Enhanced Synthesis of Alkyl Galactopyranoside by Thermotoga

Research Article pubs.acs.org/journal/ascecg

Enhanced Synthesis of Alkyl Galactopyranoside by Thermotoga naphthophila β‑Galactosidase Catalyzed Transglycosylation: Kinetic Insight of a Functionalized Ionic Liquid-Mediated System Jingwen Yang,†,‡ Bianca Pérez,‡ Sampson Anankanbil,‡ Jingbo Li,‡ Renjun Gao,*,† and Zheng Guo*,‡ †

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Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China ‡ Department of Engineering, Aarhus University, Gustav Wieds Vej 10, Aarhus 8000, Denmark S Supporting Information *

ABSTRACT: Green synthesis is of pivotal importance for environmental sustainability. This work reports a novel approach to synthesize an array of alkyl galactopyranosides using thermophilic β-galactosidase from Thermotoga naphthophila RKU-10 (TN1577) as biocatalyst and milk processing waste lactose as galactosyl donor. Ammoeng 102 (only 2.5% addition of total reaction volume), a functionalized ionic liquid (IL) containing tetraaminum cation with C18 acyl and oligoethylene glycol, is identified as the most promising one from a variety of structurally diverse ILs, affording a 2.37-fold increase in octyl galactopyranoside yield compared to the buffer system. Up to 18.2 g L−1 octyl galactopyranoside could be produced in 7 h, which is significantly higher than any previous report in terms of time-space efficiency. Kinetic study and COSMO-RS in silico predictions elucidate that the thermophilic nature of TN1577 β-galactosidase, increased solubility of substrate, suppression of hydrolysis, and excellent biocompatibility of Ammoeng 102 with enzyme (allowing TN1577 β-galactosidase to perform optimal catalysis up to 95 °C) are the main driving forces. The general applicability of the Ammoeng 102 system is verified, by which a series of alkyl galactopyranosides are successfully synthesized with n-butanol to n-tetradecanol as alkyl acceptors and lactose as galactosyl donor. KEYWORDS: β-Galactosidase, Transglycosylation, Ionic liquids (ILs), Alkyl galactopyranoside, Conductor-like screening model for real solvent (COSMO-RS)

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solvent but water presents a challenge.6 This is why enzymatic synthesis of alkyl glycosides is generally carried out in biphasic systems to ensure solubilization of substrate. However, the presence of water in the reaction system may lead to the hydrolysis of substrate lactose and hydrolysis of desired products. As illustrated in Scheme 1, enzymatic transgalactosylation of galactose from lactose to alkyl alcohols in a biphasic reaction system may also lead to two other side reactions: (i) hydrolysis of lactose to yield glucose and galactose and (ii) hydrolysis of alkyl galactopyranoside to generate alkyl alcohol and galactose.7 To increase the solubility of carbohydrate substrate, some studies suggest to use watermiscible organic solvents8 such as DMSO and DMF; however, these solvents generally deactivate glycosidases at a very low concentration.6 In addition, these solvents are toxic and harmful, which goes against the principle of eco-friendliness.

INTRODUCTION Nowadays, the production of green, eco-friendly, and biodegradable surfactants based on renewal resources with green technology is of great interest for the sustainable chemical industry.1,2 Alkyl glycosides, a type of all-natural, nontoxic, and nonionic surfactant, have better features than conventional surfactants, as they are low-foaming, nonirritating to the skin and eyes, and readily biodegradable. In addition, alkylglycosides present high compatibility, low surface tension, and wide pH adaptability, among other unique features.3,4 Alkyl glycosides are also known to have good dermatological properties, which could find potential application in the cosmetic industry for personal care.5 Alkyl glycosides can be enzymatically synthesized via β-galactosidase catalyzed transglycosylation of lactose, which is available in large quantity as a milk processing waste.6,7 However, the production of alkyl glycosides following green and sustainable synthetic approaches remains a challenging task in developing biocatalysts and creating an efficient reaction system; for example, the poor solubility of the native carbohydrates in almost any other © 2017 American Chemical Society

Received: November 27, 2016 Revised: January 2, 2017 Published: January 11, 2017 2006

DOI: 10.1021/acssuschemeng.6b02862 ACS Sustainable Chem. Eng. 2017, 5, 2006−2014

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ACS Sustainable Chemistry & Engineering

Scheme 1. β-Galactosidase (TN1577) Catalyzed Synthesis of Alkyl Galactopyranoside and Side Reaction Pathways: (A) Transgalactosylation; (B) Hydrolysis of lactose; (C) Hydrolysis of alkyl galactopyranoside

Ionic liquids (ILs), known as molten salts, can be used as solvent replacements of hazardous and volatile organic solvents in different industrial applications. Furthermore, ILs have been shown to increase the biocatalytic activity of multiple enzymes, including esterases, lipases, and proteases due to their ability to dissolve polar substrates, such as amino acids or carbohydrates, in a low-water environment.9−14 However, reports of glycosidase catalyzed synthesis in ILs are rather rare, mainly because glycosidases lose most of their activities in many types of ILs. Accordingly, it is important to identify ILs potentially valuable for glycosidase catalyzed synthesis of alkyl glycosides based on enzyme performance in different ILs.15 Therefore, study of the biocatalytic activity of β-galactosidase toward transgalatosylation in different ILs is of great interest to identify ILs which lead to enhanced production of alkyl glycosides by increasing substrate solubility, suppressing hydrolysis without losing enzyme activity.16 In this context, based on our recently identified thermophilic β-galactosidase from Thermotoga naphthophila RKU-10 (TN1577),17 we examine the biocatalytic activity of TN1577 in the synthesis of alkyl glycosides via transgalactosylation reactions in different ILs, including a group of amphiphilic tetraammonium-based salts (the Ammoeng series, containing tetraaminum cation with C18 acyl and oligoethylene glycol).9−11 Ammoeng 102 is identified as the most promising IL with significantly improved volumetric productivity, and the optimization of operation variables in Ammoeng 102 is performed using octanol as a model galactosyl acceptor. To elucidate the mechanism, a kinetic study of enzymatic transgalactosylation/hydrolysis in buffer and Ammoeng 102 systems is conducted, and an in silico prediction of substrate solubility and activity coefficients of substrates/products by a physically sound model Conductor-like screening model for real solvent (COSMO-RS) is performed, which demonstrates that increased solubility of the substrate and excellent biocompatibility of Ammoeng 102 with enzyme (allowing TN1577 β-galactosidase to perform catalysis up to 90 °C) are the main driving forces for enhanced reaction performance. Furthermore, to verify the general applicability of the Ammoeng 102 system, a series of alkyl acceptors ranging from n-butanol to n-tetradecanol were attempted using lactose as the galactosyl donor to evaluate the effect of the hydrocarbon

length of the alkyl donor on the transgalactosylation activity of the enzyme.

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EXPERIMENTAL SECTION

Materials. All the reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). ILs and silica gel plates were purchased from Solvent Innovation GmbH (Cologne, Germany) and Merck Ltd. (Darmstadt, Germany), respectively. Preparation of galactosidase. TN1577 was produced following a previously reported procedure.18 E. coli BL21(DE3) cells were preincubated in Luria-Bertani medium (LB medium, with 1 mg mL−1 ampicillin) with 180 rpm agitation at 37 °C. After the OD600 of the culture liquid reached 1.0, IPTG was added to induce enzyme expression, and the cells were grown at 25 °C for 12 h with 150 rpm agitation. The induced cells were harvested by centrifugation and washed once with 50 mmol L−1 sodium phosphate buffer (pH 7.0). A 5-g dispersion of the cells in 50 mL of 50 mmol L−1 sodium phosphate buffer (pH 7.0) was disrupted by sonication. The cellular debris was removed by centrifugation to obtain a crude lysate. The crude extract was incubated in a water bath for 10 min at 80 °C to denature the E. coli proteins. The extract was then centrifuged to separate the crude enzyme from the heat-denatured cellular components and proteins. Finally, the extract was lyophilized for subsequent experiments. β-Galactosidase activity assay. β-Galactosidase activity was measured using o-nitrophenyl-β-galactoside (o-NPG) as an artificial substrate. Kinetic parameters were determined using a UV−visible spectrophotometer 2550 (SHIMADZU, Tokyo, Japan) combined with UVProbe 2.33 software. The change in absorbance at 420 nm over 1 min was observed using 1 mL of the reaction mixture containing 0.5 mmol L−1 o-NPG, 50 mmol L−1 sodium phosphate buffer (pH 6.8), and an appropriate amount of β-galactosidase at 80 °C. One unit of enzyme hydrolytic activity was defined as the hydrolysis of 1 μmol of o-NP per minute under the defined conditions.18 Enzymatic reactions. All reactions were carried out in 2 mL reaction systems at 50−100 °C, 150U of enzyme lyophilized powder and 0.025−0.1 g of IL were added (when the reaction was performed in the presence of IL) to a mixture containing 0.2 mL of saturated lactose solution with buffer (sodium phosphate buffer, 50 mmol L−1, pH 7.0) and 1.8 mL of alkyl alcohol (from n-butanol to ntetradecanol). After 16 h of reaction, the organic phase of the reaction system and the aqueous phase were separated by centrifugation. The alkyl galactopyranosides in the organic phase mixtures were separated from other components using a silica gel column. The column was developed using as eluent first pure diethyl ether, and when the alkyl alcohol was eluted out of the column, the developing solvent was changed to ethyl acetate−methanol−water (17:2:1, v/v). Alkyl 2007


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Table 1. Molecular Structures of Ionic Liquids, and Effects on Production of TN1577 Catalyzed Transgalactosylation Investigated in This Worka

a

Reaction conditions: pH 7.0, 0.2 mL of buffer, 1.8 mL of n-octanol, 20 mg of enzyme, 75 °C, 0.05 g of ionic liquid, reaction time 16 h.

galactopyranosides were monitored by thin-layer chromatography (TLC) using ethyl acetate−methanol−water (17:2:1, v/v). TLC plates were stained with α-naphthol (2.56 g L−1) in an ethanol−sulfuric acid mixture (90:10, v/v). Alkyl galactopyranosides and carbohydrates were detected by heating the plate at 100 °C for 5 min. Structural identification of synthetic alkanyl galactopyranoside and quantitative analysis of the reaction mixture. The filtered reaction mixtures were analyzed by high-performance liquid chromatography (HPLC) using a SUPELCOSILTM LC-18 column fitted with an evaporative light scattering detector (ELSD). The temperature of the column was kept at 30 °C. Different mobile phases were used to detect the different alkyl galactopyranosides. To detect butyl galactopyranoside and hexyl galactopyranoside, the mobile phase used was methanol−water (3:2, v/v) and the flow rate was kept constant at 0.7 mL min−1; the retention times are 5.20 and 5.34 min, respectively. Detection of octyl galactopyranoside, decyl galactopyranoside, dodecyl galactopyranoside, and tetradecyl galactopyranoside

was performed using acetonitrile−water (1:1, v/v) as mobile phase and a flow rate of 0.8 mL min−1 (The resulting retention times were 4.62, 6.09, 9.13, and 17.70 min, respectively). The synthetic alkyl galactopyranosides obtained from silica gel column chromatographic separation as aforementioned were subjected to 1H NMR and ESI-MS analysis to confirm the molecular structures of the target. The identification of the compounds was determined by NMR-spectroscopy (a Bruker Avance III spectrometer) at 400 MHz for 1H. MS spectra were obtained on an instrument operating with electrospray ionization and ion-trap (ESI-IT) quadrupole detection (Bruker Daltonic GmbH, Bremen, Germany). The molecular structural information on synthetic alkyl galactopyranosides is detailed in the Supporting Information. Computational details of COSMO-RS. The molecular structures of lactose and ionic liquids (Scheme 1 and Table 1) were sketched as two-dimensional structures using Chemdraw Ultra 8.0, converted to SMILES annotations in TMoleX 16 version 4.2 (Cosmologic GmbH 2008


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ACS Sustainable Chemistry & Engineering & Co KG, Leverkusen, Germany), and then converted to threedimensional structures (3D) also in TMoleX16. The geometries of the obtained 3D structures were optimized in TMoleX16 at the AMI/ COSMO level using the inbuilt MOPAC program. Using the optimized geometries, the polarization charge densities σ of the molecular surfaces were calculated in TMoleX16 on the density functional theory (DFT) level, utilizing the BP (B88-VWN-P86) functional with a triple-ζ valence polarized basis set (TZVP). COSMO files for water and n-octanol were selected from the TZVP database in COSMOthermX C30_1602 (Cosmologic GmbH & Co KG, Leverkusen, Germany). Calculations of solubilities and activity coefficients were performed in the COSMOthermX program. Cations and anions of ionic liquids were combined into cosmo metafiles in COSMOthermX before use in calculations.

for predicting the thermodynamic properties of liquid mixtures, fluids, and solids using a statistical thermodynamics approach based on quantum chemical calculations. Previous works in our group have demonstrated the fast and reliable estimation of the solubility of flavonoids in ionic liquids using COSMO-RS,29 which agreed well with experimental data. Accordingly, COSMO-RS was used to predict the solubilities and activity coefficients of the ILs studied in this work.30 The conditions of the reaction system analyzed were a temperature of 75 °C, a 10% water content, and 2.5% ILs. The activity coefficients (γ) are calculated as In(γ ) = (μ(i) − μ(p))/RT

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(1)

where μ(p) is the chemical potential of all pure compounds and μ(i) is the chemical potential in the liquid phase. In addition, the solubilities (x) are calculated from

RESULTS AND DISCUSSION Effect of ionic liquids on the biocatalytic activity of TN1577. The ionic feature of ILs endows them with the capability to dissolve protonatable compounds such as carbohydrates by H-bonding,19 whereas the molecular structure of ILs also has a significant impact on enzymatic activity.20 Accordingly, the transgalactosylation activities of TN1577 to yield alkyl glycosides, with octyl galactopyranoside (oct-gal) as an example, were determined in different water-miscible ILs at 75 °C (Table 1). Table 1 presents the selected IL-based systems where TN1577 displays better or comparable oct-gal yield compared to the buffer system, among which the best results were obtained with Ammoeng 102 (increased 1.16−1.6 times compare to w/o IL; w/o IL standing for buffer system without addition of ILs) followed by [BMIM][BF4], and, subsequently, Ecoeng 111P. In general, these polar ILs help to dissolve more lactose, thus increasing the production of oct-gal. Comparison of product yields from [BMIM][BF4] (Conc of oct-gal = 29.87 mmol L−1) and [BMIM][PF6] (18.52 mmol L−1) suggests that a higher polarity of anions ([BF4]− > [PF6]−) gives enhanced productivity, likely due to higher solubility of lactose in the [BMIM][BF4] mediated system.10,11 Ammoeng 102 (Conc of oct-gal = 31.13 mmol L−1) displays a much higher oct-gal yield than Ammoeng 100 (Conc of oct-gal = 17.54 mmol L−1), where the major differences in their molecular structures are that Ammoeng 102 has a longer alkyl substituent (C18 vs C14), a less polar anion (EtSO4− vs MeSO4−), and a longer oligoethylene glycol, which presents a better protective effect for the enzyme.21 It has been shown that highly polar anions form strong hydrogen bonds with enzyme and promote unfolding, which leads to aggregation and precipitation of the enzyme.21 As reported elsewhere,22−25 the effects of nonfunctionalized ILs on the catalytic activity of glycosidase have been investigated. For example, Ferdjani et al.25 found that water-miscible ILs are more suitable for biocatalytic reactions using highly thermostable glycosidases. A study by Lang et al.24 observed that hyperthermostable βglycosidase CelB from Pyrococcus furiosus retained a higher transgalactosylation activity of lactose with various galactosyl donors in the presence of 45% [MMIM][MeSO4] compared to the absence of the ILs. Effect of solubility and activity coefficient predicted by COSMO-RS in the reaction mixture. To further understand the effect of these ILs on the biocatalytic activity of TN1577, the solubility and activity coefficient were studied, since they are important thermodynamic physical properties of a given compound and are affected by the environment in the reaction system.26 Klamt and co-workers27,28 have comprehensively described the theory of COSMO-RS as a priori method

log(x) = log[exp((μ(p) − μ(s) − ΔGfus)/RT )]

(2)

where μ is the chemical potential at infinite dilution and ΔGfus is the free energy of fusion. The results are summarized in Table S1 (Supporting Information). The solubility of [BMIM][BF4] (0.44 g/100 g) is higher than that of [BMIM][PF6] (0.42 g/100 g). This implies a consistency of the predicted lactose solubility in the ILs with observed oct-gal yield ([BMIM][BF4] 29.87 mmol L−1 > [BMIM][PF6] 18.52 mmol L−1) (Table 1). The lactose activity coefficient of lactose in [BMIM][BF4] (γlactose = 1.92) is lower than that of [BMIM][PF6] (γlactose = 2.04), suggesting a higher polarity of BF4, which is true in chemistry. The Ecoeng series IL displayed higher solubilities compared to the system without IL (Table S1), which is confined to the polarity of their respective anion and the structure of substituents in the cation (Table 1). The lactose solubilities were 0.42 and 0.48 g/100 g for Ecoeng 21 M and Ecoeng 111P, which agree with their respective experimental oct-gal yields: 17.66 mmol L−1 and 27.63 mmol L−1, respectively. However, Ecoeng 21 M even gives a lower yield than the no-IL system (Conc of oct-gal = 19.48 mmol L−1), which is likely due to the negative impact on enzyme activity from Ecoeng 21 M (Table 1).31 Finally, the COSMO-RS results also show that all Ammoeng series ILs were expected to increase lactose solubility compared to the no IL system (Ammoeng IL = 0.38 to 0.47g/100g vs no IL = 0.27 g/100 g, Table S1). Among Ammoeng series ILs, Ammoeng 102 displays the highest predicted solubility (Table S1) and observed oct-gal (Table 1). In brief, COSMO-RS prediction proved to be a very useful tool to help identify potential ILs for the enzymatic synthesis of alkyl galactopyranoside, as Ecoeng 111, Ammoeng 102, and [BMIM][BF4] displayed the higher theoretical solubilities (Table S1), and experimentally these ILs yielded the higher concentrations of oct-gal (from 27.63 to 31.13 mmol L−1, Table 1). Interestingly, the activity coefficients of water and n-octanol in the reaction vary in a very narrow range against alteration of ILs, which might be due to the large excess of n-octanol over water. Except for in [BMIM][BF4], [BMIM][PF6], and Ecoeng ILs, the activity coefficients of lactose in other ILs also vary in a narrow range (Table S1), again suggesting that the solubility of lactose in ILs largely determines the yield of oct-gal product (Table 1). Effect of IL concentration on the biocatalytic activity of TN1577. The concentration of IL in catalytic systems may significantly affect enzyme activity and stability.25 The effects of (s)

2009


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ACS Sustainable Chemistry & Engineering ILs and their contents can be analyzed as a function of both the mass transfer of the substrates to the active sites, and the ability to provide enzyme with an appropriate microenvironment for catalysis.32 Accordingly, the catalytic behavior of TN1577 is evaluated in different concentrations (0−5%) of the most promising ILs (Ammoeng 102 and Ecoeng 111P) and Ammoeng 111 at 75 °C in saturated lactose solution in sodium phosphate buffer (pH 7.0) and n-octanol (1:9, v/v) for the synthesis of oct-gal (Figure 1). Results show that the optimum contents of Ammoeng 102, Ecoeng 111P, and Ammoeng 111 were 2.5, 2.5, and 3.75% (v/v), respectively. For instance, the oct-gal yield increases 1.72-fold for an increase of Ammoeng 102 from 0 to 2.5% (Figure 1). Not surprisingly, the solubility of lactose in these IL systems predicted by COSMO-RS shows a corresponding increase and decrease of activity coefficient of lactose with increasing concentrations of ILs (Table S2). For example, more Ecoeng 111P dissolves more lactose (2.5% Ecoeng 111P solubility = 0.38 g/100 g vs 5% Ecoeng 111P solubility = 0.82 g/100 g) and decreasse the lactose activity coefficient (2.5% Ecoeng 111P γlactose = 1.95 vs 5% Ecoeng 111P γlactose = 1.63). However, the change of the activity coefficients of water and sn-octanol with increasing IL concentration is more complex due to the complication of structural variation in anions and cations of individual ILs. The results in Figure 1 suggest that too high a concentration of ILs impacts enzyme activity, and there is an optimal IL concentration differing from IL to IL (Figure 2). A similar observation has been reported by another group, that 25% [MMIM][MeSO4] is required for the synthesis of Nacetyllactosamine catalyzed by β-galactosidase from Bacillus circulans.33 It is worthwhile to notice that the fact that an extremely low concentration of ILs is able to perform a significant enhancement of catalytic synthesis of glycosides might represent an additional advantage over other systems.20−25,33 Effect of water content on the biocatalytic activity of TN1577. As stated above, there are three main reaction pathways which simultaneously take place during the transgalactosylation in the biphasic reaction system (Scheme 1): (1) alkylation of the galactose moiety with the alkyl alcohol; (2) hydrolysis of lactose to yield glucose and galactose; and (3) hydrolysis of the product alkyl galactopyranoside to generate galactose and alkyl alcohol. To inhibit the hydrolysis, the water content in the reaction system should be adjusted to a minimum required to maintain the enzyme activity and at the same time to solubilize the substrate. The minimum amount of water usually added ranged from 10% to 20% in the biphasic reaction system.17,34,35 Despite the high solubility of lactose in the aqueous phase of the biphasic reaction system (18.9% at 25 °C, 25.2% at 40 °C, and 37.2% at 60 °C),36 the concentration of lactose in the whole reaction system is still very low due to minimal aqueous phase. In this context, the effect of different water contents of saturated lactose solution in the absence and presence of Ammoeng 102 was investigated at 75 °C in phosphate buffer (pH 7.0) and n-octanol (Figure 2). The optimum water content of the reaction system without IL for synthesis of oct-gal yield results at 20% (Figure 2). Interestingly, the optimum water content of the Ammoeng 102 reaction system increased to 35%, suggesting that Ammoeng 102 may suppress the hydrolysis reaction. The changes of solubility and activity coefficient of the IL systems against water content were predicted by COSMO-RS and are presented in Table S3. As seen, if the water content increases,

Figure 1. Effect of different concentrations of ionic liquids on the yield of the β-galactosidase catalyzed transgalactosylation reaction. (Reaction conditions: pH 7.0 buffer: 0.2 mL, n-octanol: 1.8 mL, enzyme: 20 mg, temperature: 75 °C, reaction time: 16 h.)

the solubility of lactose and the activity coefficient of n-octanol increase in both transgalactosylation reaction systems, whereas there was a gradual decrease in activity coefficient of water and lactose in both reaction systems (w/ and w/o IL) with increasing water content until a water content of 50%. This could elegantly explain that at a low level of water content, transgalactosylation dominates the reaction over hydrolysis 2010


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Table 3. Apparent Kinetic Parameters of TN1577 Catalyzed Transgalactosylationa Reaction system

Apparent Vmax (mmol L−1 min−1)

Apparent Km (mmol L−1)

Apparent kcat (min−1)

Ea (kJ mol−1)

Without ionic liquid With 2.5% Ammoeng 102

0.39

152.34

1.34

14.45b

0.85

273.5

2.93

40.89b

a Reaction conditions: lactose: 45−100 mmol L−1, pH 7.0, buffer: 0.4 mL, n-octanol: 1.6 mL, enzyme: 20 mg, temperature: 75 °C. bEa of the reaction system was measured at 65−75 °C.

activation energy (Ea IL reaction system = 40.89 kJ mol−1 vs Ea w/o IL reaction system = 14.45 kJ mol−1), which is likely due to increased system viscosity because of addition of Ammoeng 102. Nonetheless, the IL reaction system obtains a higher Vmax (Vmax IL reaction system = 0.85 mmol min−1 L−1 vs Vmax w/o IL reaction system = 0.39 mmol min−1 L−1). Correspondingly, the Ammoeng 102 reaction system also achieves a higher kcat for transgalactosylation over the no IL system (kcat IL reaction system = 2.9 min−1 vs kcat w/o IL reaction system = 1.3 min−1), which means the transgalactosylation efficiency in the Ammoeng 102 reaction system is significantly enhanced. These findings demonstrate that the Ammoeng 102 can control the water activity to suppress the hydrolysis and thereby increase the efficiency of transgalactosylation. The hydrolysis suppression effect of Ammoeng 102 may be a result of the strong interaction between Ammoeng 102 and water molecules resulting in a low water activity, as predicted by COSMO-RS in this work (Tables S1−S4). Kaftzik’s group conducted the transgalactosylation of lactose and N-acetylglucosamine catalyzed by β-galactosidase from Bacillus circulans and also found that the use of 25% [MIMM][BF4] suppressed the secondary hydrolysis of the product and thus increased the product yield from 30% to 60%.33 Thermophilic characteristic of β-galactosidase TN1577 in alkyl galactopyranoside synthesis. Since TN1577 is from the thermophilic bacteria, it has an excellent thermostability. Thus, the temperature conditions were optimized to obtain enhanced productivity at elevated temperature due to increased solubility of lactose and improved reaction kinetics of TN1577. As shown in Figure 3, the optimum temperature of the reaction system without IL was 75−80 °C for synthesis of oct-gal; and the production of oct-gal decreased at temperature higher than 80 °C, likely due to the deactivation of TN1577 at elevated temperatures. By contrast, the optimal temperature range observed for Ammoeng 102 was broadened and enhanced (75−95 °C). More significantly, from 60 to 75 °C the Ammoeng 102 system gives a marked increase of oct-gal against increasing temperature, likely ascribed to ILassisted solubility of lactose, decreasing viscosity of the reaction system, and better mixing between octanol/aqueous phases. In

Figure 2. Effect of water content on the octyl galactopyranoside yield in the β-galactosidase catalyzed transgalactosylation reaction. (Reaction conditions: pH 7.0 buffer: 0.2−1.6 mL, n-octanol: 0.4−1.8 mL, enzyme: 20 mg, temperature: 75 °C, reaction time: 16 h. Black: with 0.05 g of Ammoeng 102, red: without ionic liquid.)

(Figure 2). Beyond 55% water and 45% n-octanol, an increase in lactose activity coefficients was observed (Table S3). This change in lactose activity coefficient may be explained by the phase diagram of water and n-octanol as predicted by COSMORS at 75 °C (Figure S1). When the water content was less than 25%, n-octanol was miscible with water; that is, there was only one phase in the reaction system. Beyond 25% water content, the reaction system gradually formed two phases, and became again one phase above 90% water content. Kinetic analysis of transgalactosylation and hydrolytic activity of TN1577. To further verify Ammoeng 102’s suppression of hydrolysis, the kinetic parameters of hydrolysis and transgalactosylation were estimated via a Lineweaver−Burk plot, respectively (Table 2 and Table 3). Results showed that the Vmax for lactose and oct-gal hydrolysis decreased upon addition of Ammoeng 102 by 3.5-fold and 3.37-fold, respectively. Also, the Km decreased after addition of Ammoeng 102. Moreover, the value of kcat for lactose and oct-gal hydrolysis in the reaction system without IL was higher than that of the Ammoeng 102 reaction system, which corroborates the assumption that Ammoeng 102 inhibits proceeding of the side reaction pathway and suppresses hydrolysis of the desired product (Scheme 1). Table 3 illustrates the results of the studies on the apparent kinetic parameters of transgalactosylation. The apparent Km of the Ammoeng 102 reaction system was higher than that of the reaction system without IL (Km IL reaction system = 273.5 mmol L−1 vs Km w/o IL reaction system = 152.34 mmol L−1). This suggests that, in the reaction system containing Ammoeng 102, a weak enzyme−substrate binding took place, thereby leading to a high Km. In addition, the high Km led to a high

Table 2. Kinetic Parameters of TN1577 Catalyzed Hydrolysis of Lactose and Octyl Galactopyranosidea Reaction system Lactose Octyl galactopyranoside

a

Without ionic liquid With 2.5% Ammoeng 102 Without ionic liquid With 2.5% Ammoeng 102

Vmax (mmol min−1 L−1)

Km (mmol L−1)

kcat (s−1)

3.08 0.88 4.58 1.36

2.44 1.03 3.09 1.02

2.96 0.85 4.40 1.31

Reaction conditions: lactose/oct-gal: 0.33−3 mmol L−1, pH 7.0, buffer: 1 mL, enzyme: 5 mg, temperature: 75 °C. 2011


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Figure 4. Time course of the β-galactosidase catalyzed transgalactosylation reaction. (Reaction conditions: pH 7.0, enzyme: 20 mg. Black: with 0.05 g of Ammoeng 102, water content: 35%, temperature: 95 °C. Red: without ionic liquid, water content: 20%, temperature: 75 °C.)

Figure 3. Effect of temperature on octyl galactopyranoside yield in the β-galactosidase catalyzed transgalactosylation reaction. (Reaction conditions: pH 7.0, enzyme: 20 mg, reaction time: 16 h. Black: with 0.05 g of Ammoeng 102, water content: 35%. Red: without ionic liquid, water content: 20%.)

improve the productivity of oct-gal at 95 °C; and it improved volumetric productivity by 2.4-fold based on a comparison of the maximum yield of each system. ILs have been described as liquid immobilization supports because multipoint enzyme-IL interactions (ionic bonds, hydrogen bonds, van der Waals, etc.) may occur, resulting in a supramolecular network able to maintain the active protein conformation at high temperatures.32 Results demonstrated that Ammoeng IL is able to provide a more efficient synthetic pathway to obtain alkyl galactopyranoside. Enzymatic synthesis of different alkyl galactopyranosides in the Ammoeng 102 reaction system. In order to verify the general applicability of the Ammoeng 102 mediated system, the synthesis of different alkyl galactopyranosides with different lengths of alkyl alcohols (from n-butanol to ntetradecanol) in the Ammoeng 102 reaction system was investigated using the aforesaid optimal reaction parameters (w/o IL reaction system, 75 °C, 2:8, v/v; IL reaction system, 95 °C, 3.5:6.5, v/v.). As shown in Figure 5, it is obvious that the

fact, the highest productivity of oct-gal was observed at 95 °C (Conc of oct-gal = 60.79 mmol L−1), which is significantly higher (2.3-fold) than the highest yield for the w/o IL system at 75−80 °C (Conc of oct-gal = 26.42 mmol L−1). Roberts et al.37 also found the optimal temperature of the reaction catalyzed by CALB in IL [BMIM][PF6] was 75 °C compared to the reaction system without IL (T = 50 °C). The oligoethylene glycol moiety in the cation of Ammoeng 102 is believed to provide a protective effect and leads to enhanced enzyme thermostability.9−11 To further analyze the results obtained, the solubilities and activity coefficients of lactose in the biphasic reaction systems at different temperatures were calculated using COSMO-RS (Table S4). Accordingly, from 50 to 100 °C, the solubility of lactose increased in both of the reaction systems. However, the solubility of lactose in the Ammoeng system is much higher than in the buffer system, e.g. at 20% water 0.64 vs 0.31 (g/100 g). This is in agreement with experimental data that shows that the system w/IL leads to a higher production of oct-gal compared to the system w/o IL. In addition, the activity coefficients of lactose and n-octanol only slightly decrease in the Ammoeng 102 reaction system when increasing the temperature from 50 to 100 °C (activity coefficients in the IL reaction system, from γlactose = 0.76 to γlactose = 0.6 and from γn‑octanol = 3.93 to γn‑octanol = 3.63) (Table S4), indicating high reactivity of both substrates is maintained. Comparably, the activity coefficients of lactose in the w/o IL reaction system decrease, from γlactose = 1.4 to γlactose = 0.98. More importantly, the solubility of lactose is increased to 1.85 g/100 g at 95 °C (Table S4), which could explain the enhanced yield of oct-gal at elevated temperature. Time course of enzymatic production of octyl galactopyranoside. As shown in Figure 4, in the Ammoeng 102 reaction system, oct-gal increased drastically in the first 6 h and reached 62.21 mmol L−1 in 7 h. On the other hand, the concentration of oct-gal in the reaction system without IL reached 25.86 mmol L−1 after 15 h and remained virtually unchanged after 15 h, suggesting a stable equilibrium state. The IL system studied could reach a similar concentration (23.85 mmol L−1) in just 2 h. Therefore, Ammoeng 102 was demonstrated to significantly shorten reaction time and

Figure 5. Effect of Ammoeng 102 on the yield of the β-galactosidase catalyzed transgalactosylation reaction with different substrates. (Octyl galactopyranoside is used as standard. Reaction conditions: pH 7.0, enzyme: 20 mg. Red: with 0.05 g of Ammoeng 102, water content: 35%, temperature: 95 °C. Black: without ionic liquid, water content: 20%, temperature: 75 °C.) 2012


Research Article

ACS Sustainable Chemistry & Engineering Table 4. Synthesis of Octyl Galactopyranoside Using Different Enzymes as Reported by Various Authors Enzyme source

Reaction conditions

Thermotoga naphthophila RKU10 Thermotoga naphthophila RKU10 Bacillus subtilis Escherichia coli

Bacillus pseudof irms Penicillium canescencs

Reaction system

75 °C,

Biphasic reaction system

pH 7.0 95 °C,

Lactose: saturated Biphasic reaction system with Ammoeng 102 Lactose: saturated Biphasic reaction system Lactose: 100 mM Amphiphilic 1,2-dimethoxyethane cosolvent Lactose: 100 mM Biphasic reaction system Lactose: 50 mM Biphasic reaction system Lactose: 174 mM

pH 7.0 25 °C, pH 7.0 37 °C, pH 7.2 40 °C, pH 9.5 60 °C, pH 5.5

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short chain alcohols (C4−C6) are better nucleophiles for transgalactosylation than those having a longer chain (C8− C12). These results agree with the Mladenoska et al.5 report that the activity of enzymes from Aspergillus oryzae, Escherichia coli, and Klyveromyces marxianus, for transgalactosylation using different alkyl alcohols (n-butanol, n-hexanol, and n-octanol), decreases as alcohol chain length increases. The Ammoeng 102 system compared to the non-IL system could reduce the reaction time to reach equilibrium and increase the yields of alkyl galactopyranosides (1.37−2.14 times) for the different alkyl alcohols, displaying an increase in catalytic efficiency in the order of n-dodecanol < n-butanol < n-decanol < n-hexanol < n-octanol. The results indicate that the Ammoeng 102-based β-galactosidase TN1577 catalyzed transgalactosylation is an effective reaction system, enabling enhanced production of a broad variety of alkyl galactopyranosides.

Reaction time (h)

Ref

25.86 (±1.04)

15

This work

62.21(±1.69)

7

This work

27.7

24

34

33.7

24

8

13

72

4

120

35

78.3

AUTHOR INFORMATION

Corresponding Authors

*(Renjun Gao) Fax: +86 43185155200; Tel: + 86 43185155212; E-mail: [email protected]. *(Zheng Guo) Fax: +45 8612 3178; Tel: +45 8715 5528; Email: [email protected]. ORCID

Jingbo Li: 0000-0001-5960-806X Zheng Guo: 0000-0002-8436-7082 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National High Technology Research and Development Program (“863” Program) of China (No. 2013AA102104) for their financial support. B.P. thanks the Danish Council for Independent Research for her postdoctoral grant 5054-00062B.

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CONCLUSION In summary, this work developed an efficient approach for enzymatic production of alkyl galactopyranosides by applying an amphilic Ammoeng 102 mediated reaction system, which offers technical advantages in improving substrate solubility, suppressing hydrolysis, and enhancing enzyme working temperature ranges (up to 95 °C), such that a maximum yield of octyl galactopyranoside of 62.21 mmol L−1 was achieved within 7 h in 35% water content using TN1577 at 95 °C with 2.5% Ammoeng 102 as cosolvent. Kinetic study and COSMO-RS in silico predictions demonstrated that increased solubility of substrate, suppression of hydrolysis of substrate and product, and excellent biocompatibility of Ammoeng 102 with enzyme are the main reasons. Compared to other reaction systems reported in the literature (See Table 4), this work significantly improved time-space efficiency (higher volumetric productivity in shorter time), demonstrating the industrial potential of the developed approach in this work.

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Maximum oct-gal yield (mmol L−1)

■

REFERENCES

(1) El-Sukkary, M. M. A.; Syed, N. A.; Aiad, I.; El-Azab, W. I. M. Synthesis and characterization of some alkyl polyglycosides surfactants. J. Surfactants Deterg. 2008, 11 (2), 129−137. (2) Yakimchuk, O. D.; Kotomin, A. A.; Petel’skii, M. B.; Naumov, V. N. Cleaning action and surfactant properties of alkyl glucosides. Russ. J. Appl. Chem. 2004, 77 (12), 2001−2005. (3) Sarney, D. B.; Vulfson, E. N. Application of Enzymes to the Synthesis of Surfactants. Trends Biotechnol. 1995, 13 (5), 164−172. (4) Das-Bradoo, S.; Svensson, I.; Santos, J.; Plieva, F.; Mattiasson, B.; Hatti-Kaul, R. Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts. J. Biotechnol. 2004, 110 (3), 273−285. (5) Mladenoska, I.; Winkelhausen, E.; Kuzmanova, S. Transgalactosylation/hydrolysis ratios of various beta-galactosidases catalyzing alkyl-beta-galactoside synthesis in single-phased alcohol media. Food Technol. Biotech 2008, 46 (3), 311−316. (6) El Seoud, O. A.; Koschella, A.; Fidale, L. C.; Dorn, S.; Heinze, T. Applications of ionic liquids in carbohydrate chemistry: A window of opportunities. Biomacromolecules 2007, 8 (9), 2629−2647. (7) Lu, W. Y.; Lin, G. Q.; Yu, H. L.; Tong, A. M.; Xu, J. H. Facile synthesis of alkyl beta-D-glucopyranosides from D-glucose and the corresponding alcohols using fruit seed meals. J. Mol. Catal. B: Enzym. 2007, 44 (2), 72−77. (8) Bae, J.; Choi, E. H.; Pan, J. G. Efficient synthesis of octyl-beta-Dgalactopyranoside by Bacillus spore-displayed beta-galactosidase using

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02862. COSMO-RS predicted results; liquid−liquid phase diagram of water and n-octanol; MS spectra; 1H NMR spectra (PDF) 2013


Research Article

ACS Sustainable Chemistry & Engineering an amphiphilic 1,2-dimethoxyethane co-solvent. Enzyme Microb. Technol. 2011, 48 (3), 232−238. (9) Devi, B. L. A. P.; Guo, Z.; Xu, X. B. Characterization of Ionic Liquid-Based Biocatalytic Two-Phase Reaction System for Production of Biodiesel. AIChE J. 2011, 57 (6), 1628−1637. (10) Guo, Z.; Chen, B. Q.; Murillo, R. L.; Tan, T. W.; Xu, X. B. Functional dependency of structures of ionic liquids: do substituents govern the selectivity of enzymatic glycerolysis? Org. Biomol. Chem. 2006, 4 (14), 2772−2776. (11) Chen, B. Q.; Guo, Z.; Tan, T. W.; Xu, X. B. Structures of ionic liquids dictate the conversion and selectivity of enzymatic glycerolysis: Theoretical characterization by COSMO-RS. Biotechnol. Bioeng. 2008, 99 (1), 18−29. (12) Persson, M.; Bornscheuer, U. T. Increased stability of an esterase from Bacillus stearothermophilus in ionic liquids as compared to organic solvents. J. Mol. Catal. B: Enzym. 2003, 22 (1−2), 21−27. (13) Kaar, J. L.; Jesionowski, A. M.; Berberich, J. A.; Moulton, R.; Russell, A. J. Impact of ionic liquid physical properties on lipase activity and stability. J. Am. Chem. Soc. 2003, 125 (14), 4125−4131. (14) Tholey, A.; Zabet-Moghaddam, M.; Heinzle, E. Quantification of peptides for the monitoring of protease-catalyzed reactions by matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrixes. Anal. Chem. 2006, 78 (1), 291−297. (15) Zhao, H.; Song, Z. Y.; Olubajo, O. High transesterification activities of immobilized proteases in new ether-functionalized ionic liquids. Biotechnol. Lett. 2010, 32 (8), 1109−1116. (16) Zhao, H. Methods for stabilizing and activating enzymes in ionic liquids - a review. J. Chem. Technol. Biotechnol. 2010, 85 (7), 891−907. (17) Yang, J. W.; Chen, X. C.; Yu, D. H.; Gao, R. J. Microwaveassisted synthesis of butyl galactopyranoside catalyzed by betagalactosidase from Thermotoga naphthophila RKU-10. Process Biochem. 2016, 51 (1), 53−58. (18) Kong, F. S.; Wang, Y. Q.; Cao, S. G.; Gao, R. J.; Xie, G. Q. Cloning, purification and characterization of a thermostable betagalactosidase from Thermotoga naphthophila RUK-10. Process Biochem. 2014, 49 (5), 775−782. (19) Ly, H. D.; Withers, S. G. Mutagenesis of glycosidases. Annu. Rev. Biochem. 1999, 68, 487−522. (20) Kim, H. S.; Eom, D.; Koo, Y. M.; Yingling, Y. G. The effect of imidazolium cations on the structure and activity of the Candida antarctica Lipase B enzyme in ionic liquids. Phys. Chem. Chem. Phys. 2016, 18 (32), 22062−22069. (21) Gorke, J.; Srienc, F.; Kazlauskas, R. Toward Advanced Ionic Liquids. Polar, Enzyme-friendly Solvents for Biocatalysis. Biotechnol. Bioprocess Eng. 2010, 15 (1), 40−53. (22) De Winter, K.; Verlinden, K.; Kren, V.; Weignerova, L.; Soetaert, W.; Desmet, T. Ionic liquids as cosolvents for glycosylation by sucrose phosphorylase: balancing acceptor solubility and enzyme stability. Green Chem. 2013, 15 (7), 1949−1955. (23) Husum, T. L.; Jorgensen, C. T.; Christensen, M. W.; Kirk, O. Enzyme catalysed synthesis in ambient temperature ionic liquids. Biocatal. Biotransform. 2001, 19 (4), 331−338. (24) Lang, M.; Kamrat, T.; Nidetzky, B. Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable beta-glycosylhydrolase celB from Pyrococcus furiosus. Biotechnol. Bioeng. 2006, 95 (6), 1093−1100. (25) Ferdjani, S.; Ionita, M.; Roy, B.; Dion, M.; Djeghaba, Z.; Rabiller, C.; Tellier, C. Correlation between thermostability and stability of glycosidases in ionic liquid. Biotechnol. Lett. 2011, 33 (6), 1215−1219. (26) Hilal, S. H.; Karickhoff, S. W.; Carreira, L. A. Prediction of the solubility, activity coefficient and liquid/liquid partition coefficient of organic compounds. QSAR Comb. Sci. 2004, 23 (9), 709−720. (27) Klamt, A. Conductor-Like Screening Model for Real Solvents - a New Approach to the Quantitative Calculation of Solvation Phenomena. J. Phys. Chem. 1995, 99 (7), 2224−2235. (28) Eckert, F.; Klamt, A. Fast solvent screening via quantum chemistry: COSMO-RS approach. AIChE J. 2002, 48 (2), 369−385.

(29) Guo, Z.; Lue, B. M.; Thomasen, K.; Meyer, A. S.; Xu, X. B. Predictions of flavonoid solubility in ionic liquids by COSMO-RS: experimental verification, structural elucidation, and solvation characterization. Green Chem. 2007, 9 (12), 1362−1373. (30) Wittekindt, C.; Klamt, A. COSMO-RS as a Predictive Tool for Lipophilicity. QSAR Comb. Sci. 2009, 28 (8), 874−877. (31) Brehm, M.; Weber, H.; Pensado, A. S.; Stark, A.; Kirchner, B. Proton transfer and polarity changes in ionic liquid-water mixtures: a perspective on hydrogen bonds from ab initio molecular dynamics at the example of 1-ethyl-3-methylimidazolium acetate-water mixturesPart 1. Phys. Chem. Chem. Phys. 2012, 14 (15), 5030−5044. (32) Lozano, P.; Garcia-Verdugo, E.; Piamtongkam, R.; Karbass, N.; De Diego, T.; Burguete, M. I.; Luis, S. V.; Iborra, J. L. Bioreactors based on monolith-supported ionic liquid phase for enzyme catalysis in supercritical carbon dioxide. Adv. Synth. Catal. 2007, 349 (7), 1077− 1084. (33) Kaftzik, N.; Wasserscheid, P.; Kragl, U. Use of ionic liquids to increase the yield and enzyme stability in the beta-galactosidase catalysed synthesis of N-acetyllactosamine. Org. Process Res. Dev. 2002, 6 (4), 553−557. (34) Kwon, S. J.; Jung, H. C.; Pan, J. G. Transgalactosylation in a water-solvent biphasic reaction system with beta-galactosidase displayed on the surfaces of Bacillus subtilis spores. Appl. Environ. Microb 2007, 73 (7), 2251−2256. (35) Kouptsova, O. S.; Klyachko, N. L.; Levashov, A. V. Synthesis of alkyl glycosides catalyzed by beta-glycosidases in a system of reverse micelles. Russ. J. Bioorg. Chem. 2001, 27 (6), 380−384. (36) Machado, J. J. B.; Coutinho, J. A. P.; Macedo, E. A. Solid-liquid equilibrium of alpha-lactose in ethanol/water. Fluid Phase Equilib. 2000, 173 (1), 121−134. (37) Roberts, N. J.; Seago, A.; Carey, J. S.; Freer, R.; Preston, C.; Lye, G. J. Lipase catalysed resolution of the Lotrafiban intermediate 2,3,4,5tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid methyl ester in ionic liquids: comparison to the industrial t-butanol process. Green Chem. 2004, 6 (9), 475−482.

2014


Enhanced Synthesis of Alkyl Galactopyranoside by Thermotoga

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