Enhanced Productivity in Glycerol Carbonate ... - ACS Publications

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: ACS Omega 2019, 4, 860−869

http://pubs.acs.org/journal/acsodf

Enhanced Productivity in Glycerol Carbonate Synthesis under Continuous Flow Conditions: Combination of Immobilized Lipases from Porcine Pancreas and Candida antarctica (CALB) on Epoxy Resins Marcelo A. do Nascimento,† Larissa E. Gotardo,† Raquel A. C. Leão,‡ Aline M. de Castro,§ Rodrigo O. M. A. de Souza,† and Ivaldo Itabaiana, Jr.*,∥

ACS Omega 2019.4:860-869. Downloaded from pubs.acs.org by 146.185.202.165 on 01/10/19. For personal use only.

†

BOSS GroupBiocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro CEP: 21941-909, Brazil ‡ Pharmacy School, Federal University of Rio de Janeiro, Rio de Janeiro CEP: 21941-170, Brazil § Biotechnology Division, Research and Development Center, PETROBRAS, Rio de Janeiro CEP: 21941-915, Brazil ∥ Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro CEP: 21941-910, Brazil S Supporting Information *

ABSTRACT: Among several value-added products from glycerol, glyceryl carbonate (GC) has gained prominence during the recent years because of its attractive physicochemical properties and the wide range of industrial applications. As a continuation of our efforts on GC production under environmentally benign conditions, in this work, we have applied a combination of two new biocatalysts containing lipases from porcine pancreas and Candida antarctica immobilized on epoxy resins for GC production via an integrated reaction between triacylglycerol and dimethyl carbonate under both batch and packed-bed reactors. Under continuous flow conditions, the 1:1 (w/w) combination of the home-made immobilized biocatalysts was able to lead to complete conversion of GC with >99% selectivity against 88% demonstrated by the commercial preparation Novozym 435. The new continuous flow biocatalytic system demonstrated a final productivity of 16 × 10−2 g of GC/h/U of the biocatalyst. properties (i.e., fp > 240 °C, bp 110−115 °C at 0.1 mmHg), nontoxicity, and water solubility. GC is a high-value-added product with a market price over 6000 US$/ton.7 To date, several catalytic processes have been developed to synthesize glyceryl carbonate with higher selectivity as well as lower reaction times, such as those that use glycerol as the alcohol source and chemicals such as CO/O2,7 organic carbonate,8 urea,9,10 or carbon dioxide11 as the carbonate source. Many strategies have been reported for GC synthesis including chemical catalysis,12,13 which can led to high conversion but with toxic waste accumulation. The most difficult step in the synthesis of glycerol esters by chemical catalysis is the removal of byproduct, as this phenomenon is important to maintain the equilibrium in the positive domain (alkyl ester formation). Also, removal of alkaline catalyst is an energy-intensive process because the treatment of highly alkaline wastewater remains a great challenge for industries.14

1. INTRODUCTION The biodiesel industry has shown significant progress in the recent years because of the threat from petroleum depletion since the last decade, and biodiesel has emerged as a leading source of renewable glycerol.1,2 In addition, the abundance of crude glycerol also had a major impact on the refined glycerol market, and as a consequence, the price of glycerol has dropped dramatically since 2006.3,4 Inevitably, the paradigm shift has attracted much attention from researchers to explore the possibilities of converting glycerol into value-added compounds such as fuel, chemical intermediates, and chemicals, where pharmaceutical, food, beverage, and personal care industries have been the largest application segments for these derivatives.5 Figure 1 shows some production routes for obtaining the main value-added chemicals originating from glycerol, including polyglycerols (1), glycerol ethers (2), glycerides (3), cyclic acetals (5), glycidol (6), epichlorohydrin (7), 1,2-propanediol (8), 1,3propanediol (9), lactic acid (10), dihydroxyacetone (11), acrolein (12), and glycerol carbonate (GC, 4), the most important product reported over the last 5 years.6 Some motivations for GC appreciation are due to its physical © 2019 American Chemical Society

Received: September 18, 2018 Accepted: December 19, 2018 Published: January 10, 2019 860

DOI: 10.1021/acsomega.8b02420 ACS Omega 2019, 4, 860−869

ACS Omega

Article

Figure 1. Main value-added chemicals originating from glycerol.

Table 1. Surface Groups and General Characteristics of the Four Supports Investigated support Epox01 Epox02 octa styr

commercial name ECR ECR ECR ECR

8205 8214 8806 1091

surface groups

immobilization form

surface area (m2/g)

pore diameter (Å)

epoxide epoxide octadecyl styrene

covalent bond covalent bond hydrophobic adsorption hydrophobic adsorption

>80 >60 >80 >450

450−600 1200−1800 500−700 950−1200

optimized through immobilization techniques, where the lipase is associated with supports to provide retention of activity, as well as greater stability and recycling potential.24−26 For industrial purposes, continuous flow systems are preferred to batch reactors as they provide greater process control, higher productivity, and improved quality/purity.27 Several types of reactors can be used in continuous operation, among which packed-bed reactors are the most popular because of their high efficiency; low cost; and ease of construction, operation, and maintenance.27,28 As a continuation of our efforts in GC production under environmentally acceptable conditions,11,29 this work presents the immobilization of lipases PPL and CALB in four distinct resinous substrates (Purolite), two with epoxide groups and two with octadecyl and styrene groups on their surfaces, through adsorption and uni- and multipoint covalent bonding for application in GC synthesis under batch and continuous flow conditions in solvent-free systems, emphasizing the additive effect of the combinations between the immobilized biocatalysts produced, as compared to that of commercial immobilized CALB Novozym 435 (N435).

Aiming at overcoming these issues, the use of biocatalysts, especially lipases (triacylglycerol ester hydrolases E.C. 3.1.1.3), has been a useful alternative for the production of GC. Among some properties of these enzymes, the non-requirement of cofactors and the ability to act in both hydrolysis and esterification reactions with high regio-, enantio-, and chemoselectivity make the lipases quite applicable in several transformations with industrial applicability. As an example, transesterification of glycerol with dimethyl carbonate (DMC) is one of the most direct and industrially feasible pathways to produce GC with high yields.8,15−17 One of the major problems to be circumvented is the intense enzymatic inhibition that occurs over the reaction because of the formation of methanol as a byproduct, which often generates dimers of alcohols in the active site of the lipases. Thus, a new transesterification process needs to be applied to take advantage of the biocatalytic potential of lipases.18 Lipases can be applied as catalysts for the cascade conversion of triacylglycerols into GC and biodiesel in a sequence of reactions including hydrolysis, esterification, and transesterification reactions.19 In this cascade reaction, hydrolysis is the initial step, which is considered as the reaction-limiting step.20 For these reasons, new biocatalysts containing lipases, which exhibit high hydrolytic activity, are needed. However, before the beginning of catalysis, many lipases need to be activated to expose their active sites to the reaction medium. Most lipases have a lid formed by a polypeptide chain that has a hydrophobic inner face that interacts with the active site. Porcine pancreatic lipase (PPL) is a digestive lipase that presents one of the largest lids in the literature, requiring interfacial activation in the presence of an emulsion to exhibit hydrolysis activity. One exception, however, is the lipase B from Candida antarctica (CALB), whose lid is smaller than that of the others, and there is no need for activation steps, which makes it one of the most reactive and industrially applicable enzymes.21−23 The interfacial activation of lipases can be

2. RESULTS AND DISCUSSION 2.1. Design of New Biocatalysts. The first phase of the work was to evaluate the behavior of lipases PPL and CALB in immobilization assays for hydrophobic adsorption of the related enzymes. It is important to note that among the four supports analyzed, two of them present surface groups that preferentially promote adsorption, whereas the other two preferentially promote covalent bonds, according to the manufacturer (Table 1). ECR 8806 and ECR 1091 supports act on the hydrophobic groups on enzyme surfaces, and epoxy resins ECR 8205 and ECR 8214 are considered as flexible supports because of their ability, at neutral pH values, to promote hydrophobic 861


ACS Omega

Article

Figure 2. Adsorption progress of lipases PPL and CALB on the four resins at 20 and 40 °C at different ionic strengths. (A and B) −20 °C; (C and D) −40 °C; (a) 0.025 M; and (b) 0.5 M.

neutral pH and low ionic strength.36,37 Thus, many papers have described that the immobilization using this type of support must be performed in two steps: In the first step, the enzyme is adsorbed onto the surface of the support. For this reason, most of the commercial supports designed for the immobilization of enzymes via epoxy groups must have highly hydrophobic characteristics. In the second step, the previously adsorbed protein is covalently attached to the epoxy groups of the support. To further investigate the effect of ionic strength, all experiments were also performed at 0.025 and 0.5 M ionic strengths and at 20 and 40 °C. Furthermore, the next step of this study was to evaluate the effect of an increase in pH (pH = 10) on enzymes previously adsorbed at pH 7 (Figure 3) and to investigate the hydrolytic activity of all preparations aiming at selecting the best biocatalysts. In Figure 3A, the results for the immobilization under milder conditions of temperature and ionic strength are shown, where the biocatalyst PPL_Epox02 is highlighted, whose activity value was 419.0 U/g against 256.0 U/g obtained by N435. The increase of pH considerably decreased the final hydrolytic activity in the new immobilized biocatalysts, except CALB_Octa. These results are in agreement with those obtained by Mateo et al.38 on the immobilization of penicillin G-acylase with support of Eupergit C epoxy groups, in addition to those obtained by Miletić et al.39 for immobilizing CALB on polystyrene nanoparticles and by Wang et al.40 with activity values close to those found for N435 (293.8 U/g). According to them, lipase immobilization in the range of pH 6.5−7.0 provided relatively high values of activity, as compared to values in alkaline medium. The ionization state of the active site of the lipase molecule is affected by the pH of the buffer used during immobilization.38 According to Chiappe et al.,41 the highest enzymatic activity can be obtained when the

adsorption of proteins and also to promote covalent bonds at higher values of pH. Based on this, it was decided to evaluate the adsorption profile of all polymers at 20 and 40 °C (Figure 2) in pH solutions with lower ionic strength (0.025 M) and higher ionic strength (0.5 M). Comparing the graphs Figure 2A,B, it is possible to verify that the increase in ionic strength could contribute to the rate of adsorption of both enzymes on the supports Epox01 and Epox02, whereas styrene and octadecyl polymers were apparently negatively influenced. When in solution, the enzymes tend to form molecular aggregates, being able to assume an inactive conformation during the immobilization, besides diminishing their solubility. The increase of the ionic strength enhances the solvation of proteins and promotes the reduction of enzyme−enzyme interactions.34 According to Mateo et al.,35 the interaction force depends on the hydrophobicity of the support and the enzyme, being controlled by pH, temperature, and buffer concentration, where the hydrophobic adsorption of proteins becomes stronger and faster on increasing the ionic strength of the medium. However, this effect can be compensated by the increase of temperature. As shown in Figure 2C,D, the combination of higher temperatures and ionic strengths demonstrated a crucial impact on the system, probably by reducing agglomerated forms and increasing the interaction of proteins with the applied supports. Epoxy resins are very stable at neutral pH and can stabilize the enzyme by multipoint covalent binding, resulting in high stabilization of the derivatives. The epoxy groups undergo nucleophilic attack of different groups on the surface of the enzyme such as amino, thiol and hydroxyl, allowing intense interactions between the enzyme and the support. However, epoxy groups are weakly reactive under mild conditions such as 862


ACS Omega

Article

Figure 3. Hydrolytic activities of enzymes CALB and PPL immobilized in different conditions of pH, temperature, and ionic strength compared to those of reference N435. Conditions: (A): adsorption at pH 7.0 (12 h) and incubation at pH 10.0 (24 h) at 20 °C and for 0.025 M phosphate buffer; (B): adsorption at pH 7.0 (12 h) and incubation at pH 10.0 (24 h) at 20 °C and for 0.5 M phosphate buffer; (C): adsorption at pH 7.0 (12 h) and incubation at pH 10.0 (24 h) at 40 °C and for 0.025 M phosphate buffer; (D): adsorption at pH 7.0 (12 h) and incubation at pH 10.0 (24 h) at 40 °C and for 0.5 M phosphate buffer.

derivative was incubated at alkaline pH (around 10.0) to effect multi-interactions aiming to obtain biocatalysts with higher stability. The proximity of the enzyme to the support could promote more rigidification of the enzyme because the epoxy supports have small spacing arms. Although it is described that the covalent multipoint bonds are more stable, in some cases, there may be an excessive stiffness, which compromises the enzymatic activity. This phenomenon may have occurred with our biocatalysts. Previous works reported that at pH 7 and low ionic strength,43 depending on the chemical characteristics of the support (in this case, free epoxy groups), there is high adsorption and, also, formation of covalent bonds unipontually with the lipase of Thermomyces lanuginosus, constituting a more simplified protocol. As the GC synthesis by transesterification involves a cascade reaction (hydrolysis followed by transesterification), all of the biocatalysts obtained by adsorption at pH 7.0 and ionic strength of 0.025 M (the ones that presented the highest hydrolysis activities) were investigated for the potential of esterification (Figure 4). In these experiments, it was observed that all biocatalysts containing PPL lipase were not able to show esterification potential under the applied conditions (data not show in Figure 4). Fatty acids with a longer chain can promote a steric hindrance that avoids the formation of the acylated enzyme

adsorption is conducted near the isoelectric point of the enzyme, leading to a decrease in the solubility of the enzyme in the enzyme solution and favoring adsorption. In this study, this fact is evidenced in Figure 3B,D, where the increase in ionic strength generated significant reduction in the hydrolytic activity. This fact may evidence that the conformations of the enzymes were also influenced by the concentration of the buffer used during immobilization. It is also important to note that the incubation at pH 10 was not attractive for any lipase in the conditions studied. When comparing the activity values obtained for adsorption at pH 7.0 and at low ionic strength (Figure 3A,C), it is observed that this condition generated biocatalysts with higher activities. PPL is a digestive lipase, active in hydrolysis reactions for which bile salts are required to emulsify fatty substrates and enhance hydrolytic activity. Thus, the surface hydrophobic adsorption process may have promoted a hyperactivating effect of the enzyme through the distortion of the lid, which is strongly influenced by the medium.42 This phenomenon is not observed in CALB, which presents a reduced lid41−44 and is not able to exhibit interfacial activation.21−23 However, the higher activity demonstrated by CALB on adsorption and not by covalent binding on the epoxide supports (Figure 3B,D) it is important to take into account that after incubation time of the enzyme at pH 7.0, the 863


ACS Omega

Article

explored by our group is transesterification of vegetable oils, in which the main idea is to add value to glycerol waste, coming from the biodiesel production chain. Taking this into account and based on the possibility to improve selectivity, the profiles of selected new biocatalysts and N435 were investigated on transesterification of soybean oil with DMC in batch and continuous flow reactors. Some combinations of the biocatalysts were also investigated, intending to verify possible improvements in the conversion (Figure 5). In this way, no more glycerol will be produced as a waste and glycerol carbonate could be easily recovered from biodiesel through azeotropic distillation.

Figure 4. Time courses of esterification of palmitic acid with ethanol catalyzed by the new CALB-immobilized biocatalysts obtained by adsorption at pH 7.0, ionic strength of 0.025 M, and 20 °C. Reaction conditions for esterification: palmitic acid/ethanol, 1:1 (100 mM) in n-heptane, 40 °C, 200 rpm, 10 U of each biocatalyst.

complex; in addition, for a short-chain fatty acid, the higher polarity also interferes with the catalysis. Kilinç et al.44 also did not obtain satisfactory esterification results with PPL immobilized on chitosan. However, because the PPL_Epox02 biocatalyst presented the best hydrolysis results and lipase CALB_Epox01 presented the best esterification results with a higher performance than that obtained by N435 (Figure 4), they were selected for the application in the synthesis of GC. The selected biocatalysts were also submitted for the desorption assay with Triton X-100 and subsequent quantification of proteins to verify the possibility of immobilization by unpunctual covalent bonds (Table 2). Table 2. Immobilization Efficiency of New Biocatalysts Compared to That of N435 biocatalyst

amount of protein (mg/g of support)a

immobilization efficiency (%)

N435 CALB_Epox01 PPL_Epox02

15 11 13

100 98

a

Measured by desorption with Triton X-100 and Lowry assay.39 Figure 5. Results of GC synthesis by applying a combination of the biocatalysts. Reaction conditions: 1:1, 1:2, or 1:3 (w/w) mixtures of each biocatalyst, with respect to the maximum concentration of 20% (w/w) of the biocatalyst relative to the vegetable oil; vegetable oil and DMC in a ratio of 1:10 (v/v); water (0.7% v/v); and 60 °C for 5−48 h at 180 rpm without organic solvents.

As shown, the new biocatalysts adsorbed lower amounts of lipases than those adsorbed by N435, which, together with the fact that they presented higher hydrolysis activities, denoted the robustness of the performed process. However, this result also shows that epoxy resins, when subjected to the immobilization process at pH 7.0, did not promote covalent bonds. This result was opposite to that obtained by our research group in immobilizing CALB on epoxy acrylate resin ECR 8205, and this difference is due to the concentration of epoxy groupings on the surface, revealing flexibility on possible interaction according to reaction conditions. 2.2. Glycerol Carbonate Synthesis. Previous publications of our group optimized the lipase-catalyzed GC synthesis by transesterification of glycerol with DMC, using Brij76 as the surfactant in a solvent-free system as well as applying N435 and CALB in different kinds of immobilization processes as biocatalysts.29,30 In all of these works, similar conversions were obtained between 24 and 48 h of reaction, with different profiles of selectivity. Another strategy to GC synthesis already

In fact, CALB was a robust lipase for transesterification of DMC with soybean oil because of the good results on conversion presented by the two biocatalysts containing this enzyme. These results are quite satisfactory for the CALB_Epox01 biocatalyst, even more because of the fact that in 48 h high selectivities (>99%, data not shown) were also reached, as with N435. If we also consider that the amount of enzyme immobilized on CALB_Epox01 was lower than that found in N435 (data already shown in Table 1), we can conclude that this home-made biocatalyst was very competitive under the conditions studied. As the synthesis of GC in this reaction involves both hydrolysis and transesterification, it was 864


ACS Omega

Article

Figure 6. Profile of reuses of the biocatalyst systems applied for GC synthesis under continuous flow reactors. Reaction conditions: vegetable oil and dimethyl carbonate in a ratio of 1:10 (v/v) in TBME, 20% (w/w) of enzyme relative to the vegetable oil, 0.7% (v/v) water, 60 °C in a column of 1.767 mL volume, and residence time of 90 min.

Table 3. Results of GC Synthesis by Applying Blends of Biocatalysts in Continuous Flow Conditionsa biocatalyst CALB_Epox01

N435

CALB_EPox01:PPL_Epox02 (1:1)

N435:PPL_Epox02 (1:1)

residence time (min)

conversion (%)

selectivity (%)

9 18 90 9 18 90 9 18 90 9 18 90

36 66 59 28 63 88 41 62 >99 43 79 78

68 >99 >99 84 >99 89 87 >99 >99 78 87 >99

a

Reaction conditons: vegetable oil and dimethyl carbonate in a ratio of 1:10 (v/v) in TBME, 20% (w/w) of enzyme relative to the vegetable oil, 0.7% (v/v) water, and 60 °C in an Omnifit column of 1.767 mL volume. In general, with all of the biocatalysts applied, shorter residence times generated lower conversions and selectivity because of the lower contact between substrates and enzymes. Greater attention should be given to reactions occurring at a residence time of 90 min (flow of 0.1 mL/min), where N435 achieved 88% conversion and selectivity below 90%. In contrast, the mixture of the home-made biocatalysts CALB_EPox01 and PPL_Epox02 reached almost 100% conversion and higher selectivity (>99%), demonstrating that the high hydrolysis activity of PPL_Epox02 is crucial for GC to be formed more quickly. This is due to optimization of the contact time between biocatalysts and substrates, thus avoiding the formation of byproducts, which also increases the selectivity values. Although the combination of PPL_Epox02 and N435 has also resulted in high selectivities, the conversions are still lower than those obtained by the home-made biocatalysts, demonstrating that this biocatalytic system is a good alternative for obtaining CG and biodiesel. These results were even better than our previous results where lipase CALB was immobilized on Accurel MP1000 and applied for CG synthesis under continuous flow conditions, where in 90 min of residence time, conversions of 71% and selectivities over 99% were obtained.29

step of the vegetable oil was limiting in the process, PPL_Epox01 could have been responsible for a more efficient hydrolysis of the oil, resulting in a faster releasing of glycerol and fatty acids for the other steps, characterizing a potentiating effect. With N435, mixtures of PPL_Epox02 were not satisfactory because the values obtained with the enzyme alone were not exceeded. As the 3:1 PPL_Epox02 and CALB_Epox01 mixtures were not proportionally better than the 1:1 mixture, it was decided to investigate the GC formation reaction under continuous flow conditions applying the ratio 1:1 at residence times between 9 and 90 min (Table 3). The biocatalyst systems applied in the continuous flow reaction were also evaluated for potential reuse for three consecutive cycles (Figure 6). As a result, the system with the home-made biocatalysts demonstrated greater preservation of activity, with lower relative loss of conversion over the cycles, compared with

expected that the PPL_Epox02 biocatalyst did not present satisfactory results for GC production because low esterification potential was detected. However, as the PPL_Epox02 biocatalyst was able to promote intense hydrolysis of the oil (for further details, see the Supporting Information), based on our objectives to construct a competitive biocatalytic system for GC synthesis and to enhance selectivity and productivity, we decided to investigate the potential effect caused by the proportional mixtures of the biocatalysts. In Figure 6, it is clearly observed that mixtures of the PPL_Epox02 biocatalyst were able to increase significantly the conversion to GC when applied together with the CALB_Epox01 catalyst, achieving 90% with a ratio of 1:1 and 94% with a ratio of 3:1, against 70% when the CALB_Epox01 catalyst was applied alone. This value is the same as that achieved with N435 alone. Due to the higher hydrolysis activity exhibited by PPL_Epox02 in comparison to that of the other biocatalysts produced, in addition to the fact that the triacylglyerols (TAG) hydrolysis 865


ACS Omega

Article

Figure 7. Analysis by scanning electron microscopy of new and commercial biocatalysts after reaction: (A) resin epox01 without enzyme; (B) CALB_Epox01; (C) resin Epox02; (D) PPL_Epox02; and (E) N435.

N435 as well as each enzyme separately, indicating an additional effect on stability. The good results obtained can be evidenced by the final microscopy structure of the biocatalysts (scanning electron microscopy, SEM). Figure 7 shows that the PPL_Epox02 and CalB_Epox01 biocatalysts, as well as the commercial enzyme N435, have a spherical structure, denoting a large surface area for contact with the enzyme and the substrates. The efficiencies of the two developed systems can be compared through productivity values (Table 4). Although in the continuous flow system larger masses of enzyme were applied, the reaction time is much lower than that in the batch system, revealing that the application of the enzyme in these reactors has been optimized. These data are important because a better contact between the enzyme and substrates at optimized times is a crucial factor for the maintenance of the

Table 4. Comparative between the Values of Productivity for Batch and Continuous Flow Systems productivity (mg of CG/h/U of biocatalyst) biocatalyst

batch

continuous flow

CALB_Epox01 N435 CALB_EPox01:PPL_Epox02 (1:1) N435:PPL_Epox02 (1:1)

0.025 0.044 0.053 0.032

0.07 0.12 0.16 0.09

selectivity. In this way, the continuous flow system was more robust than the batch system. In conclusion, we developed a robust, competitive, and stable system of biocatalysts for glycerol carbonate production from vegetable oil with consecutive hydrolysis and transesterification with DMC under batch and continuous flow approaches. Both strategies could lead to excellent selectivities 866


ACS Omega

Article

Figure 8. General Scheme of GC synthesis.29 TAG, triacylglyerols; DMC, dimethyl carbonate; BIO, biodiesel; CG, glycerol carbonate.

and yields, but the continuous flow approach was more successful when using a combination of the two home-made biocatalysts because of the additive effect of PPL on productivity.

covalent binding.31 At this stage, as no further enzyme was offered to the support, no analyses were performed for the quantification of proteins in the supernatant. The immobilizations were performed on a thermostatic orbital shaker at 20 and 40 °C and at 180 rpm for 24 h. The samples were then filtered and stored at 10 °C. The final biocatalysts were dried overnight at 25 °C. To check the covalent binding, supports were submitted for desorption with Triton X-100.32 Soluble protein contents were determined by the Bradford method using bovine serum albumin as the protein standard.45 3.2.2. Hydrolytic Activity Assay. The activities of free and immobilized lipases were determined as follows: 1 mL of extract and 10 mg of each supported enzyme were added to 19 mL of an emulsion prepared with olive oil (5% w/v) and arabic gum (10% w/v) in sodium phosphate buffer (100 mM, pH 7.0). The reactions were carried out under agitation (200 rpm) at 35 °C for 30 min. The reactions were then stopped by the addition of 20 mL of the acetone−ethanol mixture (1:1 v/v), and the fatty acids produced were extracted under agitation (200 rpm) for 10 min and titrated until end point (pH 11.0) with NaOH solution (0.04 N). The blank assays were performed by adding the extract just after the addition of the acetone−ethanol solution to the flask. One unit of lipase activity (U) was defined as the amount of enzyme that catalyzes the release of 1 μmol of fatty acids per minute, under the assay conditions. All samples were compared with the commercial preparation N435. 3.2.3. Esterification Performance. The new immobilized biocatalysts were applied in esterification reactions of lauric, palmitic, and oleic fatty acids with ethanol.30,33 The amount of each catalyst applied was adjusted to 10 U based on the data obtained from the hydrolysis assay. Solutions (10 mL) of each fatty acid in alcohol/heptane (1:1, 0.1 M) were added to 25 mL conical tubes in the presence of the biocatalysts and stirred under orbital agitation for 2 h at 200 rpm and 40 °C. Aliquots of 20 μL were collected from 5 to 120 min to study the time course reaction. All analyses were done in triplicate. The samples were derivatized with 20 μL of N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) and graded to 1.0 mL of heptane. Subsequently, the samples were analyzed in a gas chromatography unit equipped with a mass spectrometry detector (GC−MS), as described in Section 3.5. 3.3. Scanning Electron Microscopy (SEM) Analysis. The surface structure of the new supported enzymes was analyzed by scanning electron microscopy (SEM) using a Zeiss EVO 50H microscope. All micrographs were obtained from the fractured surfaces of supports coated with gold, prepared using Shimadzu sputter equipment. 3.4. Synthesis of Glycerol Carbonate from Soybean Oil. The synthesis of GC was performed by transesterification of Soybean oil and DMC catalyzed by the new immobilized biocatalysts and N435, both in batch and continuous flow conditions (Figure 8), following our previous works.11,29,30

3. MATERIALS AND METHODS 3.1. Materials. Lipase B from C. antarctica (CALB, soluble form), N435 (immobilized CALB on macroporous acrylic resin, ion exchange), and lipase from porcine pancreas (PPL) were purchased from Novozymes (Brazil). Dimethyl carbonate (DMC) was purchased from Sigma-Aldrich Co. (St. Louis). Supports of epoxy resins Purolite ECR 8205F, ECR 8214F, ECR 8806F, and ECR 1091F were purchased from Purolite International Limited (Wales, U.K.). All other reagents used were of analytical grade. 3.2. Immobilization Procedures. 3.2.1. Immobilization of Lipases CALB and PPL. Epoxy resins Purolite ECR 8205F, ECR 8214F, ECR 8806F, and ECR 1091F polymers were applied as supports for PPL and CALB immobilization both by hydrophobic adsorption and covalent binding. For this purpose, 1 mL of the enzyme solution (0.62 U/mL specific activity of both lipases) was diluted in 20 mL of phosphate buffer (25 and 50 mM, pH 7.0) and added to an appropriate support (3 g). The mixture was stirred for 4 h at 20 and 40 °C at 180 rpm, followed by vacuum filtration. At the end, the supernatant was collected and stored for protein quantification. The solid material was gravity-filtered and dried at 25 °C. In all experiments, the immobilization efficiency and yields were followed by measuring the hydrolytic activities and the protein concentrations in the supernatant solution.30 Immobilization yields (eq 1) were calculated after determining the amount of protein and enzyme units that disappeared from the supernatant and comparing with the initial protein and enzyme concentrations offered for the reaction (units per gram of support). The efficiency (eq 2) was calculated after determining the activity of the immobilized enzyme and comparing with the number of enzyme units that disappeared from the supernatant (theoretically immobilized). All samples were compared with commercial preparation N435. immobilization yield (total initial activity − total residual activity) total initial activity

(1)

immobilization efficiency observed activity = (total initial activity − total residual activity)

(2)

=

Aiming at investigating the covalent bonds of enzymes in the new supports, 1.5 g of the immobilized enzymes at pH 7.0 was also incubated in a pH 10.0 sodium phosphate buffer at concentrations of 0.025 and 0.5 M to evaluate the enzymesupport behavior in relation to the uni- and multipoint 867


ACS Omega 3.4.1. Batch Mode. In vials of 20 mL, samples of vegetable oil were mixed with DMC in a ratio of 1:10 (v/v), the immobilized enzymes (each separately or in mixtures) in a proportion of 20% (w/w) relative to the vegetable oil, and water (0.7% v/v) were added. The reaction proceeded under orbital agitation at 60 °C for 5−48 h at 180 rpm without addition of organic solvents. Final products were extracted with ethyl acetate and concentrated under vacuum. Aliquots were derivatized as described in Section 3.2.3 and analyzed by GC−flame ionization detector (FID) as described in Section 3.5. 3.4.2. Continuous Flow Mode. The same proportions of oil/DMC/water as described for the batch mode were added in a 250 mL Erlenmeyer flask, followed by addition of methyl tert-butyl ether (TBME) until the formation of a homogeneous system (120 mL). Thus, a packed-bed column of 1.8 cm diameter and 5.0 cm height (8.83 mL of reaction volume) was completely filled with 1.02 g of N435 or each new biocatalyst (separately or in the appropriate mixture). Flow rates ranging from 0.1 mL/min (residence time of 88 min) to 0.05 mL/min (residence time of 176 min) were investigated using the Asia flow system. Samples were derivatized as described in Section 3.2.3 and analyzed by GC−FID as described in Section 3.5. 3.5. Chromatography Analysis. Helium was applied as a carrier gas in all analyses. 3.5.1. Esterification Reactions of Free Fatty Acids. Analyses were performed in a GC−MS system (Shimadzu CG2010, DB 5 capillary column). Samples were prepared by dissolving 10 μL of the final product in 980 μL of heptane and 10 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The injector and detector temperatures were 250 °C, and the oven temperature was constant at 60 °C for 1 min and then increased by 10 °C/min to 250 °C, when it was held constant for 3 min. 3.5.2. Glycerol Carbonate Synthesis. Analyses were performed in a GC−FID (Shimadzu CG2010DB 5 capillary column). Each sample (1 μL) was injected at 100 °C. The oven was heated at 15 °C/min to 150 °C, at 8 °C/min to 200 °C, and at 2 °C/min to 240 °C, and then the temperature was maintained for 4 min. After this, the oven was heated until 300 °C at 15 °C/min. The detector and injector were adjusted to 280 °C.

■

ACKNOWLEDGMENTS

■

REFERENCES

Authors thank CAPES, CNPq, FAPERJ, and ANP/PETROBRAS (Process 2012/00326-0) for financial support.

(1) Abbaszaadeh, A.; Ghobadian, B.; Omidkhah, M. R.; Najafi, G. Current biodiesel production technologies: a comparative review. Energy Convers. Manage. 2012, 63, 138−148. (2) Demirbas, A. Progress and recent trends in biodiesel fuels. Energy Convers. Manage. 2009, 50, 14−34. (3) Monteiro, M. R.; Kugelmeier, C. L.; Pinheiro, R. S.; Batalha, M. O.; Cesar, A. S. Glycerol from biodiesel production: Technological paths for sustainability. Renewable Sustainable Energy Rev. 2018, 88, 109−122. (4) Ayoub, M.; Abdullah, A. Z. Critical review on the current scenario and significance of crude glycerol resulting from biodiesel industry towards more sustainable renewable energy industry. Renewable Sustainable Energy Rev. 2012, 16, 2671−2686. (5) (a) Len, C.; Delbecq, F.; Corpas, C. C.; Ramos, E. R. Continuous Flow Conversion of Glycerol into Chemicals: An Overview. Synthesis 2018, 50, 723−741. (b) Suveges, N. K.; Rodrigues, A. A.; Diederichs, C. C.; de Souza, S. P.; Leão, R. A. C.; Miranda, L. S. M.; Horta, B. A. C.; Pedraza, S. F.; de Carvalho, S. F.; Pais, K. C.; Terra, J. H. C.; De Souza, R. O. M. A. Continuous-Flow Synthesis of (R)-Propylene Carbonate: An Important Intermediate in the Synthesis of Tenofovir. Eur. J. Org. Chem. 2018, 23, 2931−2938. (6) Galy, N.; Nguyen, R.; Yalgin, H.; Thiebault, N.; Luart, D.; Len, C. Glycerol in subcritical and supercritical solvents. J. Chem. Technol. Biotechnol. 2017, 92, 14−26. (7) Deckwer, W. Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiol. Rev. 1995, 16, 143−149. (8) Dasari, M. A.; Kiatsimkul, P. P.; Sutterlin, W. R.; Suppes, G. J. Low-pressure hydrogenolysis of glycerol to propylene glycol. Appl. Catal., A 2005, 281, 225−231. (9) Quispe, C. A. G.; Coronado, C. J. R.; Carvalho, J. A., Jr. Glycerol: Production, consumption, prices, characterization and new trends in combustion. Renewable Sustainable Energy Rev. 2013, 27, 475−493. (10) Dibenedetto, A.; Angelini, A.; Aresta, M.; Ethiraj, J.; Fragale, C.; Nocito, F. Converting wastes into added value products: from glycerol to glycerol carbonate, glycidol and epichlorohydrin using environmentally friendly synthetic routes. Tetrahedron 2011, 67, 1308−1313. (11) Nogueira, D. O.; de Souza, S. P.; Leão, R. A. C.; Miranda, L. S. M.; de Souza, R. O. M. A. Process intensification for tertiary amine catalyzed glycerol carbonate production: translating microwave irradiation to a continuous-flow process. RSC Adv. 2015, 5, 20945− 20950. (12) Teng, W. K.; Ngoh, G. C.; Yusoff, R.; Aroua, M. K. A review on the performance of glycerol carbonate production via catalytic transesterification: Effects of influencing parameters. Energy Convers. Manag. 2014, 88, 484−497. (13) Jung, H.; Lee, Y.; Kim, D.; Han, S. O.; Kim, S. W.; Lee, J.; Kim, Y. H.; Park, C. Enzymatic production of glycerol carbonate from byproduct after biodiesel manufacturing process. Enzyme Microb. Technol. 2012, 51, 143−147. (14) Ochoa-Gómez, J. R.; Gómez-jiménez-aberasturi, O.; Ramírezlópez, C.; Maestro-Madurga, B. Synthesis of glycerol 1,2-carbonate by transesterification of glycerol with dimethyl carbonate using triethylamine as a facile separable homogeneous catalyst. Green Chem. 2012, 14, 3368−3376. (15) Chiappe, C.; Rajamani, S. Synthesis of glycerol carbonate from glycerol and dimethyl carbonate in basic ionic liquids. Pure Appl. Chem. 2012, 84, 755−762. (16) Rokicki, G.; Rakoczy, P.; Parzuchowski, P.; Sobiecki, M. Hyperbranched aliphatic polyethers obtained from environmentally benign monomer: glycerol carbonate. Green Chem. 2005, 7, 529−539. (17) Ochoa-Gómez, J. R.; Aberasturi, O. G. -J; Maestro-Madurga, B.; Pesquera-Rodríguez, A.; Ramírez-López, C.; Lorenzo-Ibarreta, L.;

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02420. GC−FID of the synthesis of GC by transesterification of the vegetable oil with DMC catalyzed by lipase N435, biocatalyst CalB_epox02, biocatalystsN435:PPL_epox02 (1:1), CaLB_epox02, and CALB_epox01: PPL_epox02 (1:1) and GC−FID of the vegetable oil applied for the synthesis of GC (PDF)

■

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Rodrigo O. M. A. de Souza: 0000-0002-6422-4025 Notes

The authors declare no competing financial interest. 868


ACS Omega

Article

Torrecilla-Soria, J.; Villarán-Velasco, M. C. Synthesis of glycerol carbonate from glycerol and dimethyl carbonate by transesterification: catalyst screening and reaction optimization. Appl. Catal., A 2009, 366, 315−324. (18) Simanjuntak, F. S. H.; Widyaya, V. T.; Kim, C. S.; Ahn, B. S.; Kim, Y. J.; Lee, H. Synthesis of glycerol carbonate from glycerol and dimethyl carbonate using magnesium−lanthanum mixed oxide catalyst. Chem. Eng. Sci. 2013, 94, 265−272. (19) Tudorache, M.; Protesescu, L.; Coman, S.; Parvulescu, V. I. Efficient bio-conversion of glycerol to glycerol carbonate catalyzed by lipase extracted from Aspergillus niger. Green Chem. 2012, 14, 478− 482. (20) Li, J.; Wang, T. Coupling reaction and azeotropic distillation for the synthesis of glycerol carbonate from glycerol and dimethyl carbonate. Chem. Eng. Process. 2010, 49, 530−535. (21) Zaak, H.; Siar, E. H.; Kornecki, J. F.; Fernandez-Lopez, L.; Pedrero, S. G.; Virgen-Ortíz, J. J.; Fernandez-Lafuente, R. Effect of immobilization rate and enzyme crowding on enzyme stability under different conditions. The case of lipase from Thermomyces lanuginosus immobilized on octyl agarose beads. Process Biochem. 2017, 56, 117− 123. (22) Virgen-Ortíz, J. J.; Pedrero, S. G.; Fernandez-Lopez, L.; LopezCarrobles, N.; Gorines, B. C.; Otero, C.; Fernandez-Lafuente, R. Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites. Molecules 2017, 22, 1−12. (23) Silva, M. V. M.; Bassut, J. F.; Junior, I. I.; de Souza, S. P.; Estrada, M. L. G.; Miranda, L. S. M.; de Souza, R. O. M. A. Lipase immobilization towards improved productivity on kinetic resolutions by a continuous-flow process. RSC Adv. 2015, 5, No. 102409. (24) Sánchez-Otero, M. G.; Valerio-Alfaro, G.; García-Galindo, H. S.; Oliart-Ros, R. M. Immobilization in the presence of Triton X-100: modifications in activity and thermostability of Geobacillus thermoleovorans CCR11 lipase. J. Ind. Microbiol. Biotechnol. 2008, 35, 1687−1693. (25) Reis, P.; Holmberg, K.; Watzke, H.; Leser, M. E.; Miller, R. Lipases at interfaces: A review. Adv. Colloid Interface Sci. 2009, 147− 148, 237−250. (26) Sheldon, R. A.; Woodley, J. M. Role of Biocatalysis in Sustainable Chemistry. Chem. Rev. 2018, 118, 801−838. (27) Heintz, S.; Mitic, A.; Ringborg, R. H.; Krühne, U.; Woodley, J. M.; Gernaey, K. V. Microfluidic Toolbox for the Development of InSitu Product Removal Strategies in Biocatalysis. J. Flow Chem. 2016, 6, 18−26. (28) Hessel, V.; Tibhe, J.; Noël, T.; Wang, Q. Biotechnical MicroFlow Processing at the EDGE−Lessons to be learnt for a Young Discipline. Chem. Biochem. Eng. Q. 2014, 28, 167−188. (29) Leão, R. A. C.; de Souza, S. P.; Nogueira, D. O.; Silva, G. M. A.; Silva, V. M.; Gutarra, M. L. E.; Miranda, L. S. M.; Castro, A. M.; Junior, I. I.; de Souza, R. O. M. A. Consecutive lipase immobilization and glycerol carbonate production under continuous-flow conditions. Catal. Sci. Technol. 2016, 6, 4743−4748. (30) De Souza, S. P.; De Almeida, R. A. D.; Garcia, G. G.; Leão, R. A. C.; Bassut, J.; De Souza, R. O. M. A.; Itabaiana, I., Jr. Immobilization of lipase B from Candida antarctica on epoxyfunctionalized silica: characterization and improving biocatalytic parameters. J. Chem. Technol. Biotechnol. 2017, 93, 105−111. (31) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451−1463. (32) Cabrera, Z.; Fernandez-Lorente, G.; Fernandez-Lafuente, R.; Palomo, J. M.; Guisan, J. M. Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports. J. Mol. Catal. B: Enzym. 2009, 57, 171− 176.

(33) Min, J. Y.; Lee, E. Y. Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate. Biotechnol. Lett. 2011, 33, 1789−1796. (34) Mohamad, N. R.; Marzuki, N. H. C.; Buang, N. A.; Huyop, F.; Wahabet, R. A. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol. Biotechnol. Equip. 2015, 29, 205−220. (35) Mateo, C.; Torres, R.; Fernández-Lorente, G.; Ortiz, C.; Fuentes, M.; Hidalgo, A.; López-Gallego, F.; Abian, O.; Palomo, J. M.; Betancor, L.; Pessela, B. C. C.; Guisan, J. M.; Fernández-Lafuente, R. Epoxy-Amino Groups: A New Tool for Improved Immobilization of Proteins by the Epoxy Method. Biomacromolecules 2003, 4, 772−777. (36) Grazú, V.; Abian, O.; Mateo, C.; Batista-Viera, F.; FernandezLafuente, R.; Guisan, J. M. Novel Bifunctional Epoxy/Thiol-Reactive Support to Immobilize Thiol Containing Proteins by the Epoxy Chemistry. Biomacromolecules 2003, 4, 1495−1501. (37) Mateo, C.; Palomo, J. M.; Lorente, G. F.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microbiol. Technol. 2007, 40, 1451−1463. (38) Mateo, C.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J. M. Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment. Enzyme Microb. Technol. 2000, 26, 509−515. (39) Miletić, N.; Abetz, V.; Ebert, K.; Loos, K. Immobilization of Candida antarctica lipase B on Polystyrene Nanoparticles. Macromol. Rapid Commun. 2010, 31, 71−74. (40) Wang, W.; Zhou, W.; Li, J.; Hao, D.; Zhiguo, S.; Ma, G. Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres. Bioprocess Biosyst. Eng. 2015, 38, 2107−2115. (41) Uppenberg, J.; Hansen, M. T.; Patkar, S.; Jones, T. A. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 1994, 2, 293−308. (42) Bassi, J. J.; Todero, L. M.; Lage, F. A. P.; Khedy, G. I.; Ducas, J. D.; Custodio, A. P.; Pinto, M. A.; Mendes, A. A. Interfacial activation of lipases on hydrophobic support and application in the synthesis of a lubricant ester. Int. J. Biol. Macromol. 2016, 92, 900−909. (43) Matte, C. R.; Bussamara, R.; Dupont, J.; Rodrigues, R. C.; Hertz, P. F.; Ayub, M. Immobilization of Thermomyces lanuginosus lipase by different techniques on Immobead 150 support: characterization and applications. Appl. Biochem. Biotechnol. 2014, 172, 2507− 2520. (44) Kılınç, A.; Teke, M.; Ö nal, S.; Telefoncu, A. Immobilization of pancreatic lipase on chitin and chitosan. Prep. Biochem. Biotechnol. 2006, 36, 153−163. (45) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248−254.

869


Enhanced Productivity in Glycerol Carbonate ... - ACS Publications

Recommend Documents