The Immobilization of a Cyclodipeptide Synthase Enables Biocatalysis for Cyclodipeptide Production

Cyclodipeptide synthases (CDPSs) are enzymes that use aminoacylated tRNAs as substrates to produce cyclic dipeptide natural products acting as anticancer and neuroprotective compounds. Many CDPSs, however, suffer from instability and poor recyclability, while enzyme immobilization can enhance catalyst efficiency and reuse. Here, the CDPS enzyme from Parcubacteria bacteriumRAAC4_OD1_1 was immobilized using three different supports: biochar from waste materials, calcium-alginate beads, and chitosan beads. Immobilization of active PbCDPS was successful, and production of the cyclodipeptide cyclo (His-Glu) (cHE) was confirmed by HPLC-MS. Biochar from spent coffee activated with glutaraldehyde, alginate beads, and chitosan beads activated with glutaraldehyde led to a 5-fold improvement in cHE production, with the immobilized enzyme remaining active for seven consecutive cycles. Furthermore, we co-immobilized three enzymes participating in the cascade reaction yielding cHE (PbCDPS, histidyl-tRNA synthetase, and glutamyl-tRNA synthetase). The enzymatic cascade successfully produced the cyclic dipeptide, underscoring the potential of immobilizing various enzymes within a single support. Importantly, we demonstrated that tRNAs remained free in solution and were not adsorbed by the beads. We paved the way for the immobilization of enzymes that utilize tRNAs and other complex substrates, thereby expanding the range of reactions that can be exploited by using this technology.


■ INTRODUCTION
Cyclodipeptide synthases (CDPSs) use aminoacylated tRNAs as substrates to generate a wide range of cyclic dipeptides.Cyclic dipeptides (CDPs) are natural products produced by organisms in all domains of life.Multiple reports associate these compounds with anticancer activity and neuroprotection against neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. 1CDPSs use two aminoacylated tRNAs (aa-tRNA) as substrates and therefore rely on the activity of aminoacyl-tRNA synthetases (aaRS), hijacking aa-tRNAs from central metabolism and protein synthesis. 2CDPS enzymes are promiscuous, accepting multiple different substrates and ultimately producing more than one CDP product. 3,4Despite promising advances in the engineering of CDPSs expanding substrate scope, their usage in biocatalysis is limited due to cost and lack of recyclability. 5,6mmobilization is a strategy that allows the reutilization of enzyme catalysts, resulting in the reduction of operational cost. 7,8Different approaches can be used, such as entrapment, adsorption, and covalent bonding.When a support is used, the characteristics of the material can affect the immobilization process, and the utilization of inexpensive support matrices under sustainable strategies could increase the applicability of several enzymes. 9Alginate and chitosan are natural biopolymers that serve as an excellent support for enzyme immobilization.−12 Carbon materials are also suitable supports for enzyme immobilization. 13Biochar is a carbon-rich solid derived from the thermal decomposition of biomass (i.e., pyrolysis, flash carbonization, hydrothermal decomposition, among others) from agro-industrial residues (coffee, algae, and others). 14Biochar can be easily engineered, and the process is economically sustainable. 15he immobilization of enzymes participating in natural product biosynthesis has been limited to very few examples, including fluorinase 16 and tyrosine decarboxylase. 17Prior to this work, there were no reports of CDPS immobilization.Here, we describe a facile strategy for the immobilization of CDPS enzymes using three different supports: Ca-alginate, chitosan beads, and biochar derived from various sources (macroalgae, spent coffee, distillery waste, polystyrene, and wood waste).Previously, the production of cyclic dipeptides with high yield using the CDPS enzyme from Parcubacteria bacterium RAAC4_OD1_1 (PbCDPS, GenBank: ETB63777.1)was demonstrated. 18We focused on the production of cyclo(His-Glu) (cHE), the major product from the PbCDPS-catalyzed reaction, and currently not commercially available.PbCDPS can also produce cyclo(His-Pro) (cHP) as a minor reaction product, this promiscuity being a common feature in the CDPS family. 4Developing simple enzymatic routes to synthetically challenging compounds such as histidine-containing cyclodipeptides is a pivotal step for the exploitation of these molecules as potential bioactive agents. 19,20The CDPS reaction occurs in a cascade starting with amino acids histidine (His) and glutamate (Glu), including aaRS enzymes and ATP.We initially focused on the immobilization of PbCDPS, followed by histidyl-tRNA synthetase (HisRS) and glutamyl-tRNA synthetase (GluRS).The reusability of the system was studied by evaluating the production of CDP per cycle.The effect of quenching and loading in the support matrix was determined.We successfully immobilized PbCDPS and carried out the complete enzymatic cascade for CDP production (PbCDPS, HisRS, and GluRS), demonstrating the feasibility of including immobilization and catalyst recycling in a complex cascade involving aa-tRNA and three distinct enzymes.Our work sets the stage for future studies on CDPSs and other enzymes that utilize tRNAs as substrates and for complex biocatalytic cascade reactions leading to the biosynthesis of cyclic dipeptide natural products, enabling the production of valuable natural products.
PbCDPS and aaRS Production and Purification.PbCDPS was produced and purified following the protocol of Sutherland et al. 3 The gene encoding PbCDPS was cloned into a pJ411 expression vector with a C-terminal hexahistidine tag and transformed into E. coli BL21(DE3) competent cells (NEB).Cells were grown at 37 °C until the OD 600 reached 0.6, and protein expression was induced with IPTG (1 mM).The cells were then grown at 16 °C overnight.After harvesting, the resultant cell pellet was resuspended in 30 mL per 1 L of grown culture in lysis buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 20 mM imidazole, 5% glycerol).Cells were lysed using a high-pressure cell disruptor (Constant Systems) and centrifuged at 51000 g for 30 min at 4 °C.The lysate was filtered through a 0.8 μm membrane and loaded onto a 5 mL HisTrap  2) Process for PbCDPS immobilization in beads: (c) Ca-alginate beads, (d) alginate coated-chitosan beads, and (e) chitosan beads.Prioritization of the different immobilization strategies used for PbCDPS; starting with two different types of supports, the immobilization was made through entrapment, adsorption, and covalent bonding.Supports with more than 30% of immobilization were selected for the measurement of product formation (cHE), and reusability was tested only for the supports with the higher concentration of cHE.
HP column (GE Healthcare), pre-equilibrated with lysis buffer.The column was washed with 20 column volumes (CV) of lysis buffer, and the adsorbed proteins were eluted using elution buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 300 mM imidazole, 5% glycerol) with stepped increasing concentrations of imidazole (10%, 20%, and 100%, 10 column volumes each).Proteins of interest were dialyzed into dialysis buffer (20 mM HEPES, pH 7, 250 mM NaCl, 5 mM 2mercaptoethanol) overnight at 4 °C.PbCDPS was further purified via size exclusion chromatography using a Superdex 200 Increase 16/60 column pre-equilibrated with dialysis buffer.Fractions containing pure PbCDPS were pooled, concentrated to 10 mg/mL, flash-frozen in aliquots, and kept at −80 °C for future use.Enzyme concentration was measured using the Nanodrop DeNovix (DS-11 FX) spectrophotometer/fluorometer.Identity of the cHE product was confirmed using a Waters ACQUITY UPLC liquid chromatography system coupled to a Xevo G2-XS QTof mass spectrometer equipped with an electrospray ionization (ESI) source.10 μL of each sample was loaded onto an HSS-T3 column (2.1 × 100 mm, 1.8 μm, Waters Acquity) at 40 °C for 9 min.A gradient mobile phase from 1% B to 50% B was used, where the mobile phases consist of A-0.1% formic acid in water and B-0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min.The capillary voltage was set at 2.5 kV in positive ion mode.An MSE scan was performed between 50 and 700 m/z.The mass expected and observed for cHE was 267.1088 and 267.1093, respectively (ppm deviation: < 2).
The aminoacyl-tRNA synthetases of interest to this project− GluRS and HisRS−were purified from E. coli as detailed above following the protocol of Sutherland et al. 3 The purification buffers for GluRS contained 50 mM HEPES, pH 8, 500 mM NaCl, and 20 or 300 mM imidazole, while HisRS buffers contained 50 mM HEPES-KOH, pH 7.6, 10 mM MgCl 2 , 2 mM 2-mercaptoethanol, and 10 or 400 mM imidazole.aaRS enzymes were concentrated to 10 mg/mL, flash-frozen in aliquots, and kept at −80 °C for future use.
tRNA Pool Extraction.For the extraction of the pool of all tRNAs produced by E. coli, a protocol described by Sutherland et al. 3 was used without further modification.
Synthesis of Biochar.The biochar utilized in this study was prepared from various waste sources by using a conventional low-temperature pyrolysis methodology.These sources included (I) spent coffee obtained from a local coffee shop at St. Andrews, Scotland; (II) a mixture of polystyrene and wood, composed of 95% wood and 5% polystyrene waste; (III) dry distillery waste; and (IV) dried macroalgae sourced from the North Sea (Guardbridge, St. Andrews, Scotland) (Figure 1.1).
Production of biochar was conducted by using a pyrolysis reactor designed in-house.The reactor was a vertical-tubular design constructed with Stainless Steel 316 material.To ensure temperature homogeneity inside the reactor, the waste material was placed on an SS316 disk mesh that allowed the gas to cross the samples in the center of the reactor.Temperature monitoring was achieved by using an internal thermocouple.Prior to the initiating of the pyrolysis process, nitrogen gas was purged into the reactor to eliminate any trace of oxygen.The waste products were subjected to pyrolysis at a temperature of 550 °C, with a ramp rate of 10 °C min −1 and a residence time of 30 min under a nitrogen flow of 50 mL min −1 .Following the 30 min duration at 550 °C, the samples were rapidly cooled to prevent alterations in the residence time.Subsequently, the pristine biochar derived from the different waste sources underwent physical modification using various methods.This involved subjecting the biochar to thermal treatment at high temperatures in the presence of oxidizing agents, such as carbon dioxide or steam.Additionally, depending on the specific biochar source, acid or hydrogen peroxide pretreatment was employed.
For the biochar derived from distillery waste, polystyrenewood waste, and coffee waste, a quartz tube was used to contain the material within the furnace.Two quartz-wool plugs were placed on either end of the tube to maintain the material in the center.The biochar was heated under a nitrogen atmosphere until it reached 920 °C.At this temperature, the gas atmosphere was switched from nitrogen to carbon dioxide.The physical modification process was carried out at 920 °C for 1 h with a flow rate of 40 L min −1 of carbon dioxide.This treatment was used to modify the structure and porosity of the biochar, resulting in increased pore dimensions and surface areas as well as carbon: oxygen ratio in the final biochar due to further thermal treatment a 920 °C under N 2 .As for the biochar obtained from macroalgae, a different approach was necessary due to its fine powder form.At 920 °C, carbon dioxide was capable of completely gasifying the biochar.Therefore, hydrogen peroxide (H 2 O 2 ) was employed as an oxidizing agent.The biochar was subjected to a temperature of 150 °C, which is the boiling point of hydrogen peroxide, for 1 h in a reflux reactor.For the immobilization of PbCDPS on biochar, all sources were used with and without activation.
PbCDPS Immobilization on Biochar.Adsorption.A preliminary experiment to select the time of immobilization was carried out, analyzing the protein remaining in the supernatant following immobilization.Based on this, the optimal time for immobilization was 30 min for adsorption methods.For immobilization by physical adsorption of PbCDPS on biochar, 15 mg of biochar was mixed at room temperature, by gently stirring with 15 μg of PbCDPS in the presence of buffer (HEPES 100 mM, KCl 100 mM, and MgCl 2 10 mM at pH 7).After adsorption, immobilized PbCDPS was separated from the solution by centrifugation and washed with the same buffer to remove the unattached protein from the support.Protein in the supernatant and buffer after washing were pooled and quantified to evaluate immobilization efficiency (in % PbCDPS immobilized).
Adsorption on Activated Biochar.To increase the number of functional groups on the support surface and allow a covalent link between the enzyme and the support, biochar was impregnated with glutaraldehyde. 21Impregnation was made by mixing the support with a glutaraldehyde solution (2% v/v) for 2 h at room temperature.Subsequently, the biochar was washed with deionized water to eliminate excess glutaraldehyde.Following this, 15 mg of modified biochar was mixed with 100 μg of PbCDPS for 30 min at room temperature.After immobilization, the support was washed to remove the excess protein.Protein in the supernatant and buffer after washing were pooled and quantified to evaluate immobilization percentage.Immobilization percentage of PbCPDS on biochar was determined from the difference between the initial protein and protein detected in the supernatant and washes.The total protein concentration was assessed by using the Bradford method at a wavelength of 595 nm.
PbCDPS Immobilization in Alginate or Chitosan Beads.Alginate Beads.PbCDPS was entrapped in alginate beads by mixing 115 mg of enzyme with 1 mL of sodium alginate solution (3% w/v), following a reported protocol. 22he mixture was stirred and dropped through a syringe into 10 mL of CaCl 2 0.2 M. After, PbCDPS alg beads were washed with a HEPES/KCl/MgCl 2 at pH 7 buffer to remove free enzyme.CaCl 2 and solutions from washes were collected to evaluate immobilization percentage and efficiency.PbCDPS alg beads were activated with 2% v/v glutaraldehyde for 2h at 4 °C, and these beads were subsequently referred to as PbCDPS alg beads glut .Alginate-Coated Chitosan Beads.Alginate-coated chitosan beads were prepared according to the optimal conditions described by a reported protocol. 23A solution containing a 3% w/v solution of sodium alginate and 115 mg of PbCDPS were added as drops using a syringe into a solution containing 10 mL of CaCl 2 0.2 M, 0.2% chitosan, and 1.5% acetic acid at pH 5. Subsequently, PbCDPS alg:chi beads were washed with buffer (HEPES/KCl/MgCl 2 at pH 7) to remove the free enzyme.The chitosan/acetic acid/CaCl 2 solution and washes were collected to evaluate the immobilization percentage and efficiency.PbCDPS alg:chi beads were further activated with 2% v/v glutaraldehyde for 2 h at 4 °C, and these beads were subsequently referred to as PbCDPS alg:chi beads glut .Chitosan Beads.Chitosan beads were prepared according to a protocol previously described 24 with some modifications.A solution of 2.5% w/v of chitosan and 1.5% acetic acid was prepared and extruded dropwise into 2 M NaOH solution at room temperature.After, chitosan beads were collected and washed with deionized water to remove excess NaOH.Following this, the chitosan beads were activated using 2% v/v glutaraldehyde for 2 h at room temperature.Excess glutaraldehyde was removed by washing the activated beads with deionized water.Finally, the beads were incubated with PbCDPS overnight at 4 °C for immobilization.Activated chitosan beads (PbCDPS chi beads glut ) were washed with buffer (HEPES/KCl/MgCl 2 at pH 7) to eliminate free enzymes.All buffers and solutions from washes were collected to evaluate the immobilization percentage and efficiency.
Characterization of Biochar and Beads.The functional groups of biochar and beads were evaluated using the Fourier transform infrared technique (FTIR: Shimadzu IR Affinity 1S IR Spectrometer−for solid or liquid samples).Specific surface area (Brunauer−Emmett−Teller, BET) and pore size and volume (BJH) were determined using Micromeritics Tristar ii Surface Area and Porosity Instrument with VacPrep 061 Degasser by adsorption and desorption of nitrogen at 77 K.The morphology was analyzed using scanning electron microscopy (SEM) EVO MA 25 ZEISS.
Determination of Enzyme Activity and Product Formation cHE.Production of cyclic dipeptide product by PbCDPS free in solution as well as after immobilization was detected using HPLC assays, and product identity was further verified by liquid chromatography mass spectrometry (LC-MS) as was mentioned before.The determination of the product formation cHE was made by following the protocol previously described. 3Reaction buffer contained 100 mM HEPES, pH 7, 100 mM KCl, 10 mM MgCl 2 , 5 mM ATP, 10 mM DTT, 500 μM histidine, 500 μM glutamate, and 50 μM tRNA pool.DEPC-treated water was added to achieve the final volume of 50 or 100 μL.Finally, 5 μM of each aaRS enzyme (HisRS and GluRS) and PbCDPS were added, and the reaction proceeded overnight at room temperature.Cold methanol was added to a final volume of 80% to quench the reaction.Samples were incubated at −80 °C for 15 min and centrifuged for 10 min at room temperature.The supernatant was transferred to a second Eppendorf tube and dried using nitrogen.Finally, LC-MS grade water was used to reconstitute samples to the same initial reaction volume.The production of cHE was monitored using high-performance liquid chromatography (HPLC, Shimadzu CMB-20A).Samples (20 μL) were injected onto a Waters XSelect Premier HSS-T3 column (4.6 × 50 mm, 2.5 mm) and run at 40 °C for 30 min.A gradient mobile phase from 1% B to 50% B over 5 min at a flow rate of 1 mL min −1 was used, where mobile phase A = 0.1% trifluoroacetic acid in water and mobile phase B = 100% acetonitrile.The absorbance at 214 and 254 nm was monitored.A large-scale reaction (5 mL total volume) was set up to produce a cHE standard to be used for quantification.After the reaction proceeded overnight, the mixture was separated using a 10 kDa filter membrane to eliminate other molecules present in the reaction.After that, the mixture was centrifuged to remove precipitate and then dried under nitrogen.The subsequent residue was resuspended in LC-MS grade water and this solution injected on the HPLC.A Shimadzu CMB-20A equipped with a fraction collector and a Waters XSelect Premier HSS-T3 column (4.6 × 50 mm, 2.5 mm) were used to purify cHE.The samples were injected and run at 40 °C for 30 min.A gradient mobile phase from 1% B to 50% B over 5 min at a flow rate of 1 mL min −1 was used, where mobile phase A = 0.1% trifluoroacetic acid in water and mobile phase B = 100% acetonitrile.The selected fractions were collected, then combined, and dried by lyophilization.Finally, this 0purified powder was used for high-resolution MS and for a standard curve on HPLC.
Optimization of Loading and Quenching during Immobilization.Different concentrations of protein were tested to determine the optimal amount of PbCDPS to be immobilized in each support (alginate and chitosan beads, biochar from spent coffee), as well as the effect in the production of cHE.For alginate beads, 9.2, 13.9, 17.3, and 23.1 μg of PbCDPS were immobilized per mL of alginate.For biochar from spent coffee, 10, 15, and 20 μg of PbCDPS were used, and finally, for chitosan beads, 7.3, 14.6, 29.2, and 58.4 μg of PbCDPS were immobilized.The immobilization efficiency was measured as in the previous section, and the ratio of cHE produced with the immobilized PbCDPS per condition was compared.Additionally, to investigate if the support could retain or adsorb product after each reaction, we compared cHE obtained when the complete system was quenched (liquid and support) and when only the liquid was quenched.
Reusability of Immobilized PbCDPS.Reusability of immobilized PbCDPS was evaluated only with supports that showed higher cHE production.After each overnight cycle, supports under evaluation were recovered, and a new assay was started omitting PbCDPS but with other components as specified under "Determination of Enzyme Activity and Product Formation cHE".
Co-Immobilization of the Enzymes Involved in the cHE Production.cHE is the final product of a cascade reaction between PbCDPS, HisRS, and GluRS.Ideal conditions for PbCDPS were employed to immobilize the two other enzymes taking part in the cascade for cHE production.Simultaneous immobilization of PbCDPS, HisRS, and GluRS was performed as described in the previous sections.Bradford was used to determine the amount of free protein and to qualitatively estimate the immobilization efficiency.

■ RESULTS AND DISCUSSION
Synthesis and Characterization of Biochar.Biochar produced from different sources by pyrolysis was used as a carrier for the immobilization of PbCDPS.Biochar properties can be modified using an additional activation process after pyrolysis.Recent studies have shown that the activation with CO 2 could increase the specific surface area, pore structure, and functional groups on the surface. 15However, in previous experiments optimizing immobilization conditions (Table S1), high quantities of enzyme immobilized in the nonactivated biochar resulted in limited cHE product formation.This could be due to the reduction of the pore size after the activation (Table S2), which limits diffusion of other reaction components such as aa-tRNA in and out of the biochar pores. 25TIR measurements were performed using biochar samples to verify the chemical groups present at the material surface (see Figure S1).FTIR spectra between 3600 and 3200 cm −1 reveal peaks corresponding to OH bonds of phenol or alcohol groups.However, the relatively small size of these peaks is due to the loss of moisture caused by the high temperatures reached in the gasification process. 26The absorbance peak at 3000−2800 cm −1 represents the aliphatic C−H stretch vibration.They are poorly pronounced in all samples due to the degradation of aliphatic compounds at high gasification temperatures, resulting in a higher peak in mild pyrolysis temperatures.The absorbance peaks at 800 and 1600 cm −1 are attributed to the aromatic C−H stretch and the aromatic C� C stretch, respectively.In all biochar samples, peaks attributed to cellulose and hemicellulose (3200−3000 cm −1 for OH and 3100−3000 cm −1 for CH) are absent, due to the hemicellulose and cellulose being completely thermally degraded in biochar. 27Double bonds could be due to the condensate aromatic structure observed in graphene.Representative peaks for C−H stretching (750−900 and 3050−3000 cm −1 ), C�C (1380−1450 cm −1 ), and C−C and C−O stretching (1580− 1700 cm −1 ) are present. 28Bands between 1800 and 1500 cm −1 can be attributed to the C�O bond stretch of the carboxylic acids and ketones.Figure S1e−g show the FTIR spectra for beads.The broad peak at 3367 cm −1 was assigned to free hydroxyl groups.Characteristic peaks of alginate were 1606 and 1425 cm −1 for the C�O bond.The characteristics peaks of chitosan were at 1664 cm −1 for amide I and 1544 cm −1 for amide II. 11rom scanning electron microscopy (SEM) studies, the biochar samples have different structural characteristics due to the different biomass morphologies as shown in Figure 2. The majority of the waste analyzed has a porous fiber structure typical of biomass except for the spent coffee (Figures 2c and  3d), where the structure was not as well-defined as in other samples, and deeper cavities suitable for the adsorption of molecules were observed.This result agrees with the pore size study (Table S2), as biochar from spent coffee was the material with the highest pore size.Biochar from macroalgae and distillery waste (Figure 2a,e) has a honeycomb pore structure.Biochar from polystyrene and wood waste (Figure 2h) has fewer pores produced due to the complex lignin cellulose structure typical of the wood.Additionally, images show that in general biochar has a high degree of macroporosity, which could permit a suitable adsorption of the protein and an effective interaction between enzymes and substrates. 28mmobilization of PbCDPS.Ca-Alginate Beads.PbCDPS was immobilized in alginate beads by an entrapment technique.With this approach, no PbCDPS was detected in the supernatant after the immobilization in all systems under comparison (Table 1), indicating a promising candidate for the activity assay of PbCDPS.Alginate is commonly used due to its stability, nontoxicity, and low cost.−31 Previously, horseradish peroxidase (HRP) was immobilized in Ca-alginate beads, and a maximum immobilization of 89 ± 5% was reported, 32 considerable but likely lower to what was achieved with PbCDPS alg beads and PbCDPS alg beads glut .Two approaches for immobilization were employed, with and without functionalization, and in both cases, no protein was detected after immobilization (Table 1).High immobilization percentage has been reported previously with this system, such as 75% immobilization achieved for acrylamidase in chitosan-coated alginate beads. 23Immobilization of HRP in Ca-alginate beads using glutaraldehyde was achieved with an immobilization of 87%. 33Reports indicate that the mechanical strength is higher in coated beads than in simple alginate beads. 23Additionally, the free hydroxyl groups in chitosan can react with other groups, which could explain the high levels of immobilization obtained.Alginate-Coated Chitosan Beads.Two approaches for immobilization were employed, with and without functionalization, and in both cases, no protein was detected after immobilization (Table 1).High immobilization percentage has been reported with this system, such as 75% immobilization achieved for acrylamidase in chitosan-coated alginate beads. 23mmobilization of HRP in Ca-alginate beads using glutaraldehyde was achieved with an immobilization of 87%. 33ports indicate that the mechanical strength is higher in coated beads than in simple alginate beads. 23Additionally, the free hydroxyl groups in chitosan can react with other groups, which could explain the high levels of immobilization obtained.
Chitosan Beads.The chitosan beads activated with glutaraldehyde provided a biocompatible support surface, leading to a maximum immobilization of 83.27% (Table 1).This result is in agreement with previous reports for this support. 34In this study, manganese peroxidase was immobilized onto glutaraldehyde active chitosan beads, and 81.4% of immobilization was achieved.Glutaraldehyde functionalization is an important step for enzyme immobilization, as during the reaction between glutaraldehyde with chitosan generation of aldehyde groups on the bead surface could lead to side reactions with amino groups of the enzyme. 35Additionally, it has been reported that chitosan beads could improve their mechanical resistance after functionalization due to the crosslinking of the polymeric chains of chitosan.In the present work, in the absence of glutaraldehyde, only 9.72% of the enzyme was immobilized, and therefore, the enzyme is likely mainly attached to the functional groups that glutaraldehyde provides and not to the support itself.
Biochar.In preliminary experiments (Table S3), the glutaraldehyde concentration and immobilization time were established.The immobilization of PbCDPS on the biochar systems ranged between 17.03% and 45.74%.Comparing the immobilization time of chitosan beads (8 h) with the biochar (0.5 h), it is apparent that in a short-time frame, high loadings were not achieved, probably since PbCDPS will be mostly immobilized on the surface of beads, whereas more surface areas are available with biochar and immobilization is completed with less time.Although PbCDPS was immobilized in the biochar systems, the precise mechanism underlying the adsorption is unknown.However, we hypothesize that the enzyme is supported in the biochar by van der Walls forces.Multiple enzymes have been successfully immobilized using this approach on different materials. 21,36,37For example, pepsin was immobilized on biochar obtained from pupuhna palm by  adsorption and covalent bonding.A high immobilization efficiency (>95%) was obtained, possibly due to the porosity and pore diameter of the biochar and low molecular weight (35 kDa) of the enzyme. 38o determine whether immobilized PbCDPS maintained activity after the immobilization process, a cutoff for immobilization efficiency was set to >30% (Figure 1(3)), and the activity assay for PbCDPS was performed only for the selected systems marked green in Table 2. PbCDPS trapped on alginate beads (PbCDPS alg beads ) exhibits greater catalytic potential compared to other beads systems under evaluation, as this system had the highest concentration of cHE product after the reaction.The high content of cHE produced with PbCDPS alg beads could be due to high porosity of alginate beads allowing necessary interactions between the PbCDPS and the aa-tRNA substrates. 39aa-tRNAs are large molecules (average molecular weight of ∼25 kDa), and therefore, production of cHE was highly affected by the porosity of the beads.However, adding glutaraldehyde to immobilization or coating alginate beads with chitosan resulted in a decreased product production.This could be due to glutaraldehyde acting as a cross-linking agent, as it was previously reported to provide a reinforcement to alginate beads through cross-linking within the matrix. 40Glutaraldehyde could also lead to protein crosslinking and interfere with active site availability.
In the case of PbCDPS chi beads glut , chitosan would be positively charged under the conditions employed, while tRNAs would be negatively charged, increasing the chance of electrostatic attraction between tRNA and the support.Glutaraldehyde concentration and biochar surface area could permit the immobilization of PbCDPS on the surface and not inside the material, and this immobilization would likely facilitate interactions between the enzymes and substrates participating in cHE production.
In the activity assays, a cascade reaction produces the final product cHE.If the aaRS enzymes (HisRS and GluRS) are free in solution, they will generate aa-tRNA products, which then must act as substrates for the entrapped enzyme.This reaction was confirmed by HPLC-MS (Figure S2).The PbCDPS alg beads system was improved by varying the concentration of PbCDPS in beads.Figure S3a shows that the maximum ratio of cHE/ PbCDPS concentration was achieved with 13.9 μg of PbCDPS, as larger amounts of immobilized PbCDPS did not translate to increased cHE formed.Similarly, in the case of the immobilization on biochar from spent coffee (Figure S3b), different loadings of PbCDPS were tested, and the maximum activity was obtained when 15 μg was employed.In the case of PbCDPS chi beads glut , the immobilization percentage was similar for all the loadings tested.Diffusional obstacles may be expected using larger enzyme loadings, and that could reduce the observed activity of immobilized protein. 41We hypothesize that this could be due to the large size of aa-tRNAs and the fact that aaRS enzymes are not embedded in the support, and therefore, aa-tRNAs would have to diffuse in and out of the support, decreasing the amount of product generated.
An RNA denaturing gel was run to determine whether the tRNA pool employed remains free in solution or is adsorbed to beads during and after the reaction in the three selected supports (see Figure S4).After the reaction, the tRNA pool concentration was higher in the supernatant than in the support.This could be due to the large molecular weight of tRNA, requiring large pores to enter into the beads or the biochar.Additionally, because tRNA was mostly free in solution, other proteins in the cascade also needed to remain free in order to complete the sequence of reactions leading to cHE.Only in the chitosan beads was the concentration of the tRNA pool similar in the beads and in the supernatant, likely due to electrostatic interactions between tRNA and chitosan.Additionally, protein gel electrophoresis was employed to test whether aaRS enzymes were free in solution or adsorbed to beads during and after the reaction.Figure S5 shows that HisRS and GluRS were present mainly in the supernatant of all systems.Consequently, a successful support cannot hinder contact between substrates and enzymes, and therefore, the physical properties of the support employed will play an important role in the selection of an ideal support.
Reusability of PbCDPS Immobilized.Reusability of immobilized PbCDPS was studied for the selected systems in batch reactions (PbCDPS alg beads ,PbCDPS ).The number of cycles in which immobilized PbCDPS retained activity is a marker of immobilization success.In PbCDPS alg beads , the amount of cHE detected increased as cycles progressed (Figure 33a).This could be due to the product entrapped in the support after each reaction cycle, so a quenching experiment was performed (Figure S6) comparing the product recovered after the system was completely quenched and when only the supernantant, free of beads, was recovered.Product yield was higher when the system was completely quenched until the fourth cycle.This could be due to bead saturation with the product.A similar experiment evaluating cHE product entrapment in the support was carried out for chitosan beads and biochar (Figure S6).The amount of product after the reaction was similar when the supernatant or entire system was analyzed.This was confirmed in the reusability experiments with biochar and chitosan (Figure 3a).In a similar study, Bilal et al. 24 studied the reusability of manganese peroxidase (MnP) immobilized onto chitosan observing that immobilized enzymes retained up to five cycles of their initial activity, whereas Santos et al. 38 reported an efficiency of 85% after seven cycles in the immobilization of pepsin on biochar.Immobilized laccase on biochar from wood maintained up to 80% of its initial activity after five cycles.
Co-Immobilization of PbCDPS and aaRS Enzymes.The production of the cyclic dipeptide involves a cascade reaction between three enzymes: two aaRS and the PbCDPS.We therefore aimed to immobilize the three enzymes on the same system.First, the two aaRS were immobilized on the selected systems (alginate beads, chitosan beads, and biochar from spent coffee).The activity of cHE was detected in the three systems when HisRS and GluRS were immobilized and PbCDPS was free in the reaction (Figure 3b).Then, the immobilization of the three enzymes (PbCDPS, HisRS, and GluRS) involved in the cascade reaction for the cyclic dipeptide production was carried out (Figure 3c).In the alginate beads, the amount of the cHE product was similar to when only PbCDPS was immobilized.In the case of biochar from spent coffee and the chitosan beads, cHE was obtained at lower amounts than when only PbCDPS was immobilized (Figure 3c).Alginate beads have been used successfully for the co-immobilization and recycling of other enzymes.Arana-Pena et al. 41 reported that in porous materials, co-immobilization could be troublesome, since some enzymes could be localized in the micropores and others on the surface of the material, causing mass transfer problems between the substrates and enzyme catalysts taking part in the reaction.
When the reusability of the co-immobilized enzymes was tested (Figure 3c), alginate beads were demonstrated to be a promising option for immobilization.However, after the third reaction cycle, enzymatic activity showed signs of decline.In this case, as previously observed, some cHE was trapped inside the beads after the first cycle.The findings presented in Figure S5 suggest that the decline in activity over the cycles could be attributed to challenges in mass transfer to and from the beads, rather than the loss of enzyme activity within the reaction.This is because tRNA remains outside the support structure after the reaction, making it more challenging to access the active sites of the enzymes involved.

■ CONCLUSIONS
In this investigation, the immobilization of PbCDPS leading to the production of the cyclodipeptide cHE was established.PbCDPS is involved in the biosynthesis of cyclic dipeptide natural products with pharmaceutical applications, requiring two large aminoacyl-tRNAs as substrates.Although immobilization has been used extensively, its application to enzymes involved in the biosynthesis of natural products is scarce.When free in solution, PbCDPS is an unstable enzyme, catalyzing a few turnovers before denaturation.Immobilization improved enzyme stability, as after seven reaction cycles, the enzyme maintained catalytic turnover.Additionally, we showed that co-immobilization of PbCDPS and other enzymes participating in the cascade reaction to produce cyclic dipeptides (HisRS and GluRS) could be performed.tRNA synthetases are widely employed in transcription/translation commercial kits and have extensive applications in molecular and chemical biology.Therefore, our work demonstrates the feasibility of enzyme immobilization, alone or in a cascade, when large complex substrates such as tRNAs are required.While the successful immobilization of PbCDPS represents a significant advancement in stabilizing enzymes for cyclic dipeptide synthesis, further enhancements in immobilization techniques are needed to optimize its efficiency.Additionally, exploring the scalability of this process for industrial applications holds immense promise.Advancing this approach toward industrial-scale implementation could revolutionize pharmaceutical production, offering a pathway for the efficient, sustainable, and large-scale synthesis of cyclic dipeptide natural products with diverse pharmaceutical applications.
FTIR spectra for different supports employed for immobilization, standard curve for cHE quantification, details on biochar particle size and immobilization efficiency, gels showing location of proteins and tRNA during the PbCDPS catalyzed reaction, and effect on different quenching strategies in calculating cHE yield (PDF)

■ AUTHOR INFORMATION
Corresponding Author

Figure 1 .
Figure 1.Workflow describing the evaluation and prioritization of different immobilization strategies.(1) Biochar production from spent coffee, macroalgae, polystyrene, wood, and distillery waste; (a) PbCDPS immobilization on biochar through adsorption; and (b) covalent bonding.(2) Process for PbCDPS immobilization in beads: (c) Ca-alginate beads, (d) alginate coated-chitosan beads, and (e) chitosan beads.Prioritization of the different immobilization strategies used for PbCDPS; starting with two different types of supports, the immobilization was made through entrapment, adsorption, and covalent bonding.Supports with more than 30% of immobilization were selected for the measurement of product formation (cHE), and reusability was tested only for the supports with the higher concentration of cHE.

Figure 3 .
Figure 3. (a) Comparison of reusability of PbCDPS immobilized in Ca-alginate beads by entrapment, chitosan beads by covalent bonding, and biochar from spent coffee by covalent bonding with the free PbCDPS.(b) Comparison of reusability for the co-immobilization of PbCDPS, HisRS, and GluRS in alginate beads by entrapment, chitosan beads by covalent bonding, and biochar from spent coffee by covalent bonding.(c) Co-immobilization of HisRS and GluRS by entrapment in alginate beads, covalent bonding in chitosan beads, and covalent bonding in biochar from spent coffee.All the experiments were performed in triplicate, and all data are reported average ± SEM.Each color represents an additional cycle for the cHE production.

Table 1 .
Immobilization of PbCDPS on Biochar from Polystyrene and Wood Waste, Macroalgae, Spent Coffee, and Distillery Waste and in Beads from Alginate, Alginate-Coated, and Chitosan a a Rows in green highlight the accepted systems.

Table 2 .
cHE Production from PbCDPS Was Immobilized Using Different Strategies a a Rows in green are successful immobilization systems.