Bioresource Technology 144 (2013) 135–140

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Bioresource Technology

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Enzyme-catalyzed synthesis of heptyl-b-glycosides: Effect of watercoalescence at high temperature

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.06.080

⇑ Corresponding authors. Tel.: +52 55 5622 5310; fax: +52 55 5616 1868.E-mail addresses: mina_montiel@comunidad.unam.mx (C. Montiel), ebg@u-

nam.mx (E. Bárzana).

Carmina Montiel a,⇑, Ismael Bustos-Jaimes b, Eduardo Bárzana a,⇑a Department of Food Science and Biotechnology, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City 04510, Mexicob Department of Biochemistry, Faculty of Medicine, National Autonomous University of Mexico, Mexico City 04510, Mexico

h i g h l i g h t s

�Water coalesces producingsubstrates, products and catalystdifferential partition.� Partition produce undesirable side-

reactions including catalystinactivation.� Side reactions can be minimized by

modifying water content of thesystem.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 March 2013Received in revised form 18 June 2013Accepted 20 June 2013Available online 28 June 2013

Keywords:Heptyl-b-glycosidesWater coalescenceMaillard reactionHyperthermophilic b-glycosidase

a b s t r a c t

Alkyl glycosides can be synthesized by glycosidases in organic media with limited amounts of water.These systems, however, limit the solubility of the sugar substrates and decrease reaction yields. Hereinwe report the enzymatic synthesis of heptyl-b-glycosides in heptanol catalyzed by a hyperthermophilicb-glycosidase at 90 �C. Our results indicate that dispersion of water in heptanol changes with timeproducing coalescence of water at the bottom of the reactor, playing a key role in the reaction yield.Water-soluble substrate, enzyme and products are concentrated in the aqueous phase, according to theirpartition coefficients, promoting side reactions that inactivate the enzyme. Reaction yield of heptyl-b-glycosides was 35% relative to lactose, at 7% water. The increase in the water phase to 12% diminishedthe enzyme inactivation and increased the heptyl-b-glycosides yield to 52%. Surface-active compounds,SDS and octyl glucoside, increased water dispersion but were unable to prevent coalescence.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The long-chain alkyl glycosides display high surface-activeproperties useful in washing and cleansing agents. Thesecompounds are widely used in cosmetic products and other indus-trial product formulations (Bhatia et al., 2002; Cerón-Camachoet al., in press). Alkyl glycosides can be synthesized by severalchemical methods (Rybinski and Hill, 1998). A drawback of the

chemical synthesis is the purity of alkyl glycosides. Chemical reac-tions for alkyl glycosides syntheses usually produce a mixture ofmono-, di- and tri-glycosides, as well as oligosaccharides. Theseproduct mixtures are called alkyl polyglycosides. Selectivealkylation of sugars requires complicated steps of protection anddeprotection of reactive hydroxyl groups, the activation of theanomeric carbon of carbohydrates and the preexistence of a or banomers, as well as the use of heavy metals as catalysts (Rybinskiand Hill, 1998). The enzymatic synthesis of alkyl glycosides, incontrast with the chemical synthesis, provides regio- and stereose-lective advantages. Therefore, high purity products can be obtainedin one-step reactions (Panintrarux and Adachi, 1995). Enzymatic

136 C. Montiel et al. / Bioresource Technology 144 (2013) 135–140

synthesis can employ natural sugar wastes or by-products as sub-strates, for example lactose, which is an attractive substrate for al-kyl glycoside synthesis, as it constitutes approximately 70–75% ofthe solids in the sub-products from cheese manufacture (Hanssonand Adlercreutz, 2002). The synthesis of alkyl glycosides is cata-lyzed by glycosidases, either by direct condensation of a monosac-charide and an alcohol (reverse hydrolysis) (Ismail and Soultani,1999) or by transglycosylation between a reactive acyl donor, likean ester, an amide group or a glycosidic bond, and an alcohol (Gar-cía-Garibay et al., 2000).

Enzymatic reactions in non-aqueous media have evolved dra-matically during the last decades as a key approach for the produc-tion of a wide variety of materials and chemicals, ranging fromdrugs to polymers and food additives, to name a few. Several re-ports of enzymatic transglycosylation in organic media, using di-verse strategies and different b-glycosidases have been published(Vic and Thomas, 1992; Panintrarux and Adachi, 1995; Vic andCrout, 1997; García-Garibay et al., 2000; Hansson and Adlercreutz,2001, 2002; Zou et al., 2012; Krisch et al., 2012). The use of long-chain alcohols as substrate and the solvent is an appealing strat-egy. One limitation in these systems is the poor solubility of carbo-hydrates in the long-chain alcohols. Besides single-phase organicmedia, biphasic aqueous-organic systems have been employedfor the enzymatic synthesis of alkyl glycosides. In these systems,the partition of compounds and the alcohol solubility in the aque-ous phase play a critical role in the alkyl glycosides production(Hansson and Adlercreutz, 2002). Moreover, the alkyl glycosideproduct is also a substrate for the enzyme and, therefore, its hydro-lysis may take place in the aqueous phase (Papanikolaou, 2001).The use of water miscible organic co-solvents to increase the solu-bility of the sugars resulted in low conversions and large reactiontimes because of the relative low concentrations of alcohol andsugars (Otto et al., 1998; Ismail et al., 1999; García-Garibay et al.,2000). The use of thermophilic b-glycosidases and high tempera-tures is a reasonable alternative to increase sugars concentrationsin the limited amount of water available, thus increasing the over-all alkyl glycosides production (Vulfson et al., 1990; García-Garibayet al., 2000; Turner et al., 2007; Krisch et al., 2012). Previous stud-ies have demonstrated that synthesis of alkyl glycosides is highlydependent on water content in the reaction media, independentlyof the enzyme source (García-Garibay et al., 2000). However, othervariables that may influence the reaction are the water coalescenceand chemical modification of the enzyme by carbohydrates at hightemperatures. Maillard reaction, a complex browning processinvolving proteins and reducing sugars, has been found responsibleof enzyme inactivation even for thermophilic b-glycosidases (deRoode et al., 2001; Zou et al., 2012). The understanding of the roleof water on the enzymatic reaction will allow the prediction of theoptimum amount of water needed to support competitivereactions.

In this research, we studied the macroscopic aggregation stateof water and its evolution during the time course of an enzyme-catalyzed transglycosylation at high temperature. We found thatwater aggregation has a strong effect on both, enzyme stabilityand reaction yield.

2. Methods

2.1. Chemicals and biochemicals

The hyperthermophilic b-glycosidase (CloneZyme Gly-001-02)was kindly provided by Diversa, Sharon Hill, PA. Heptyl glucoside,heptyl galactoside, octyl glucoside, heptanol, methanol, acetoni-trile, glucose and lactose were purchased from Sigma–Aldrich inthe highest purity available.

2.2. Enzymatic reaction

The enzymatic reaction of transglycosylation between lactoseand heptanol was performed in jacket glass reactors sealed withscrew caps. Reactors were magnetically stirred and temperaturewas adjusted to 90 �C. Each reactor contained 40 mL, where 93%(v/v) was constituted by dry heptanol (0.01–0.03% water by coulo-metric titration) and 7% (v/v) water. Lactose, or glucose when indi-cated, was added as a solid in a quantity to obtain a concentrationof 40 mM. The reaction was started by the addition of 127 lL of b-glycosidase solution (17 mg/mL in 0.1 M acetate buffer pH 5). Ali-quots of 1 mL were withdrawn from the reaction at different timesand transferred into an ice bath to stop the reaction. Concentra-tions of heptyl glycosides and carbohydrates were analyzed as de-scribed in Section 2.3.

2.3. Heptyl glycosides, carbohydrates and protein analyses

Heptyl glycosides were separated from heptanol by evaporationunder vacuum. Under these conditions, a fraction of heptyl glyco-sides may be lost by steam distillation. To quantify the loss of hep-tyl glycosides, a know concentration of octyl glucoside was addedto the sample as internal standard and its loss (usually 2.9%) wasused to correct concentration data. Standards of heptyl glucosideand heptyl galactoside were used to determine retention times ofheptyl glycosides. Samples were quantified by HPLC (HP 1100 ser-ies) equipped with a light scattering detector (HPLC-PR-ELSD). AC18 column (Waters, 150 mm � 3.9 mm, 5 lm) and a Hypersil pro-pylamino (Waters, 250 mm � 4.6 mm, 5 lm) were utilized for theseparation of heptyl glycosides and carbohydrates respectively.The mobile phase consisted of methanol–water (75:25) for hep-tyl-glycosides and methanol–acetonitrile–water (60:22.5:17.5)for carbohydrates. All the analyses proceeded at room temperaturein the HPLC column. The conditions in the detector were 100 �C inthe evaporator and 70 �C in the nebulizer. The nitrogen flow ratewas 0.8 mL min�1.

Total protein concentration was determined by a modifiedmethod of Lowry (Peterson, 1977), using bovine serum albumin(BSA) as standard. To determine if the enzyme suffered any changeafter the reaction of transglycosylation the samples were analyzedby SDS–PAGE electrophoresis (Laemmli, 1970) in 8% acrylamidegel. The enzyme samples were taken after four hours of reactioneither in presence of glucose or lactose. A sample of fresh b-glyco-sidase was used as a control. All results, from this and the followingsections, represent the mean of triplicate experiments.

2.4. Partitioning of compounds between aqueous and organic phases

The partition of heptyl glycosides and carbohydrates was deter-mined using the equilibrium concentration in organic media. In 3different reactors containing heptanol with 7% (v/v) water wereadded individually lactose, lactose and heptyl glucoside, and glu-cose and heptyl glucoside. All reactors were stirred for 8 h at90 �C, and then without stirring for 24 h at this temperature. Final-ly, the concentration on each component was analyzed by HPLC insamples withdrawn from the center of the reactors. Partition coef-ficients in the reaction conditions, K, were calculated according toEq. (1),

K ¼ Ci½ �O= Ci½ �W ð1Þ

where [Ci] is the concentration of compound i, either in organic (O)or water (W) phase.

The partition of compounds when enzymatic reaction occurredwas also evaluated. In this case, samples from the center and thebottom of the reactor were analyzed. The reaction was carried

Fig. 1. Time course of lactose transglycosylation at 90 �C in the center of thereactor. Lactose (s), heptyl glycoside (d), glucose or galactose (h). Initialconcentration of lactose was 40 mM, heptanol 93% (v/v) and 7% (v/v) water.

C. Montiel et al. / Bioresource Technology 144 (2013) 135–140 137

out for 4 h with stirring at 90 �C. Heptyl glycosides, monosaccha-rides, disaccharides and soluble protein were quantified.

2.5. Water dispersion in heptanol

In an attempt to increase the solubility of water in heptanol,20 mM of either SDS or octyl glucoside were added to the reactionmedia. Reactors were stirred at 90 �C for 9 days, and the water con-tent in the center of the reactors was measured by coulometrictitration (Aquatest 8; Photovolt, Indianapolis, IN). Samples of med-ia were taken 2 h after reaching 90 �C under continuous stirring. Asan aid to observe the two-phase system, the water-soluble diso-dium salt of 6-hydroxy-5-[(4-sulfophenyl) azo]-2-naphthalene-sulfonate (Sunset Yellow FCF) was added to a final concentrationof 0.1 mg/L (2 mg/L in the aqueous phase). This concentration ofdye in the reduced volume of water is enough to produce notice-able color in the water phase (Ghoneim et al., 2011).

2.6. Non-enzymatic browning

In order to dissect the two possible causes of non-enzymaticbrowning, either carbohydrate caramelization or Maillard reaction,4 controls prepared as the enzymatic reaction were used. Thesecontrols contained enzyme, or lactose, or lactose and enzyme, allwith 93% of heptanol and 7% of water at 90 �C, except for one reac-tor with enzyme whose temperature was set to 25 �C. Concentra-tions of heptyl glycosides and carbohydrates were analyzed byHPLC. Integrity of the enzyme was analyzed by SDS–PAGE as de-scribed previously. Residual activity of the enzyme was measuredfor the hydrolysis of lactose in 0.1 M acetate buffer, pH 5.0, at 90 �C.The aqueous phase was recovered by centrifugation at 3000 rpmby 5 min and 50 lL were used to assay the hydrolysis of lactose.A sample of fresh enzyme was included as control. Consumptionof lactose and production of monosaccharides were measured byHPLC as described in Section 2.2. Maillard reaction was evaluatedby spectrophotometry of colored compounds produced at 420and 490 nm according to Bruins et al. (2003). Absorbance for theaqueous phase was measured in the range from 200 to 700 nm,showing a maximum at 420 nm. This maximum is used to measurethe final step of Maillard reaction, that is characterized by nitro-gen-containing unsaturated polymers, besides the early stage ofthe reaction where non-colored compounds are produced and ab-sorbed at 280 nm like hydroxymethylfurfural and pyrazines (Lericiet al., 1990).

2.7. Effect of water in enzyme stability

The influence of water on transglycosylation of lactose in organ-ic media was studied by setting up enzymatic reactions with differ-ent water contents (3%, 5%, 7%, 9%, 12% and 14% v/v) while the restof the components remained constant. The concentrations of hep-tyl glycosides and carbohydrates were analyzed as describedbefore.

3. Results and discussion

3.1. Imbalanced concentrations of transglycosylation substrates andproducts

Enzymatic transglycosylation of lactose with heptanol, actingboth as reactant and continuous reaction media, was carried outat 90 �C using a hyperthermophilic b-glycosidase as catalyst. Earlyresults indicated that yields of transglycosylation fluctuate withthe distance from the bottom at which samples were taken. Inthe center of the reactor, the lactose concentration decreased from

40 mM to 2 mM in the first minutes of reaction (Fig. 1), even in thecontrol reaction without enzyme. However, the concentration ofheptyl glycosides did not increase following this sharp decreaseof lactose. Instead, transglycosylation reaction progressed for 3 huntil fully stopped without appearance of the stoichiometricamount of heptyl glycosides corresponding to the lactose con-sumed during the process. Glucose and galactose, both productsof transglycosylation, were produced at the same rate of the glyco-side during the first hour of reaction. After 2 h, monosaccharideconcentration in the center of the reactor diverged from the con-centration of heptyl glycosides, suggesting that monosaccharidesare consumed in other process (Fig. 1). García-Garibay et al.(2000) attributed this decrease to the utilization of glucose by b-glycosidase, as this enzyme performs the direct glycosylation ofglucose. In agreement with this hypothesis, the glycosidase usermanual for this enzyme indicates that it can use different sugarsas substrates, including derivatives of b-D-cellobiose, b-D-galactose,b-D-glucose, b-D-fucose, b-D-xylose, a-L-arabinopyranoside, b-D-lactose and b-D-mannose. Nevertheless, if the enzyme were cata-lyzing the direct glycosylation of glucose, or galactose if present,the final concentration of heptyl glycosides should exceed thestarting molar concentration of lactose. In contrast, we observedthe complete stop of heptyl glycosides production after 3 h, at amolar ratio smaller than 1:1 of lactose to heptyl glycoside. In addi-tion, the notorious decrease of lactose concentration during thefirst minutes of reaction may be attributable to water coalescenceand the formation of two phases in an apparent homogeneoussystem.

3.2. Two phases detection, partition and simultaneous reactions

In the presence of a biphasic medium, partition of substratesand products is critical for productivity. In this system, having alow water amount, the appearance of a second phase is notstraightforward. In order to elucidate the presence of two phasesin these conditions, different reaction media were set up withlactose or lactose plus heptyl glucoside, or glucose plus heptyl glu-coside, in absence of the enzyme. After stirring at high tempera-ture, the center of the reactor was analyzed showing that heptylglucosides showed high affinity for heptanol as 97% of the initialconcentration was detected in the center of the reactor. On theother hand, the concentrations of carbohydrates detected in thecenter of the reactor were only 5% (2 mM) for lactose and 41%(15.4 mM) for glucose, either in presence or absence of heptylglucoside. These results indicate that, although the system at thebeginning is apparently homogeneous, the water in heptanolcoalesces and forms a second phase in which the water-soluble

Table 1Partition coefficients, K, of substrates, products and b-glycosidase in the biphasicsystem heptanol:water 93:7, at 90 �C.

Component Ka

Lactose 0.06Glucose 0.90Galactose 0.97Heptyl-b-glucoside 32.33Heptyl-b-galactoside 35.42b-Glycosidase 0.00

a All determinations were made by triplicate and the error is lower than 5%.

Fig. 2. Time course of lactose transglycosylation at 90 �C in the center (a), and thebottom of reactor (b). Lactose (s), heptyl glycoside (d), glucose or galactose (h) andb-glycosidase (N) were analyzed. The initial concentration of lactose was 40 mM.

Fig. 3. Time course of oligosaccharides formation in samples from the bottom of thereactor. The analyzed signal (d) corresponds to the peak with a retention time of6.4 min shown in the inset. The inset corresponds to a representative HPLCchromatogram of a sample taken from the bottom of reactor at 0.5 h. Peak 1, heptyl-b-glycoside; peak 2, monosaccharides; peak 3, lactose; peaks 4 and 5,oligosaccharides.

138 C. Montiel et al. / Bioresource Technology 144 (2013) 135–140

compounds may concentrate. Though the second phase becomesevident after 8 h at 90 �C, just by stopping the stirring, the aqueousphase has to appear during the first minutes of stirring to explainthe sharp drop of the initial concentration of carbohydrates. It isreasonable that enzyme will also be concentrated in the aqueousphase. Thus, water coalescence could be related to the cease of al-kyl glycoside production after two hours of reaction despite thehigh substrate concentration remaining in the whole system. Ta-ble 1 shows the partition coefficients for the substrates and prod-ucts of the enzymatic reaction, as well as the partition of theenzyme. Noticeably, most of lactose remains in the water phase,as the enzyme does. On the other hand, monosaccharides are welldistributed in both phases, while heptyl glycosides are mainlypresent in the organic phase.

Partition of substrates and products of the reaction was alsostudied along a real transglycosylation reaction. Samples fromthe center and bottom positions of reactors were analyzed. Behav-ior in the center of the reactor (Fig. 2a) was similar to the previ-ously observed. In this experiment, the concentration of theenzyme was monitored and, as expected for a biphasic system,its concentration decreased in time up to 85% after 4 h. In contrast,in the bottom of the reactor, enzyme concentration did not in-crease. A mass balance is not a good analysis tool because samplesfrom the bottom of reactor do not accurately represent the aque-ous phase. Although water is supposed to concentrate at the bot-tom of reactors due to its density, it is expected to be in the formof droplets of heterogeneous size.

The reaction time course in the center and bottom of reactorshowed a similar behavior during the first hour of reaction. How-ever, when water coalescence increased, hydrolysis of lactose pre-dominates over transglycosylation in the bottom of the reactor,thus increasing the monosaccharide concentration (Fig. 2b). Inthe hydrolysis reaction, water competes with heptanol for the ac-tive site of the enzyme, and because of the high concentrationsof water and lactose in the bottom of reactor, hydrolysis is favored(Hansson et al., 2001). This increase of lactose hydrolysis coexistswith a carbohydrate-only transglycosylation, which in turn pro-duces oligosaccharides from the available monosaccharides andlactose as demonstrated by HPLC analyses (Fig. 3). Oligosaccha-rides were not found in samples from the center of the reactor,where monosaccharides and heptyl glycosides keep an apparent1:1 stoichiometry during the first 2 h of the reaction (Fig. 2a). Is-mail et al. (1999) reported the existence of these simultaneousreactions during the synthesis of alkyl glycosides. In these experi-ments, an additional browning reaction, later identified as Maillardreaction, occurs in parallel to transglycosylation and hydrolysis.Browning becomes evident at the bottom of the reactor after 2 hof reaction. Fig. 4 resumes the possible reactions taking place un-der our reaction conditions.

3.3. Aqueous dispersion and water content in heptanol

To evaluate the water dispersion inside the reactor, a hydro-philic dye was added as a visual aid. This experiment demonstrated

that the water phase exists in heptanol from the very beginning, inthe form of droplets, visible to the naked eye, which slowly coa-lesce forming a ring on the reactor walls, following the propellingforce of stirring. As the mass of water increases with time, the ringmoves to the bottom of the reactor. The ring formation was evidentafter 1.5 h of reaction and moved downward reaching the bottomafter 4 h (data not shown). These results strongly suggest that is

Fig. 4. Possible way of the reactions that occurring in the system heptanol–water.Lac, lactose; G1, monosaccharide (glucose or galactose); Hepgly, heptylglycosides;E-G1, enzyme-monosaccharide complex; E, enzyme; NEBP, non enzymatic brown-ing products.

Fig. 5. Relative color development of Maillard reaction in different reaction systemsat 2 (light gray) and 6 h (dark gray) at 90 �C. Reactors contained 7% water inheptanol and either enzyme (0.05 mg/mL), lactose (40 mM) or enzyme and lactose.

C. Montiel et al. / Bioresource Technology 144 (2013) 135–140 139

possible that carbohydrates and enzyme segregate from the bulkheptanol into this growing water phase.

Taking in mind the observed poor stability of water dispersion inheptanol, 2 surface-active compounds were added to increase thestability of emulsion: sodium dodecyl sulfate (SDS) as ionic surfac-tant and octyl glucoside as non-ionic surfactant, in addition to thewater-soluble dye. The systems and a control without surfactantwere stirred at 90 �C for 4 h. Water dispersions in the presence ofsurfactants were more stable than the control, observing coales-cence at longer times. Dispersion in presence of octyl glucosideresulted in smaller water droplets than those formed with SDS.Unexpectedly, the content of water in the center of the reactordid not increase significantly with surfactants at short times(<1000 min). In all reactors, a drastic fall in the water content wasobserved during the first minutes, and then the water contentdecreased slowly until phase separation. Finally, after 18 h, theequilibrium was reached in the center of the reactor, where only2% of water was conserved (data not shown). In the presence ofSDS or octyl glucoside, the yield of heptyl glycosides was not im-proved. Even the initial rates of formation of heptyl glycosides didnot change with the surfactants (Supplementary material Fig. 1).

Fig. 6. Effect of water over transglycosylation (d) and Maillard reaction (N). Theproducts of the final stage of Maillard reaction were measured at 420 nm after 4 hof reaction.

3.4. Non-enzymatic browning

Taking into account the high concentration of carbohydratesand enzyme in the aqueous phase and the high temperature, twodifferent reactions might occur: thermal carbohydrate degradationor Maillard reaction. Exploration of the conditions producingbrowning indicated that it appears quickly when lactose, a reduc-ing sugar, was in presence of the enzyme. This browning is causedby the reaction between lysine e-amino groups from proteins andreducing sugar (Bruins et al. 2003). Browning was also visible after6 h in the reactor containing lactose only, as product of carameliza-tion, while the reactor containing only enzyme did not developnoticeable color either at 25 or at 90 �C. This indicates that thereaction of Maillard predominates during the first 2 h of reaction,and that the contribution of the caramelization appears later fromlactose and doubtless from monosaccharides. The latter reactionexplains the reduction of monosaccharides observed after 2 h ofreaction in Fig. 2.

Spectrophotometric evaluation of Maillard reaction clearly indi-cated the strong Maillard reaction in the reactor containing bothlactose and enzyme (Fig. 5) In addition, it shows that carameliza-tion also contributes to browning at 6 h in the reactor that onlycontains lactose. Taking into account the color formed in the reac-tor containing lactose and enzyme at 2 h, it is clear that the pre-dominating reaction is Maillard and not caramelization.

3.5. Effect of water in non-enzymatic browning

Water plays a double critical role in transglycosylation reac-tions: (1) it increases lactose solubility that enhances transglycosy-lation rate, and (2) at high concentrations of water, it may promoteproduct hydrolysis because water is also a reaction partner for theenzyme (García-Garibay et al. (2000). We evaluate the effect ofincreasing water concentration from 3% to 14% (v/v) on the trans-glycosylation rate and presence of Maillard reaction. As expected,an increase from 3% to 12% resulted in enhancements of transgly-cosylation rate and decreased at water contents higher than 12%(Fig. 6). The molar yield of the reaction was increased from 35%to 52%, relative to lactose (Fig. 2 in Supplementary material). We

140 C. Montiel et al. / Bioresource Technology 144 (2013) 135–140

found that Maillard reaction was also affected by water content,favoring browning at low water contents. This information andthe results on water dispersion presented here, suggest that Mail-lard reaction is promoted by the high concentration of enzyme andcarbohydrates in the aqueous phase. It is reasonable that participa-tion of the enzyme in browning results in its inactivation. Previousreports (Hansson et al., 2001; Bruins et al., 2003) confirm the inac-tivation of enzymes by Maillard reaction. A qualitative evaluationof the chemical modification of the enzyme caused by Maillardreaction is the change of its mobility in SDS–PAGE. A fraction ofthe enzyme incubated with 40 mM lactose for 4 h at 90 �C, notice-ably reduced its electrophoretic mobility (Fig. 3 in Supplementarymaterial). This change in the apparent molecular weight of the en-zyme is explained both by the anomalous migration of the chemi-cally modified protein and by the formation of compoundscorresponding to the final stage of the Maillard reaction, knownas advanced glycation end products (AGEs), which are able tocrosslink the proteins through covalent bonds (Miller et al.,2003). AGEs can be produced by degradation of Amadori’s productsand compounds like glyoxal and methylglyoxal, produced from anintermediate formed between glucose and amino-groups of the en-zyme during the Strecker degradation. In addition, melanoidins,another product of the Maillard reaction, react with free amino-groups of lysine and massively modify the protein, thus diminish-ing its mobility in the gel. These results are in agreement with theobserved inactivation of the enzyme for the hydrolysis of lactose.The enzyme recovered from the transglycosylation reactor lost77% of its activity after 2.5 h of incubation at 90 �C in presence oflactose and heptanol as solvent. This enzyme shows high thermo-stability, retaining 92% of its activity after 145 h at 90 �C, accordingto the manual of the enzyme. Therefore, a normal degradation ofthe enzyme is not supposed to occur within the lapse of time ofthe experiment.

4. Conclusions

Alkyl glycosides are surface-active compounds of high indus-trial interest. They can be synthetized through the catalytic actionof glycosidases in biphasic media, in which the organic phase isalso the alcohol substrate for the enzyme. The low yields of trans-glycosylation found in the system described here, are produced bytwo main factors: (1) coalescence of water, which reduces heptanolconcentration in the aqueous phase where enzyme and carbohy-drates concentrate, and (2) enzyme inactivation by Maillard reac-tion. Optimization of water content and stabilization of theenzyme could help to circumvent these problems. Enzyme cata-lyzed synthesis of alkyl glycosides remains highly promising andis worth to continue research in this field.

Acknowledgement

The authors acknowledge the excellent technical assistance ofLaura Álvarez-Añorve.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.06.080.

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