Immobilization and Stabilization of an Engineered Acyltransferase for the Continuous Biosynthesis of Simvastatin in Packed-Bed Reactors

1 Immobilization and Stabilization of an Engineered Acyltransferase for the Continuous Biosynthesis of Simvastatin in Packed-Bed Reactors Guillermo García-Marquinaa,b, Judith Langerb,c, Mercedes Sánchez-Costab, Gonzalo JiménezOsésd,e* and Fernando López-Gallegob,e* aDepartamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, E-26006 Logroño, Spain. b Center for cooperative Research in Biomaterials (CIC biomaGUNE) - Basque Research and Technology Alliance (BRTA) Paseo de Miramón, 182, 20014 Donostia-San Sebastián, Spain. cBiomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBERBBN), 20014 Donostia-San Sebastián, Spain dCenter for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain. elkerbasque, Basque Foundation for Science, Plaza Euskadi 5, 48009 Bilbao, Spain. * Corresponding authors. Fernando López-Gallego: [email protected] Gonzalo Jiménez-Osés: [email protected] Page 1 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 KEYWORDS: biocatalysis, protein engineering, protein immobilization, continuous flow reactor, acyltransferase, simvastatin. ABSTRACT Simvastatin is a top-selling cholesterol-lowering drug traditionally obtained through a semi-synthetic process starting from lovastatin. However, this process is cost-demanding and makes use of chemical reagents that can generate considerable waste. The sustainability concerns underlying the current semi-synthetic process encouraged us to immobilize an engineered acyltransferase LovD-BuCh2 on different porous carriers to develop an innovative and sustainable process for the continuous biomanufacturing of simvastatin. We systematically assessed the effect of the immobilization on the functional and structural properties of the immobilized enzyme, the enzyme spatial distribution across the solid material, and the thermal stability of the immobilized biocatalysts. After screening several immobilization carriers, we selected LovD-BuCh2 immobilized on the controlled porous glass particles functionalized with Fe3+-catechol complexes (EziG1) as the most suitable heterogeneous biocatalyst to optimize the continuous synthesis of simvastatin. This immobilized enzyme was four times more thermally stable than its free counterpart and maintained > 60% product yield during 5 operational cycles in batch. Under the optimal flow conditions, we achieved 100% of simvastatin yield with a space-time yield of 4.61 g L-1 h-1 and specific productivity of 17 mgproduct x mgenzyme-1 L-1. This flow-biocatalysis system improves some green chemistry metrics such as the reaction mass efficiency (RME) and the atom economy when compared to previous studies of simvastatin manufacturing. Page 2 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 INTRODUCTION In the last decades, the high demand for active pharmaceutical ingredients (APIs) like the cholesterol lowering drug simvastatin (SVA)1, 2 encourages the development of novel technologies that shorten the time-to-market of pharmaceuticals. In this context, flow chemistry emerges as one of the most useful approaches to speed APIs discovery and manufacturing.3-5 Besides, biocatalysis is bursting into the API manufacturing model due to the excellent properties of enzymes as catalysts to advance toward more sustainable processes. In the last decade, chemical engineers have bridged biocatalysis and flow chemistry to intensify the production of pharmaceuticals.6, 7 From the successful cooperation of these two disciplines, flow-biocatalysis is born to enhance the efficiency, sustainability, and safety of chemical manufacturing. All these reasons entail flow biocatalysis as a key enabling technology in green and sustainable chemistry. However, the integration of enzymes into flow reactors undergoes some limitations such as solubility, lability, and/or substrate inhibition that are inherent to the biological nature of the biocatalysts and must be tackled if a biochemical continuous process wants to be implemented. One of the most adopted solutions to implement enzymes into flow-reactors is their immobilization on solid materials which enables their separation from the products and often enhances the stability and the reusability of the immobilized biocatalysts.8-10 In fact, a packed-bed reactor (PBR) formed by a column packed with an immobilized enzyme is one of the most widely used configurations in flow-biocatalysis.11-13 Furthermore, enzymes can also be directly attached to the walls of microreactors, to the porous surface of thin films forming the membrane reactors, and to the inner surface of porous monoliths as part of the monolithic reactors.14-17 In all these cases, the enzymes can be considered heterogeneous biocatalysts where the catalytic sites are in a different phase (solid) than the reactants. Hence, the retention of enzyme activity and stability upon Page 3 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 the immobilization process is not trivial as the enzymes bound to solid surfaces may undergo negative structural distortions and suffer from mass transport restrictions of the reactants. When the immobilization strategy is well designed and the resulting heterogeneous biocatalyst profoundly characterized, one can maximize the activity/stability balance, giving rise to efficient heterogeneous biocatalysts that can be continuously operated in a flow-reactor.18-22 Acyl transferases have been successfully applied in flow during the last decade for the manufacturing of fragrances, flavors, and pharmaceuticals.23 Remarkably, an acyl transferase from Mycobacterium smegmatis has been immobilized on agarose porous microparticles functionalized with aldehydes and packed into a PBR for the continuous synthesis of melatonin and other tryptamine derivatives with an extraordinary space-time yield in an unprecedented short retention time.24 This example demonstrates that enzymatic processes can be intensified in flow up to reaching the productivity and operational stability demanded by the industrial sector. In particular, the intensification of acyl transferase driven processes is quite challenging as these enzymes catalyze the transacylation reaction of different esters using different nucleophiles (alcohols, amines, and thiols) through a kinetically controlled synthesis where synthetic and hydrolytic activities compete.25, 26 An especially interesting case is the biosynthesis of SVA catalyzed in vitro by an engineered acyl transferase LovD9 from Aspergillus terreus using α-dimethylbutyryl-Smethylmercaptopropionate (DMB-SMMP) as acyl donor and yeast-fermented Monocolin J acid (MJA) as acyl acceptor.27 Unfortunately, this enzyme-driven acylation step suffers from substrate inhibition by MJA and two unwanted side reactions underlying the mechanism of LovD9: the hydrolysis of the acyl-enzyme complex ultimately converting DMB-SMMP into 2,2Page 4 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 dimethylbutanoic acid (thioesterase activity) and the hydrolysis of the final product back to MJA (esterase activity) (Scheme 1). Scheme 1. Mechanism of Simvastatin acid (SVA) synthesis and its side-reactions. DMB-SMMP: -dimethylbutyryl-S-methyl-mercaptopropionate; DMB: dimethylbutyric acid; MJA: Monacolin J acid; SVA: Simvastatin acid. MJA inhibits the formation of the acyl-enzyme complex. The acylenzyme complex can be hydrolyzed by water (thioesterase activity), producing the dead-end product DMB. The product (SVA) can be hydrolyzed by the esterase activity of the enzyme to MJA and DMB (dead-end product). In recent work,2 we dissected the kinetically controlled synthesis of SVA catalyzed by LovD and created a new minimalist variant with only 14 mutations (LovD-BuCh2) and similar activity and stability properties as the in vitro evolved LovD9 (featuring 29 mutations);28the most efficient acetyl transferase variant ever reported for SVA biosynthesis. Despite having an excellent enzyme to catalyze such industrially relevant synthesis, this reaction has never been intensified Substrate inhibition Thioesterase activity Esterase activity H2OH2O Acyl-enzyme complex Page 5 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 through enzyme immobilization and engineering of a continuous process. In this work, we have optimized the immobilization of LovD-BuCh2 variant by testing different carriers and immobilization chemistries. The resulting heterogeneous biocatalysts were profoundly characterized at both structural and functional levels to find the optimal immobilization protocol that maximizes the activity/stability balance of the immobilized enzymes. This optimization and characterization voyage have enabled us to fabricate a heterogeneous biocatalyst (LovDBuCh2@EziG1) that was successfully integrated into a PBR for the continuous synthesis of SVA. Tuning the flow conditions and substrate concentrations allowed us to reach 100% yield of SVA and minimize product hydrolysis that was limiting the SVA yield under steady-state conditions. EXPERIMENTAL SECTION Materials Substrates Monacolin J acid (MJA) and -dimethylbutyryl-S-methyl-mercaptopropionate (DMB-SMMP) were kindly donated by Prof. Yi Tang laboratory (UCLA, USA). Simvastatin hydroxyl acid ammonium salt 98% was purchased from Toronto Research Chemicals (Toronto, Canada). 2,2’-Dithiodipyridine 100%, Amicon Ultra-0.5 centrifugal filter units (10 kDa), NaBH4, CoCl2, iminodiacetic acid (IDA), rhodamine B isothiocyanate (RhB) and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St.Louis, IL, USA). Agarose microbeads 4BCL (AG, particle size 50-150 μm, pore size 50 nm) with (30 μmol of Co2+ g support -1) (AG-Co2+) and without cobalt chelates were purchased from Agarose Bead Technologies (Madrid, Spain). Porous glass microbeads EziG1 (particle size 75-125 μm, pore size 50 nm, 10 μmol Fe3+ g support -1) were kindly donated by EnginZyme (Stockholm, Sweden). His-tagged LovD-BuCh2 variant (Table S1) was overexpressed in E. coli BL21 strain as previously described Page 6 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 (see Supporting Methods)2. Only when LovD-BuCh2 was labelled with fluorophores, we used a pure enzyme solution which was purified through immobilized metal-affinity chromatography using AG-Co2+ for 1 h at 4 °C (see supporting information).2,29. Functionalization of agarose microbeads with epoxide groups and cobalt chelates (AGCo2+/E) 1 g of agarose 4BCL was diluted in 1.6 mL of acetone and 44 mL of water containing 0.12 M of NaBH4 and 0.1 M of NaOH at 4 °C. 11 mL of (±)-epichlorohydrin (≥ 99% purity) were added very slowly at the same temperature to avoid epoxide hydrolysis. The mixture was incubated for 16 h at room temperature with mild stirring. Finally, the suspension was filtered and washed with 10 mL of water. Then, 1 g of epoxy-agarose was modified by adding 0.5 M of iminodiacetic acid (IDA) in water at pH 11 and stirring at room temperature for 1 h with mild agitation. The suspension was filtered and washed with 10 mL of water. Under these conditions, the activation degree is 20.2 μmol g-1 of IDA and 18.5 μmol g-1 of epoxides according to the titration of remaining diols before and after functionalizing the epoxy-agarose with IDA. For quantification, the remaining diols in the agarose carriers were stoichiometrically oxidized with periodate, and the remaining periodate in solution was determined by the colorimetric oxidation of KI in basic media (water saturated with HCO3-). 1 g of IDA-epoxy-agarose was additionally modified by adding 10 mL of a CoCl2 solution (30 mg mL-1 in water) and incubated for 1 h at 25 °C with mild stirring. Finally, the suspension was filtered, washed with 10 mL of water, and stored at 4 ºC. Enzymatic spectrophotometric assay Page 7 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 Enzyme assay to determine the activity of both free and immobilized enzymes was performed in 96-well plates for 30 min at 37 °C using 3 mM MJA, 2 mM DMB-SMMP and 2 mM 2,2’- dithiodipyridine (2-DTDP) in 50 mM HEPES 10 mM MgCl2 (pH 8) and 10% dimethyl sulfoxide (DMSO). The thiol formed (methyl mercaptopropionic acid, MMP) after the enzymatically driven thiolysis of DMB-SMMP spontaneously reacts with 2-DTDP, generating 2thiopyridone that absorbs at 343 nm with an absorption coefficient of 7600 M-1 cm-1 (Scheme S1).30 1 unit of enzyme is defined as the amount of enzyme needed to produce one micromole of MMP per minute under the assay conditions. Immobilization and one-step purification of LovD-BuCh2 on different carriers 5 mL of the clear crude extract containing His-tagged LovD-BuCh2 was diluted with 50 mM HEPES at pH 8 until reaching 0.11 mg mL-1 LovD-BuCh2 (enzyme concentration was estimated by SDS-PAGE, see supporting information). This enzyme solution was then incubated with 0.5 g of AG-Co2+, AG-Co2+/E and EziG1. A similar procedure was followed for the immobilization of the fluorophore-labelled pure enzyme. Upon the immobilization on AG-Co2+/E, the immobilized sample was incubated for 24 h at room temperature and pH 8. Next, the beads were filtered and incubated with 1 M glycine solution in phosphate buffer 50 mM at 4 °C and pH 8 for 2 h, to react with and remove the remaining epoxide groups, thus avoiding unspecific covalent interactions during enzyme storage and operational time. Then, the beads were washed with 10 mL of 50 mM sodium phosphate buffer, vacuum dried, and stored at 4 °C for further use.31 A fraction of the immobilized enzyme was eluted with 250 mM imidazole and 50 mM HEPES at pH 8 to assess further assays with both soluble and immobilized enzymes. Immobilization yield for each carrier was quantitatively assessed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using bovine serum albumin (BSA) as titration protein curve (Figure Page 8 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9 S1) under reducing conditions, and through the Bradford protein assay,32 calculating the difference of protein concentration between the supernatant before and after contacting it with the carriers. The immobilization parameters were determined as described in supporting information. Microscopy and biophysical characterization of LovD-BuCh2 on different carriers The protein distribution of the enzyme across the surface of the different carriers was determined by Confocal Laser Scanning Microscopy (CLSM) (see supporting information). To that aim the pure enzyme (see supporting information for purification details) was first labelled with a fluorophore (Rhodamine B or fluorescein). For more details, see supporting information. Moreover, the enzyme spatial distribution was also studied by Raman spectroscopic microscopy using the unlabeled enzyme (see supporting information). Structural rearrangements and mobility of the free and immobilized protein were analyzed by intrinsic protein fluorescence and protein fluorescence anisotropy of the immobilized enzymes (see supporting information). Finally, the immobilized and free enzymes were thermally inactivated at different temperatures. For further experimental details, see supporting information. Operational stability of LovD-BuCh2 immobilized on AG-Co2+/E and EziG1 in batch Batch reaction time courses were measured for free LovD-BuCh2, LovD-BuCh2@AGCo2+/E, and LovD-BuCh2@EziG1 at 3 mM MJA, 2mM DMB-SMMP in 50 mM HEPES and 10% dimethyl sulfoxide (DMSO) at pH 8 (reaction mixture). Reactions were initiated in a 1.5 mL column by mixing either 65 mg of LovD-BuCh2@AG-Co2+/E (1.06 mg g-1support) or 203 mg of LovD-BuCh2@EziG1 (0.34 mg g-1support) with 600 µL of the reaction mixture. The reaction was incubated a 25 ºC and samples were withdrawn at 1, 2, 4, 8, and 24 h by filtering the reaction mixture in order to separate the immobilized enzyme from the reaction crude. For the reusability Page 9 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 Figure 2. (a-c) 40X magnification confocal microscope fluorescent images (20X in the right corner, red field) of LovD-BuCh2 labeled with Rhodamine isothiocyanate (RhB) and immobilized onto cobalt-activated agarose microbeads (AG-Co2+) (a), AG-Co2+ functionalized with epoxide groups (AG-Co2+/E) (b), and porous glass functionalized with Fe3+-catechol complexes (EziG1) (c). Infiltration distance values were calculated from fluorescence profiles of 20 single beads of similar size (see Figure S2) (d). d ab cd AG-Co2+ AG-Co2+/E EziG1 0 5 10 15 20 25 30 35 40 Infiltration distance (mm) Page 16 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 17 To validate the distribution results obtained with fluorescence microscopy and to study the enzyme/carrier interactions, we applied Raman microscopy and spectroscopy as reported for other immobilized enzymes.39-43 To this aim, we acquired Raman maps of LovD-BuCh2@EziG1 and applied True Component Analysis (TCA) to spatially and chemically resolve the different chemical species within the Raman hyperspectral imaging data.44 Analyzing the averaged spectra and Raman maps of free and immobilized LovD-BuCh2@EziG1 by TCA, we identified the protein Raman spectrum as one of the components in both free and immobilized enzyme (Figure 3a). From this spectra, we identified a peak at 1447 cm-1 corresponding to the deformation of C-H bonds of the protein (δ-CH) and the typical 1656 cm-1 peak assigned to the amide I region of proteins.40 TCA generates a component map that shows how the same protein Raman spectrum shown in Figure 3a is located at the outer surface of the particles (Figure 3b). Looking closer to the Raman spectrum (range 1200-1750 cm-1) (Figure 3c), we observed an increase in the signal ratio between 1275 cm-1 and amide I peak when the N-3 atom of the imidazole ring binds to the metal. On the other hand, the C4=C5 bond signal of the imidazole ring experiences a shift from 1600 cm-1 when it is free to 1580 cm-1 when it is bound to the metal. These spectroscopic signatures agree with previous studies that report how histidine coordination to divalent metals can be detected in the Raman spectrum.45 Therefore, the Raman spectrum of the immobilized LovDBuCh2@EziG1 revealed that the enzyme is mainly located at the outer surface of the carrier in agreement with the spatial distribution elicited by CLSM for the same sample (Figure 2c). Furthermore, the differences found between the Raman spectra of the free and immobilized enzyme suggest that some conformational changes occur in the protein upon the immobilization. Page 17 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 Figure 3. (a) Averaged Raman spectra of free LovD-BuCh2 and LovD-BuCh2@EziG1 (1003800 cm-1 range). (b) LovD-BuCh2 immobilized on EziG1. Top: bright-field images, and bottom: Raman 2-D map obtained with True Component Analysis (TCA) of the Raman spectrum of the immobilized enzyme (blue line in panel a). (c) Zoom in of panel (a) to better visualize the spectral differences of the averaged Raman spectra of free LovD-BuCh2 (red) and LovDBuCh2@EziG1(blue) in the range from 1200 to 1750 cm-1. Thermal stability analysis The thermal stability of enzymes is a key parameter for industrial processes in biocatalysis to guarantee the biocatalyst operability under drastic conditions and long times. It is well b a δ-CH Amide I σ-CH Free LovDBuCh2 LovDBuCh2EziG1 0 50 100 150 200 CDC (cts) 500 1000 1500 2000 2500 3000 3500 Raman Shift (cm-1) cAmide I δ-CHHis C4=C5 0 6 12 18 1260 1350 1440 1530 1620 1710 CDC (cts) Raman Shift (cm-1) Page 18 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 established that the rigidification of the enzyme backbone as a consequence of its binding to solid carriers upgrades its resistance against inactivating agents. However, this immobilizationmediated stabilization is generally linked with the decrease of the immobilized enzyme specific activity (iSA).46, 47 To assess the stability of the three heterogeneous biocatalysts described in the previous section, we incubated them at different temperatures for 1 h. Figure 4a reveals that LovDBuCh2@AG-Co2+/E and LovD-BuCh2@EziG1 retained more than 80% of their activity after incubation at temperatures up to 40 °C, whereas LovD-BuCh2@AG-Co2+ and the free enzyme retained only 50% of their initial activity upon 1 h incubation under the same temperature. Under thermal incubation, we suggest that the free enzyme may experience protein aggregation or conformation changes due to undesired protein interactions that alter their optimal secondary and tertiary structure.48 As expected from the mesophilic origin of this enzyme, all the soluble and heterogeneous biocatalysts were almost fully inactivated upon incubation at 50 °C. Hence, we set 40 °C as the inactivation temperature to determine the thermal deactivation kinetics and the half-life time (t1/2) of the different heterogeneous biocatalysts. Figure 4b confirms that the soluble enzyme was the most unstable preparation with a t1/2 = 27.6 minutes and an inactivation constant ki = 1.497, whereas LovD-BuCh2@AG-Co2+, LovD-BuCh2@EziG1, and LovD-BuCh2@AG-Co2+/E keep more than 50% activity for more than 50 minutes with ki = 0.744, 0.374 and 0.286, respectively (Table S2). These results indicate that the site-directed and multivalent irreversible immobilization of LovD-BuCh2 on AG-Co2+/E enhances the enzyme stability by a factor of 4, a similar stabilization factor to that achieved with the EziG1 carrier where the enzyme was attached only through reversible interactions. The surface properties and the functionalization of EziG1 carriers may promote additional non-covalent (electrostatic) Page 19 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20 interactions with the enzymes compared to the AG-Co2+ which only binds the enzyme through the His-tag. Therefore, the stabilization of LovD-BuCh2 in both EziG1 and AG-Co2+/E may be related to the multivalent binding (of different nature) between the enzyme and the surfaces of these carriers that would protect the immobilized enzymes against thermal inactivation. A similar trend was observed for the inactivation of these immobilized and free biocatalysts in DMSO; the solvent typically used to dissolve the acyl donor (DMB-SMMP) herein used for the acetylation reaction catalyzed by LovD-BuCh2 (Figure S4). Figure 4. (a) Thermal inactivation of free and immobilized LovD-BuCh2 on different carriers at different temperatures for 1 h. The residual activity (%) was calculated based on the initial activity at 25 °C of each biocatalyst. (b) Inactivation time courses of LovD-BuCh2 immobilized on different carriers and its free counterpart incubated at 40 °C. All the activity assays to calculate the residual activity upon thermal incubation were performed with 3 mM MJA, and 2 mM DMBSMMP. a b 25 37 40 45 50 0 20 40 60 80 100 120 Residual activity (%) Temperature (ºC) AG-Co2+ AG-Co2+/E EziG1 Free 050 100 150 200 250 0 20 40 60 80 100 120 Residual activity (%) Time (min) AG-Co2+ AG-Co2+/E EziG1 Free Page 20 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 21 Mobility and conformational studies of immobilized LovD-BuCh2 To better understand the molecular origin of the enzyme stabilization promoted by immobilization, biophysical techniques were employed to elucidate the structural rearrangements of the protein when immobilized on different carriers before and after thermal inactivation. Protein aromatic residues show intrinsic fluorescence that can be detected upon excitation at 280 nm and provide information about the protein folding/conformation states.49, 50 Therefore, structural changes in the protein when it is immobilized on a carrier or subjected to thermal shock can be detected via changes in its fluorescence spectrum. Figure 5a and Figure S5 show a slight hypsochromic shift of the maximum emission wavelength (λmax) from 335 nm to 330 nm when LovD-BuCh2 was immobilized in all the carriers and incubated at 25 °C compared to the free enzyme, unveiling a reduced solvent exposure of the aromatic residues upon immobilization process that suggests conformational changes in the enzyme when interacting with the carriers. The presence of those new conformations may explain the lower specific activity of the immobilized enzymes compared to their free counterpart. When the free and immobilized biocatalysts were incubated at 40 °C for 4 hours, the free enzyme exhibited a 50 nm bathochromic shift in λmax because thermal unfolding induces a higher solvent exposure of the aromatic residues. The extent of that shift was significantly lower in the immobilized preparations: LovDBuCh2@AG-Co2+ underwent a 5 nm red-shift in λmax, while LovD-BuCh2@AG-Co2+/E and LovD-BuCh2-EziG1 maintained their λmax upon incubation at 40 °C. Thus, when the enzyme is immobilized, the thermal shock impacts the protein folding less than when it is in solution. Another relevant observation is that besides the alleged rigidification provided by covalent binding in LovD-BuCh2@AG-Co2+/E that explains protein stabilization, EziG1-enzyme interactions also seemed to provide a more protected protein towards thermal shock. This fact agreed with the Page 21 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 22 results obtained from thermal deactivation assays at 40 °C (Figure 4. b), which revealed that EziG1 affinity immobilization improves enzyme thermostability when compared to AG-Co2+. Besides conformational changes in tertiary structure, the reduction in mobility undergone by the enzymes after the immobilization process can also be assessed by determining their rotational tumbling by measuring the anisotropy of fluorophores conjugated to the protein structure. To that aim, we labeled LovD-BuCh2 with fluorescein isothiocyanate (FITC) and immobilized it on the three carriers above described. The protein fluorescence anisotropy of the labeled and immobilized enzymes was measured as described elsewhere51 (see Experimental section). The anisotropy of the immobilized enzymes was higher than that of the free ones in all cases, demonstrating that the immobilization process consistently reduced enzyme rotational tumbling, an indication of enzyme rigidification upon immobilization (Figure 5b, Table S3). LovD-BuCh2@AG-Co2+ exhibited an anisotropy two times higher than the free enzyme, whereas the enzyme anisotropy in LovD-BuCh2@EziG1 and LovD-BuCh2@AG-Co2+/E was 2.45 and 2.94 times higher than in the free enzyme, respectively. When the relative anisotropy was plotted as a function of the thermal half-life at 40 °C, we found a linear positive correlation between those two parameters, supporting the idea that thermostability is enhanced by reducing the protein mobility when anchored to a solid surface.52, 53 Irreversible and multipoint covalent immobilization promoted by AG-Co2+/E explains the higher compactness and the lower mobility of the immobilized enzymes according to Trp fluorescence and anisotropy studies, entailing the high stability of this biocatalyst. This correlation between the valency of the attachment and the protein deformation and its increasing effect on protein stability under denaturing conditions has already been reported for other immobilized proteins.54, 55 Page 22 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 23 Figure 5. (a) λmax of each biocatalyst after incubation at 25 °C and 40 °C for 4 h. (b) Normalized anisotropy as a function of the half-life time at 40 °C. The anisotropy of free LovD-BuCh2 was set to a reference value of 1. Operational stability of LovD-BuCh2@EziG1 and LovD-BuCh2@AG-Co2+/E In addition to thermal stability, operational stability is fundamental to assessing the potential of any heterogeneous biocatalysts. Herein, the two most thermostable heterogenous biocatalysts LovD-BuCh2@AG-Co2+/E and LovD-BuCh2@EziG1 were exploited for the batch synthesis of SVA (Figure 6a). As expected from the immobilization parameters, immobilized enzymes produced SVA slower than the free enzyme. The yields and production rates were determined by UPLC-MS (Figure S6). As observed in Figure 6a, the free enzyme reached 100% yield of SVA production in 8 h and was the fastest biocatalyst with a specific productivity (SP) of 0.58 mg SVA mgenzyme -1 h-1 after 8 h of reaction. In contrast, LovD-BuCh2-EziG1 and LovD-BuCh2-AG-Co2+/E exhibited SP of ab EziG1 AG-Co2+/E AG-Co2+ Free 275 300 325 350 375 400 lmax (nm) 25 ºC 40 ºC Page 23 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 24 0.20 and 0.07 mg SVA mg enzyme -1 h-1 after 8 h, and SVA yields of 75% and 50% after 24 h, respectively. The rate of SVA production was significantly slower using the enzyme irreversibly immobilized on AG-Co2+/E, in accordance with the low specific activity of this immobilized enzyme (Table 1). Interestingly, we observed a 15% reduction of the product yield after 8 h using the free enzyme as a biocatalyst, which suggests that the free enzyme hydrolyzes the accumulated product. Although we did not observe any transient product yield decay when using the heterogeneous biocatalysts, we cannot discard the existence of hydrolytic activity in those biotransformations as the time courses never reached a plateau. Finally, the two heterogeneous biocatalysts herein tested were recycled in consecutive 24 h cycles (Figure 6. b). As a result, both LovD-BuCh2@AG-Co2+/E and LovD-BuCh2@EziG1 exhibited a considerably high operational stability. While the initial product yield achieved with LovD-BuCh2@EziG1 diminished from 75% to 50 % after 8 operational cycles, the lower SVA yield obtained with LovD-BuCh2@AG-Co2+/E after the first cycle (50%) remained constant during the same 8 cycles. Therefore, the heterogeneous biocatalyst with the highest thermal stability also exhibits the highest operational stability although its productivity was the lowest. Covalent immobilization in epoxide, glyoxyl, or glutaraldehydeactivated supports has already proven successful to stabilize protein structure, ensuring its reusability in several cycles of operation.56-58 However, LovD-BuCh2@EziG1 demonstrated that covalent attachment is not always necessary to obtain a reusable biocatalyst, as other factors such as surface distribution or electrostatic interactions can also guarantee high productivity for long operation times. Therefore, the excellent operational stability and the higher productivity of LovD-BuCh2@EziG1 encouraged us to exploit it for the continuous synthesis of SVA in a PBR. Page 24 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25 Figure 6. (a) Batch reaction time courses for free LovD-BuCh2, LovD-BuCh2@AG-Co2+/E, and LovD-BuCh2@EziG1 using 3 mM Monacolin J acid (MJA) and 2 mM α-dimethylbutyryl-Smethyl-mercaptopropionate (DMB-SMMP). (b) 24 h batch cycles under the same reaction conditions. Continuous synthesis of SVA with a LovD-BuCh2@EziG1 packed-flow reactor To set a continuous synthesis of SVA to have better control of the reaction outcome, we packed a bed of 1 g of 0.34 mg g-1 LovD-BuCh2@EziG1 in a 1.5 cm3 column and tested the reaction with 1 mM MJA and 2 mM DMB-SMMP at different flow rates. Figure 7a shows that the higher the flow rate, the higher the SVA yield (reaching the maximum yield at 100 μL min-1). This is a counter-intuitive fact because high flow rates normally decrease the residence times lowering the product yields. However, this reasoning is only valid for those systems where a single reaction takes place at a time, which is not the case of LovD-BuCh2 that catalyzes the kinetically controlled synthesis of SVA where two unwanted side hydrolytic reactions (product and acyl donor hydrolysis) occur simultaneously to the target synthetic reaction. We hypothesize that the a b 0 5 10 15 20 25 0 20 40 60 80 100 SVA yield (%) Time (h) AG-Co2+/E EziG1 Free 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 SVA yield (%) Cycles AG-Co2+/E EziG1 Page 25 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 32 once immobilized. Mobility and conformational studies together with the microscopic characterization of the immobilized enzyme revealed its spatial distribution across the surface of the carrier. After evaluating the activity and the stability of the three heterogeneous biocatalysts herein described, we conclude that LovD-BuCh2@EziG1 presents the best activity/stability balance for their use in cell-free SVA biosynthesis. This heterogeneous biocatalyst exhibited an excellent operational stability which allowed us to implement it in a continuous SVA synthesis that was ultimately intensified by optimizing the flow rate, as well as the concentration and ratio of the substrates. Remarkably, this work illustrates the potential of immobilizing engineered enzymes for the process development of biotransformations. Finally, we assessed the sustainability of the process herein reported in comparison with fermentative and chemical SVA synthetic processes. The sustainability metrics revealed an outstanding mass efficiency and atom economy of the process when using LovD-BuCh2@EziG1 operated in continuous under the optimal conditions. However, the extensive use of water in this biotransformation was the major limitation to achieving an E factor lower than 1000. As far as we know, this work is the first demonstration of a heterogeneous biocatalyst for the continuous biomanufacturing of SVA. Further research of continuous operational conditions is required to minimize the hydrolytic side reaction, as well as the development of a downstream process of simvastatin purification and crystallization coupled to the continuous flow process. We are confident that the lessons learned in this work will be highly useful to implement other biocatalytic kinetically controlled syntheses in flow. Page 32 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 33 ASSOCIATED CONTENT Supporting Information. Supporting methods, control experiments, and additional data including SDS-PAGE analysis, UPLC-MS chromatograms , CLSM profiles and protein intrinsic fluorescence spectra that support the main results are provided in the supporting information. AUTHOR INFORMATION Corresponding Authors Fernando López-Gallego - Heterogeneous Biocatalysis Laboratory. Center for Cooperative Research in Biomaterials (CIC biomaGUNE) – Basque Research and Technology Alliance (BRTA). Miramon Pasealekua, 182, 20014 Donostia, Gipuzkoa, Spain. Email: [email protected] Gonzalo Jiménez-Osés - Center for Cooperative Research in Biosciences (CIC bioGUNE) – Basque Research and Technology Alliance (BRTA). Bizkaia Technological Park, Building 800, 48160 Derio, Bizkaia, Spain. Email: [email protected] Authors Guillermo García-Marquina - aDepartamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, E-26006 Logroño, Spain. Center for Cooperative Research in Biomaterials (CIC biomaGUNE) – Basque Research and Technology Alliance (BRTA). Miramon Pasealekua, 182, 20014 Donostia, Gipuzkoa, Spain. Email: [email protected] Page 33 of 39 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 34 Judith Langer – Center for Cooperative Research in Biomaterials (CIC biomaGUNE) – Basque Research and Technology Alliance (BRTA). Miramon Pasealekua, 182, 20014 Donostia, Gipuzkoa, Spain. Email: [email protected] Mercedes Sánchez-Costa - Center for Cooperative Research in Biomaterials (CIC biomaGUNE) – Basque Research and Technology Alliance (BRTA). Miramon Pasealekua, 182, 20014 Donostia, Gipuzkoa, Spain. Email: [email protected] ACKNOWLEDGMENT This research was funded by Agencia Estatal de Investigación of Spain (projects CTQ2015-70524R and RTI2018‐099592‐B‐C22 to G.J.O. and predoctoral fellowship to G.G.M.). G.J.O. and F.L.G. acknowledge support from Ikerbasque. This work was performed under the Maria de Maeztu Units and Severo Ochoa Centers of Excellence Program from the Spanish State Research Agency (grants MDM-2017-0720 to CIC biomaGUNE and SEV-2016-0644 to CIC bioGUNE). We thank Prof. Yi Tang for kindly providing us with MJA, and DMB-SMMP. Finally, we are very grateful to K. E. Cassimjee (EnginZyme AB) for kindly donating us the EziG carriers and to Dr. Elin Stridfeldt, Dr. Ashley Mattey, Dr. Alden Clemments, and Dr Alexey Volkov (all from EnginZyme AB) for their fruitful discussion of the results related to the immobilization on EziG. 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