All chemicals were of analytical grade purity and obtained from Merck KGaA (Darmstadt, Germany), ABCR (Karlsruhe, Germany), Roth (Karlsruhe, Germany), VWR (Hannover, Germany), and Bachem (Bubendorf, Switzerland). Phage-resistant Escherichia coli BL21 (genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS [λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5) was obtained from New England Biolabs (Ipswich, USA). The plasmids coding for TA-3FCR, TA-3HMU, and TA-3N5M were available in our lab collection (Steffen-Munsberg et al. 2013a, 2013b, 2016). The chaperone plasmid kit was purchased from TaKaRa Bio Inc., Shiga (Japan).
Functional expression of proteins
The codon-optimized genes (see SI) encoding the putative transaminases TA-1 to TA-10 of this study were ordered from Gen9 and obtained in the cloning vector pG9m-2 (Cambridge, USA) (see Table 1 for the GenBank accession numbers). The genes were subcloned by digestion with NdeI and BamHI and ligated into the pET22b or pET28b plasmids, which were digested with the same enzymes. After verifying the final constructs by sequencing, transformed E. coli BL21 cells were grown in TB-medium supplemented with 50 µg mL−1 kanamycin (pET28b) or 100 µg mL−1 ampicillin (pET22b) and induced at an optical density (OD600) ≈ 1.0 with 1 mM isopropyl-d-thiogalactopyranoside (IPTG). The protein expression was optimized by varying inducer concentration, temperature, OD at induction and expression time, and by coexpressing chaperones from the TaKaRa chaperone plasmid kit. E. coli cultures with plasmids coding for TA-1, TA-5, and TA-10 were grown in 200 mL TB medium at 37 °C and 160 rpm until the OD600 reached 1.0–1.4. Afterwards, protein expression was induced with IPTG and the cultures were shaken at 20 °C for 16–20 h and then centrifuged for harvesting the cells. The resuspended pellets were disrupted by ultra-sonication (two cycles 30 s, 40% pulse, 50% intensity at 0 °C), the suspension was centrifuged (13,000 × g, 1 min, 4 °C), and the amount of soluble and insoluble protein was investigated by SDS-PAGE. The enzyme TA-9 was co-expressed with the chaperones groES-groEL, promoter Pzt-1, encoded on plasmid pG-KJE8. The chaperone gene expression was induced with 10 ng mL−1 tetracycline 1 h before adding the inducer IPTG.
The cell pellet was washed with 40 mL of lysis buffer (Buffer A, sodium phosphate 50 mM, pH 8.0) and then resuspended in 20 mL of loading buffer (Buffer B, sodium phosphate 50 mM, pH 8.0, containing 0.3 M NaCl, and 0.1 mM PLP). After disruption by sonication at 0 °C for 10 min, the suspension was centrifuged at (8500 × g, 1 h) and the supernatant was passed through a 0.45 µm filter prior to chromatography. Chromatography was performed using an Äkta Purifier. As the recombinant proteins contained a His6-tag, a 5 mL Ni–NTA (nickel nitrilotriacetic acid) column (GE Health care) was used for purification. After washing the column with 60 mL of binding buffer (Buffer C, sodium phosphate 50 mM, pH 8.0; containing 0.3 M NaCl, 0.1 mM PLP, and 0.03 M imidazole) the crude extract was loaded. The enzymes were eluted by elution buffer (Buffer D, sodium phosphate 50 mM, pH 8.0; containing 0.3 M NaCl, 0.3 M imidazole, and 0.1 mM PLP; 5 mL min−1 flow rate) and the fractions containing the desired protein were collected. For desalting, size exclusion chromatography with 5 mL Sephadex desalting columns (GE Healthcare) using Buffer A was performed. The enzyme solutions were stored at 4 °C and the protein concentrations were determined by absorption at 285 nm using the Nano Quant method.
Molecular weight determination
Analysis of protein samples was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) employing a 4.5% stacking gel and a 12.5% separating gel. Samples were mixed with a twofold stock of SDS sample buffer (100 mM Tris HCl pH 6.8, SDS 4% (w/v), glycerol 20% (v/v), β-mercaptoethanol 2% (v/v), 25 mM EDTA, bromophenol blue 0.04% (w/v)) and were denatured by incubation at 95 °C for 10 min. Unstained protein molecular weight marker (Thermo Scientific, Waltham, MA, USA) was used as reference. Protein staining was done with Coomassie Brilliant Blue. Nondenaturing (native) gel electrophoresis was run in the absence of SDS to analyze the oligomerization state of the enzymes. Approximately 0.025 mg mL−1 of purified proteins were diluted 1:1 with twofold stock of sample loading buffer (glycerol 20% (v/v), bromophenol blue 0.0025% (w/v), dissolved in water) and loaded on the 4.5% stacking gel (5 mL of 0.125 M Tris HCl, 1.5 mL of 30% (w/v) acrylamide/bisacrylamide, 3.41 mL of dH2O, 100 µL of ammonium persulfate 10% (w/v), 10 µL of TEMED (tetramethylethylenediamine)). The 7.5% separating gel consisted of 5 mL of 0.75 M Tris HCl, 2.5 mL of 30% (w/v) acrylamide/bisacrylamide, 2.5 mL of dH2O, 100 µL of ammonium persulfate 10% (w/v), and 10 µL of TEMED. The running buffer consisted of 0.05 M Tris, 0.038 M glycine, pH 8.3. For comparison, the following protein standards were applied: TA-3HMU and TA-3FCR (both 98 kDa as dimers), catalase (232 kDa as tetramer), and TA-3N5M (212 kDa as tetramer).
Size exclusion chromatography was performed to analyze the size and oligomerization of the proteins using a HiPrep 16/60 Sephacryl S-100 HR (GE health care) column mounted in an Äkta Purifier using Buffer A (see Fig. S4 for chromatograms and calibration details).
Enzyme activity assays
The photometric acetophenone assay was used to determine the pH and temperature profile and the amino acceptor substrate spectrum (Schätzle et al. 2009). The assay solution contained 2.5 mM (S)-1-phenylethylamine ((S)-PEA), 1 mM pyruvate (or other amino acceptors for determining the amino acceptor spectrum), 2.5% (v/v) DMSO, and 50 mM HEPES pH 8.0. The reaction was started by adding enzyme (1 µg–1 mg) at 30 °C in UV-transparent 96-well plates (Greiner) in a final volume of 200 µL. The acetophenone formed was quantified at 245 nm for 10 min (interval of 30 s). Three control reactions were included where enzyme, amine donor, or amino acceptor was replaced by buffer. Measurements were done in triplicates with enzymes from two independent batch purifications. Activities were calculated by using the formula enzyme activity (U/mL) = (slope*60)/5.23). The slope from the assay reaction expressed in ΔAbs/min was corrected by substracting the blank of the control without amine substrate. One unit of activity is defined as the formation of 1 µmol acetophenone per minute. For determining the pH profiles, measurements were performed employing 0.1 M Davies buffer (Davies 1959) at pH values between pH 5 and 12.
The alanine dehydrogenase (AlaDH) assay was performed to determine the amino donor spectrum as described earlier (Steffen-Munsberg et al. 2016). The reaction was carried out in HEPES (50 mM pH 8.0) and contained 2.5 mM amino donor (an amine or amino acid), 1 mM pyruvate, 2.5% (v/v) DMSO, 0.3 mg/mL recombinant AlaDH from Thermus thermophilus (Steffen-Munsberg et al. 2016; Vali et al. 1980), 5 µM methoxy-PMS, 1 mM NAD+, 0.3 mM XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) salt. The reaction was started by adding the putative transaminase enzyme at 30 °C. Two control reactions were included where AlaDH or the putative transaminase protein were substituted by buffer. Measurements were done in triplicates using transaminase proteins from two independent batch purifications. Activities were calculated by using the formula enzyme activity (U/mL) = (slope*60)/12.64). The slope from the assay reaction expressed in ΔAbs/min was corrected by substracting the absorbance of the control without transaminase. One unit of activity is defined as the formation of 1 µmol of the formazane dye per minute. With slight modifications, this assay was also used with α-ketoglutarate (1 mM) as the co-substrate, as a replacement for pyruvate. The generated L-glutamate is than oxidatively deaminated by glutamate dehydrogenase (type I, ammonium sulfate precepitation from bovine liver, 0.3 mg/mL), yielding the same color reaction (referred to as glutamate dehydrogenase assay).
The thermal stability, also referred to as “resting stability” or “storage stability” of the enzymes was investigated by incubating the purified enzyme at 40 °C or temperatures between 60 and 80 °C for a defined time in sodium phosphate buffer containing varying PLP concentrations. Samples of the enzyme solution were collected at respective time intervals, cooled down to 30 °C, and the activity was measured by acetophenone assay as described above at 30 °C.
For assaying the stability under operating conditions similar to a biocatalysis reaction (operating stability), the proteins were incubated with 200 mM β-alanine as amino donor, 20 mM cyclohexanone as the amino acceptor, 2.5% (v/v) DMSO, and 50 mM HEPES buffer (pH 8.0) containing varying PLP concentrations at temperatures between 30 and 80 °C). Before incubation at elevated temperatures, the initial activities at t0 were determined at 30 °C. While incubating the enzyme at different temperatures, samples were collected at different time intervals and enzyme activities were measured by acetophenone assay. The assay solution contained 2.5 mM (S)-PEA, 1 mM pyruvate and 2.5% (v/v) DMSO, 50 mM HEPES pH 8.0. The measurement was started by adding the enzyme sample and followed for 10 min with a 30 s kinetic cycle at 30 °C. The ratios of volumetric activities after and before heat treatment were than calculated to obtain the relative activities of the enzymes at operating conditions.
The Tm (melting temperatures) of the proteins were determined by measuring the intrinsic fluorescence signal changes of proteins during temperature-dependent unfolding employing the Nano temper device (Prometheus). The fluorescent signal is plotted against the temperature (20–95 °C). The purified proteins of concentrations between 0.1 and 1.0 mg mL−1 (in 50 mM HEPES buffer, pH 8.0) were loaded to the Prometheus capillaries (Prometheus™ NT.48), and the temperature was increased by 0.5 °C per minute. The Tm of all the proteins was measured at different concentrations of PLP (0.01, 0.1, and 1.0 mM). The enzyme samples were prepared by adding PLP stock solution and incubating them at 25 °C for 1 h prior to the measurement. In the same way, the Tm was also measured under operating conditions after incubating with different PLP concentrations (0.01, 0.1, 1.0 mM), 200 mM L-alanine as amino donor, 20 mM cyclohexanone as amino acceptor, 2.5% (v/v) DMSO, and 50 mM HEPES buffer 1 h prior to the measurement.
For optimizing the pH for TA-10, reactions were performed at a 1 mL scale using 2 mL Eppendorf tubes at different pH values (HEPES; 6.0–8.0 Bicine; 8.0–9.5) at 30 °C and 800 rpm. To find the optimal isopropylamine (IPA) and ketone concentrations, catalytic reactions were carried out with IPA (0.05–2 M) and 4-phenyl-2-butanone (10–500 mM) at 30 °C, pH 7.5, and 800 rpm shaking. After 24 h and 48 h, 100 µL reaction samples were collected and quenched by adding 10 µL of 10 M NaOH, and extracted with 300 µL of dichloromethane (DCM). The organic layers were dried using anhydrous MgSO4 and taken for GC analysis using the Hydrodex-β-TBDAc column (Machrey and Nagel). For the analysis, the following conditions were used: initial temperature 100 °C, kept for 5 min, temperature raise: 10 °C/min, target temperature 220 °C, kept for 10 min, column flow 1.65 mL·min−1. As TA-10 was found to be a thermostable enzyme, asymmetric synthesis was carried out at 60 °C, pH 7.5, and 800 rpm. The reaction mixture contained the optimal concentrations of 0.75 M IPA and 30 mM 4-phenyl-2-butanone and 1.0 mg/mL TA-10. Samples were collected over the 70 h reaction and were processed and subjected to GC analysis as described above.