase wereconnected in series and substrate solution composed of10 mM -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 RPC

RPC analyses of reaction mixtures passed throughimmobilized-transaminase columns were performed onKTAmicro chromatography system. The RPC column(RPC C2/C18 ST 4.6/100) was equilibrated at 1 ml/min with5 CV of an eluent composed of 20% acetonitrile and 0.1%trifluoroacetic acid. Aliquots of 10 l were applied usingAutosampler A-905 and followed by isocratic elution with15 CV of the same eluent as for equilibration. A standard

curve for subsequent analysis was prepared by sequentialapplication of 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 10 mMacetophenone in 100 mM sodium phosphate buffer with0.5 mM PLP, pH 7. Acetophenone is the product of the modelreaction used for evaluation of transaminase columns. Ina separate run, a mixture of -MBA (18 mM) and sodiumpyruvate (30 mM) in 0.1 M Tris-HCl, 0.5 mM PLP, pH 7.65,was applied to determine the elution positions of thesesubstances that are substrate and co-substrate, respectively,in the model reaction. Acetophenone shows a strongabsorbance at 245 nm (4) and the UV detector was thereforeset to 245 nm, together with 254 nm and 280 nm.

ResultsPurification

Transaminase was purified from freeze-dried E. colicellsusing Ni-IMAC with step elution (Fig 5 and 6) followed bydesalting 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 theenzyme (M

r57 000) behaves as an elongated dimer or possibly

a trimer (apparent Mrof 160 000). The purity of the preparationwas 86% as judged from UV peak integration, which wasconfirmed by SDS-PAGE (data not shown). The preparation didnot contain significant amounts of aggregates as determinedby GF analyses. The specific activity was determined usinga model reaction (-MBA and pyruvate as substrates) tobe 56 U/mg. Aliquots of the purified transaminase weresubjected to buffer exchange in solution suitable for couplingof transaminase on HiTrap NHS-activated HP columns.

Fig 5. Workflow for the purification.

Cell harvest

Cell disruption

Clarification

IMAC

Desalting


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

Fig 7. Buffer exchange of purified transaminase on HiPrep 26/10 Desaltingcolumn 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/min

UV cell: 2 mmSystem: KTA 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: SuperloopSample volume: 4.5 mlSample: Eluate from IMAC purificationCoupling buffer: 200 mM NaHCO

3, 0.5 mM PLP, 500 mM NaCl, pH 8

Flow: 5 ml/minUV cell: 2 mmSystem: KTA pure equipped with Column valve V9-C,

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

Immobilization on NHS-activated SepharoseFast Flow: Condition screening

Transaminase was coupled in a 96-well filterplate forscreening of coupling conditions. A full factorial DoE set upwas used with pH and amount of protein as factors, andactivity as response (Fig 8). Different amounts of protein(5 to 20 mg/ml medium) at pH 7.5 to 9.5 were coupled inwells containing the equivalent of 55 l of NHS-activated

Sepharose Fast Flow. Activity in each well was determinedusing the model reaction for transaminase based on -MBAas substrate. Three series of wells were set up to allowthree measurements (at different times) for each activitydetermination (each experimental point).

E

lutionbuffer(%)

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

NHS-activated Sepharose 4 Fast Flow

Four conditions and center point = seven reactions

Three time points used for activity assay after coupling

Center points

1 2

1

2

A)

B)

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

l

l

pH

Protein

load

High

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Excluded

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HCl

Apply transaminase

Incubate 30-120 min

Deactivate using ethanolamine

Multiple wash-cycle

Immobilization

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

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

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0R2 ReproducibilityModel

validityQ2

1.5

1.0

0.5

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-0.5Conc.

(A)

(B)

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 forthe coupling of transaminase. Phases in the UNICORN method are shownon the right.

Method outline

Immobilization on HiTrap NHS-activated HP:Robustness testing

A protocol for coupling of transaminase on HiTrap NHS-activatedSepharose HP 1 ml was designed (Fig 10) based on the findingsfrom the condition screening experiment and using theUNICORN predefined method, NHS coupling (Fig 11). Theexperiment was run on KTA pure equipped with Loopvalve (V9-L) and Column valve (V9-C) to allow automaticruns of five experimental points at a time. A robustness test

based on DoE was performed with three factors; couplingpH (8 to 9), load concentration (3 to 5 mg/ml medium) andincubation time (30 to 120 min), and with coupling efficiencyas response.


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R2 ReproducibilityModelvalidityQ2

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-6Enz Enz*Inc Enz*pH Inc*pHpHInc

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

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

(A)

(B)

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: KTA pure equipped with Column valve V9-C,

UV-monitor U9-M, Loop valve V9-L

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Alternating washeswith Buffer B and C

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 beforeuse. Transaminase solutions were applied on the column andleft to react for different periods of time. After the incubation,deactivation using Tris buffer (pH 8.5), and wash with acetatebuffer (pH 4) was performed three times in sequence. Thehigh peak early in the chromatogram (flowthrough) is causedby N-hydroxysuccinimide (NHS) leaving the activated groupsin the medium upon reacting with amine groups on theprotein surface. The flowthrough peaks were analyzed asdescribed below. Additional minor squared peaks correspondto the deactivation and washing steps.

The coupling efficiency was determined by the desaltingmethod 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 aHiTrap Desalting column to separate NHS from uncoupledprotein, using absorbance at 280 nm for quantitation. Thecoupling efficiencies obtained were in the range of 87.5% to98.3% for the robustness test experiment. The R2 and Q2parameters in the Summary-of-fit and the Coefficient plotshowed weak or no relationship between the factors andthe 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 ofthe model obtained from the DoE results. For each parameter, the value1 means perfect fit of model to experimental data.

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

Q2(Goodness of prediction) shows how well the model predicts tentative

new data. This parameter is often considered a more realistic and usefulperformance indicator than R2, since it can better measure how themodel can accurately predict the outcome of data points. Q2 should begreater than 0.1 for a model to be significant and greater than 0.5 fora good model. Q2 is negative in this model. In robustness testing theoptimal result for Q2 is close to zero. A negative Q2 indicates weak or norelationship between the factors and the response.

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

Reproducibilityis the variation of the response under the same

conditions (pure error), often at the center points, compared to the totalvariation of the response. A reproducibility value of 1 represents perfectreproducibility.


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Fig 14. Principle of on-column (HiTrap NHS-activated HP) transaminaseactivity 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 transaminaseon 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 reactions

The model reaction based on -MBA and pyruvate assubstrates (Fig 4) was used for evaluation of the activityof the immobilized transaminase in the 1-ml HiTrap NHS-activated HP column (4 mg immobilized transaminase/mlmedium, Fig 14). The experiments were performed usingKTA pure system combined with a column oven heatedto 40C (Fig 15). The column was equilibrated in buffer

with the cofactor PLP, and reaction mixtures containingvarious concentrations of -MBA and pyruvate in excesswere applied on the column. Figure 16 shows a resultingchromatogram. Effluent was collected using a fractioncollector and analyzed for content of acetophenone (theproduct) by RPC on KTAmicro chromatography system (Fig 17).

Apply substrate and cofactor (PLP)

Factors tested:

Substrate concentrationFlow rate (residence time)TemperaturepHIonic strengthStabilizers

Collect and analyze flowthrough

Column: HiTrap NHS-activated HP 1 ml with immobilizedtransaminase

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: KTA pure and external column oven (40C)

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

40C

Reaction mixture

Collection of reaction product

Steel capillary loop for conditioningof reaction mixture

4 mg transaminase/ml medium


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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 -MBA and 100 mM sodiumpyruvate. Sodium pyruvate (first peak) eluted at 1.07 ml and -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 mMacetophenone applied in a series of runs for the preparation of standard curve. (C) Enlargement of acetophenone peaks in B. (D) Acetophenone standard curve.

Column: RPC 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: KTAmicro

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Acetophenone concentration (mM)

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

(C) Product peaks from a series of analyses with differentconcentrations of product (D) Acetophenone standard curve

A condition screening was performed with concentrationof -MBA and flow rate as factors with yield (concentrationof the product, acetophenone) and conversion factor asresponses (Fig 18). Center points were not included. Forvisualization of the screening results yield and substrate

conversion factor was subjected to multiple linear regressionanalysis and preparation of contour plots using SigmaPlotsoftware (Fig 19). The highest yield (2.0 mM acetophenone)was unexpectedly obtained using the low level of substrateconcentration combined with low flow rate. This may indicateproduct inhibition effects, which can be a drawback withasymmetric synthesis (5). The conversion factor was alsohighest for the same data point. As expected the conversionfactor plot indicated that a high substrate concentrationreduces the conversion factor of the reaction. The highest

conversion factor obtained was 20%. It was hypothesizedthat a longer (larger) column will increase the yield andconversion factor since this corresponds to an increasedresidence time on the column. This was tested by connectingfive transaminase columns in series and applying a reaction

mixture with 10 mM -MBA and 17 mM sodium pyruvateat 0.1 ml/min. The yield was 3.9 mM acetophenone andthe substrate conversion factor was 40%. The dramaticincrease in yield upon increasing residence time (from 10 to50 min) indicates that the reaction rate or mass transportof substrate in the chromatographic medium is limiting theyield. Further work will be performed to investigate this.


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

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

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(B)

(B)

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

ConclusionsHistidine-tagged transaminase could be purified in a singlestep on KTA pure system using Ni-IMAC giving a yield of5 mg transaminase/g dried E. coli cells with a purity of 86%.The specific activity was 56 U/mg in solution as determinedusing a model enzyme reaction. The transaminase enzyme(M

r57 000) behaved as a dimer or possibly a trimer as

determined from GF analyses (apparent Mrof 160 000)

under the conditions used. The preparation did not containsignificant amounts of aggregates. By buffer exchange ona HiPrep 26/10 Desalting column, purified enzyme could bequickly transferred into buffers suitable for NHS coupling toSepharose medium. Conditions that gave a robust methodfor NHS coupling on HiTrap NHS-activated Sepharose HP1 ml were determined, which allowed the immobilization of4 mg transaminase/ml packed medium bed. Investigationof on-column reactions based on catalysis by immobilizedtransaminase showed that passage through the column of10 mM -MBA (the substrate) at 40C with a residence timeof 10 min gave a yield of 2 mM acetophenone (the product).

This yield corresponds to a substrate conversion factor of20%. The conversion factor was doubled to 40% by using fiveHiTrap NHS-activated HP 1 ml columns in series (residencetime 50 min) under the same conditions.

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

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

2. Percudani, R. and Peracchi, A. A genomic overview ofpyridoxal-phosphate-dependent enzymes.EMBO Reports4, 850854 (2003).

3. Martin, A. R., et al.. Characterization of free and immobilized(S)-aminotransferase for acetophenone production.

Appl. Microbiol. Biotechnol.76, 843851 (2007).

4. Schtzle, S., et al. Rapid and Sensitive Kinetic Assay forCharacterization of omega-transaminases.Anal. Chem. 81,82448248 (2009).

5. Shin J.-S. and Kim B.-G. (1999). Asymmetric synthesis ofchiral amines with omega-Transaminase.Biotechnol.Bioeng.65, 206211.

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


11/1229-0211-99 AA 11

Ordering information

Product Code number

KTA 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

KTAmicro 28-9483-03Deep 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) 17-5087-01NHS-activated Sepharose 4 Fast Flow (25 ml) 17-0906-01

IEF Sample application pieces 18-1129-46

ImageQuant TL 28-9380-94

RPC 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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