Improvement of Efficiency in the Enzymatic Synthesis of Lactulose

Article pubs.acs.org/JAFC

Improvement of Efficiency in the Enzymatic Synthesis of Lactulose Palmitate Claudia Bernal,* Andres Illanes, and Lorena Wilson Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2085, P.O. Box 4059, Valparaíso, Chile S Supporting Information *

ABSTRACT: Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/ water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source, and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS), were used. The highest lactulose palmitate yields were obtained at 47 °C in acetone, for all biocatalysts, while the best lactulose:palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30−50%). KEYWORDS: immobilization, lipase, lactulose, silica support, sugar fatty acid ester

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INTRODUCTION Surfactants are amphiphilic compounds containing both hydrophobic and hydrophilic moieties that in oil/water or air/water mixtures can reduce the interfacial tension forming emulsions.1 The development of new surfactants, with innocuous properties and low cost, is an urgent need, both in the food and pharmaceutical industry. Sugar esters are nontoxic, odorless, and biodegradable molecules exhibiting a wide range of hydrophilic−lipophilic balance values, which allows their use as additives, emulsifiers, and antimicrobial agents in such industries.2,3 There are several conventional methods for the commercial production of sugar esters, most of them based on chemical catalysis, using environmentally offensive reactants and leading to complex mixtures of products.4 Sugar esters synthesis by enzyme catalysis is more selective and environmentally acceptable,5 so there is current interest in the production of these compounds by biocatalysis.6 Lipases are promising biocatalysts for such reactions owing to their ability to utilize a wide spectrum of substrates;7 however, the use of nonaqueous solvents as reaction media is required to increase the yield and productivity of the esterification reaction while enzyme immobilization will allow a better control of the reactivity and selectivity also increasing the operational stability of the biocatalyst.8 Reaction conditions for sugar ester synthesis have been studied from several precursors (sugar and fatty acid) and with different lipases,9,10 but the optimal conditions always depend on substrates and biocatalysts so that no guidelines for the enzymatic synthesis of sugar esters have been established. Furthermore, the lipase source and the immobilization methodology used to obtain the biocatalyst have shown to strongly influence the biocatalyst performance in the reaction.11−13 © 2015 American Chemical Society

Sugar esters are formed by the esterification reaction between carboxylic acids and sugars, lauric acid being the most commonly used acid11,14 while many different sugars have been tested.15 The number of carbon atoms of the carboxylic acid and the molecular size of the sugar moiety will determine the hydrophilic hydrophobic balance of the sugars esters synthesized.16 Therefore, the synthesis of lactulose palmitate monoester seems interesting because palmitic acid has a large hydrophobic chain widely used in foods, while lactulose is an interesting sugar due to its prebiotic properties.17 In a previous work,13 we reported the influence of the chemical surface of the support on the immobilization of lipases from Alcaliges sp. and Pseudomonas stuzeri, evaluating the expressed activity and thermal stability. This report refers only to palmitate lactulose synthesis under typical conditions (acetone, 40 °C and molar ratio of substrates of 1:2). The main purpose of this work is the evaluation of the influence of four solvents in the expressed activity and thermal stability of lipases immobilized to silica supports activated with octyl or octyl-glyoxyl groups with the aim of improving the catalytic performance of the biocatalyst in the synthesis of palmitate lactulose, evaluating the monoester yield obtained in different organic solvent media, at different temperatures and molar ratio of substrates.

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MATERIALS AND METHODS

Chemicals. The following analytical grade reagents were purchased from Merck (Darmstadt, Germany) and used without further modification: ethyl acetate (EtAc), sodium silicate (25−29% SiO2 Received: Revised: Accepted: Published: 3716

November 1, 2014 March 17, 2015 March 23, 2015 March 23, 2015 DOI: 10.1021/jf505222x J. Agric. Food Chem. 2015, 63, 3716−3724

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Journal of Agricultural and Food Chemistry and 7.5−9.5% Na2O), sodium periodate, and sulfuric acid (98%). Sodium borohydride, cetyltrimethylammonium bromide (CTAB), trimethoxy(octyl)silane (OTMS; 96%), glycerin, glycidyloxypropyltrimethoxysilane (GPTMS 99.7%), p-nitrophenol (pNP), p-nitrophenyl butyrate (pNPB), acetone, propylene carbonate, methyltetrahydrofuran (Me-THF), tert-butanol (t-BuOH), palmitic acid, ascorbyl palmitate, and TritonX-100 (laboratory grade) were purchased from Sigma−Aldrich (St. Louis, MO, USA). Lactulose was purchased from Carbosynth (Berkshire, United Kingdom). Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), under the trade names Lipase TL and Lipase QL respectively, were kindly donated by Meito-Sangyio Ltd. (Fuchu, Japan). All other reagents and solvents were of the highest available purity and used as purchased. Synthesis and Characterization of Supports. Silica Synthesis. Synthesis and functionalization of silica was carried out according to a previous work.18 A typical reaction of synthesis is described below: a mixture with the following molar proportion of reagents: SiO2:Na2O:CTAB:EtAc:H2O = 1:0.3:0.24:7.2:193 was heated at 80 °C for 48 h under quiescent conditions. Then the solid was calcined at 540 °C (heating rate: 1.5 °C min−1) for 3 h and derivatized according to the requirements of the chemical surface. Octyl Silica Derivatization (OS). One g of silica was activated under vacuum then was mixed with 30 mL of 10% OTMS in toluene solution and gently stirred under reflux for 6 h. After filtration, the solid was washed with acetone and abundant water (conditioning the silica surface for the enzyme) and finally dried at room temperature.13 Octyl-glyoxyl Silica Derivatization (OGS). The octyl and glyoxyl cobonded silica (OGS) particles were prepared in a similar way than OS, using a 10% GPTMS-OTMS mixture in a 1:1 proportion. After washing and filtration, the derivatized silica was oxidized by contacting the support with 0.1 M NaIO4 solution for 2 h at 25 °C. Details of the grafting reaction can be found in Bernal et al.13,19 Biocatalyst Preparation. AsL and PsL were immobilized in the supports according to their chemical surface. For OS, the support was prepared at pH 7 to favor its hydrophobic interaction with the lipase, while for OGS preparation, the enzyme was first contacted with the support at pH 7 to favor hydrophobic interaction but, after that, the pH of the suspension was increased to 10 with the aim of favoring covalent bond formation between the glyoxyl groups of the activated silica and the lysine residues of the enzymes. Immobilization was carried out offering 160 and 200 IU of PsL and AsL per gram of support, respectively. More details about the immobilization process can be found in previous works.13,18 Assay of lipase activity was performed by measuring the increase in absorbance at 348 nm produced by the released p-nitrophenol in the hydrolysis of 0.4 mM p-nitrophenyl butyrate (pNPB) in 25 mM sodium phosphate buffer at pH 7.0 and 25 °C. One international unit of lipase activity (IU) was defined as the amount of enzyme that hydrolyzes 1 μmol of pNPB per minute under the conditions described above. Expressed activity was determined in IU per gram of biocatalyst. Stability of Biocatalysts. Stability of the biocatalysts and the soluble lipases was evaluated under two nonreactive conditions: in 25 mM sodium phosphate buffer at pH 7.0 and 60 °C, and in 100% acetone, t-BuOH, Me−THF, and propylene carbonate at 40 °C without the addition of surfactant or activity stabilizers in both cases. Aliquots were withdrawn periodically under stirring in order to have a homogeneous biocatalyst suspension. The residual activity of the enzymes was measured as described previously, and half-life (period of time at which the enzyme have lost one-half of its initial activity) was determined. Lactulose Palmitate Synthesis. Lactulose palmitate was synthesized at a 10 mL scale by esterification of lactulose (5 mM) with palmitic acid (2.5 mM) using different organic solvents: acetone, t-BuOH, propylene carbonate, and Me−THF at 40 °C. The reaction mixture contained 15 mg/mL of biocatalyst. Substrates and products of reactions were determined by HPLC using a C-18 column (Waters Symmetry C18, 5 μm 4.6 mm × 150 mm) and a UV−vis spectrophotometer detector (JASCO model AS-2089). Separation

was achieved by eluting with acetonitrile:water mixtures as mobile phase at a flow rate of 1 mL/min with the following gradient program: 60:40 (v/v) for 6 min and then 95:5 (v/v) for 18 min. Detection was performed in a UV detector at 270 nm and the monoester of lactulose palmitate was analyzed by HPLC-MS (model 2020, Shimadzu, Kyoto, Japan) with photodiode detector (SPD M 20A) and mass analyzer (MS 2020) with electrospray ionization (ESI) system. The monoester yield was calculated according to eq 1: Y(%) =

[monoester] × Vreactor × MWlactulose × 100 Wilactulose

(1)

where [monoester] is the lactulose palmitate monoester molar concentration, Vreactor is the reactor volume, MWlactulose is the molecular weight of lactulose, and Wilactulose is the initial mass of lactulose in the reaction mixture. Specific productivity (SP) was calculated according to eq 2: SP =

[monoester] × MWmonoester × V t × Wbiocatalyst

(2)

where [monoester] is the lactulose palmitate monoester molar concentration, MWmonoester is the molecular weight of the monoester, V is the reactor volume, t is the time of reaction, and Wbiocatalyst is the mass of biocatalyst in the reaction mixture. Purification of lactulose monoester was carried out by scaling-up the HPLC procedure in a preparative column of Silica C-18 (particle size, 40−75 μm; and pore size, 70 Å). Reaction mixture was dissolved in acetone and injected into the column. Mobile phase consisted of acetonitrile and water mixtures at different ratios (60:40 for monoester and 95:5 for other products and residual palmitic acid). The flow rate was kept at 2.0 mL/min, and the injection volume was 0.5 mL. Fractions were analyzed by HPLC according to the methodology described above. Collected peak fractions were evaporated to dryness under reduced pressure at 55 °C. Experimental Design. With the aim of evaluating the effect of reaction conditions in the lactulose palmitate monoester yield and specific productivity, an experimental design was carried out. For the 23 factorial design, temperature and substrates molar ratio were selected as variables: each of them were tested at 5 levels (high, centered, low, and two axials) coded −1.41, −1, 0, +1, and +1.41. The uncoded and coded values for each level of these variables are given in Table 1. The experimental setting (11 duplicate runs) was generated

Table 1. Factors and Their Levels Selected for the 23 Full Factorial Design variable

temperature (°C) lactulose:palmitic acid molar ratio temperature (°C) lactulose:palmitic acid molar ratio

levels −1.41 −1.00 Acetone 26.5 30.0 0.60 1.00

0.00

1.00

1.41

38.5 2.00

47.0 3.00

50.5 3.41

Propylene Carbonate 54.1 50.0 40.0 0.60 1.00 2.00

30.0 3.00

26.0 3.41

by Design-Expert 6.0.8 software. The reaction media were analyzed after 48 h by HPLC, as described above, analyzing the monoester yield (eq 1). Response surface analysis was carried out from these experiments, and ANOVA analysis, the model adjustment for the final equations, and their graphic representation were done with the Design-Expert 6.0.8 software. Error obtained between experimental and modeled data (Ey) was calculated according to eq 3:

Ey = 3717

experimental yield × 100 predicted yield

(3) DOI: 10.1021/jf505222x J. Agric. Food Chem. 2015, 63, 3716−3724

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Journal of Agricultural and Food Chemistry

Table 2. Stability and Expressed Activity in Organic Solvents of Pseudomonas stutzeri (PsL) and Alcaligens sp. (AsL) Lipases Immobilized in Porous Octyl-silica (OS) and Octyl-glyoxyl-silica (OGS)a aqueous media biocatalyst

EA IU g−1

soluble-PsL OGS-PsL OS-PsL soluble-AsL OGS-AsL OS-AsL

2486*b 87.0 100 1355*b 58.0 60.0

propylene carbonate EA UI g−1 96.0 300 31.0 264

t1/2 (h) 2.40 4.00 2.80 3.20 18.2 9.20

t-BuOH

acetone EA IU g−1 150 365 58.0 108

t1/2 (h) 2.60 30.3 2.60 1.90 5.00 3.80

EA IU g−1 163 410 50.0 214

Me−THF t1/2 (h) 3.20 91.3 33.6 3.20 11.7 46.0

EA IU g−1 21.0 75.0 14.0 18.0

t1/2 (h) 3.60 47.0 21.0 8.00 36.2 5.00

EA: specific activity per unit mass of biocatalyst. t1/2: half-life for every solvent at 40 °C. b*Enzyme activity of soluble enzyme expressed in IU per gram of commercial powder. a

enzyme surface to the glyoxyl groups of the support.13 The highest thermal stability was obtained in the presence of tBuOH. This is due to its high viscosity that can decrease the conformational mobility of the protein, stabilizing its tridimensional structure.17 Palmitate Lactulose Synthesis. Sugar ester synthesis was carried out in the presence of pure solvent with the aim of favoring esterification over hydrolysis. Propylene carbonate, acetone, t-BuOH, and Me−THF were selected because polar solvents favor the solubility of lactulose, one of the main problems of synthesis of sugar fatty acid esters.21 After 48 h of reaction, all biocatalysts catalyze the conversion of palmitic acid to palmitate ester (Figure 1); however, only the conversion to

All experiments were done in triplicate and the standard deviation (σx) was calculated according to σx = ± 0.5 ∑ (x i − x 2)

(4)

where xi is the individual value for every measure and x is the average value in the experiment. Biocatalyst Reuse. Effect of time and biocatalyst:palmitic acid mass ratio was evaluated with the aim of optimizing the batch reaction of synthesis. At the selected conditions after the experimental design, reaction was carried out during 48 h with 100 IU/g palmitic acid or during 24 h with 200 IU/g palmitic acid. In both cases, the yield of monoester and specific productivity were evaluated (eqs 3 and 4). Operating performance in successive batches was evaluated with the obtained biocatalysts under the optimum reaction conditions established by the experimental design. At the end of each batch, one aliquot was taken and analyzed by HPLC as previously described in order to determine the monoester yield and specific productivity. Between cycles, the reacted medium was filtered out and the retained biocatalyst washed with acetone and dried at 25 °C to remove residual substrate, products, and solvent prior to using it in the subsequent batch.

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RESULTS AND DISCUSSION Biocatalysts Characteristics. Synthesis of lactulose palmitate was performed with lipases from Pseudomonas stutzeri and Alcaligenes sp. because they exhibited good esterase activity when immobilized in octyl silica and glyoxyl-octyl silica.13 Their expressed activities in aqueous media are shown in Table 2, indicating that the orientation into the support influenced the expressed activity and immobilization yield, which was reported in a previous work.13 Considering that esterification reactions are conveniently conducted in organic solvents, expressed activity and stability at 40 °C was evaluated in acetone, tBuOH, Me−THF, and propylene carbonate. Despite the fact that the activity of lipases in soluble form cannot be measured in organic solvent (the proteins precipitate), it can be seen that immobilization in octyl and octyl-glyoxyl silica favored the expressed activity of both enzymes (in comparison with expressed activity of the biocatalyst in aqueous medium) except when the activity was measured in Me−THF (Table 2). This behavior can be explained by the physicochemical characteristics of acetone, propylene carbonate, and t-BuOH, which could favor the opening of the lipase lid and preclude the essential water layer around the active site of the enzyme from being stripped off.20 Thermal stability of biocatalysts in pure organic solvent (with water content corresponding to the equilibrium moisture) at 40 °C was increased by immobilization in most of cases, it being higher for the heterofunctional support OGS (Table 2). This trend is due to the extra stiffening caused by the covalent attachment of the lysine residues of the

Figure 1. Monoester yield on lactulose palmitate synthesis with Pseudomonas stutzeri (PsL) and Alcaligens sp. (AsL) lipases immobilized in porous octyl-silica (OS) and octyl-glyoxyl-silica (OGS) at 40 °C after 48 h with lactulose:palmitic acid molar ratio of 1:2 in the presence of acetone (black), t-BuOH (hatched white), Me−THF (gray), and propylene carbonate (cross-hatched gray). Experiments were carried out by triplicate.

monoester was quantified because this molecule has better hydrophilic−lipophilic balance than di- and triester (synthesis yield of di- and triester under evaluated conditions was less than 2%). Monoester identification was reported in a previous work.13 Immobilized lipases in OS exhibited a higher yield toward monoester synthesis in comparison with OGSbiocatalysts, probably because the lid of lipase in OS support is more open than in OGS silica, which promotes the catalysis. Yield of lactulose palmitate monoester synthesis depended on the solvent used as reaction medium (Figure 1), acetone 3718

DOI: 10.1021/jf505222x J. Agric. Food Chem. 2015, 63, 3716−3724

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Table 3. Experimental Design and Results (Experimental and Predicted) Obtained Using a 23 Factorial Design for the Optimization of Yield Lactulose Palmitate Monoester Synthesis Catalyzed by Lipase from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL) Immobilized in Porous Octyl-silica (OS) and Octyl-glyoxyl-silica (OGS), Using Acetone As Solventa OGS-PsL

a

OS-PsL

OGS-AsL

OS-AsL

T

r

E (%)

M (%)

E (%)

M (%)

E (%)

M (%)

E (%)

M (%)

47.0 47.0 50.5 38.5 30.0 38.5 38.5 30.0 38.5 38.5 26.5

3.00 1.00 2.00 2.00 1.00 2.00 3.40 3.00 0.60 2.00 2.00

39.6 36.1 36.2 16.4 14.5 16.4 37.4 10.0 14.8 19.6 13.1

39.7 36.2 36.0 17.5 14.6 17.5 37.2 10.2 14.6 17.5 12.9

50.1 41.7 41.8 18.6 4.10 19.1 44.4 4.80 6.60 21.0 2.50

49.4 41.0 42.5 20.1 3.50 20.1 45.1 4.20 7.30 20.1 3.20

14.4 20.6 14.4 6.40 6.22 6.20 4.60 6.00 18.6 6.40 4.60

11.0 20.5 16.6 6.40 8.35 6.40 7.60 4.80 16.7 6.40 3.60

16.5 14.5 16.5 12.5 4.10 13.6 13.6 4.10 27.1 14.0 2.20

18.5 13.1 18.5 13.6 6.80 13.6 13.6 7.30 25.8 13.6 1.60

T, temperature (°C); r, lactulose:palmitic acid molar ratio; E, experimental data; M, value predicted by model.

Figure 2. Response surface and contour graphics for the effect of temperature and lactulose:palmitic acid molar ratio on lactulose palmitate monoester yield with lipase from Pseudomonas stutzeri (PsL) and from Alcaligenes sp. (AsL) immobilized in porous octyl-silica (OS) and octylglyoxyl-silica (OGS), using acetone as solvent. (A) OGS-PsL, (B) OS-PsL, (C) OGS-AsL, (D) OS-AsL.

t-BuOH and Me−THF are used, probably because these solvents have high dielectric constants, similar to water (Table S1, Supporting Information), which favors hydrolysis22,23 while slowing down esterification (Figure 1). Effect of Reactions Conditions on Lactulose Palmitate Monoester Yield. Considering the results of monoester yield (Figure 1), the experimental design was done only with acetone and propylene carbonate according to the variables specified in Table 1.

being the best solvent in all cases, probably because the balance between high polarity and low viscosity (Table 1) favors the persistence of the essential water layer around the active site of the lipase, the solubility of lactulose and the diffusion of substrates and products into and out of the biocatalyst. In propylene carbonate, the monoester yield was the second highest obtained, possibly because its viscosity is seven times higher than acetone, which reduces the diffusion rates of substrates and products. On the other hand, the reaction of palmitic acid to lactulose palmitate monoester is so slow when 3719


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Table 4. Experimental Design and Results (Experimental and Predicted) Obtained Using a 23 Factorial Design for the Synthesis Optimization of Lactulose Palmitate Monoester Synthesis Catalyzed by Lipase from Pseudomonas stutzeri and Alcaligenes sp. Immobilized in Porous Octyl-silica (OS) and Octyl-glyoxyl-silica (OGS), Using Propylene Carbonate As Solventa OGS-PsL

a

OS-PsL

OGS-AsL

OS-AsL

T

r

E (%)

M (%)

E (%)

M (%)

E (%)

M (%)

E (%)

M (%)

30.0 50.0 30.0 50.0 26.5 54.0 40.0 40.0 40.0 40.0 40.0

1.00 1.00 3.00 3.00 2.00 2.00 0.60 3.40 2.00 2.00 2.00

0.39 2.00 2.25 1.50 0.60 1.83 1.48 1.70 2.32 2.13 2.25

0.34 2.18 1.94 1.41 0.82 1.74 1.36 1.95 2.25 2.25 2.25

3.83 5.07 4.86 6.12 2.84 5.40 3.92 4.83 5.54 5.80 5.83

3.43 4.95 4.26 5.80 3.40 5.56 4.15 5.33 5.76 5.76 5.76

2.20 3.23 2.70 5.33 1.00 4.73 2.57 5.33 4.06 4.33 4.28

1.86 3.29 2.69 5.72 1.26 4.42 2.78 5.08 4.15 4.15 4.15

3.14 5.75 3.63 5.90 2.84 6.68 2.35 2.70 4.25 3.60 4.11

2.61 5.36 3.06 5.47 3.42 7.07 2.81 3.21 4.03 4.03 4.03

T, temperature (°C); r, lactulose:palmitic acid molar ratio; E, experimental data; M, value predicted by model.

ANOVA analysis for the behavior of biocatalyst of lipase from Alcaligenes sp. are presented below (eqs 7−8). OGS-AsL in acetone

The evaluation of interactions between temperature and substrates molar ratio indicates that lipase from P. stuzeri exhibited a similar dependence on these variables, independently from the support used for its immobilization, when the esterification reaction is carried out in acetone. Values of monoester yield obtained are presented in Table 3. The adjustment of data with ANOVA analysis, using the DesignExpert 6.0.8 software, shows that they can be properly modeled by a third-order polynomial in the case of monoester yield (R2: 0.9945 and 0.9968 for OGS-PsL and OS-PsL, respectively) Some terms were eliminated from the model because its statistical interaction could be irrelevant according to the pvalue (T2r and r2T), allowing obtaining of the polynomial equations that model the reaction behavior (eqs 5−6): OGS-PsL in acetone

Y (%) = 28.5 − 1.1T − 8.3r + 0.04T 2 + 2.9r 2 − 0.18Tr (7)

OS-PsL in acetone Y (%) = −30.8 + 2.6T − 26.4r − 0.04T 2 + 3.2r 2 + 0.5Tr (8)

where Y is the monoester yield, r is the lactulose:palmitic acid molar ratio, and T is the temperature. In the case of OS-AsL, the best reaction conditions were similar to those found with OGS-PsL and OS-PsL: 47 °C and molar ratio of substrate 1:3, although a lower value of yield was obtained (Y = 29.5%). In the case of OGS-AsL, best reaction conditions were 47 °C and a molar ratio of substrate 1:1, at which 20.4% yield was obtained. Differences obtained among biocatalyst can be attributed to the fact that the lipase from Alcaligenes sp. is less sensitive to changes in its 3D structure caused by the immobilization by adsorption into hydrophobic surface because this lipase, different from the one from P. stutzeri, has only one lid.26,27 The values are consistent with the response surface for every biocatalyst (Figure 2), showing that the maximum values are unique in both cases. When propylene carbonate was used as solvent, the monoester yield trend obtained with both biocatalysts could be properly modeled by a second-order polynomial (eqs 10−12) that according to ANOVA analysis represent a good statistical adjustment between predicted and experimental values, as shown in Table 4. OGS-PsL in propylene carbonate

Y (%) = 425.9T + 64.7r + 0.9T 2 − 45.3r 2 + 0.2Tr − 7.5 × 10−3T 3 + 8.2r 3

(5)

OS-PsL in acetone Y (%) = 515.6T + 103.7r + 1.3T 2 − 63.6r 2 + 0.2Tr − 0.01T 3 + 11.1r 3

(6)

where Y is the monoester yield, r is the lactulose:palmitic acid molar ratio, and T is the temperature. Data optimization using response surface methodology allowed obtaining of the conditions that maximized monoester yield, as presented in Figure 2 for all biocatalysts.24,25 The graphical analysis indicates that yield is positively affected by temperature and molar ratio. These models predict that the highest values of monoester yield are obtained for both PsL biocatalysts when the reaction is carried out at 47 °C and lactulose:palmitic acid molar ratio of 1:3, with predicted values of 39.7% and 49.4% for OGS-PsL and OS-PsL, respectively. Biocatalysts obtained with lipases from Alcaligenes sp. exhibited a similar trend for yield, independently of the immobilization support, but those from P. stutzeri showed a different behavior when the reaction was carried out in acetone (Figure 2). In the latter case, the monoester yield was adjusted to a second-order polynomial (R2: 0.9044 and 0.9447 for OGSAsL and OS-PsL, respectively). Experimental and predicted data are shown in Table 3. The model equations obtained by

Y (%) = − 13.20 + 0.54T + 3.77r − 4.88 × 10−3T 2 − 0.38r 2 − 0.06Tr

R2 = 0.938

(9)

OS-PsL in propylene carbonate Y (%) = − 10.37 + 0.59T + 2.44r − 6.39 × 10−3T 2 − 0.51r 2 − 4.11 × 10−4Tr

R2 = 0.867

(10)

OGS-AsL in propylene carbonate 3720


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Figure 3. Response surface and contour graphics for the effect of temperature and lactulose:palmitic acid molar ratio on lactulose palmitate monoester yield with lipase from Pseudomonas stutzeri (PsL) and from Alcaligenes sp. (AsL) immobilized in porous octyl-silica (OS) and octylglyoxyl-silica (OGS), using propylene carbonate as solvent. (A) OGS-PsL, (B) OS-PsL, (C) OGS-AsL, (D) OS-AsL.

Table 5. Monoester Yield (Y) and Specific Productivity (P) Obtained in the Synthesis under Optimum Conditions of Lactulose Palmitate Monoester, With Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL) Immobilized in Porous Octylsilica (OS) and Octyl-glyoxyl-silica (OGS)a acetone

a

propylene carbonate

biocatalyst

T (°C)

r

Y (%)

P

Ey (%)

T °C

A:B

Y (%)

P

Ey (%)

OGS-PsL OS-PsL OGS-AsL OS-AsL

47.0 47.0 47.0 47.0

1:3 1:3 1:1 1:3

38.3 48.3 20.7 30.9

0.06 0.08 0.04 0.05

3.50 2.30 1.50 3.30

44.0 46.0 37.8 50.0

1:2.0 1:2.4 1:3.0 1:2.0

2.20 5.80 4.40 5.60

0.004 0.01 0.007 0.01

4.40 4.90 6.40 5.10

r, lactulose:palmitic acid molar ratio; P, specific productivity (g day−1 g catalyst−1); Ey, yield error.

is reflected by the fact that the type of solvent and the reaction conditions (temperature and substrates molar ratio) at which the highest yield was obtained are different for each biocatalyst (Table 5); however, they were similar when lipases were immobilized in OS, which does not occur when acetone is used as reaction medium, showing the strong effect of the high viscosity of propylene carbonate in P. stutzeri lipase activity reducing the yield obtained with this enzyme more strongly than with the enzyme from Alcaligenes sp. In the same way, the yield was the lowest when the reaction was catalyzed by OGSPsL and lower in propylene carbonate than in acetone, also showing the marked effect of viscosity on the performance of this lipase (Table 5). Considering the results obtained at optimum conditions (Table 5), the effect of reaction time and biocatalyst to substrate mass ratio were evaluated. When the reaction time was decreased to one-half (down to 24 h), and the biocatalyst dose was doubled (up to 200 IU per gram of palmitic acid),

Y (%) = −9.64 + 0.55T − 0.34r − 6.53 × 10−3T 2 2

− 0.11r + 0.04Tr

2

R = 0.967

(11)

OGS-AsL in propylene carbonate Y (%) = 5.60 − 0.34T + 2.52r + 6.09 × 10−3T 2 − 0.51r − 8.57 × 10−3Tr

R2 = 0.884

(12)

where Y is the monoester yield, r is the lactulose:palmitic acid molar ratio, and T is the temperature. Figure 3 shows the response surfaces for the yield of all biocatalysts during palmitate lactulose synthesis using propylene carbonate. In this case, a different response surface was obtained for each biocatalyst, indicating that the high viscosity of propylene carbonate (Table S1, Supporting Information) produced a more significant effect on the catalytic behavior of the immobilized lipases due to the extra stiffness in the 3D structure when they were immobilized into OGS support. This 3721


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Figure 4. Monoester yield (filled symbols) and hydrolytic expressed activity (empty symbols) during reaction with lipase from Pseudomonas stutzeri (A) and from Alcaligenes sp. (B) immobilized in octyl-silica (OS) (triangles) and in octyl-glyoxyl-silica (OGS) (squares) in successive batch operation (24 h of operation in each batch) in the synthesis of lactulose palmitate monoester under optimal conditions. Experiments were carried out in triplicate.

similar monoester yields and specific productivities were obtained than those achieved in the experimental design, showing that the performance of the biocatalyst is determined by the enzyme dose and the reaction time, as expected. Differences in the values of yield and productivity between both conditions were lower than 5% (data not shown). Operating Performance in Successive Batches. Considering the lactulose palmitate monoester yields obtained with all biocatalyst in acetone and propylene carbonate, the biocatalyst reuse was tested in successive 24 h batch operation, using acetone as reaction medium at the optimum reactions conditions (Table 5) with 200 IU per gram of palmitic acid. Yield and cumulative productivity of palmitate monoester synthesis obtained are presented in Figures 4 and 5. After 10 successive batches, monoester yields obtained with all lipase biocatalysts from P. stutzeri progressively decreased with each batch. (Figure 4A); however, in the last evaluated batch, the relative hydrolytic activity of OGS-PsL biocatalyst remained higher than the one of OS-PsL in the same batch (44.1% and

26.9% with respect to the initial batch, Figure 4A). On the other hand, lipase from Alcaligenes sp. exhibited a different behavior, showing similar values for monoester yield after each batch when immobilized in OGS support (Figure 4B), probably because in this case the additional stiffness provided by covalent bonding to the support precluded its inactivation; the opposite happened when this lipase was immobilized only by adsorption in OS support (67.4% and 28.6% respect to initial, OGS-AsL and OS-AsL, respectively, Figure 4B). This behavior was the expected for the lipases immobilized in two steps: adsorption by the hydrophobic pocket and then covalent bond formation with the lysine residues near to the active pocket.13 Evaluation of specific productivity in successive batches showed a similar trend regardless of the lipase (Figures 5). In this case, the biocatalysts obtained with octyl silica had a better behavior batch after batch, generating a higher increase in the cumulative productivity in comparison with the biocatalyst obtained with glyoxyl-octyl silica. This behavior can be explained considering the initial hydrolytic activity because 3722


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Figure 5. Cumulative productivity (filled symbols) and hydrolytic expressed activity (empty symbols) during reaction with lipase from Pseudomonas stutzeri (A) and from Alcaligenes sp. (B) immobilized in octyl-silica (OS) (triangles) and in octyl-glyoxyl-silica (OGS) (squares) in successive batch operation (24 h of operation in each batch) in the synthesis of lactulose palmitate monoester under optimal conditions. Experiments were carried out in triplicate.

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biocatalysts immobilized by adsorption expressed a higher activity (OS-PsL and OS-AsL), making the increase in productivity faster than in the case of OGS-AsL and OGSPsL (Figures 5). However, it was expected that the cumulative productivity of OGS-AsL biocatalyst will be higher than OS-AsL after many batches because in this case the operational stability was significantly higher that the OS-AsL (Figure 5B). Lipases from Alcaligenes sp. and P. stutzeri immobilized in silica with different functional groups were used for the synthesis of lactulose palmitate monoester with very good yields and specific productivities. Solvents, temperature, and substrates molar ratio influenced the biocatalyst performance, best results being obtained with both lipases when acetone was used as solvent. Enzymatic immobilization influenced also the monoester yields and productivities obtained with each biocatalyst, showing that covalent immobilization into glyoxyl-octyl silica could produce a fairly active and robust biocatalyst able to be reused several times.

ASSOCIATED CONTENT

S Supporting Information *

Physicochemical properties of organic solvents. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +56 322372025. Fax: +56 322273803. E-mail: [email protected], /[email protected]. Funding

Work financed and postdoctoral fellowship by Chilean Fondecyt grant 3130321. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Generous donation of lipases from Pseudomonas stutzeri and Alcaligenes sp. from MEITO-SANGYO. Ltd. is acknowledged. 3723


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Improvement of Efficiency in the Enzymatic Synthesis of Lactulose

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