Enzyme-Catalyzed Synthesis of Glycerol Carbonate in Solventless Liquid One Phase Conditions: Role of Reaction Medium Engineering on Catalytic Performance David Gonzalez-Miranda, Diego Carballares, Tomás Pedregal, Roberto Fernandez-Lafuente, Miguel Ladero,* and Juan M. Bolivar* Cite This: Ind. Eng. Chem. Res. 2023, 62, 15798−15808 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: This study pursues the efficient synthesis of glycerol carbonate (GC) using immobilized lipases on octyl agarose by means of interfacial activation under solventless conditions. The monophasic system formed between glycerol and ethylene carbonate at elevated temperatures displays low viscosity and minimizes mass transfer hindrances, which represent promising novel conditions in this biocatalytic process. Immobilized lipases from Candida rugosa and Thermomyces lanuginosus on octyl agarose were identified as suitable catalysts. While thermally denatured biocatalysts lost their catalytic capacity completely, biocatalysts with the catalytic Ser irreversibly inhibited showed significant enzymatic activity, suggesting that the catalytic Ser may not play a key role in the reaction. The influence of various operational variables and conditions: temperature, catalyst concentration, and reaction setup (2 mL vials and 100 mL round-bottom flasks with magnetic stirring were used) were studied with the aim of achieving high conversions (>99%) and yields (>99%) in short reaction times. Among the tested lipases, immobilized C. rugosa lipase exhibited the highest turnover numbers. The use of immobilized lipases allowed for multiple reaction cycles without a significant decrease in the initial reaction rate or the final conversion, highlighting the potential for catalyst reuse. This research achieved remarkable results in terms of reaction productivity, product concentration, atom efficiency, and turnover number compared to any previous study, emphasizing the significance of reaction engineering approaches in developing efficient sustainable processes for synthesizing valuable chemicals. The study emphasizes the potential of lipases as catalysts for GC synthesis and underscores the importance of considering monophasic solventless conditions at high temperatures for improved reaction efficiency and high reaction atomic efficiency. ■ INTRODUCTION The shift toward renewable feedstocks in the chemical industry, as part of the circular bioeconomy and sustainable development goals, has led to the promotion and implementa- tion of platform chemicals or building blocks.1,2 Biorefineries, which can be created through thermochemical and biochem- ical routes, are being used to produce purer mixtures of products such as polyphenols, triglycerides, phospholipids, proteins, and monosaccharides in the C3−C6 range.3−5 The increasing production of biodiesel has resulted in the increased production of glycerol, a C3 platform chemical that accounts for 10% of the original mass of triglycerides in oils and fats.6 While glycerol is used in various industries, its total production can be reduced by integrating it into the final diesel product, producing ecodiesel.7−9 Additionally, sustainable bio-/chemo- processes can be used to upgrade glycerol and produce valuable products.6,10−13 Glycerol carbonate (GC) is a highly valuable product derived from glycerol, considered a “green chemistry” product due to its numerous benefits in various fields.12,14−17 The transesterification of glycerol with alkyl or cyclic carbonates is a useful method for producing GC. Among these methods, the reaction of glycerol with dimethyl carbonate is commonly used.12,15,16 As an alternative, the reaction with ethylene carbonate (EC) offers high substrate reactivity, producing valuable byproducts like ethylene glycol.15,18−20 To maximize product concentration, conversion, productivity, and atom efficiency, and to minimize excess carbonate, solvent waste, and subproduct formation (high selectivity), careful catalyst design and reaction medium engineering are necessary.15,18−20 Received: June 25, 2023 Revised: September 10, 2023 Accepted: September 12, 2023 Published: September 26, 2023 Articlepubs.acs.org/IECR © 2023 The Authors. Published by American Chemical Society 15798 https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 This article is licensed under CC-BY 4.0 D ow nl oa de d vi a U N IV C O M PL U T E N SE D E M A D R ID o n N ov em be r 6, 2 02 3 at 1 8: 37 :0 7 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. https://pubs.acs.org/action/doSearch?field1=Contrib&text1="David+Gonzalez-Miranda"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Diego+Carballares"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Toma%CC%81s+Pedregal"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Roberto+Fernandez-Lafuente"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Miguel+Ladero"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Miguel+Ladero"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Juan+M.+Bolivar"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.iecr.3c02128&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?goto=articleMetrics&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?goto=recommendations&?ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?goto=supporting-info&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=tgr1&ref=pdf https://pubs.acs.org/toc/iecred/62/39?ref=pdf https://pubs.acs.org/toc/iecred/62/39?ref=pdf https://pubs.acs.org/toc/iecred/62/39?ref=pdf https://pubs.acs.org/toc/iecred/62/39?ref=pdf pubs.acs.org/IECR?ref=pdf https://pubs.acs.org?ref=pdf https://pubs.acs.org?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as https://pubs.acs.org/IECR?ref=pdf https://pubs.acs.org/IECR?ref=pdf https://acsopenscience.org/researchers/open-access/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ In particular, reaction medium engineering is crucial to minimize solvent usage and maximize product concentration during GC production.18,19 The thermomorphic properties of the ethylene carbonate-glycerol mixture offer an advantage since the mixture transitions from monophasic to biphasic systems depending on the temperature. To perform this process in the biphasic regime, which takes place below 76 °C, requires cosolvents that can dissolve both substrates, as widely reported in the literature, employing both chemical19−22 and biological catalysis.3,13,23−29 Enzyme catalysis has been ex- plored in these cosolvent-based monophasic systems at temperatures under 76 °C but, mainly, the researchers employed dimethyl carbonate as a reagent.3,13,23−29 This approach proceeds with long reaction times (>24 h) and produces only moderate concentrations of GC (<1 M). Above 76 °C, a monophasic regime is reached, allowing the utilization of solventless systems that enable high substrate concen- trations and an intense substrate−catalyst contact.20,21 Although the monophasic regime has been studied for noncatalytic thermal reactions above 100 °C,20 there are no reported examples at the threshold of the monophasic phase regime (76−80 °C), where no thermal reaction exists.20 High selectivity of the reaction avoiding the accumulation of reaction intermediate and subproduct formation in the presence of EC excess is also of crucial importance.18,20,21 The combination of exquisite selectivity of lipases and the solventless process is therefore an unexplored strategy. In this promiscuous reaction of transcarbonation, lipases have been the utilized enzymes.30−33 Lipases are versatile biocatalysts that can catalyze various reactions, making them useful in many industries, including energy, fine chemistry, food technology, and pharmaceutical production.30,31,34−38 The harnessing of the biocatalytic potential and monophasic regimes requires high enzyme performance under the high temperatures used, which can cause enzyme inactivation. Therefore, it is necessary to utilize biocatalysts that can remain active under these harsh conditions. Lipases are interfacial enzymes with a unique catalytic mechanism called interfacial activation, which involves a polypeptide chain that can isolate or expose the active center to the medium production.31,34−36 The large hydrophobic pocket in lipases open form allows them to become adsorbed to the substrate droplets and to act at the oil−water interface3636. Although lipases can be used in their free form, immobilized lipases have advantages such as easy recovery and reuse, and possibility of using various reactor configurations.29,30,39,40 However, an appropriate immobiliza- tion protocol is necessary to improve enzyme selectivity, specificity, activity, and response to inhibitors and harmful chemicals.41−43 Controlled immobilization protocols are essential to achieve the full benefits of immobilization, as an unsuitable immobilization protocol can lead to a biocatalysts with even worse performance than that of the free enzyme.43− 48 In this paper, we have selected lipases immobilized by means of its interfacial activation on octyl-agarose31,36 due to the multiple advantages of this immobilization method. First of all, the lipases immobilization by its interfacial activation on the hydrophobic surface of the support permits in a single step to immobilize, purify, stabilize, and have the open and monomeric form of the enzyme.31,36,49 This latter feature enables a clear exposition of the enzyme active site to the medium; that way, the immobilized enzymes can interact with substrates, covalent inhibitors, etc. (in fact, the increase of the covalent inhibition rate of the lipases immobilized in this way was used as a proof of the mechanism of immobiliza- tion).31,36,49 Additionally, the enzyme immobilization on octyl-agarose has been described to produce significant stabilization of the lipases,31,36 this stabilization may be a key point in the performance of the enzymes under the high temperatures required to have a monophasic solvent free system, and we expect that can be enough to permit the utilization of lipases in this kind of systems. Moreover, glycerol has been described as an enzyme, and lipase, stabilizing compound,50,51 and its use as a substrate can also present some positive effects. Another advantage of this immobilization system is that it guarantees that the enzyme is immobilized in a monomeric form. Among the utilized lipases, lipase B from Candida antarctica is a lipase bearing a small lid, unable to isolate the active site from the medium even in its closed form.52,53 However, CaLB is a lipase able to act in the interface of the drops of their natural substrates (oils and fats) previous adsorption on the substrates drops.54−59 That way, CaLB is also able to become adsorbed to most hydrophobic supports,56,60−66 even bearing a small lid, because the full hydrophobic pocket surrounding the active center is quite large. Lipases have been described to have a strong tendency to give bimolecular aggregates involving two open form of the lipases, leaving in many instances partially blocked active centers, and altering the enzyme features.52−56 In this case, CaLB is an exception, the lid is too small to give a large enough hydrophobic surface to immobilized other lipase molecule.67 These interactions may also rise with other hydrophobic components (e.g., hydrophobic proteins) of the lipase extract, making complex the understanding of the crude enzyme features.68−70 The objective of this work is to investigate and report a biocatalytic method to produce GC by studying the catalytic potential of different lipases under solvent-free monophasic conditions. A key aspect of this study is the emphasis on reaction medium engineering, specifically working with a liquid phase system enabled by tunable temperature-dependent phase behavior. Additionally, the utilization of lipases immobilized on octyl-agarose, known for their high stability, further enhances the overall performance. Through this research, the focus is on achieving not only high yield and productivity but also maintaining total selectivity, paving the way for an efficient and sustainable approach to GC synthesis. ■ MATERIALS AND METHODS Materials. Different commercial lipase solutions were used: Palatase 20,000 L (lipase from Rhizomucor miehei, RML, 2.9 mg of protein/mL),71 Lecitase Ultra (a chimeric phospholi- pase, LeU, 21.85 mg of protein/mL),72 Lipozyme B (lipase B from C. antarctica, CaLB, 5.57 mg of protein/mL),62,73 NovoCor ADL (lipase A from C. antarctica, CaLA, 7.63 mg of protein/mL),74 Lipozyme TL (lipase from Thermomyces lanuginosus, TLL, 14.72 mg of protein/mL),75 Eversa Trans- form 2.0 (lipase produced by the evolution of the lipase from T. lanuginosus, ETL, 27.50 mg of protein/mL),33,50,76 and Novozym 435 (commercial immobilized CaLB, N435)77 were purchased from Novozymes (Spain). Lipomod 34MDP (powder) from Candida rugosa (containing 3.3% of protein), CRL38 was obtained from Biocatalysts Inc. (UK), Amano (lipase from Pseudomonas fluorescens, PFL, containing 13.2% protein) was obtained from Sigma-Aldrich. All protein concentrations were determined by the Bradford method.78 Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15799 pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as The list of other materials and chemical use can be found in the Supporting Information. Methods. Biocatalyst Preparation and Characterization. Determination of the Enzyme Activity. The hydrolysis of p- nitrophenyl butyrate (p-NPB) was used as a standard hydrolytic assay to characterize free enzyme preparations, follow immobilization, parametrize their results, and stand- ardize the obtained immobilized enzymes. It was carried out as previously described79 with some modifications (Supporting Information). One U of activity is defined as the amount of enzyme able to produce one μmol of p-nitrophenol per minute under the previously mentioned conditions. Enzyme Immobilization. Immobilization of the enzymes on octyl-agarose and octyl-agarose activated with divinyl sulfone was carried out as reported previously with slight modifications described in the Supporting Information.41,79 Maximum loading was pursued. Enzyme immobilization was followed by protein/activity balance following reported immobilization procedures80 and reported protocols for ETL,50 CRL,81 TLL,82 PFL,79 RML, LeU, CaLA, and CaLB.82 Data of the mass of the solid carrier are expressed in wet matter unless mentioned otherwise. The protein loading and the measured specific activities of the different immobilized enzyme preparations referred to the p-NPB hydrolysis are shown in Table S1. Irreversible Inhibition of Immobilized Lipases by D-pNPP. The immobilized enzymes were submitted to irreversible inhibition to erase their natural activity based on the covalent blocking of the catalytic serine. For that purpose, diethyl p− nitrophenyl phosphate (D-pNPP) was used. D-pNPP is a lipase irreversible inhibitor, specifically reacting with the catalytic Ser.83 It was performed following reported procedures with slight modifications.84,85 Different lipase-immobilized preparations (1 g) were suspended in 20 mL of 100 mM sodium phosphate buffer solution at pH 7.0 and 25 °C. D- pNPP was added stepwise every 30 min (adding new amounts of solid D-pNPP to reach a concentration of 10 mM each time). Samples of biocatalyst suspensions were withdrawn periodically after each addition, and their activities were measured using the p-NPB assay until no activity was found. Experimental Setup of the Transcarbonation Reaction. Reaction of Transcarbonation in a Thermal Mixer. The reaction of transcarbonation to evaluate the different lipases was monitored using a thermal mixer (Eppendorf Thermo- mixer) at 76, 78, or 80 °C and 800 rpm (glycerol: ethylene carbonate = 1:2). Glycerol (660 mg) and the indicated amount of catalyst (w/w) were poured inside a 2 mL vial and submitted to ultrasound with a probe sonicator (Fisherbrand 505 Sonicator with Probe) for 15 min. Afterward, the mixture was introduced into the thermal mixer until the desired temperature was reached. The stabilizing effect of glycerin should preserve the enzyme activity in this process.34 1.29 g of ethylene carbonate previously heated under the reaction condition was added to start the reaction. The initial concentration of the glycerol, which is the limiting reagent, is set at 4.76 M. Samples of 0.1 mL of the initial and 24 h reaction mixture were taken in duplicate and diluted 25 times in an 8 g/L citric acid solution, then filtered, and prepared for HPLC analysis. The data shown are the averages of multiple experiments performed (N ≥ 3). In parallel, a control reaction without a catalyst was carried out. The same reaction setup was used to evaluate the reaction background of the liquid medium containing commercial lipases. Commercial lipases (200 μL) were filtered with centrifugal filters of nominal molecular weight limit (NMWL = 10,000 Da) while centrifuging at 13,300 rpm for 20 min. The reaction was then compared by utilizing the same equivalent amount of the retained fraction (lipase) and the filtered fraction (liquid medium) of each one of the enzymes as catalysts. Control reactions using the support without enzyme were also carried out. Reaction of Transcarbonation in a Round-Bottom Flask. A round-bottom flask (100 mL) was utilized as the recipient of the reaction (glycerol: ethylene carbonate = 1:2). This was submerged inside a beaker (250 mL), located on a heating plate, at 78 °C and 800 rpm, until the reaction temperature was reached. The round-bottom flask, containing glycerol (9 g) and the indicated amount of catalyst (0.80 wet g, 4.52 wet g, and 5.32 wet g) was previously ultrasounded in a sonicator with probe (Fisherbrand 505 Sonicator with Probe) for 15 min. 17.6 g of ethylene carbonate previously heated to the reaction condition was added to start the reaction under strict control of the temperature. The reactor temperature was continuously monitored and controlled. Initial glycerol concentration, which is the limiting reagent, was 4.76 M. An initial sample was taken, and then, at different reaction time values (1, 4, 7, 9, 21, and 24 h), new samples were withdrawn in duplicate and diluted 25 times in an 8 g/L citric acid solution and then filtered and prepared for HPLC analysis. In parallel, the same reaction was monitored without the catalyst as a negative control. The data shown are the average of multiple performed experiments (N ≥ 3). Biocatalysts Recovery. After the first use, the content of the reaction was vacuum filtered and washed up with Milli-Q several times and the catalyst was collected. Once we knew the amount of catalyst that we had lost by difference of weighing, we recalculated the amount of substrates to be added to the flask to maintain the same concentration inside the reaction flask. Glycerol was then poured into the flask with the catalyst and submitted to ultrasounds with a probe sonicator (Fisherbrand 505 Sonicator with Probe). Then, the flask was set at the desired temperature with adequate stirring, and the reaction was monitored since ethylene carbonate was poured. This procedure was repeated throughout the next experiments. Analytical Methods. Reaction samples were diluted in a 7.68 g/L citric acid solution, used as an internal pattern, and their composition was determined by high-performance liquid chromatography (HPLC) using a modular device (Jasco PU- 2089, AS-2059, CO-2060, RI-2031, and MD-2015), employing a REZEX ROA-Organic Acid H+ column and as mobile phase 0.005 N H2SO4 prepared in Milli-Q water using a flow rate of 0.5 mL/min at a temperature of 60 °C. Following the oven, a refractive index detector (RID) at 55 °C is used to detect the different compounds. The retention times were as follows: citric acid, 10.4 min; glycerol, 16.7 min; GC, 19.8 min; ethylene glycol, 20.8 min; and ethylene carbonate, 28 min. ■ RESULTS AND DISCUSSION Catalytic Performance of Free Lipases in Solventless Conditions for GC Formation. Initially, the feasibility of a solventless process was evaluated using free lipases with a molar substrate ratio of 1:2 (Gly/EC) following the procedure described in the Methods Section. The results of this experiment are summarized in Table 1, which shows the production of GC (M) after 24 h. All the preparations of the lipases tested in this study produced GC, but with varying yields. The lipase preparation from ETL showed the highest production, followed by TLL Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15800 https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as and LeU, with TLL producing 2.72 M at 78 °C and 3.03 M at 80 °C (57.1 and 63.7% yield of GC, respectively) and LeU producing 2.82 M at 78 °C and 2.77 M at 80 °C (59.2 and 58.2% yield of GC, respectively). Conversely, the lipases from C. antarctica produced lower yields of GC, regardless of the temperature. Based on the results of this evaluation, the ETL extract was found to be the most effective lipase extract for the formation of GC. This study is the first to demonstrate the use of free lipase extracts under monophasic solvent-free reaction conditions for the formation of GC. Previous studies in the literature have used free lipases extracts in transcarbonation reactions with both ethylene carbonate and dimethyl carbonate in the presence of cosolvents, resulting in low yields (42% for GC) after 24 h.23 To investigate whether the observed activities are compro- mised by components in commercial lipase preparations, an additional experiment was conducted. The complex media composition of the commercial preparations made it necessary to design an experiment that could eliminate the possibility of other components contributing to the observed GC production. Centrifugal filtration (10 kDa cutoff) was used to obtain enzyme-free solution, what we called permeate (it was checked a lack of activity vs p-NPB), which was then added to the transcarbonation reaction in an amount equivalent to that shown in Table 1. Samples were taken after 24 h, and the production of GC was recorded in Table 1. The results demonstrated that the permeate fractions of the lipase extract solutions had a significant contribution to the observed transcarbonation reaction. These findings suggest that some of the components of commercial lipase solutions, including inorganic or organic compounds, might act as catalysts of the transcarbonation reaction.19,21 Therefore, immobilization of the enzymes seems to be a reasonable way to study the reaction without the artifacts identified when using free enzymes. Immobilization would allow for the removal of all of the compounds present in the lipase suspensions through deep washing of the immobilized enzymes. Catalytic Performance of Enzyme-Catalyzed GC Formation in Solventless Heterogeneously Catalyzed Reactions. A new evaluation was conducted using immobi- lized lipases. For comparison, the commercial immobilized lipase Novozym 435 was used as a benchmark biocatalyst. Temperature conditions were explored at 78 and 80 °C, to work in the threshold of the monophasic regime, and the reaction was performed on a 1 mL scale with orbital shaking and a molar ratio of 1:2 (Gly/EC) in a thermal mixer. The amount of catalyst was set to 3% (w/w), expressed in the wet weight of the solid support. The results are shown in Figure 1. Immobilized CRL surpassed all of the other catalysts [1.85 M GC at 78 °C (38.9% yield)], followed by TLL [1.04 M GC at 78 °C (21.8% yield)] and ETL [0.92 M GC at 78 °C (19.3% yield)]. CaLB preparations displayed low levels of GC production. The increase in temperature to 80 °C produced a significant increase in the product yields, suggesting good stability of the biocatalysts under the reaction conditions. The GC production of all other enzymes stayed under 1.00 M at the two studied temperatures. To the best of our knowledge, this is the first example of the application of immobilized lipases under monophasic solvent-free reaction conditions for this reaction, since in previous literature studies, lipases were studied for the transcarbonation reaction with both ethylene carbonate and dimethyl carbonate in the presence of cosolvents (Table S2). The stability of enzymes under relatively harsh reaction conditions is crucial for this reaction. ETL, a variant of TLL designed for biodiesel production,86 is already a stable enzyme, and it is reasonable that their catalytic capacities are similar to that of the parent enzyme. Both enzymes are thermostable, and studies have reported that ETL is even more stable, which favors its performance in this reaction.50 CRL produced a higher yield of GC, and it has been reported that its thermal stability increases in polar solvents,34,87 which may explain its good performance in these reaction conditions. Additionally, its immobilization on octyl agarose has been reported to produce very stable biocatalysts of lipases, as the open and adsorbed form of the lipase is more stable than the enzyme in open/close equilibrium.31 These three catalysts were chosen for further studies. Assessment of the Biocatalytic Nature of the Formation Reaction of GC. To assess the role of the active center of the enzymes in the catalysis of the reaction, CRL, ETL, and TLL covalently immobilized on octyl-vinyl sulfone Table 1. GC Formation (M) in the Reactions Catalyzed by Free Lipases under Solventless Conditionsa GC concentration (M) free 78°C free 80°C permeate 78°C CaLA 1.25 1.24 0.67 CaLB 2.07 2.37 Nd ETL 4.74 4.97 4.20 TLL 2.72 3.03 0.89 LeU 2.82 2.77 0.54 aGlycerol initial concentration was 4.76 M at a molar ratio glycerol: ethylene carbonate of 1:2 and a catalyst concentration of 3% w/w (weight of wet support/weight of reactants). Temperatures of free enzymes studied: 78 and 80 °C. Figure 1. Formation of GC using different immobilized lipases on octyl-agarose, lipases A and B from C. antarctica (CaLA and CaLB); Eversa Transform lipase (ETL), lipase from T. lanuginosus lipase (TLL); C. rugosa lipase (CRL), Lecitase Ultra lipase (LeU), P. fluorescens lipase (PFL), and R. miehei lipase (RML), and commercial immobilized lipase Novozym 435 (N435) at different temperatures (78 °C in orange and 80 °C in green) and 800 rpm. Glycerol initial concentration was 4.76 M at molar ratio glycerol/ethylene carbonate = 1:2. Sampling was performed 24 h after the beginning of the reaction. Catalyst concentration was established at 3% w/w (weight of wet support/weight of reactants). Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15801 https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig1&ref=pdf pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as supports (to prevent enzyme release while maintaining the interfacial activation as the primary cause of immobilization) were subjected to thermal denaturation and selective irreversible covalent inhibition procedures to erase their activity. Using thermally inactivated enzymes, no GC formation activity was observed for all lipases. These results suggest that the specific conformation of the lipase structure plays a fundamental role in catalyzing the reaction. To analyze the involvement of the catalytic Ser in the reaction, we covalently inhibited the biocatalysts Ag-octyl- CRL, Ag-octyl-ETL, and Ag-octyl-TLL by D-pNPP, irrever- sibly blocking the active site. This was chosen instead of inhibiting free lipases, as it has been reported that it guarantees full inactivation of the immobilized enzyme.84,85,88,89 The inhibited catalysts were fully unable to hydrolyze p-NPB. The inhibited catalysts were then used in the transcarbonation reaction, and Figure 2 shows that while the yields of GC were significantly decreased for CRL (from 1.17 to 0.02 M), the product formation was inhibited only by 50% in the case of TLL and ETL. Therefore, blocking the catalytic Ser decreased the activities of the different enzymes in this reaction but did not eliminate them, confirming that this Ser is not directly responsible for this biocatalyst activity. Similar experimental procedures and observations have been reported for other lipases49,84,90 and could be explained by the existence of an alternative group of the enzyme able to catalyze this promiscuous reaction.49,84,91,92 Under the solventless and high-temperature conditions assayed, the possibility of some catalytic groups with activity dependence on the enzyme structure (as the activity is lost after heating) cannot be discarded. The fact that the activity is decreased in all the enzymes suggests that even not being the Ser related to the catalytic activity, some near group could be responsible, and this group exits in two very unrelated lipases (perhaps some group of the catalytic triad). Further studies, such as protein engineering through site-directed mutagenesis to remove specific amino acids (catalytic Ser, Asp, or His of the catalytic triad), substrate engineering, and computational studies based on molecular dynamic analyses, would be of interest to further investigate the exact mechanism, but this exceeds the target of this paper, and only the produced enzymes were available in this research, not the genes. Scaled-Up Biocatalytic Synthesis of GC by Means of Transcarbonation Reaction Using Immobilized Lipases. The biocatalytic production of GC was conducted in a stirred scaled-up round-bottomed flask at 80 °C and 800 rpm for 24 h using the three best lipase biocatalysts. To minimize external mass transfer resistances, stirring conditions were suited. The time courses formation of GC were analyzed over time and the results, depicted in Figure 3, demonstrate an increased production of GC with a GC formation yield and glycerol conversion of up to 99% after 24 h. The obtained results exceed those achieved on a small scale under orbital shaking, likely due to better mixing in this reactor performance. Notably, a slight acceleration of the reaction was observed after a lag phase. This lag phase was attributed to the time required for the ethylene carbonate impregnation of the porous particles initially filled with glycerol. Obviously, only when ethylene carbonate is inside the biocatalyst particle can the reaction start, and only after reaching the enzyme saturation concentrations can the biocatalyst give the maximum reaction rate. This issue could be addressed with the enhancement of internal mass transfer provided by catalyst engineering and new reactor technologies (e.g., microreactor technology).93−95 There is no evidence of reaction deceleration due to catalyst inactivation as the reaction continued to proceed after 24 h with only a slight concavity in the data curve. Based on this promising activity and stability of the catalyst, the formation of the product was intensified by increasing the catalyst concentration. With that purpose, reactions containing 22.5− 45.04% w/w (weight of wet support/weight of reactants) were implemented. To minimize the amount of water introduced in the system (agarose is a very hygroscopic material) and to Figure 2. Formation of GC using different inhibited immobilized lipases on octyl-agarose. The figure shows the comparison in the final concentration of GC between noninhibited immobilized lipases (orange on the left) and inhibited immobilized lipases (green on the right). Operation conditions: 78 °C and 800 rpm; the initial concentration of glycerol was 4.76 M. Molar ratio glycerol/ethylene carbonate = 1:2. Catalyst concentration was established at 3% w/w (weight of wet support/weight of reactants). Figure 3. GC formation employing enzymatic catalysis using lipases. (Lipase from C. rugosa (CRL), Eversa Transform 2.0 (ETL) lipase from T. lanuginosus (TLL. Operation conditions: 78 °C and 800 rpm, the initial concentration of glycerol was 4.76 M. Molar ratio glycerol/ ethylene carbonate = 1:2. Catalyst concentration was established at 3% w/w (weight of wet support/weight of reactants). Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15802 https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig3&ref=pdf pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as facilitate the initial access of the reactive mixture into the porous particles, a previous treatment of the catalyst in glycerol was carried out to replace the intrapore water by glycerol as described in the Methods Section. Results are shown in Figure 4. Increasing the catalyst concentration resulted in a significant improvement in the reaction rate, with conversions of 72.7% for CRL, 36.1% for ETL, and 26.3% for TLL achieved after just 2 h. After 6 h, almost quantitative conversion (higher than 99%) and GC yield (higher than 99%) were obtained for CRL, maintaining excellent selectivity. However, the use of 45% w/w (weight of wet support/weight of reactants) resulted in significantly lower performance in terms of conversion, which may be attributed to an underperforming stirring under conditions causing an inadequate dispersion of the solid biocatalyst in the liquid phase. Additionally, the high hygroscopic character of the solid carrier might cause an increase in the presence of water not fully replaced by glycerol. This is manifested not only in the conversion−time profile obtained but also in the initial reaction rate (Table 2). CRL enabled a higher conversion compared to ETL and TLL. In terms of volumetric productivity activity (μmol/min/mL) and specific productivity (μmol/min/mg protein), Table 2 shows the initial reaction rate at 22.52 and 45.04% of wet catalyst. CRL shows a better performance at 22.52% compared to a wet catalyst concentration of 45.04% due to plausible mass transfer limitations. Limitations in batch studies at high enzyme loads could be addressed both from biocatalyst engineering (internal mass transfer resistances) and from reactor engineering to mitigate mixing efficiency. For example, continuous process development using (micro)reactor technology, which would be beneficial for the future work on intensified process design.93− 95 Based on the results presented in Figures 3 and 4, no catalyst inactivation was observed in a single batch reaction. To investigate the potential for catalyst reuse, several repeated batches of the reaction with varying catalyst reuses were performed. Figure 5 displays a representative series of these experiments. While some degree of data scattering was observed, it is likely due to accidental mixing control issues (e.g., experiment 2) rather than catalyst inactivation. The results suggest that the biocatalyst can be reused at least three times without a significant decrease in the initial reaction rate or final conversion and GC yield. Comparative Analysis of the Catalyst Productivity and Catalyst Efficiency. To situate the results of the formation of GC reported in this paper with other results of enzyme catalysis in this reaction under other conditions,23− 29,96−104 we made an effort to extract the literature data (reaction time, conversion, and catalyst concentration) and compiled it in Table S2. The resulting data on reaction productivity, final product concentration, and turnover number are presented in Table S3. Reaction productivity is defined as moles produced per unit of time and it shows both scale and reaction rate. Dimensionless turnover number (TN) is shown in Table S3 to inform about the number of catalytic turnovers per active site. It was calculated referring to one reaction cycle once known the molecular weight of the enzymes.50,105 As it can be seen in the tables, most of the previous biocatalytic attempts of GC formation lie in the use of a large excess DMC Figure 4. GC formation employing enzymatic catalysis using lipases [lipase from C. rugosa (CRL), lipase Eversa Transform 2.0 (ETL), and lipase from T. lanuginosus (TLL) for both panels]. Panel A exhibits a reaction course with a 22.52% w/w (weight of wet support/weight of reactants) and panel B with a 45.04% w/w (weight of wet support/weight of reactants). Operation conditions: 78 °C and 800 rpm, the initial concentration of glycerol was 4.76 M. Table 2. Initial Reaction Rate and Average Reaction Rate of the Transcarbonation Reaction from Glycerol to GC Using Different Lipasesa r0 (mol·h−1·L−1/μmol·min−1·mg−1) r (mol·h−1·L−1/ μmol·min−1·mg−1) lipase 3% wet catalyst 22.52% wet catalyst 45.04% wet catalyst 3% wet catalyst 22.52% wet catalyst 45.04% wet catalyst CRL 0.08/2.67 1.73/7.68 1.307/2.88 0.09/1.66 0.83/2.058 0.83/1.02 ETL Nd 0.86/3.82 0.95/2.11 0.03/0.55 0.72/1.77 0.72/0.88 TLL Nd 0.62/2.75 0.73/1.62 0.01/0.28 0.59/2.18 0.75/1.39 aNote: C. rugosa lipase, (CRL), Eversa Transform lipase, (ETL), and Lypozyme TL 100 L (T. lanuginosus lipase), (TLL). Reaction conditions: 78 °C and 800 rpm. The initial reaction rate was calculated from the obtained concentrations at 2 h and the average reaction rate was calculated at 6 h from the beginning of the reaction. Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15803 https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig4&ref=pdf pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as as the carbonate source, temperatures between 50 and 70 °C, and long reaction times. Since high cosolvent concentrations are used, concentrations of GC lower than 1 M are obtained even at high conversions. The only previous attempt of a lipase-catalyzed reaction using EC also leads to lower product concentration and significantly lower reaction productivity than the results reported in this paper. As for immobilized CRL, regardless of the amount of catalyst used, TN values are the highest compared to their analogous values for ETL and TLL, which means a more efficient reaction. Immobilized ETL presented slightly higher values than TLL. Immobilized ETL and TLL decreased TN values when the amount of catalyst increased to a certain value, and the efficiency was lost when a larger amount of catalyst is used. Reaction intensification expressed in terms of productivity and product concentration reported in this paper is also accompanied by TN of 105, the highest reported to the best of our knowledge. Finally, we have calculated the E factor (for details see the Supporting Information), resulting in valued <1 for the lab-scale intensified reaction (calculated for 1 batch reaction and all the catalyst as waste). ■ CONCLUSIONS In conclusion, immobilized lipases on octyl agarose by interfacial activation have been shown to be efficient catalysts to synthesize GC under solventless conditions. The use of a thermally induced single liquid phase and the optimization of reaction medium engineering parameters, such as temperature, catalyst concentration, and reactor setup, have allowed high conversions and yields to be achieved with short reaction times. The obtained turnover numbers are among the highest reported for enzyme-catalyzed reactions, especially for CRL at a high atom efficiency. These results highlight the potential of sustainable chemistry and engineering approaches for the development of efficient and environmentally friendly processes to synthesize value-added chemicals, such as GC. Besides catalyst design and reaction medium engineering, substrate, reactor, and process engineering are crucial for the development of an efficient and sustainable process.93−95,106 In that manner, reactor engineering (e.g., continuous flow technology) and process integration (involving downstream) are interesting future venues for the current biocatalytic system. ■ ASSOCIATED CONTENT *sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128. Details of materials used, details of methods used, enzyme activity, enzyme immobilization, determination of reaction metrics, characterization of immobilized enzymes, literature summary of the catalyst and reaction conditions, and literature survey of reaction and catalyst performance (PDF) ■ AUTHOR INFORMATION Corresponding Authors Miguel Ladero − FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid 28040, Spain; orcid.org/0000-0002-9146-3830; Email: mladerog@ ucm.es Juan M. Bolivar − FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid 28040, Spain; orcid.org/0000-0001-6719-5082; Email: juanmbol@ ucm.es Authors David Gonzalez-Miranda − FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid 28040, Spain; orcid.org/0000-0001-5381-9325 Diego Carballares − Departamento de Biocatálisis, ICP-CSIC, Madrid 28049, Spain Tomás Pedregal − FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid 28040, Spain Roberto Fernandez-Lafuente − Departamento de Biocatálisis, ICP-CSIC, Madrid 28049, Spain; orcid.org/0000-0003- 4976-7096 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.iecr.3c02128 Author Contributions DGM, DC, and TP performed the experiments, and RFL, ML, and JMB designed and supervised the research and secured funding. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Funding J.M.B. acknowledges funding from the Government of Community of Madrid (2018-T1/BIO-10200) and from UCM-Santander (PR108/20). Support from MINECO and AEI through projects CTQ2017-84,963-C2-1-R, PCI2018- 093,114, and PID2020-114365RB-C21 is gratefully acknowl- edged. RFL acknowledges the support from MINECO and AEI through project PID2022-136535OB-I00 Notes The authors declare no competing financial interest. Figure 5. GC formation employing enzymatic catalysis using the lipase from C. rugosa (CRL) several and sequential times. The catalyst amount was 22.5%% w/w (weight of wet support/weight of reactants). Operation conditions: 78 °C and 800 rpm, with an initial concentration of glycerol was 4.76 M. Industrial & Engineering Chemistry Research pubs.acs.org/IECR Article https://doi.org/10.1021/acs.iecr.3c02128 Ind. Eng. Chem. Res. 2023, 62, 15798−15808 15804 https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?goto=supporting-info https://pubs.acs.org/doi/suppl/10.1021/acs.iecr.3c02128/suppl_file/ie3c02128_si_001.pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Miguel+Ladero"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://orcid.org/0000-0002-9146-3830 https://orcid.org/0000-0002-9146-3830 mailto:mladerog@ucm.es mailto:mladerog@ucm.es https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Juan+M.+Bolivar"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://orcid.org/0000-0001-6719-5082 https://orcid.org/0000-0001-6719-5082 mailto:juanmbol@ucm.es mailto:juanmbol@ucm.es https://pubs.acs.org/action/doSearch?field1=Contrib&text1="David+Gonzalez-Miranda"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://orcid.org/0000-0001-5381-9325 https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Diego+Carballares"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Toma%CC%81s+Pedregal"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Roberto+Fernandez-Lafuente"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://orcid.org/0000-0003-4976-7096 https://orcid.org/0000-0003-4976-7096 https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig5&ref=pdf https://pubs.acs.org/doi/10.1021/acs.iecr.3c02128?fig=fig5&ref=pdf pubs.acs.org/IECR?ref=pdf https://doi.org/10.1021/acs.iecr.3c02128?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as ■ ABBREVIATIONS CaLA lipase A of C. antarctica CaLB lipase B of C. antarctica CRL C. rugosa lipase DMC dimethyl carbonate EC ethylene carbonate ETL Eversa Transform 2.0 lipase GC glycerol carbonate Gly glycerol HPLC high-performance liquid chromatography LeU Lecitase ultra lipase MR molar ratio N435 Novozym 435 NMWL nominal molecular weight limit PFL lipase from P. fluorescens p-NP p-nitrophenol p-NPB p-nitrophenyl butyrate RID refractive index detector RML lipase from R. miehei TLL Lypozyme TL 100 L (T. lanuginosus lipase) TN turnover number ■ REFERENCES (1) Ucal, M.; Xydis, G. 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