A novel multi-enzymatic high throughput assay for transaminase activity

A novel multi-enzymatic high throughput assay for transaminase activity
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  A novel multi-enzymatic high throughput assay for transaminase activity Katrin Weinhandl a , Margit Winkler a , Anton Glieder a , Andrea Camattari b , * a  Austrian Centre of Industrial Biotechnology GmbH (ACIB), Petersgasse 14, 8010 Graz, Austria b Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria a r t i c l e i n f o  Article history: Received 30 March 2012Received in revised form 1 June 2012Accepted 14 June 2012Available online 21 June 2012 Keywords: BCAT ilvE  L  -Glutamate oxidase Pichia pastorisEscherichia coli a b s t r a c t Aminotransferases (ATs) are crucial enzymes for prokaryote and eukaryote metabolism, and have at-tracted industrial attention for large scale synthesis of amino acids and chiral amines.A high throughput screening procedure for branched chain aminotransferases (BCATs) was developed toidentify optimal expression on a large number of expression clones.  Escherichia coli  BCAT, encoded by the ilvE   gene, was expressed in  E. coli  and  Pichia pastoris . This simple colorimetric assay procedure allowedthe identi fi cation of optimal clones for  ilvE   expression and enabled the testing of its activity in cell lysatesor on whole cell catalysis.   2012 Elsevier Ltd. All rights reserved. 1. Introduction Aminotransferases (ATs; alternative name: transaminases TAs)are crucial enzymes for prokaryotic and eukaryotic organisms,being involved in both amino acid catabolism (transamination of amino acids represents the  fi rst step of the pathway leading thecarbon backbone to pyruvate) and anabolism (several amino acidsare synthesized by amino transfer from an amine donor to thecorresponding ketoacid, providing building blocks for proteinsynthesis). Transaminases have been objects of intensive study,given their potential application in chiral amine or amino acidsynthesis; although typically characterized by an unfavorableequilibrium constant, which hinders industrial large scale appli-cations of TAs, several examples of TA-based processes are docu-mented in literature. 1 e 4 In particular, branched chain aminotransferase (BCAT) from Escherichia coli , encoded by the  ilvE   gene, 5 has been extensivelycharacterized in terms of substrate and co-substrate preference,kinetic characteristics and possible applications. 6 Overexpressionin  E. coli  was reported in literature: 7 however, transaminase ex-pression has been scarcely tested with other expression hosts.Yeasts, and in particular the methylotrophic yeast  Pichia pastoris ,are increasingly applied in both laboratory and industrial practicefor their high biomass yield and consistent protein expression ca-pability, 8 fi nding applications as hosts, e.g., for  Vibrio  fl uvialis  u -transaminase expression. 9 Recently, the 3D structure of IlvEp was elucidated, 10 allowingrationalization of the reaction mechanism and enabling active siteengineering and rational mutant generation. In order to attemptlibrary screening for improved or modi fi ed IlvEp mutants, func-tional high throughput screening is a necessity; screening strate-gies for transaminase activity have been explored, 11 e 13 althoughexamples were given mostly for partly puri fi ed enzyme prepara-tions. In order to overcome possible interferences from complexmixtures (possibly present in a sample like a crude lysate), ideallythe screening method should be speci fi c and robust. Herein wereport a photometric assay, which couples aminotransferase ac-tivity to byproduct oxidation, and subsequent chromophore oxi-dation. Commercially available  L  -glutamate oxidase from Streptomyces  sp. was selected because of its high af  fi nity to  L  -glu-tamate, 14 the co-product formed after BCAT catalyzed trans-amination. In the course of   L  -glutamate oxidation, hydrogenperoxide is formed; in the presence of horseradish peroxidase,hydrogen peroxide promotes the oxidation of ABTS, a colorimetricsubstrate (Fig. 1). The present study illustrates the application of a novel transaminase assay for screening of BCAT activity in eithercell lysates or intact cells, which permitted the identi fi cation of optimal expression clones. 2. Results2.1. Assay development and validation A schematic representation of the assay is shown in Fig. 1.  L  -Leucine was chosen as model substrate to detect BCAT activity.  L  - *  Corresponding author. Tel.:  þ 43 316 873 4077; e-mail address: (A. Camattari). Contents lists available at SciVerse ScienceDirect Tetrahedron journal homepage: 0040-4020/$  e  see front matter    2012 Elsevier Ltd. All rights reserved. Tetrahedron 68 (2012) 7586 e 7590  Glutamate, formed during the transamination reaction with 2-oxoglutarate as amino group acceptor, is oxidized by  L  -glutamate oxi-dase with subsequent release of ammonia and hydrogen peroxide.The latter serves as the electron donor for HRP-based oxidation of ABTS. Given the strict dependence on H 2 O 2  for the reliability of theassay, two commerciallyavailable horseradish peroxidase mixtureswere tested for the H 2 O 2 -dependent reaction (Fig. 2). In absence of hydrogenperoxide,anHRPTypeIIpreparationshowedasigni fi cantbackground signal, and was therefore ruled out for furtherutilization.To prove the assay reproducibility and reliability, an in vitrovalidation was carried out. In the absence of either H 2 O 2 ,  L  -gluta-mate,  L  -glutamate oxidase or HRP (class VIa), no signal was detec-ted; on the contrary, using  L  -glutamate as substrate and withoutexternal addition of H 2 O 2 , absorbance of ABTS at 420 nm wasdetected in the presence of   L  -glutamate oxidase and horseradishperoxidase. The glutamate oxidase assay was tested by HPLC todetermine its detection limit. It was possible to measure the spe-ci fi c accumulation of   L  -glutamate in concentrations as little as25  m M; additionally, an acceptable linearity of the response be-tween the concentration of   L  -glutamate and the assay signal wasmeasured (Fig. 3). 2.2. Measuring IlvEp activity in  E. coli In order to benchmark the screening procedure with a crudelysate sample, the  ilvE   gene was cloned in an expression vector for E. coli . SDS-PAGE analysis of cell lysates obtained from cultures of   E.coli  BW25113 overexpressing  ilvE   under an IPTG-inducible tacpromoter showed a band, accumulating over time, consistent withIlvEp molecular weight (data not shown). The proposed  L  -gluta-mateoxidase assay was used to detect transaminase activity. In theabsence of any external addition of   L  -glutamate to the assay mix-ture, only an  E. coli  lysate preparation containing IlvEp showeda signi fi cantly higher signal compared to the background (Fig. 4). 2.3. Screening for IlvEp activity in  P. pastoris To achieve an optimal expression in a heterologous host like  P. pastoris ,  ilvE   was manuallycodon-optimized, cloned and expressed Fig. 1.  Schematic representation of the BCAT screening method based on  L  -glutamate oxidase. Fig. 2.  HRPs dependency for H 2 O 2 . Two different commercially available horseradishperoxidases were tested for H 2 O 2  dependence, incubating HRP VIa (black bars) or HRPII (white bars) in the presence or absence of hydrogen peroxide (H). Fig. 3.  HPLC assay validation. K. Weinhandl et al. / Tetrahedron 68 (2012) 7586  e 7590  7587  under the control of either a constitutive or a methanol-induciblepromoter.  P. pastoris  CBS7435 Mut S was transformed with approx-imately 2  m g of linearized plasmid. Such a conspicuous amount of DNA was used to increase the possibility of identifying clones car-rying multiple copies of   ilvE  , thus improving the chance of achievinghighlevelsofexpression.Singlecolonieswereinoculatedin deep well plates (96 well format) and screened with the abovedescribed assay after up to120 h of culturing (constitutive pro-moter) or by a methanol induction procedure (methanol-induciblepromoter, see Experimental section). After detergent treatment,  P. pastoris  cell lysates were screened for transaminase activity asdescribed. The screening results are depicted in Fig. 5; as shown, itwas possible to identify and select highly active clones for furthercharacterization.A particularly active clone (named D4) was selected after screen-ingofapproximately100clones.Copynumberdeterminationfortheintegrated  ilvE   expression cassette in clone D4 indicated 3 e 4  ilvE  copiesinthe P. pastoris genome,inaccordancewiththehighamountofDNAused.Thespeci fi cactivityofIlvEpfor L  -leucinetransamination,expressed in  E. coli  or  P. pastoris , was 10.36  0.39  m mol min  1 mgprotein  1 or 13.35  1.23 m mol min  1 mg protein  1 . Cell lysates fromboth expression hosts were tested for  L  -leucine conversion; lysatesfrom P.pastoris providedaslightlyhigherspeci fi cactivityafter72hof culture,signi fi cantlyhigherthanthe6hofinductionrequiredtoreachthe highest conversion activity for  E. coli , which re fl ects the differentgrowingandproductionbehaviorsofthesetwohosts.Tocon fi rmthepotential of the identi fi ed  P. pastoris ilvE  -expressing clone, bio-conversionofthesubstratecouple L  -leucineand2-oxoglutarateto4-methyl-2-oxo pentanoic acid and  L  -glutamate were performed witheither whole cells or cell lysates (Fig. 6A and B). As expected, uponnormalization on the biomass amount, whole cell transaminationprovided lower conversions compared to the lysate preparation; cellpermeabilization for the substrates/products represents a majorhindrancetoachievehighperformance.Ontheotherhand,wholecellcatalysis proved to be simpler in setting and handling, compared tothelysatepreparation.Thedecreaseinconversioncapacityovertimeof cultivation, evidently shown by the decreasing amount of   L  -gluta-mate measured (Fig. 6A) can be explained considering that cell in-corporation of amino acids, like the substrate  L  -leucine, typicallydecreases as long as the starvation phase proceeds. The relativelyconstant transamination activity for the corresponding lysates(Fig. 6B) is consistent with this hypothesis. 3. Conclusion The presented assay, based on the application and high speci- fi city of   L  -glutamate oxidase for  L  -glutamate, was developed andtested to detect transaminase activity. To validate the assay, thebacterial gene  ilvE  , encoding a branched chain aminotransferase(BCAT) was overexpressed in both  E. coli  and  P. pastoris , and its Fig. 4.  IlvEp-mediated absorbance signal in  E. coli  lysate. Transaminase activity of celllysates from  E. coli  BW25113 overexpressing  ilvE   (black bars) were compared withlysates from  E. coli  BW25113 carrying the empty plasmid (white bars). Fig. 5.  Screening of   ilvE   expression in  P. pastoris . Cell lysates from deep well platecultivation of   P. pastoris  CBS7435 Mut S expressing  ilvE   under the control of the AOX1promoter (black dots, 72 independent clones) or the constitutive GAP promoter (whitedots, 92 independent clones) have been tested for transaminase expression. Thedashed line represents the detected value for the parental strain. Fig. 6.  Bioconversion of   L  -leucine to 4-methyl-2-oxo pentanoic acid with  P. pastoris CBS7435-Mut S (clone D4) as whole cell transamination (black bars,  ilvE  -overexpressingD4clone;whitebars,CBS7435-Mut S parentalstrain)(A).Bioconversionof  L  -leucineto4-methyl-2-oxo pentanoic acid with  P. pastoris  CBS7435-Mut S (clone D4) with cell lysatescorresponding tothe same samples in Fig. 6B (black bars, ilvE  -overexpressingD4 clone;the signal for CBS7435-Mut S parental strain fell below the detection level) (B). K. Weinhandl et al. / Tetrahedron 68 (2012) 7586  e 7590 7588  capability for detecting speci fi c transaminase activity has beencon fi rmed. In particular, in the case of   P. pastoris  expression, char-acterized by a typical heterogeneity of protein expression amongdifferent clones, having the possibility to screen for a large numberof clones in a simple and effective manner is considerably useful. A P. pastoris  expression clone carrying approximately four copies of  ilvE   integrated in its genome was identi fi ed using the presentedassay, and its transamination capacity was tested in either cell ly-sates or as a whole cell catalyst. Even if further development foroptimizing the assay performance is expected, the presentedscreening method shows a signi fi cant potential for a fast assess-ment of transamination activity, with the option of testing at thesame time different amine acceptor. 4. Experimental section4.1. Synthetic genes and materials Thegenesequenceof   ilvE  wasin-houseoptimizedfor E. coli  or P. pastoris  codon usage, using a modi fi ed version of publicly availablecodon usage tables ( and synthe-sized by DNA 2.0 (Menlo Park, CA, USA); DNA 2.0 provided also theBW25113  E. coli  strain transformed with the  ilvE   expression vector.Oligonucleotides were purchased from IDT Integrated DNA Tech-nologies BVBA (Leuven, Belgium), DNA-modifying enzymes wereobtained from Fermentas GmbH (Burlington, Ontario, Canada) andInvitrogen Corp. (Carlsbad, CA, USA). Recombinant  L  -GlutamateOxidase from  Streptomyces  spp., horseradish peroxidase type II andVI-A, as well as all chemicals for the assay were purchased fromSigma Aldrich (St. Louis, MO, USA). 4.2. Cloning of   ilvE   in  P. pastoris  expression vectors  A P. pastoris  codon optimized  ilvE   gene was cloned in the in-house  E. coli e P. pastoris  shuttle vectors pPpT4_S andpPpT4_GAP_S; both pPpT4_S and pPpT4_GAP_S are similar topPpT2, 15 except two sequent point mutations in PEM72 promoter(see below).  Eco RI and  Not  I restriction sites were added, during thegene design process, at the 5 0 and 3 0 of   ilvE   coding sequence. TheDNA-fragments were digested with  Eco RI and  Not  I and ligated intothe vectors consisting of an srcin of replication of   E. coli  pBR322,a promoter region containing a  Smi I site (AOX in T4_S, GAP inT4_GAP_S) for linearization, multiple cloning site for  Eco RI,  Spe I,  Asc  Iand Not  Irestriction,AOX1transcriptiontermination signalandan antibiotic resistance marker cassette for Zeocin resistance(bacterial promoter EM72,  P. pastoris  ILV5 promoter and a codon-optimized zeocin resistance gene  ble  from  Streptoalloteichus hin-dustanus ). 16 E. coli  TOP10F ’  (Invitrogen Corp.) served as an in-termediate host for all cloning steps. P. pastoris  Mut S derived from the wild type strain CBS7435 (TUGraz strain collection number 3445) was transformed by electro-poration following a condensed protocol for preparation of elec-trocompetent cells. 17 Expression cassettes were linearized with Smi I, puri fi ed via QIAquick PCR Puri fi cation Kit (Qiagen, Germany)and 200 ng (or 2000 ng, respectively) of DNA were transformed inice-cold cuvettes (0.2 cm, Cell projects Ltd., Kent, UK) by pulsing at200  U , 25  m F and 1.5 kV. For the subsequent regeneration phase,1 mL of a special medium (50% YPD, 50% 1 M sorbitol) was addedimmediately and the cell suspension was transferred into a sterile12 mL polypropylene tube (Greiner, Frickenhausen, Germany) andincubated for 2 h at 30   C without agitation. Afterward, aliquots of 100 and 200  m L were plated to YPD-Zeocin Agar plates (100 mg/L Zeocin) and incubated at 28   C.Copy number determination for  ilvE   expression cassette in-tegrated in  P. pastoris  genome was performed as previouslyreported. 18 4.3.  ilvE   expression in  P. pastoris For an initial activity screening, transformants were cultivatedin 96-deep well plates over a period of 96 h in 600  m L BMD1 (1%glucose)incaseofGAPpromoterdriven expression.Toinducegeneexpression driven by the AOX1 promoter, transformants were ini-tially grown in 250  m L BMD1 for 60 h, then induced with 250  m L BMM2 and additionally with 50  m L BMM10 after 70, 90 and 110 htotal cultivation time. 19 Around 50 h after the  fi rst induction, cellswere harvested by centrifugation (Eppendorf 5810R 4000 rpm,4   C,10 min) and lysed by addition of 150 m LY-PER   Plus (ThermoScienti fi c, Rockford, USA) and incubated on a Heidolph Titramax1000 shaker (Heidolph Instruments, Schwabach, Germany) at1200 rpm for 45 min. Supernatants of a further centrifugation stepwere ready to use in the screening assay.The three best GAP promoter clones were chosen after rescre-ening in 250 mL shake  fl asks, containing 50 mL BMD1 media. Cellswere harvested after 72 h cultivation time by centrifugation(Eppendorf 5810R 4000 rpm, 4   C, 10 min) and lysed by a shortglassbeadextractionmethodmodi fi edfromgeneralcelldisruptionprotocols. 20 The pellet of 1 mL aliquot of the culture was resus-pended in 900  m L cell lysis buffer (50 mM potassium phosphatebuffer pH 7.9, 5% glycerol, 1 mM phenylmethanesulfonyl  fl uoride,1 mM EDTA, 2 mM dithiothreitol). Finally, glass beads(425 e 600  m m, 1/3 of the total volume) were added and the tubeswere vortexed at maximum speed on a Vortex-Genie  (adaptedwith a 12-tubes vortexing rack) for 30 s. Afterward, partially dis-rupted cells were incubated on ice for 30 s. This procedure wasrepeated 10 times until a  fi rst visible foam generation appeared,indicating cell lysis. Cell debris was removed by centrifugation(4000 rpm, 5 min, 4   C) and the cleared supernatant was used forthe assay.To induce  ilvE   expression in  E. coli , a single colony of   ilvE  -expressing benchmark clone was cultivated in 50 mL Luria Brothmedium and induced with 1 mM IPTG at OD 600 ¼ 1. Cells wereharvested after 6 h of growth and lysed byglass beads as describedabove. 4.4. Enzymatic assays IlvEp activity was determined in a microtiter plate format. Thereaction mix (100  m L) contained 10 mM amino acid ( L  -leucine),10 mM 2-oxo glutarate, 25  m M pyridoxal-5-phosphate (PLP),2.5 mM 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS), 10 mU/mL   L  -glutamate oxidase, 10  m g/mL horseradish per-oxidase (HRP) and 100 mM sodium phosphate buffer pH 7.5 toa fi nalvolumeof100 m L.Thereactionwasstartedbyadding20 m Lof the crude lysate and then incubated for 30 min at room tempera-ture. The samples were immediately measured at 420 nm witha Synergy-Mx Biotek plate reader (Biotek GmbH, Germany).Crude lysates generated from shake  fl asks were subjected to ki-netic measurements (30 min at 25   C), which started immediatelyafter adding the crude lysate. For calculation of the speci fi c activitya molar extinction coef  fi cient of 36,000 L mol  1 cm  1 was used. 21 The detection limit of the assay was determined by measuringdifferent  L  -glutamate concentrations (0.0125 e 5 mM) in the usualassay mix (2.5 mM ABTS, 25  m M PLP, 10 mU/mL   L  -glutamate oxi-dase, 10  m g/mL HRP, 100 mM sodium phosphate buffer pH 7.5).20  m L crude extract of the wild type (CBS7435 Mut S ) withouttransaminase activity was added to simulate the suspected back-ground of a crude lysate. 4.5. HPLC method and conditions For a veri fi cation of the coupled assay results on crude lysates,thesamesampleswerealsoanalyzedbyHPLC.Reactionscontained K. Weinhandl et al. / Tetrahedron 68 (2012) 7586  e 7590  7589  25 mM  L  -leucine, 25 mM 2-oxo glutarate, 50  m M pyridoxal-5-phosphate, 100 mM sodium phosphate buffer pH 7.5 and 20  m L of crudeextract.After30minofincubation,proteinswereprecipitatedbyheattreatment(96  Cfor10min)andremovedbycentrifugation.The supernatants were analyzed by HPLC using pre-column de-rivatization in an Agilent 1200 rapid Resolution HPLC system.Therefore, the samples were mixed with 0.1 M sodium tetraboratebuffer pH 10.5, 2-mercaptoethanol and OPA ( o -phthaldialdehyde)according to Davis et al. 22 A Lichrochart 250-4 Purospher Star RP-18e 5  m M column was used as stationary phase at a  fl ow rate of 0.7 mL/min (mobile phase: 60% KH 2 PO 4  20 mM, pH 8, and 40%acetonitrile). Signaldetectionwas at338nm,the retentiontimesof  L  -glutamate and  L  -leucine were at 3.14 and 6.75 min, respectively. 4.6. Whole cell transamination of   L  -leucine For whole cell conversion, an  ilvE   expressing  P. pastoris  CBS7435Mut S strain was cultivated in duplicate in 2 L shake  fl asks with200 mL BMD1 media. Aliquots of 1 mL culture were sampled after24, 32, 48, 72, 80 and 96 h of expression. After centrifugation(4000 rpm, 10 min, 4   C), cell pellets were washed once with PBSbuffer and then resuspended in 500  m L of a substrate solutioncontaining 25 mM  L  -leucine, 25 mM 2-oxo glutarate,0.025 mM PLPand 100 mM sodium phosphate buffer pH 7.5. After an incubationtime of 60 min at room temperature, cells were removed by cen-trifugation (4000 rpm, 10 min) and 50  m L of the supernatant wereadded to 50  m L enzyme/ABTS-solution (5 mM ABTS, 1  m g HRP,0.001 U  L  -glutamate oxidase) in a microtiter plate. Samples weremeasured immediately at 420 nm for 45 min.On the same microtiter plate a  L  -glutamate standard with con-centrations of 5, 2,1, 0.5, 0.2, 0.1, 0.05, 0.025, 0.02, and 0.0125 mMwasplacedtobeabletoestimateconversionef  fi ciency.Valueswerenormalized on dry cell weight.  Acknowledgements This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traf  fi c, Innovation and Technology (bmvit), the Styrian BusinessPromotion Agency SFG, the Standortagentur Tirol andZIT e Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research PromotionAgency FFG.The authors wish to thank Kateryna Lypetska (Institute of Or-ganic Chemistry, Graz University of Technology) for the invaluablehelp on the HPLC method development. References and notes 1. Chanatry, J. A.; Schafer, P. H.; Kim, M. S.; LeMaster, D. M.  Anal. Biochem.  1993 ,  213 , 147 e 151.2. Patel, R. N.  Biomol. 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