Optimization of butylgalactoside synthesis byb-galactosidase fromAspergillus oryzae

Ali Ismaila, Michel Linderb, Mohamed Ghoula,*aLaboratoire des Sciences du Genie Chimique, Centre National de la Recherche Scientifique-INPL, Vandoeuvre, France

bLaboratoire de Physico-Chimie et Genie Alimentaire, Ecole Normale Superieur INPL, Vandoeuvre, France

Received 29 July 1998; received in revised form 7 January 1999; accepted 10 February 1999

Abstract

Response surface methodology (RSM) was used to optimize the enzymatic synthesis of butylgalactoside from lactose catalyzed byb-galac-tosidase fromAspergillus oryzae. The empirical models developed by using RSM were adequate to describe relationships between the operatingconditions (temperature, water-to-butanol volume ratio, lactose concentration, enzyme concentration) and the responses (butylgalactoside con-centration, conversion yield). Based on contour plots and canonical analysis, optimal conditions for maximizing butylgalactoside concentrationwere: temperature (45°C), water-to-butanol volume ratio (44%), lactose concentration (134 g/l), and enzyme concentration (1.5 g/l). Experimentaldata indicated that up to 24 g/l were produced at the optimum point. Maximum conversion yield of 79.5% was obtained at: temperature (46°C),water-to-butanol volume ratio (18%), lactose concentration (10 g/l), and enzyme concentration (1.5 g/l). The models were verified experimentally.Synthesis at a large scale was successful. © 1999 Elsevier Science Inc. All rights reserved.

Keywords:Alkylglycosides; Butylgalactoside;b-galactosidase; Doehlert matrix; Optimization; Response surface methodology

1. Introduction

For several years, there has been a great interest in theenzymatic preparation of alkylglycosides [1–5]. These typesof nonionic surfactants have several interesting properties indetergency, foaming, wetting, emulsification, and antimi-crobial effect [6,7]. Moreover, they can be used as rawmaterials for sugar fatty acid ester synthesis [8]. Producedfrom renewable agricultural resources, they are nontoxic,non-skin-irritating, and very biodegradable [7,9,10]. Forthese reasons, they have great potential application in manydiversified areas such as the pharmaceutical, chemical, cos-metic, food, and detergent industries.

Research efforts are mainly limited to alkylglycosideswith high alkyl groups (C$ 6) having foaming properties.No in-depth attention is given to the synthesis of alkylgly-cosides produced from short alcohol (e.g. butanol). Never-theless, these molecules could be successfully used as flu-idifiers or emulsifiers or as substrates for sugar fatty acidester synthesis [11–13].

A recent paper reported on the result of the synthesis ofseveral butylglycosides by glycosidases [14]. The highestconcentration of butylglycosides was obtained when lac-tose was used as a glycosyl donor. However, only limiteddata about the use of lactose for the synthesis of alkyl-glycosides are available [15,16]. Moreover, neither spe-cific information on the interaction effects of reactionparameters nor any detailed optimal conditions have beengiven.

Response surface methodology (RSM) can evaluate theeffects of multiple parameters, alone or in combination,on response variables [17]. It has been successfully ap-plied for optimizing conditions in food, chemical, andbiological processes [18 –20] but has been rarely reportedon for optimizing the enzymatic synthesis of alkylglyco-sides [21].

In this work, we perform the enzymatic synthesis of butyl-galactoside byb-galactosidase fromAspergillus oryzaebyusing lactose as the glycosyl donor and butanol as thealcohol. The aim of this study was to investigate andoptimize the process parameters by using RSM accordingto the Doehlert uniform shell design for four factors:temperature, water-to-butanol volume ratio, lactose, andenzyme concentrations.

* Corresponding author. Tel.:133-83-595-892; fax:133-83-595-796.E-mail address:mohamed.ghoul@ensaia.inpl-nancy.fr (M. Ghoul)

Enzyme and Microbial Technology 25 (1999) 208–213

0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S0141-0229(99)00028-9

2. Materials and methods

b-Galactosidase fromA. oryzae(EC 3.2.1.23, 4.5 U/mg)and lactose were purchased from Sigma. Silica gel 20320 3 0.5 cm thin-layer-chromatography plates were pro-vided from Merck. Butanol and other reagents (purity.99.5%) were purchased from Fluka.

2.1. Butylgalactoside synthesis

In a typical experiment, enzyme was first dissolved insodium acetate buffer (pH 4.5, 10 mM). Then, butanol andlactose were added to the medium. The total reaction volumewas 115 ml. Incubation was carried out in a stirred thermo-stated-jacketed reactor. At regular intervals, 100ml of themedium were withdrawn and heated in boiling water for 10min to stop the reaction synthesis. Reaction was monitored bythin-layer chromatography, and compounds were quantified byhigh-performance liquid chromatography.

2.2. Analytical procedures

Thin-layer chromatography was performed by using sil-ica gel plates eluted with chloroform/methanol/acetic acid/water (80/15/8/2 v/v). Compounds were revealed by spray-ing the plate witha-naphtol solution (1.59 g ofa-naphtolwas dissolved in 51 ml of ethanol and then added to 4 ml ofwater and 6.5 ml of 18 M sulfuric acid). Products wereobtained by carbonization at 105°C (5.5 min) and quantifiedat 545 nm by photodensimetry by using Shimadzu CS-9000apparatus.

High-performance liquid chromatography was carriedout with Merck System (LaChrom, Merck) equipped with arefractive index detector (L-7490, Merck). A sugarpak col-umn (3003 6.5 mm, Waters) was used. The flow rate of100% water eluent was 0.6 ml/min at 90°C. The concentra-tions of compounds were determined from the peak areas.

2.3. Purification of butylgalactoside

After evaporation of butanol under vacuum, butylgalac-toside was purified on a silica gel liquid chromatographycolumn (703 3 cm, Merck) by using dichloromethane/methanol solution (8/2 v/v) with a 4 ml/min flow rate.Fractions containing butylgalactoside were collected andsolvent was evaporated.

2.4. Experimental design and statistics

A Doehlert experimental design [22] was used withNEMRODt software [23]. This experimental matrix dis-plays a uniform distribution of the points within the exper-imental domain. Factors investigated were temperature, wa-ter-to-butanol volume ratio, lactose, and enzymeconcentrations. A feature of Doehlert design is that thenumber of levels of each experimental factor is not the

same. A Doehlert design with four factors uses five levelsfor the first parameter, three levels for the last one, andseven levels for others. One should use the design so that theparameter with the most-complex relationship is modeledwith the largest number of levels. Most reported studies onthe enzymatic synthesis of alkylglycosides indicated that thewater amount and the concentration of glycosyl donor hadimportant roles in the performance of the reaction [1–5].Temperature influences the enzyme activity and the equi-librium of reaction [24], whereas the enzyme concentrationslightly affects the reaction synthesis [3,25]. Therefore, thehighest number of levels (7) were used to model water-to-butanol volume ratio and lactose concentration, whereastemperature had five levels and enzyme concentration hadthree levels.

The total number of points for four factors is 21 (N 5 k2

1 k 11). Experiment 21 performed at the center of theexperimental field was repeated three times to estimate pureerror (Table 1).

Performance of the process synthesis was evaluated byanalyzing two responses: maximum concentration of butyl-galactoside produced (C) expressed in g/l; and conversionyield (Y) expressed as the butylgalactoside concentrationsynthesized over the theoretical concentration of butylgal-actoside produced from lactose.

A full quadratic model containing 14 coefficients includ-ing interaction terms was used to describe relationshipsbetween responses and experimental factors:

h 5 b0 1 Oi51

4

b i Xi 1 Oi51

4

biiXi2 1 O

i51

3 Oj5i11

4

bij Xi Xj (1)

whereh is the response,b0 is the constant coefficient,Xi

(i 5 1 to 4), are uncoded variables,bis are the linearcoefficients,biis are the quadratic coefficients, andbijs (iandj 5 1 to 4) are the second-order interaction coefficients.

2.5. Data analysis

Data were computed by using NEMRODt software [23]including ANOVA and canonical analysis to obtain inter-action data between the process variables and responses.

2.6. Optimization and verification of models

Optimization of the reaction conditions in terms of tem-perature, water-to-butanol volume ratio, lactose, and en-zyme concentrations was calculated by using the predictivemodels from RSM. The synthesis of butylgalactoside wascarried out at the optimal conditions. Butylgalactoside con-centration and conversion yield were analyzed and com-pared with predictive values.

2.7. Scale-up synthesis

Reaction for scale-up synthesis was carried out at theoptimal conditions determined for optimization of the bu-

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tylgalactoside concentration. The reaction volume was in-creased by a factor of 10. Reaction was incubated in a 1.5-lreactor. Butylgalactoside concentration was analyzed andcompared with predictive data.

3. Results and discussion

3.1. Synthesis kinetics

The time course of the transglycosylation reaction oflactose showed that butylgalactoside and glucose concen-trations increased at the beginning of the reaction (Fig. 1).Only a small amount of residual galactose was noticed.After a few hours, when most of the lactose was consumed,butylgalactoside concentration reached a plateau, thendropped. This decrease in butylgalactoside concentrationmay be due to its hydrolysis, as it can be observed with anincrease of galactose concentration.

The butylgalactoside concentration obtained at the pla-teau depended upon the operating conditions, primarily thewater amount in the system and lactose concentration (Ta-ble 2). The mechanism of the butylgalactoside synthesis istoo complicated to devise a theoretical model, as severalparameters can influence this reaction. Therefore, an empir-ical model may be useful, particularly if responses need

only be approximated over a range of variables [17]. In thispresent work, RSM was used to model the enzymatic syn-thesis of butylgalactoside. Models thus obtained could al-low the study and the optimization of the four processparameters: lactose concentration, temperature, water-to-butanol volume ratio, and enzyme concentration.

Table 1Operating variables, levels, and experimental data used in the Doehlert design

Experimentno.a

Operating variables Responses

TemperatureX1

(°C)

Water to butanolvolume ratioX2

(%)

LactoseconcentrationX3

(g/l)

EnzymeconcentrationX4

(g/l)

ProductconcentrationC(g/l)

ConversionyieldY(%)

1 70 20 50 1.5 3.1 9.02 30 20 50 1.5 15.0 43.53 60 35 50 1.5 12.0 34.84 40 5 50 1.5 2.2 6.45 60 5 50 1.5 1.0 2.96 40 35 50 1.5 13.8 40.07 60 25 90 1.5 19.0 30.68 40 15 10 1.5 5.2 75.49 60 15 10 1.5 5.1 73.9

10 50 30 10 1.5 4.7 68.111 40 25 90 1.5 16.0 25.812 50 10 90 1.5 5.1 4.313 60 25 60 2.5 14.0 33.814 40 15 40 0.5 11.5 41.715 60 15 40 0.5 11.0 39.916 50 30 40 0.5 13.0 47.117 50 20 80 0.5 13.0 23.518 40 25 60 2.5 13.0 31.419 50 10 60 2.5 7.6 18.420 50 20 20 2.5 9.0 65.221a 50 20 50 1.5 14.3 43.521b 50 20 50 1.5 15.0 46.421c 50 20 50 1.5 16.0 41.4

a Treatments were run in a random order.

Fig. 1. Time course of the enzymatic synthesis of butylgalactoside. Glucose(F), galactose (Œ), butylgalactoside (l), lactose (f). Temperature550°C; water-to-butanol volume ratio5 20%; lactose concentration5 18g/l; enzyme concentration5 1.5 g/l.

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3.2. Statistical analysis

ANOVA indicated that the quadratic models derivedfrom RSM could adequately be used to describe the butyl-galactoside concentration (C) and the conversion yield (Y)under a wide range of operating conditions (Table 3). Forthe two models, there was no lack of fit and a satisfactorycoefficient of determination (R2 5 0.896 and 0.954 forCandY, respectively).

Moreover, statistical analysis showed that temperature,water-to-butanol volume ratio, and lactose concentrationwere the most important factors becauseC and Y weresignificantly affected (Table 4). Enzyme concentration hadno significant effect on the reaction synthesis (P 5 0.180and 0.679 forC andY, respectively).

After elimination of nonsignificant coefficients (P .0.05), the reduced models forC and Y can be written asfollows:

C 5 229.1201 1.1773 X1 1 0.8613 X2 1 0.095

3 X3 2 0.0133 X12 2 0.0253 X2

2 1 0.0103 X2

3 X3 2 X32 (2)

Y 5 213.3211 3.6863 X1 1 2.4963 X2 2 1.655

3 X3 2 0.0403 X12 2 0.0743 X2

2 1 0.0223 X2

1 X3 1 0.006X32 (3)

The simplest way to study the relationships betweenresponses and process factors is to construct an isoresponsesurface. These are generated by plotting responses versustwo variables (when all other factors are held constant).

3.3. Isoresponse representations

From 30–55°C, temperature did not have a significanteffect on butylgalactoside concentration (Fig. 2). At tem-peratures above 55°C, butylgalactoside concentrationdropped progressively.

From Fig. 2, it can be also seen that the water-to-butanolvolume ratio significantly affected butylgalactoside concen-tration (C). Indeed,C increased with the increase in thewater content, then dropped at high levels of water. Theseresults agree with other works, which report that a minimumwater amount is needed to perform glycosylation reaction[1,2,4,5,14,26]. This minimum is indispensable for the hy-drate state of the enzyme. The augmentation of water con-tent leads to activating the enzyme. However, as the butyl-galactoside concentration depends upon the magnitude ofsynthesis and hydrolysis reactions, at high water content,the hydrolysis is more favorable and, therefore, the butyl-galactoside concentration decreases [14,26].

Table 2Effect of lactose concentration and water-to-butanol volume ratio onbutylgalactoside concentration (C) and conversion yield (Y) obtained atthe plateaua

Lactoseconcentration(g/l)

Water-to-butanolvolume ratio(%)

C(g/l)

Y(%)

15 10 5.2 50.215 40 0.5 4.860 25 14.3 34.590 25 19.2 30.9

aTemperature5 50°C; enzyme concentration5 1.5 g/l.

Table 3NOVA for process variables pertaining to butylgalactoside concentration(a) and conversion yield (b) responses

a. Butylgalactoside concentration

Source Sum of squares d.f. Mean squareF-ratio P-value

Model 517.72 15 34.51 47.82 0.021

Residual 59.82 8Lack of fit 58.36 6 9.73 13.33 0.072Pure error 1.46 2 0.73

R2 0.896

b. Conversion yield

Source Sum of squares d.f. Mean squareF-ratio P-value

Model 9183.83 15 612.25 97.20 0.010

Residual 441.33 8Lack of fit 428.72 6 71.45 11.35 0.083Pure error 12.61 2 6.30

R2 0.954

Table 4Regression coefficients of second-order polynomials representingrelationships between indicated responses (CandY) and independentvariables of temperature (ior j 5 1), water-to-butanol volume ratio(i or j 5 2), lactose concentration (ior j 5 3), and enzymeconcentration (ior j 5 4)

Coefficient C Y

Model P-value Model P-value

b1 1.271 0.026 3.738 0.022b2 1.046 0.004 3.307 0.005b3 20.036 0.007 22.033 0.001b4 20.062 0.180 1.847 0.697b11 20.015 0.016 20.043 0.016b12 20.001 0.759 20.003 0.767b13 0.002 0.208 0.004 0.324b14 0.022 0.677 0.076 0.625b22 20.028 0.008 20.082 0.008b23 0.009 0.018 0.022 0.028b24 0.001 0.983 20.078 0.664b33 20.002 0.020 0.006 0.022b34 0.033 0.210 0.071 0.323b44 21.119 0.127 22.688 0.174

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However, the effect of water on the reaction synthesisdepended upon the lactose concentration (Fig. 3). Indeed, theoptimum water-to-butanol volume ratio increased with theaugmentation of lactose concentration (e.g. for lactose concen-tration 5 40 g/l, optimum water-to-butanol volume ratio5

25%, whereas for lactose concentration5 70 g/l, optimumwater-to-butanol volume ratio5 31%). Therefore, several op-tima could be found depending on the concentration of lactose.

On the other hand, the influence of lactose concentrationon the product concentration depended upon the water con-tent of the system. Indeed, at a low water-to-butanol volumeratio (e.g. 5%), the increase of the lactose concentration ledto a little decrease inC, whereas at a high water-to-butanolvolume ratio (e.g. 25%),C rose progressively with theincrease of lactose concentration.

The predicted conversion yield (Y) was plotted versus wa-ter-to-butanol volume ratio and lactose concentration (Fig. 4).Y increased with the increase of water content, then dropped,whereas the increase of lactose concentration led to a decreasein Y regardless of water level in the medium. However, com-pared with the decrease at high water content, the diminutionof Y was more spectacular at low water levels.

All previously reported works relating to alkylglycosideproduction have attempted to optimize process factors byusing a “one-variable-at-a-time” technique. However, thistechnique does not take into account any interaction effectson the response and may fail to find the region of optimalresponses. In the present study, we adopted response surfacemethodology to locate the co-optimal levels of processfactors and to gain further insight into the relative impor-tance and interactions of these factors.

3.4. Optimization and verification of models

Optimal conditions were determined by canonical anal-ysis to derive the stationary points. Optimization of the

Fig. 2. Response surface contours for butylgalactoside concentration (C) asa function of temperature (X1) and water-to-butanol volume ratio (X2).Lactose concentration5 50 g/l; enzyme concentration5 1.5 g/l. Numbersrepresent data predicted by RSM at the maximum point.

Fig. 3. Response surface contours for butylgalactoside concentration (C) asa function of water-to-butanol volume ratio (X2) and lactose concentration(X3). Temperature5 50°C; enzyme concentration5 1.5 g/l.

Fig. 4. Response surface contours for conversion yield (Y) as a function ofwater-to-butanol volume ratio (X2) and lactose concentration (X3). Tem-perature5 50°C; enzyme concentration5 1.5 g/l.

212 A. Ismail et al. / Enzyme and Microbial Technology 25 (1999) 208–213

butylgalactoside concentration was performed by takinginto account temperature, water-to-butanol volume ratio,and lactose concentration. Enzyme concentration was heldconstant at 1.5 g/l. Optimal conditions were: temperature(45°C), water-to-butanol volume ratio (44%), and lactoseconcentration (134 g/l). Predicted value indicated 22.861.3 g/l. Experimental data were 24.1 g/l.

Optimization of the conversion yield was carried out bytaking into account both temperature and water-to-butanolvolume ratio. Lactose concentration was kept constant at itslowest value used in the experimental field (10 g/l) asYincreased with the decrease of lactose concentration. En-zyme concentration was also kept constant at 1.5 g/l. Opti-mal temperature and water-to-butanol volume ratio were46°C and 18%, respectively. PredictedY determined byRSM was 80.06 3.8%. Experimental value was 79.5%.

These results clearly show that there is no appreciabledifference between experimental and predicted responses.Empirical models derived from RSM can successfully beused to describe the enzymatic synthesis of butylgalactosideby A. oryzaeb-galactosidase.

3.5. Scale-up synthesis

To explore whether the predictive model from RSMcould be applied to a large scale synthesis, the reactionvolume was increased by a factor of 10. Reaction wascarried out at the optimal conditions determined by RSM forthe optimization of the butylgalactoside concentration. Re-sults gave 22.3 g/l of butylgalactoside, which indicated nosignificant difference between large-scale and small-scalesynthesis.

Acknowledgments

This work was supported by research grants from theEuropean Union Project No. AIR2-CT-94-2291.

References

[1] Chahid Z, Montet D, Pina M, Graille J. Effect of water activity onenzymatic synthesis of alkylglycosides. Biotechnol Lett 1992;14:281–4.

[2] Ljunger G, Adlercreutz P, Mattiasson B. Enzymatic synthesis ofoctylglucoside in octanol at controlled water activity. Enzyme MicrobTechnol 1994;16:751–5.

[3] Panintrarux C, Adachi S, Araki, Y, Kimura Y, Matsuno R. Equilib-rium yield of n-alkyl-b-D-glucoside through condensation of glucoseand n-alcohol byb-glucosidase in a biphasic system. Enzyme MicrobTechnol 1995;17:32–40.

[4] Vic G, Biton J, Le Beller D, Michel JM, Thomas D. Enzymaticglucosylation of hydrophobic alcohol in organic medium by the

reverse hydrolysis reaction using almond-b-D-glucosidase. Biotech-nol Bioeng 1995;46:109–16.

[5] Vulfson EN, Patel R, Beecher JE, Andrews AT, Law BA. Glycosi-dases in organic solvents: I. Alkyl-b-glucoside synthesis in a water-organic two-phase system. Enz Microb Technol 1990;12:950–4.

[6] Balzer D. Alkylpolyglucosides, their physico-chemical properties andtheir uses. Tenside Surf Det 1991;8:419–27.

[7] Matsumura S, Imai K, Yoshikawa S, Kawada K, Uchibori T. Surfaceactivities, biodegradability, and antimicrobial properties of n-alkylglucosides, mannosides, and galactosides. J Am Oil Chem Soc 1990;67:996–1001.

[8] Mutua LN, Akoh CC. Synthesis of alkyl glycoside fatty acid esters innon-aqueous media byCandida sp. lipase. J Am Oil Chem Soc1993;70:43–6.

[9] Busch P, Hensen H, Kahre J, Tesmann H, Alkylpolyglycosides—anew cosmetic concept for mildness and care. Agro-Food-IndustryHi-Tech 1994;9:23–8.

[10] Madsen T, Petersen G, Seiero C, Torslov J. Biodegradability andaquatic toxicity of glycoside surfactants and a nonionic alcoholethoxylate. J Am Oil Chem Soc 1996;73:929–33.

[11] Adelhorst K, Bjorkling F, Godtfredsen SE, Kirk O. Enzyme-catalyzed preparation of 6-o-acylglucopyranosides. Synthesis 1990;2:112–5.

[12] Monsan PF, Paul E, Pelenc P, Boures E. Enzymatic production ofa-butylglucoside and its fatty acid esters. Ann NY Acad Sci 1996;799:633–41.

[13] Milius A, Brancq B. Alkylpolyglucosides: new orientations. Agro-Food-Industry Hi Tech 1996;9:17–20.

[14] Ismail A, Ghoul M. Enzymatic synthesis of butylglycosides by gly-cosidases. Biotechnol Lett 1996;18:1199–1204.

[15] Stevenson DE, Stanley RA, Furneaux RH. Oligosaccharide and alkylb-galactopyranoside synthesis from lactose withCaldocellum saccharo-lyticumb-glycosidase. Enzyme Microb Technol 1996;18:544–9.

[16] Stevenson DE, Stanley RA, Furneaux RH. Optimization of alkylb-D-galactopyranoside synthesis from lactose using commerciallyavailableb-galactosidases. Biotechnol Bioeng 1993;42:657–66.

[17] Box GEP, Draper NR. Empirical model-building and response sur-faces. New York: Wiley, 1987.

[18] Chen HC, Chang CC, Chen CX. Optimization of arachidonic acidproduction byMortierella alpina Wuji-H4 isolate. J Am Oil ChemSoc 1997;74:569–78.

[19] Linder M, Fanni J, Parmentier M, Sergent M, Phan-Tan-Luu R.Protein recovery from veal bones by enzymatic hydrolysis. J Food Sci1995;60:949–52.

[20] Shieh CJ, Akoh CC, Koehler PE. Four-factor response surface opti-mization of the enzymatic modification of triolin to structured lipids.J Am Oil Chem Soc 1995;72:619–23.

[21] Ismail A, Soultani S, Ghoul M. Optimization of the enzymatic syn-thesis of butylglucoside using response surface methodology. Bio-technol Prog 1998;14:874–78.

[22] Doehlert DH. Uniform shell design. Appl Stat 1970;19:231–9.[23] Mathieu D, Phan–Tun–Luu R. NEMRODt: New Efficient Method-

ology for Research Using Optimal Design. Marseille (LPRAI), Uni-versity of Aix, 1992.

[24] Vulfson EN, Patel R, Law BA. Alkyl-b-glucoside synthesis in awater-organic two-phase system. Biotechnol Lett 12:397–402.

[25] Rodriguez M, Gomez A, Gonzalez F, Barzana E, Lopez–Munguia A.Selectivity of methyl-fructoside synthesis withb-fructofuranosidase.Appl Biochem Biotechnol 59:167–75.

[26] Laroute V, Willemot RM. Glucoside synthesis by glucoamylase orb-glucosidase in organic solvents. Biotechnol Lett 1992;14:169–74.

213A. Ismail et al. / Enzyme and Microbial Technology 25 (1999) 208–213