Mild hydrolysis of chemically stable valerolactams by a biocatalytic ATP-dependent system fueled by metaphosphate

Medium-sized 5- and 6-membered ring lactams are molecules with remarkable stability, in contrast to smaller β-lactams. As monomers, they grant access to nylon-4 and nylon-5, which are alternative polyamides to widespread caprolactam-based nylon-6. Chemical hydrolysis of monocyclic γ- and δ-lactams to the corresponding amino acids requires harsh reaction conditions and up to now, no mild (enzymatic) protocol has been reported. Herein, the biocatalytic potential of a pair of heterologously expressed bacterial ATP-dependent oxoprolinases – OplA and OplB – was exploited. Strong activity in the presence of excess of ATP was monitored on δ-valerolactam and derivatives thereof, while trace activity was detected on γ-butyrolactam. An ATP recycling system based on cheap Graham's salt (sodium metaphosphate) and a polyphosphate kinase allowed the use of catalytic amounts of ATP, leading to up to full conversion of 10 mM δ-valerolactam at 30 °C in aqueous medium. Further improvements were obtained by co-expressing OplA and OplB using the pETDuet1 vector, a strategy which enhanced the soluble expression yield and the protein stability. Finally, a range of phosphodonors was investigated in place of ATP. With acetyl phosphate and carbamoyl phosphate, turnover numbers up to 176 were reached, providing hints on a possible mechanism, which was studied by 31P-NMR.

Electronic Supplementary Material (ESI) for Green Chemistry.This journal is © The Royal Society of Chemistry 2023

Individual cloning of OplA and OplB
The synthetic gene coding for 5-oxoprolinase A (OplA) from Pseudomonas putida KT2440 with Uniprot accession number Q88H50 flanked with NdeI at N-terminus and HindIII at C-terminus restriction sites and codon optimized for expression in E. coli and the gene coding for 5-oxoprolinase B (OplB) from Pseudomonas putida KT2440 with Uniprot accession number Q88H51 flanked with NdeI at N-terminus and HindIII at C-terminus restriction sites and codon optimized for expression in E. coli were ordered and cloned in pET28a(+) using standard molecular biology protocols.Transformation in competent cells E. coli NEB5α was followed by transformation in E. coli BL21(DE3).

Expression at 30 °C after induction with IPTG
Overnight cultures were prepared form the glycerol stocks in 250 mL baffled autoclaved shaking flasks.100 mL LB medium were mixed with 100 μL of 50 mg/mL kanamycin stock solution (50 μg/mL final concentration), and 300 μL of glycerol stock of PpOplA or PpOplB.The flasks were incubated at 37 °C and 120 rpm overnight.For growing the cells, 2 L baffled shaking flasks were used.Each flask was filled with 500 mL of autoclaved LB medium, 500 μL of 50 mg/mL kanamycin stock solution (50 μg/mL final concentration) and 5 mL of overnight culture.Incubation was done at 37 °C and 120 rpm.At an OD600 of 0.6, 500 μL of 1 M IPTG stock (1 mM final concentration) was added to each flask and incubation was done at 30 °C and 120 rpm overnight.The next day, the cells were harvested by centrifugation (8000 rpm, 20 min, 4 °C) and washed two times with dH2O.
For determination of over-expression level, the pellets were resuspended in lysis buffer (50 mM sodium phosphate buffer containing 300 mM NaCl, 8% glycerol, 10 mM imidazole, pH 7.5, 10 mL buffer per g of pellet).Then the cells were disrupted by ultrasonicating the suspension on ice [(30% amplitude, 2 sec pulse on, 4 sec pulse off for 5:30 min) x 2].Afterwards the centrifugation was done (18000 rpm, 20 min, 4 °C) to remove cell debris.The Bradford assay was used to measure the protein concentration of each sample.The expression level of PpOplA and PpOplB was analyzed from the supernatant and the pellet samples by SDS-PAGE (10% SDS-gels, using MOPS as running buffer, 15 μg protein loading on each cavity, Figure S1).The molecular weight of the PpOplA is about 75 kDa and PpOplB is about 63 kDa.

Expression by autoinduction
PpOplA and PpOplB were expressed using autoinduction medium.The following solutions were prepared: 20x NPS containing 900 mL dd H2O, 66 g (NH4)2SO4, 136 g KH2PO4 and 142 g Na2HPO4 and 50x 5052 containing 250 g glycerol, 700 mL dd H2O, 25 g glucose and 100 g α-lactose monohydrate.Mixing was done in the given sequence and after stirring and heating for dissolving the components, the volume was set to 1 L. Afterwards, 20x NPS solution was autoclaved and 50x 5052 solution was filter sterilized using 0.22 μm syringe filters under laminar flow.Solutions of 40% glucose, 1 M MgSO4 and 1000x trace metal mixture were prepared and filter sterilized using 0.  In case of resuspension of the cell pellet in Tris-HCl (100 mM, pH 7.5 containing 4 mM MgCl2), low recovery of soluble OplA was observed: After 48 h, the cells were harvested by centrifugation (8000 rpm, 20 min, 4 °C) and washed with Tris-HCl (100 mM, pH 7.5 containing 4 mM MgCl2).200 mg of the pellets were taken up into 1.5 mL micro reaction tubes and resuspended in 800 μL Tris-HCl (100 mM, pH 7.5 containing 4 mM MgCl2).Then the cells were disrupted by ultrasonicating the suspension on ice (30% amplitude, 2 sec pulse on, 4 sec pulse off for 5:30 min).Afterwards the centrifugation was done with bench top centrifuge at room temperature (1460 rpm, 2 x 5 min) to remove the cell debris.Then SDS-PAGE from the supernatant and the pellet samples of PpOplA and PpOplB was done (Figure S3).

Purification of individually expressed PpOplA and PpOplB
For the purification, all the buffers were filtered using 0.2 μm filter papers and degassed before using.
The enzymes were purified separately using 5 mL His-Trap TM FF columns.The column was washed first with 50 mL ddH2O then with 50 mL binding buffer (ammonium bicarbonate, 50 mM, pH 8.5, containing 300 mM NaCl, 50 mM imidazole and 8% glycerol).Afterwards the sample was loaded onto the column and washed again with 50 mL binding buffer to remove protein impurities.The elution of the enzyme was done using 10 to 15 mL of elution buffer (ammonium bicarbonate, 50 mM, pH 8.5, containing 300 mM NaCl, 250 mM imidazole and 8% glycerol).The Bradford assay was done from all the collected fractions from PpOplA and PpOplB purification and SDS-PAGE was run from these samples (Figure S4).The eluted fractions were concentrated using VIVASPIN tubes (MWCO 10 kDa, 4,688 x g at 4 °C).After concentration, the samples were desalted by using PD-10 desalting columns and eluted with ammonium bicarbonate buffer (50 mM, pH 8.5, containing 4 mM MgCl2).The purified enzymes were directly used in the biotransformation reactions and the leftovers were stored at 4 °C.

Cloning
For co-expression of PpOplA and PpOplB, the pETDuet-1 vector was used.Both genes were codonoptimized for expression in E. coli.

Overexpression and preparation of cell-free extracts in ammonium bicarbonate
E. coli BL21 (DE3) cells were used and the expression plasmid pETDuet-1_PpOplAB (pEG 732) was introduced via heat shock transformation.An overnight culture with 10 mL of LB medium (100 μg/mL ampicillin) was inoculated with a single colony and incubated at 37 °C and 120 rpm.500 mL TB medium (100 μg/mL ampicillin) in a 2 L baffled flask were inoculated with 5 mL overnight culture and incubated at 24 °C and 120 rpm for 24 h.The autoinducing properties of TB medium resulted in overexpression of PpOplAB.Cultures were harvested by centrifugation (20 min, 12000 x g, 4 °C), resuspended in ammonium bicarbonate buffer (50 mM, pH 8.5, 10 mM MgCl2) and again centrifuged (45 min, 3166 × g, 4 °C).The resulting pellets were resuspended in 6 mL ammonium bicarbonate buffer (50 mM, pH 8.5, 10 mM MgCl2) per g cell mass, the cells were disrupted by sonication (Branson Ultrasonics™ Sonifier™ SFX250, 30% amplitude, 1 sec pulse on, 4 sec pulse off, total on 2:30 min, two times), and cell debris were removed by centrifugation (30 min, 18000 rpm, 4 °C) to deliver CFEs.

Purification of PpOplAB
Protein purification by Ni-NTA affinity chromatography was performed using an ÄKTA chromatography system and a HisTrap HP 5 mL column with ammonium bicarbonate buffer (50 mM, pH 8.5, 10 mM MgCl2, buffer A) and ammonium bicarbonate buffer (50 mM, pH 8.5, 10 mM MgCl2, 300 mM imidazole, buffer B).The column was equilibrated with 8% buffer B (5 CV) and the lysate was applied.After washing with 8% buffer B (4 CV), bound PpOplAB was eluted with 100% buffer B (4 CV).The enzymecontaining fractions were pooled and concentrated before being desalted on a PD-10 column.PpOplAB was obtained in ammonium bicarbonate buffer (50 mM, pH 8.5, 10 mM MgCl2) and protein concentration was determined by Bradford assay.Cell lysis and purification process were analyzed via SDS-polyacrylamide gel electrophoresis (main text, Figure 1).

Densitometric analysis for protein content in CFE
The software ImageJ was used to compare the pixel density of the total protein lane (15 µg applied on SDS gel) with the spots of the target enzymes.A photograph of the SDS gel was opened with ImageJ and the image was first changed to black and white (Image→Type→32-bit) and then inverted (Edit→Invert Image), which reduces the picture to bright and dark elements.The whole lanes were selected and their integrated density values were measured (Table S1).The process was repeated for the bands corresponding to SmPPK2, PpOplA and PpOplB.PpOplA: 3.847 µM (6.6% w/w); PpOplB: 2.927 µM (5.0% w/w), corresponding to a molar ratio of 1.1:1.

Biocatalytic reactions using CFEs of PpOplA and PpOplB expressed individually
The pellets of PpOplA and PpOplB were separately resuspended in lysis buffer (ammonium bicarbonate, 50 mM, pH 8.5 containing 4 mM MgCl2), disrupted by sonication with a Branson Sonifier® using following settings: [(30% amplitude, 1 sec pulse on, 4 sec pulse off for 2:30 min) x 2].Afterwards the centrifugation was done (17000 rpm, 30 min, 4 °C) to remove cell debris.Then the protein concentration (PpOplA and PpOplB) was measured using Bradford assay.Reactions were performed in 2 mL micro reaction tubes and the samples were shaken horizontally for 24 h at 30 °C and 140 rpm.Two blank reactions were run in parallel in the absence of (i) enzyme and (ii) substrate.After this time, the mixture was filtered via centrifugation and the supernatant was split into two 2 mL micro reaction tubes (each 450 μL).One sample was lyophilized to be used for amino acid derivatization (see section 2.3.1) and further measurements, and the other tube was used for lactam extraction.

Biocatalytic reactions using lyophilized cells of PpOplA & PpOplB expressed individually
The pellets obtained by autoinduction of PpOplA and PpOplB were lyophilized and tested in the hydrolysis of 1a-c.Ammonium bicarbonate buffer (50 mM, pH 8.5) containing 4 mM MgCl2 was used for the rehydration of the pellets.The reaction took place in ammonium bicarbonate (50 mM, pH 8.5) containing the lyophilized cells (25 mg/mL for OplA and 55 mg/mL for OplB in 1 mL reaction volume), ATP (5 mM and 12.5 mM final concentration), MgCl2 (4 mM), the substrate (2 mM and 5 mM final concentration).The reaction mixtures and blanks were shaken 48 h (120 rpm, 30 °C, horizontally).Results showed that with using pellets, lower conversions compared to CFE (data not shown).It is worth to mention that when the same pellets in combination with potassium phosphate buffer (50 mM, pH 7.5) as the reaction buffer were used, no conversion was observed in any case (data not shown).

Biotransformations with CFEs of co-expressed PpOplAB
Biotransformations were performed on an analytical scale (500 µL if not stated otherwise) in 1.5 mL micro reaction tubes that were incubated in an Eppendorf Thermomixer at 900 rpm.All components were combined, as indicated in the table footnotes.25 µL reaction volume were used for analysis by LC-MS (see section 9.2 for details).

Synthesis of 2d-g as hydrochloride salts
100 mg of 1d-g were dissolved under stirring in 1 mL concentrated HCl in a microwave reaction tube (13 mL).The tube was sealed and the mixture heated at 80 °C for 5 days.After completion of the reaction (no lactam present, as monitored by LC-MS), the reaction mixture was cooled down to room temperature and concentrated using a speed vac and dried at 80 °C in the oven overnight.The obtained salts or oily substances still contained water.The water content was determined by 1 H-NMR by using acetic acid as internal standard (2 µL, 2 mg, 33.3 µmol, added to 700 µL sample in D2O, Table S3).The purity of the samples was taken into account for the preparation of the calibration curves.

Derivatization
The biotransformation samples were filtered and dried by lyophilization in 2 mL micro reaction tubes.After lyophilization, the resulting residue was taken up in 700 μL MeOH containing 5% DMAP, and 150 μL ethyl chloroformate was added.Then samples were incubated at 50 °C (horizontal shaking, closed vials, 650 rpm).After 0.5 h, 75 μL ethyl chloroformate was added to each tube and the tubes were shaken for another 0.5 h (vertical shaking, open vials, 550 rpm).Afterwards, the solvent was removed under air flow and 700 μL of 2% aq.HCl solution was added.The extraction was done with ethyl acetate (4 x 400 μL) containing 5 mM behenic acid methyl ester as internal standard.The combined organic phases were dried using anhydrous Na2SO4 and then air flow till complete dryness.Then the residues were redissolved in 150 μL ethyl acetate and transferred to plastic GC-vials and measured by GC-FID and GC-MS.GC-MS: 7890A GC System (Agilent Technologies, Santa Clara, CA, USA), equipped with a 5975C mass selective detector and an HP-5MS column (5% phenylmethylsiloxane, 30 m x 320 μm x 0.25 μm, J&W Scientific, Agilent Technologies) using He as carrier gas.Injector temperature: 250 °C; Injection volume: 5 μL; Split ratio: 90:1; Flow rate: 0.7 mL/min; Temperature program 1: 100 °C, hold time 0.5 min, 10 °C/min to 300 °C, hold time 0 min; EI mode, energy 70 eV, MS Source: 230 °C, MS Quadrupole: 150 °C.The retention times of different compounds are given in Table S4.S5.

Calibration curves for GC-FID
To create the calibration curves, various samples with different concentrations (0.5 mM-10 mM) from 100 mM stock solutions of reference compounds were prepared.For example, for a sample with 2 mM concentration, 20 μL from stock was added into 980 μL potassium phosphate buffer (50 mM, pH 7.5).Each sample was split into two 2 mL micro reaction tubes (each 450 μL).The content of one of the tubes was lyophilized to be used for amino acid derivatization (see 9.1.1.),and the other tube was used for lactam extraction.For this purpose, the samples were first saturated with NaCl then extracted with acetonitrile (4 x 400 μL) containing 2 mM behenic acid methyl ester as internal standard.The combined organic phases were dried using anhydrous Na2SO4 and then air flow till complete dryness.Then the residues were redissolved in 150 μL ethyl acetate and transferred to the GC-vials and measured by GC-FID and GC-MS.      1, entries 2-3) in the presence of 2 mM (blue) and 5 mM (red) ATP.Only 2b can be seen with that method (8.7 min).

Analysis of enantioselectivity
The synthesis of the reference amino acid rac-2e was performed as indicated section 8, p. 10.After 8 days, the reaction was cooled down, and then the pH was brought to 6-7 using NaOH before the sample using a speedvac (vacuum concentrator).

Derivatization of 2e
The reference amino acid rac-2e was derivatized to rac-5-(((benzyloxy)carbonyl)amino)-3methylpentanoic acid for analysis on chiral phase HPLC.The synthesis was adapted from the literature. 30 mg of N-(Benzyloxycarbonyloxy)succinimide (0.89 mmol, 1 equivalent) was dissolved in 4.47 mL of THF with 10 vol% water (447 µL), employing magnetic stirring.Subsequently, triethylamine (Et3N) (187.2 µL, 1.34 mmol, 1.5 equivalents) was added, and the reaction mixture was kept at 0 °C with an ice bath for 10 minutes.Following this, 223 mg (0.89 mmol, 1 equivalent) of 2e as ammonium chloride salt was introduced in one portion, and after an additional 10 minutes of stirring, the ice bath was removed, allowing the reaction to reach room temperature.Confirmation of reaction completion was made after 2 hours by HPLC-MS analysis using the method of an isocratic gradient 80:20 ACN (+0.1% formic acid) and water (+0.1% formic acid) at a ratio of 80:20 for 8 minutes.The reaction mixture was acidified with 2 vol% HCl until the pH reached below 3, followed by four extractions with 15 mL of ethyl acetate.The collected organic phases were then dried using sodium sulfate (Na2SO4), and the solvent was evaporated under reduced pressure, resulting in 510.9 mg of crude product.The reaction crude was purified by solubilization in 2 mL of acetonitrile and subsequent injection into the preparative HPLC system.The system employed a gradient elution procedure featuring a Phenomenex Luna® 5 µm C18(2) 100 Å column sized at 21.2 mm x 250 mm (00G-4252-P0-AX), with a flow rate of 30 mL/min.The purification method followed these conditions: Initially, a gradient of 60:40 water (with 0.1% TFA) to ACN (with 0.1% TFA) was applied for 2 minutes.Over 35 minutes, the gradient transitioned to a composition of 40:60 water (with 0.1% TFA) to ACN (with 0.1% TFA).Subsequently, after 40 minutes, the composition was adjusted to 100% ACN and kept for 5 minutes.The collected fractions were dried under reduced pressure and subjected to analysis, isolating the desired product as a white solid weighing 146.7 mg (62.2 % yield).

Analysis of the biotransformations
The biotransformation of 1e was performed as indicated in the substrate screening, Table 3 (only difference: 8 mg/mL PpOplAB CFE).After incubation of the reaction, the samples were basified by adding 50 µL of a 10 M NaOH solution in water.The pH was checked, and an additional 50 µL base was added as needed to ensure the pH was above 10.
For the analysis of 1e, the substrate was extracted from the biotransformation mixture using ethyl acetate: 200 µL of EtOAc was added to the samples, followed by centrifugation at maximum speed for 10 minutes.From the upper organic phase, 150 µL was carefully extracted.This extraction process was repeated three times, but in the last repetition, only 100 µL of EtOAc was added.The combined organic phases were then dried with sodium sulfate (Na2SO4).Finally, 150 µL of the extracted material was transferred into a plastic GC vial and analyzed.The determination of the enantiomeric excess of 1e was conducted as reported in the literature 4 using Gas Chromatography with Flame Ionization Detection (GC-FID) and the following conditions: HYDRODEX β-6TBDM column, 50 m, 0.25 mm ID, carrier gas: H2, flow rate 1 mL/min, injection volume 2 µL with a Split/Splitless ratio of 30:1, injection temperature 230°C; oven temperature 50°C, heating rate: 5°C/min to 80 °C, then 0.   For the analysis of 2e, to the remaining aqueous phase, 150 µL of a 150 mM stock solution N-(benzyloxycarbonyloxy)succinimide in acetonitrile (ACN) was added. 5The reaction was then allowed to react for 1.5 h in a Benchtop thermomixer at 50 °C and 600 rpm.Following the reaction, the Eppendorf tubes were allowed to cool down to room temperature.Subsequently, 700 µL of a 4 M HCl solution in water was added, and the pH was checked and adjusted to be less than 3 by adding more acid if necessary.The mixture was brought to dryness using a SpeedVac.The whitish solid, with a few brownish residues, was resuspended in 200 µL of ACN.The resuspension was vortexed, and sometimes, a pipette tip was used to ensure complete dissolution.The mixture was then incubated at 50 °C and 600 rpm for 10 min.The mixture was centrifuged at maximum speed for 10 min.After centrifugation, 150 µL of the supernatant was transferred into a plastic GC vial.The presence of the desired peak (264 m/z using the negative ionization method) was confirmed using High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) analysis with the Luna(R) 5µm C18(2) 100 Å column (250 x 4.6 mm).The analysis was conducted using an isocratic elution method with a ratio of 80:20 ACN (+ 0.1% Formic Acid) to water (+ 0.1% Formic Acid) for 8 minutes.The analysis was carried out at a constant temperature of 30 °C, with a flow rate set at 0.6 mL/min, and samples were injected using a 5 µL volume.Comparison with elution of the authentic reference material confirmed that derivatized 2e was present.The enantiomeric excess analysis of the product was conducted using a Shimadzu HPLC-DAD (High-Performance Liquid Chromatography with Diode Array Detection) employing a Chiralpak column IA (from DAICEL) with dimensions of 4.6 mm ID and 250 mm L with a particle size of 5 µm (part no.80325).The analysis was performed in isocratic elution technique using a heptane:2-propanol ratio of 94:6.The analysis was conducted at a constant temperature of 30 °C, with a flow rate set at 1 mL/min, and samples were injected using a 10 µL volume.The two enantiomer peaks (analyzed using a wavelength of 210 nm), as obtained with the synthesized reference, eluted at 23.8 min and 25.5 min.Based on the consumption of (R)-1e, the major enantiomer was attributed to (R)-2e at 25.5 min.

NMR
NMR measurements were performed on a Bruker Avance NEO 500 MHz NMR equipped with a Double Resonance Broadband Probe (BBI).

31 P-NMR
Biotransformations to be analyzed by 31 P-NMR were prepared on 1 mL scale, analogously to the experiments for investigation of PpOplAB substrate scope.After 1 h, the reaction mixture was vortexed to denaturate the enzyme, which was pelleted by centrifugation (2 min).25 µL of the supernatant was used for LC-MS analysis to determine conversion and samples for NMR analysis were prepared by mixing 630 µL of the supernatant with 70 µL D2O.12. Safety concerns -Butyrolactam 1a and -valerolactam 1b are not innocuous compounds.Their safety datasheets indicate hazard pictograms GHS07 (exclamation mark: irritant, sensitising, harmful) and GHS08 (health hazard: long-term health hazard) for 1a and GHS07 (exclamation mark: irritant, sensitising, harmful) for 1b.
22 μm syringe filters under laminar flow.The 1000x trace metal mixture was containing 50 mM FeCl3, 20 mM CaCl2, 10 mM MnCl2 and 10 mM ZnSO4, and 2 mM CoCl2, 2 mM CuCl2, 2 mM NiCl2, 2 mM Na2MoO4, 2 mM Na2SeO3, and 2 mM H3BO3.The trace metal mix was assembled from autoclaved stock solutions of the individual components except for FeCl3, which was added from the 0.1 M solution in ~0.12 M HCl.For preparation of pre-culture, 4.635 mL of LB medium was mixed with 5 μL of 1 M MgSO4, 100 μL of 40% glucose, 250 μL of 20x NPS and 10 μL of 50 mg/mL kanamycin stock solution, then 2 mL of this mixture was transferred into a 15 mL falcon Sarstedt tube and 5 μL of glycerol stocks from PpOplA or PpOplB was added to this mixture then incubation was done at 37 °C and 120 rpm overnight.For the main culture, 926 mL of LB medium was mixed with 2 mL of 1 M MgSO4, 20 mL of 50x 5052, 50 mL of 20x NPS, 2 mL of 50 mg/mL kanamycin stock solution and 200 μL of trace metal solution.Then 450 mL of this mixture was transferred into a 2 L baffled flask and 900 μL from pre-culture of PpOplA or PpOplB was added.Incubation was done for 48 h, 17 °C and 120 rpm.After 48 h, the cells were harvested by centrifugation (8000 rpm, 20 min, 4 °C) and washed with Tris-HCl (100 mM, pH 7.5 containing 4 mM MgCl2).3.5 g of the fresh pellets were resuspended in ammonium bicarbonate buffer (50 mM, pH 8.5, containing 4 mM MgCl2, 10 mL buffer per g pellet).Then the cells were disrupted by ultrasonicating the suspension on ice [(30% amplitude, 1 sec pulse on, 4 sec pulse off for 2:30 min) x 2].After centrifugation (18000 rpm, 20 min, 4 °C), the cell free extracts (CFE) were obtained.The expression level of PpOplA and PpOplB was analyzed by SDS-PAGE (10% SDS-gels, using MOPS as running buffer, 15 μg protein loading on each cavity, FigureS2).

Figure S15 .
Figure S15.GC trace from the biotransformation of 2 mM 1b to 2b (Table1, entries 2-3) in the presence of 2 mM (blue) and 5 mM (red) ATP.Only 2b can be seen with that method (8.7 min).

Figure S38 .
Figure S38.Trace obtained from the biotransformation of rac-1e on chiral phase GC indicating enrichment of (S)-1e with 8% ee.

Figure S39 .
Figure S39.Overlay of trace obtained from derivatized rac-2e (green) and trace obtained from the biotransformation of rac-1e after derivatization (cyan) on chiral phase HPLC indicating formation of (R)-2e with 68% ee.

Figure S40 .
Figure S40.Spectrum from the biotransformation of 10 mM 1b by PpOplAB in the presence of 12.5 mM ATP.

Figure S41 .
Figure S41.Spectrum from a sample of PpOplAB in the presence of 12.5 mM ATP without substrate.

Figure S42. 1 H
Figure S42. 1 H NMR spectrum of 2d in D2O with acetic acid as internal standard.

Figure S43. 1 H
Figure S43. 1 H NMR spectrum of 2e in D2O with acetic acid as internal standard.

Figure S44. 1 H
Figure S44. 1 H NMR spectrum of 2f in D2O with acetic acid as internal standard

Table S1 .
Estimation of concentration of PpOplAB and SmPPK2 in CFEs used in the biotransformation of 50 mM 1b (see main text, Table6, entry 6 and Figure3) based on densitometry (integrated density) (see FigureS6).

Table S3 .
Mass of amino acid hydrochlorides added to NMR samples and calculated water content.

Table S4 .
The retention times (RT) of lactams and derivatized amino acids analyzed by GC-MS

Table S5 .
The retention times (RT) of lactams and derivatized amino acids analyzed by GC-FID

Table S6 .
MS settings used for lactam and amino acid analysis.

Table S7 .
m/z values [M+1] and retention time (RT) of lactams and corresponding amino acids.