Optimization of Lipase Performance in Detergent Formulations for

ARTICLE pubs.acs.org/IECR

Optimization of Lipase Performance in Detergent Formulations for Hard Surfaces Encarnacion Jurado,* Miguel García-Roman, German Luzon, Deisi Altmajer-Vaz, and Jose Luis Jimenez-Perez Chemical Engineering Department, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, 18071, Granada, Spain

bS Supporting Information ABSTRACT: Lipases have been added to detergents in an attempt to develop more environmentally friendly and energy efficient products for triglyceride-based stains. However, more research is needed to improve the efficiency of lipase-containing detergents. In this work we have examined all the factors affecting lipase performance in detergent formulations, particularly for hard surface cleaning. Several issues, such as thermal and hydrodynamic stability, kinetic aspects, and surfactant compatibility, both in solution and at the oil/water interface, have been addressed and the conditions for an optimal performance have been established. The role of some stabilizers in preventing lipase deactivation has also been considered, and some insight on the mechanisms of lipase deactivation under washing conditions is provided. Finally, washing tests conducted in a continuous flow device which simulates a clean-in-place (CIP) system have demonstrated that, under optimal conditions, a reduction of the surfactant dosage was possible upon the incorporation of lipases in detergent formulations.

1. INTRODUCTION Lipases (E.C. 3.1.1.3) are enzymes which in natural systems catalyze the conversion of triglycerides in diglycerides, monoglycerides, and free fatty acids. However, in microaqueous environments they are also able to catalyze the reverse reaction, i.e. esterification. This capability makes lipases an extremely versatile tool and consequently applications for them have been found in a wide number of fields, such as those put forward in the numerous reviews on this subject published so far.16 The use of lipases as functional ingredients in detergents is included among this wide range of applications. Although proteases were the first enzymes to be introduced in commercial laundry detergents and are currently the most widely used enzymes for cleaning purposes, lipases are also of particular interest for detergent producers, as fats and oils (mainly triglycerides) are difficult to remove from textiles and hard surfaces, mainly at low temperatures.7 The removal of triglyceride-based stains by lipases is thought to be accomplished by the decomposition of fatty materials into more hydrophilic substances,7,8 or by the release of surface active molecules, such as free fatty acids, which can induce an advantageous change in the properties of the soil/water interface.9,10 Lipolase, a fungal lipase from Thermomyces lanuginosus developed by Novozymes,11 was the first commercial lipase designed to be used as a detergent additive. It was launched in 1988 and incorporated in commercial detergents in Japan, the United States, and Europe from 1988 to 1991. Since then, Novozymes have marketed three genetically modified variants of Lipolase (LipoPrime, Lipolase Ultra, and Lipex) which show improved washing performance in the first wash cycle or at low temperatures.12,13 Very recently another detergent lipase, LipoClean, has also been introduced by Novozymes, who claim that, as part of a multienzyme solution, LipoClean can effectively replace 25% r 2011 American Chemical Society

of the surfactant content of commercial detergents.14 Not only Novozymes, but other important enzyme producers, such as Genencor, Gist-Brocades (now part of DSM), and the Japanese Showa-Denko have also developed lipases for detergents. However, neither of the detergent lipases developed by these companies is still being marketed in 2011, according to the information offered through their corporative websites. Additionally, in the last decade a considerable number of lipases with potential use as detergent additives have also been produced and characterized by academic research groups, mainly in India and China. Lipases both from bacterial1518 and fungal1925 origins have been tested with successful results as regards surfactant compatibility, resistance against oxidizing agents, and activity at low temperature. However, despite the promising perspectives and laboratory results, some authors8 and even enzyme producers,26 have acknowledged that lipase performance, particularly in the first wash cycle, is not as good as expected. This fact, together with the traditionally high prices of enzyme ingredients, may explain the lower occurrence of lipases in commercial detergent formulations, as compared to that of proteases and amylases. Actually, among the major enzyme producers only Novozymes is currently offering and developing lipases for detergents, whereas Genencor, DSM, and Showa Denko have retired their equivalent products from the market. Several reasons can explain the reduced (compared to that expected) efficiency of lipases under washing conditions: (a) Lipases are particularly active at low water activity (between 10 and 40% of water content), which enhances Received: July 11, 2011 Accepted: September 11, 2011 Revised: September 5, 2011 Published: September 11, 2011 11502

dx.doi.org/10.1021/ie201486a | Ind. Eng. Chem. Res. 2011, 50, 11502–11510

Industrial & Engineering Chemistry Research

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Table 1. Structural and Physicochemical Characteristics of the Surfactants Used in This Studya as1 2/ (Å molc)

MW, surfactant

CMC, g/L

12

288.4

2.0

1013

343.4

3.3

structural formula R SO4

SDS

g/ mol

C6 H5 SO3

R

Γ1, mol/m2 1.16 106

143

supplier Panreac, Barcelona, Spain Cepsa Química, San Roque, Spain

LAS

R

G600

R O ðC6 H11 O5 Þ1:2

11.7

377.0

0.015

3.41 106

48.7

Henkel KgaA, D€usseldorf, Germany

C12EO10

R ðO CH2 CH 2 Þ10 OH R N þ ðCH3 Þ3

12

627.0

0.043

2.79 106

59.5

Sigma, St. Louis,MO, USA

12

308.4

5.6

DTAB

Sigma, St. Louis, MO, USA

a

R: number of carbons in the alkyl chain. MW: average molecular weight, calculated according to the information provided by the suppliers. CMC: critical micelle concentration. Γ1: surface concentration. as1: area per molecule at the surface.

(b)

(c)

(d)

(e)

lipid hydrolysis during the drying stage, better cleaning results being achieved in subsequent washing cycles.8 This issue is particularly important for laundry cleaning, but it does not apply to hard-surface cleaning, due to the absence of a real drying stage. The increasing tendency towards washing at ambient temperatures (below 20°C), particularly in domestic laundry cleaning,27 which reduces lipase activity and also affects the state of aggregation of fatty soils. However, this is not the case for hard surface cleaning, as higher temperatures are frequently used for this purpose. Lipases can be destabilized by surfactants or interact with them at the oil/water interface,10,28 due to the interfacial nature of their catalytic action. Additionally, these enzymes can undergo thermal29 or even interfacial deactivation,3033 which implies the loss of hydrolytic activity of the lipase as a result of its adsorption at the air/water interface. The initial reaction rate of enzymatic lipolysis mostly depends on enzyme concentration and on the specific interfacial area between the oily soil and the aqueous medium.34 Experiments conducted by Jurado et al.29 on a bench-scale device which simulates a clean-in place system demonstrated that the interfacial area between the oily soil and the washing bath increases sharply throughout the first 5 min of the washing cycle. Consequently lipases cannot exert their full catalytic action from the beginning of the washing cycle. Furthermore, as the reaction proceeds, the interface becomes saturated with reaction products such as free fatty acids, diglycerides, and monoglycerides, which causes an abrupt decrease of the reaction rate.34 Some authors have also put forward the influence of the kinetic aspects of lipolysis on the washing efficiency of lipases.8,35 Nevertheless, the cleaning action of lipases does not require a complete hydrolysis of the fatty material being cleaned, but rather the production of a sufficient amount of surface active substances which can lower the interfacial tension and thus facilitate the removal of the fatty deposits. The degree of ionization of the fatty acids released by hydrolysis of the fatty soil has also a strong influence on the removal of lipid films, as have been suggeested by Snabe et al.36,37 Finally, and unlike other detergent enzymes, lipases perform the same role as surfactants, i.e., they act specifically against oily soils. Thus, the only justification for introducing lipases in a detergent formulation is to reduce the amount of surfactant needed to achieve satisfactory cleaning

results, with the additional benefit of lipases being a more biodegradable and environmentally compatible agent than surfactants. In the present work we have examined all the factors affecting lipase performance under washing conditions, particularly for hard surface cleaning which is a scarcely addressed issue in the existing literature. Thermal and interfacial stability, surfactant compatibility, and kinetic aspects have been studied and several stabilizers tested in order to find the optimal conditions which lead to the best washing results using lipases. To conclude, washing experiments have been conducted in a continuous flow device which simulates an industrial clean-in-place (CIP) system, to find out whether, under optimal conditions, a reduction of the surfactant dosage is possible upon the incorporation of lipases in detergent formulations.

2. MATERIALS AND METHODS 2.1. Enzymes and Chemicals. Two commercial lipases, named Lipolase 100 (L100) and Lipex (LX), from Novozymes (Bagsvaerd, Denmark), were used. Both enzymes are genetically engineered variants of the lipase from the fungus Thermomyces lanuginosus. Remarkably, Lipex was designed to exhibit an enhanced adsorption to oil/water interfaces.37 Surfactants belonging to three different groups were tested to assess their compatibility with lipases: two anionic (sodium dodecyl sulfate, SDS, and a linear alkylbenzene sulfonate, LAS), two nonionic (an alkyl glucoside with commercial name Glucopone 600, G600, and a polyoxyethylene alkyl ether, C12EO10), and one cationic (dodecyl-trimethyl ammonium bromide, DTAB). The chemical structure, average molecular weight, and supplier of each surfactant are summarized in Table 1, together with some of their physicochemical properties such as the critical micelle concentration (CMC), the surface concentration, Γ1, and the area per molecule at the surface, as1. All the surfactants chosen differ in their head groups but posses the same number of carbons in their hydrophobic alkyl tail (≈12). The calculated values of Γ1 and as1 are in good agreement with those found in literature for the same or similar substances. Three different substances with a known capacity to stabilize enzymes by different mechanisms38,39 were chosen as stabilizers: (a) a polyalcohol, ethylene glycol (10% w/w), (b) an amino acid, glycine (0.1 M), and (c) a fatty alcohol, 1-dodecanol (0.01% w/w). The latter was chosen because of its strong capacity to reduce the surface tension of water, even when a trace amount of this substance is present. All these substances, of analytical grade, were provided by Panreac (Barcelona, Spain). 11503

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Figure 1. Schematic plot of the bath-substrate-flow device.

A commercial fat, pork lard, from “El Pozo” (Murcia, Spain), was used as the model soil for the washing tests. It was purchased from a local supplier and stored at 4 °C until use. 2.2. Physicochemical Characterization of the Surfactants Tested. The critical micelle concentration of all the surfactants used in our study was determined from the break in their surface tension vs log of concentration curves. Surface tension measure€ ments were conducted with a Kr€uss K11 tensiometer (KRUSS GmbH, Hamburg, Germany), using the Wilhelmy plate method. The surface concentration, Γ1, and the area per molecule at the surface, as1, were calculated when possible using the Gibbs equation in the following form: 1 ∂γ mol , Γ1 ¼ nRT ∂ln C1 T cm2

ð1Þ

where γ represents the surface tension (N/m), C1 the surfactant concentration (mol/L or g/L), R is the gas constant (J/mol 3 K), and T is the absolute temperature. The value of n is 1 for dilute solutions of a nonionic surfactant or for a 1:1 ionic one in the presence of a swamping amount of electrolyte, whereas n = 2 for a 1:1 ionic surfactant in the absence of any other solutes.40 On knowing the surface concentration, it is possible to calculate the area per molecule at the surface from eq 2. 2

as1 ¼

1020 Å , 6:023 1023 Γ1 molec

ð2Þ

2.3. Lipase Activity Determination. Lipolytic activity was determined by the pHstat method34 at pH 7.0 and 30 °C using tributyrin (Panreac, Barcelona, Spain) emulsions as the substrate. Its value was expressed in lipase units (LU), with 1 LU being the quantity of enzyme capable of releasing one micromole of butyric acid per minute under assay conditions. The emulsification of tributyrin was achieved by mechanical dispersion using an UltraTurrax stirrer (IkaWerke, Staufen, Germany) at 13 000 rpm for 1 min. 2.4. Deactivation Experiments. Three different types of deactivation experiments were carried out: (a) Thermal stability tests: Lipase solutions of 0.01 g/L were kept unstirred at temperatures between 30 and 60 °C in a screw-cap tube. Samples were withdrawn at different time

intervals, and their lipolytic activity measured. The residual activity was expressed as a percentage of the initial one. (b) Interfacial stability tests: Lipase solutions of 0.01 g/L were magnetically stirred in an open beaker at stirring rates between 300 and 1000 rpm at room temperature. Care was taken to avoid trapping air bubbles in the lipase solution while stirring. Lipolytic activity was measured at specific time intervals, and the residual activity was expressed as described above. (c) Stability tests under washing conditions: These experiments were performed in the bath-substrate-flow (BSF) device, a continuous flow apparatus which simulates the washing process of hard surfaces in CIP like systems. A simplified scheme of this device is shown in Figure 1, whereas a more detailed description of the same can be found elsewhere.10,29,41 L100 solutions (0.01 g/L) were initially placed in the stirred tank A (see Figure 1) and then circulated with the aid of the peristaltic pump C through the packed column B for 30 min. At the end of each run, the residual lipase activity was measured. A flow rate of 50 L/h, the maximum attainable in the experimental device, was used in all the experiments, which gives a Reynolds number of 1965 (almost transition flow). The tests were conducted according to a two-level factorial design, with two factors (stirring rate in tank A and temperature) and one response variable (residual activity after 30 min.). The analysis of the results was made with the help of the software package Modde 6.0 from Umetrics (Umea, Sweden). 2.5. Surfactant Compatibility Tests. The effect of surfactants on the activity and stability of the detergent lipases L100 and LX was studied in a range of situations. To do that, several kind of experiments were planned and conducted: (a) Stability tests: 10 g/L solutions of both lipases were incubated for 24 h at 25 °C in the presence of two different concentrations of surfactant: one below the CMC (0.2CMC) and one above (2CMC). The residual activity of the lipase solution was determined at the end of the experiment. (b) Activity tests: The activity of both lipases was assayed using tributyrin emulsions stabilized with the various surfactants studied, and the results were compared to that of a control test with gum arabic, a hydrocolloid with a widely recognized compatibility with lipases.34 In order to evaluate the effect of the specific interfacial area of the emulsions prepared with gum arabic and surfactants on lipase activity, the droplet size distribution of the emulsions used for the activity experiments was measured by laser diffraction using a Coulter LS230 instrument (Beckman-Coulter, Miami, USA). (c) Desorption tests: A calculated amount of surfactant, high enough to guarantee the full covering of the whole surface of the emulsion droplets, was added to the reaction medium (always a gum arabic-stabilized tributyrin emulsion) 5 min after the enzyme, i.e., when the enzymatic hydrolysis is already taking place. The capacity of a particular surfactant to desorb or interact with the enzyme at the interface can be directly deduced from its effect on the reaction rate. Consequently we have defined the desorption capacity of a surfactant (DC) as in eq 3. DC ¼ 11504

r2 1 r1

100, %

ð3Þ



Figure 2. Deactivation kinetics of the enzyme L100 at temperatures between 30 and 60 °C. The deactivation data have been fit to a first-order model (solid lines).

where r1 and r2 are the hydrolysis reaction rates before and after the addition of the surfactant, respectively. In every case, the enzyme concentration was chosen to ensure linear kinetics throughout the experiment. 2.6. Washing Tests. To complete our study, washing tests were conducted with the BSF device which was described above (Figure 1). The aim of these experiments is to establish the capacity of the enzyme, under optimal conditions, to reduce the amount of surfactant needed to achieve a successful cleaning. Commercial pork lard was used as the fatty soil and borosilicate glass spheres, 6 mm in diameter, were used as the substrate (material to be soiled). In a typical experiment, the soiled substrate (with approximately 0.1 g of soiling material per gram of substrate) was placed in the packed column (B, Figure 1) and the washing solution, initially placed in the stirred tank A, was then circulated through the column for 10 min. The pH of the washing solution was monitored throughout the experiment and adjusted when required with the help of a pH-electrode partly immersed in tank A (see Figure 1). The soil remaining on the substrate at the end of the experiment was extracted with i-octane and determined spectrophotometrically. To make this possible, the fatty soil was previously stained with the fat-soluble pigment Sudan III (0.02% w/w), so it could be determined by measuring its absorbance at 500 nm. The washing performance or detergency (De) was calculated according to eq 4. mi mf 100, % ð4Þ De ¼ mi where mi stands for the total amount of fatty material initially deposited over the substrate and mf represents the amount of soil which remained adhered to it at the end of the experiment.

2. RESULTS AND DISCUSSION 3.1. Thermal Stability of Detergent Lipases. The deactivation curves of the enzyme L100 are plotted in Figure 2, where the experimental points have been fitted to a first-order model. The enzyme seems to be pretty stable when incubated below 60 °C, which is in agreement with previous studies conducted by our research group.29 Thus, the thermal stability of the enzymes L100 and LX was studied at 60°C both alone and in the presence some stabilizers. As expected, both L100 and LX underwent a moderate but significant deactivation at 60 °C presenting residual activities of 80.6 ( 4.3% and 72.5 ( 8.2% after 1 h of incubation,

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Figure 3. Residual activity of lipase L100 after 1 h incubation at 60 °C in the presence of some stabilizers. Error bars represent the standard deviation of three replicates. Glycine was used as an additive (*), without adjusting the pH of the solution, and as a buffer (**), at pH 10.8.

Figure 4. Residual activity of lipases L100 and LX after stirring at 1000 rpm for 2 h at ambient temperature, alone and in the presence of several stabilizers. Only those substances which gave positive results with the enzyme L100 were also tried with LX. Error bars represent the standard deviation of three replicates. Glycine was used as an additive (*), without adjusting the pH of the solution, and as a buffer (**), at pH 10.8.

respectively. However a t-test for the comparison of the means indicates that there are no significant differences between the residual activities of both enzymes, suggesting that their susceptibility to thermal deactivation is similar. The residual activity of the enzyme L100 after incubation for 1 h at 60 °C in the presence of several stabilizers is plotted in Figure 3. Glycine buffer and ethylene glycol seem to stabilize the enzyme, but an ANOVA of the experimental results revealed no significant difference with the control experiment. Similar results were obtained with the enzyme LX. In any case, it can be concluded that both ethylene glycol and glycine buffer (0.1 M, pH 10.8) could be a suitable option to protect detergent lipases from thermal deactivation. 3.2. Mechanical Stability of Detergent Lipases. We have studied the behavior of lipases L100 and LX when subjected to stirring at 1000 rpm for 2 h alone (control) and in the presence of several stabilizers. As can be observed in Figure 4, both lipases become deactivated under our experimental conditions. However, the residual activity of LX is significantly lower than that of L100, which is compatible with the interfacial nature of enzyme deactivation, as the enzyme LX is more surface-active than L100, and this possibly enhances its susceptibility to elevated surface tensions. The ANOVA of data presented in Figure 4 together with the multicomparison tests to determine differences among groups indicate that dodecanol (0.01% w/w) and glycine buffer (0.1 M pH 10.8), can significantly enhance the stability of the enzyme L100. However, only the glycine buffer significantly protected the enzyme LX from deactivation. 11505



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Figure 6. Stability of the detergent lipase LX in the presence of surfactants. Residual activity was measured after 24 h incubation at two different surfactant concentrations. The results of a control test, in the absence of surfactant, are also included. Error bars represent the standard deviation of three replicates. Figure 5. Deactivation kinetics of enzyme L100 as a function of the stirring speed at ambient temperature. The solid line corresponds to the fitting of all data points to a pseudo-first-order model (eq 5).

Although both glycine and dodecanol proved to be good stabilizers, the stabilization mechanism is presumably different. Dodecanol probably acts by drastically reducing surface tension, and thus, avoiding lipase denaturation at the interface. Conversely, glycine cannot reduce surface tension appreciably. The mechanism behind enzyme stabilization by glycine may be related to the protection of enzyme structure against denaturation, as suggested by Arakawa and Timasheff.38 However, glycine by itself produced negligible improvement of lipase stability (see Figure 4), which suggests that the alkaline conditions of glycine buffer may play an important role in stabilization. Vilchez et al.42 also found that high pH values increased protease stability even in the presence of other stabilizers. The interfacial nature of lipase deactivation caused by hydrodynamic stress is also supported by the analysis of the deactivation curves of the enzyme L100 at different stirring speeds which are plotted in Figure 5. The experimental points were fitted to a pseudo-first order model, given by eq 5: Ar ¼ ð1 Af Þexpðkd tÞ þ Af

ð5Þ

where Ar represents the residual lipase activity at any time, expressed as a percentage of the initial one, and Af and kd are the kinetic parameters of the model, i.e., the final residual activity and the deactivation constant, respectively. The activity of the enzyme decreased with time at every stirring speed, until a final constant value (Af) was reached. However, no clear tendency of the model parameters with the stirring speed was found (see Figure 5), and because of that, all experimental points could be fitted together to the model (solid line in Figure 5). Considering that the effect of shear stress must be very low under our experimental conditions, the observed behavior may reflect that stirring is able to enhance interfacial deactivation, probably by promoting the adsorption of the enzyme at the interface, but is not the controlling factor in it. On the other hand, the amount of interfacial area and the interfacial tension, which were constant under our experimental conditions, are likely to play a determining role on the interfacial deactivation of lipases, as suggested by previous studies.32,33 3.3. Surfactant Compatibility. The effect of the head group type on the lipase compatibility of different surfactants was studied from the points of view of lipase stability and lipasesurfactant interaction at the interface.

Figure 7. Desorption capacity (eq 1) of the different surfactants tested. The results of a control experiment are also included. Error bars represent the standard deviation of three replicates.

To check the stability of lipases L100 and LX, both of them were incubated for 24 h in the presence of two different surfactant concentrations (below and above the CMC). The results for LX are shown in Figure 6 (completely analogous results were found with L100). An ANOVA of the residual activity data showed that, for surfactant concentrations below CMC (0.2CMC), both anionic surfactants LAS and SDS caused a significant lipase destabilization, whereas the nonionic surfactants C12EO10 and G600 could even be considered as stabilizers, yielding a better residual activity than that of the control test. Similar conclusions can be drawn from the results at higher surfactant concentration (2CMC), but a much more pronounced effect of LAS and SDS was observed as well as a slight denaturing effect of DTAB. These results are in good agreement with previously reported data on enzyme destabilization caused by surfactants.28,43 Anionic surfactants are generally recognized as protein-denaturing agents, due to the strong electrostatic interactions between the surfactant hydrophilic group and positively charged amino acid side chains, in addition to the hydrophobic interactions between the surfactant alkyl chain and the long aliphatic chains of both lysine and arginine.44 Another conclusion which can also be drawn from the results of the stability tests is that some nonionic surfactants, such as G600 or C12EO10, can be used to prevent lipase interfacial deactivation, as they simultaneously preserve lipase stability and reduce the interfacial tension. As regards the lipasesurfactant interaction, two different tests were conducted: desorption tests, in which the surfactant was added to the reaction medium 5 min later than the enzyme, 11506



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Table 2. Experimental Design to Test Lipase Stability under Washing Conditionsa

Figure 8. Activity of lipases L100 and LX measured on tributyrin emulsions stabilized with surfactants. The results of a control experiment with gum arabic as stabilizer are also included. Error bars represent the standard deviation of three replicates.

and activity tests, in which the lipase activity was determined using tributyrin emulsions stabilized by the different surfactants tested. The results of the desorption experiments can be seen in Figure 7, where the desorption capacity (DC), calculated according to eq 3, is presented for every surfactant. The amount of surfactant added was calculated using data in Table 1 in order to guarantee the full covering of the emulsion droplets. All surfactants tested exerted a significant effect on the lipolysis rate, and notably LAS and SDS led to negative DC values, which imply that both surfactants are somehow enhancing the hydrolytic capacity of the adsorbed lipase, may be promoting its adsorption or, which is more probable, forming an especially active complex with it, as suggested by Xia et al.45 Similar results have already been reported by our research group10 when the enzymatic hydrolysis of tributyrin in the presence of LAS was monitored. The high DC of C12EO10 is also noteworthy, which means that this surfactant strongly interacts with the adsorbed lipase causing its desorption or maybe forming an inactive complex with it. On the other hand both G600 and DTAB showed a significant but moderate DC which seems compatible with their joint application with lipases. Although both lipases presented a very similar behavior, the enzyme LX proved to be slightly more resistant to desorption than L100, as revealed by the significantly lower DC values of surfactants G600, C12EO10, and DTAB. This observation can be attributed to the higher surface activity of LX compared to that of L100. It may seem surprising that the results of the desorption experiments did not match at all with those of the stability experiments; however, this shows that lipase surfactant interaction largely depends on lipase conformation which is different in solution and on the interface.46 When the activity of both lipases was measured on a surfactant-stabilized tributyrin emulsion (Figure 8), a severe reduction of activity compared to that of the control was recorded for all surfactants tested, except for G600. The nonionic surfactant C12EO10, which presents a high DC, also proved to be a powerful inhibitor of lipase adsorption, leading to an almost complete reduction of lipase activity. It was not possible to produce a stable tributyrin emulsion with DTAB, and because of that, no result for this cationic surfactant was obtained in this test. As regards anionic surfactants, both LAS and SDS prevent lipase action when they are already present at the interface, which agrees with previous observations by Skagerlind et al.47 The

a

exp number

temperature, °C

stirring rate, rpm

res act (30 min), %

1

30

100

120.2

2 3

60 30

100 300

72.1 96.9

4

60

300

59.0

5

45

200

86.4

6

45

200

81.9

7

45

200

92.1

8

45

200

93.6

The result of each experiment is also shown.

specific interfacial area of the surfactant-stabilized tributyrin emulsions proved to be very similar to that of the control emulsion prepared with gum arabic as demonstrated by laser diffraction measurements (data not shown), which excludes any influence of this parameter on the activity values determined with surfactant-stabilized emulsions. For a lipase to be compatible with a particular surfactant the two following conditions must be fulfilled: (a) The lipase should be stable in the presence of the surfactant throughout the whole storage life of the detergent product. This is an issue of key importance for liquid detergent formulations, whose global market share is steadily increasing.48 (b) The surfactant should not prevent or delay lipase adsorption onto the oil/water interface, as this is an essential step in the enzymatic hydrolysis of triglycerides. In view of the results of the surfactant compatibility tests, we can conclude that the nonionic surfactant G600 is the one which satisfies the two former conditions to a larger extent and, consequently, will be further used for the washing tests. 3.4. Washing Tests. As a previous stage to the performance of washing tests with lipase in the BSF device, the simultaneous effect of both temperature and hydrodynamic stress on enzyme stability was evaluated in the system. Experiments were programmed and conducted according to a two-level factorial design with two factors (temperature and stirring rate) and one response variable (residual activity after 30 min.). All the experiments performed and their corresponding results are presented in Table 2. The evaluation of the raw data shows that the replicate error is acceptable and the results are normally distributed. The results were fitted to a linear model with interaction terms, which according to the R2/Q2 diagnostic tool and the lack-of-fit test49 proved valid to describe the experimental results and useful to make predictions within the experimental region. The effect of each factor on lipase stability was evaluated through the coefficient plot,10 revealing that both the temperature and the stirring rate have a significant effect on lipase stability in our experimental device. However the effect of temperature is the most significant one, and the interaction between temperature and stirring rate is not significant. It is remarkable that despite the hydrodynamic conditions in the system not being drastic (low stirring rate and moderate Reynolds number), they were able to affect lipase stability, particularly at 60 °C. At this temperature, bubble nucleation and growing in the system are favored, thus increasing the interfacial area and causing lipase to destabilize more quickly. We have already established a set of conditions (temperature, hydrodynamic conditions, stabilizers, surfactants) which should 11507



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Table 3. Results of the Washing Experiment Conducted under Optimized Conditions (See Text)a detergency, % surfactant

a

concentration (g/L)

enzyme

T, °C

soil

pH

mean

SD

reference

Glucopone 600

0.5

Lipex, 1 g/L

pork lard

45

6.410.2

99.0

0.2

this work

Berol LFG 61

1

no

oleic acid

45

neutral

69.6

3.6

41

mixtureb

1

no

fatty acid mixturec

45

neutral

83.4

0.7

50

Glucopone 600

1

no

triolein

45

8

98.8

Berol LFG 61

1

no

fatty acid mixturec

45

neutral

79.6

0.9

51

29

The results of several experiments without enzyme are also shown for ease of comparison. b Glucopone 650, Findet 1214N/23, Cellesh 100 (14:70:16 w/w). Stearic, palmitic, and oleic acid (12:12:76 w/w).

c

to a different time interval of the washing process. Despite a significant removal of the fatty soil being obtained by the enzymesurfactant solution at acidic pH (Figure 9b), complete removal and a better emulsification of the soil in the washing solution were achieved after raising the pH to 10.2 (Figure 9c). It should also be considered that the results of the washing test were obtained with pork lard as the fatty soil, which makes them particularly satisfactory, as pork lard has a higher melting point than oleic acid or triolein, thus being a much more difficult-toremove substance. Thus, the results of the washing test suggest that, under the appropriate conditions, it is possible to partly reduce the amount of surfactant in a detergent formula, by replacing it by lipases. Figure 9. Appearance of the packed column of the BSF device containing the soiled substrate at three different time intervals of the washing process: (a) 0 min, pH = 6.4; (b) 6 min, pH = 6.4; (c) 10 min pH = 10.2.

lead to an optimized washing performance of lipase containing detergent formulations. To confirm this assumption, a washing test with a detergent solution containing the surfactant G600 (0.5 g/L) and the lipase LX (1 g/L) was conducted. The surfactant dosage was reduced by 50% compared to that typically used in previous tests in the BSF device29,41,50,51 in order to find out whether, under optimized conditions, the presence of a lipase would allow a reduction of the amount of surfactant needed to achieve satisfactory cleaning results. Glycine (0.1 M) was used as enzyme stabilizer, and mild hydrodynamic conditions (flow rate of 30 L/h; 200 rpm stirring rate) and temperature (45°C) were chosen to preserve lipase stability, according to our previous results. Additionally, 6 min after the start of the experiment, the pH of the washing solution was raised by NaOH addition from 6.4 to 10.2, i.e., from below to above the pKa of oleic or stearic acids. This sudden increase of pH can enhance the removal of lipid films up to a 50%, according to a recent study by Snabe at al.37 The results of the washing test conducted as described above are presented in Table 3 where they are also compared to those of tests carried out at higher surfactant concentrations. As can be seen in Table 3, the washing solution containing lipase and 0.5 g/L of surfactant led to very good detergency results (almost complete cleaning), better than those of tests conducted with higher surfactant dosages. An outstanding increase in detergency was observed when the pH of the washing solution was raised above 9.5, i.e., when the fatty acids released by the hydrolysis of the fatty soil became fully ionized, which agrees with the findings of Snabe and co-workers.36,37 This effect can be clearly appreciated in the three images of the packed column of the BSF device which are plotted in Figure 9, each one corresponding

3. CONCLUSIONS The best conditions for the application of the detergent lipases L100 and LX to the removal of fatty residues from hard surfaces have been investigated. Both enzymes underwent a significant destabilization when exposed to temperatures above 50°C, hydrodynamic stress, and anionic surfactants (LAS and SDS). Thermal deactivation followed first-order kinetics and could be prevented by ethylene glycol or a glycine buffer. On the other hand, mechanical deactivation followed pseudo-first-order kinetics and seems to have an interfacial nature, as it could be prevented by small amounts of surface active substances such as dodecanol. Nonionic surfactants (alkyl glucosides and polyoxyethylene ethers) were able to stabilize the enzyme under storage conditions (bulk enzymesurfactant interaction). However, when the lipasesurfactant interaction was studied at the oil/ water interface, only the alkyl glucoside proved to be compatible with both lipases. Washing tests conducted with a lipase-containing formulation under optimized conditions yielded outstanding cleaning results, even with a reduced surfactant dosage, suggesting that lipases can partly replace surfactants in detergent products, when the washing conditions are properly chosen. ’ ASSOCIATED CONTENT

bS

Supporting Information. Summary of the results of the surfactant compatibility tests (Table 1s). This information is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: +34-958243307. Fax: +34-958248992. E-mail: ejurado@ ugr.es. 11508



’ ACKNOWLEDGMENT The authors are grateful for the financial support provided by the Spanish and Andalusian Governments through the projects CTQ2006-12089 and P07-TEP-02603, respectively. ’ REFERENCES (1) Barros, M.; Fleuri, L. F.; Macedo, G. A. Seed lipases: sources, applications and properties - a review. Braz. J. Chem. Eng. 2010, 1, 15. (2) Fernandez-Lafuente, R. Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst. J. Molec. Catal. B 2010, 3-4, 197. (3) Gandhi, N. N. Applications of lipase. J. Am. Oil Chem. Soc. 1997, 6, 621. (4) Hasan, F.; Shah, A. A.; Hameed, A. Industrial applications of microbial lipases. Enzyme Microb. Technol. 2006, 2, 235. (5) Houde, A.; Kademi, A.; Leblanc, D. Lipases and their industrial applications: An overview. Appl. Biochem. Biotechnol. 2004, 1-3, 155. (6) Jaeger, K.; Ransac, S.; Dijkstra, B. W.; Colson, C.; van Heuvel, M.; Misset, O. Bacterial lipases. FEMS Microbiol. Rev. 1994, 1, 29. (7) Olsen, H.; Falholt, P. The role of enzymes in modern detergency. J. Surfactants Deterg. 1998, 4, 555. (8) Aaslyng, D.; Gormsen, E.; Malmos, H. Mechanistic studies of proteases and lipases for the detergent industry. J. Chem. Technol. Biotechnol. 1991, 3, 321. (9) Rubingh, D. N.; Bauer, D. M. Catalytic behavior of lipases under laundry conditions. Tenside, Surfactants, Deterg. 1998, 4, 6. (10) Jurado, E.; Bravo, V.; Luzon, G.; Fernandez-Serrano, M.; García-Roman, M.; Altmajer-Vaz, D.; Vicaria, J. M. Hard-surface cleaning using lipases: Enzyme-surfactant interactions and washing tests. J. Surfactants Deterg. 2007, 1, 61. (11) Huge-Jensen, I. B.; Gormsen, E. Enzymatic detergent additive. Eur. Patent EP0258068, March 2, 1988. (12) Herbots, I.; Kottwitz, B. Enzymes in Nonfood Applications. In Enzymes in Industry; Aehle, W., Ed.; Wiley-VCH: Germany, 2004; pp 155244. (13) Vind, J.; Svendsen, A.; Patkar, S.; Andersen, K.; Halkier, D.; Bojsen, K. Lipase Variant. WIPO Patent WO0060063, October 12, 2000. (14) Friis-Jensen, S. Wash clean and green with Novozymes Lipoclean. Biotimes 2009, 3, 6. (15) Rathi, P.; Saxena, R. K.; Gupta, R. A novel alkaline lipase from Burkholderia cepacia for detergent formulation. Process Biochem. 2001, 2, 187. (16) Bora, L.; Kalita, M. C. Production of thermostable alkaline lipase on vegetable oils from a thermophilic Bacillus sp. DH4, characterization and its potential applications as detergent additive. J. Chem. Technol. Biotechnol. 2008, 5, 688. (17) Saisubramanian, N.; Sivasubramanian, S.; Nandakumar, N.; Indirakumar, B.; Chaudhary, N. A.; Puvanakrishnan, R. Two step purification of Acinetobacter sp. lipase and its evaluation as a detergent additive at low temperatures. Appl. Biochem. Biotechnol. 2008, 2, 139. (18) Wang, H. K.; Liu, R. J.; Lu, F. P.; Qi, W.; Shao, J.; Ma, H. J. A novel alkaline and low-temperature lipase of Burkholderia cepacia isolated from Bohai in China for detergent formulation. Ann. Microbiol. 2009, 1, 105. (19) Gulati, R.; Isar, J.; Kumar, V.; Prasad, A. K.; Parmar, V. S.; Saxena, R. K. Production of a novel alkaline lipase by Fusarium globulosum using neem oil, and its applications. Pure Appl. Chem. 2005, 1, 251. (20) Suresh Kumar, S.; Kumar, L.; Sahai, V.; Gupta, R. A thiolactivated lipase from Trichosporon asahii MSR 54: Detergent compatibility and presoak formulation for oil removal from soiled cloth at ambient temperature. J. Ind. Microbiol. Biotechnol. 2009, 3, 427. (21) Liu, R.; Jiang, X.; Mou, H.; Guan, H.; Hwang, H.; Li, X. A novel low-temperature resistant alkaline lipase from a soda lake fungus strain Fusarium solani N4-2 for detergent formulation. Biochem. Eng. J. 2009, 3, 265.

ARTICLE

(22) Dos Prazeres, J. N.; Cruz, J. A. B.; Pastore, G. M. Characterization of alkaline lipase from Fusarium oxysporum and the effect of different surfactants and detergents on the enzyme activity. Brazilian J. Microbiol. 2006, 4, 505. (23) Romdhane, I. B. B.; Fendri, A.; Gargouri, Y.; Gargouri, A.; Belghith, H. A novel thermoactive and alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochem. Eng. J. 2010, 1, 112. (24) Saisubramanian, N.; Edwinoliver, N. G.; Nandakumar, N.; Kamini, N. R.; Puvanakrishnan, R. Efficacy of lipase from Aspergillus niger as an additive in detergent formulations: A statistical approach. J. Ind. Microbiol. Biotechnol. 2006, 8, 669. (25) Thirunavukarasu, K.; Edwinoliver, N. G.; Durai Anbarasan, S.; Gowthaman, M. K.; Iefuji, H.; Kamini, N. R. Removal of triglyceride soil from fabrics by a novel lipase from Cryptococcus sp. S-2. Process Biochem. 2008, 7, 701. (26) Fuglsang, C. C.; Okkels, J. S.; Petersen, D. A.; Patkar, S. A.; Thellersen, M.; Svendsen, A.; Borch, K.; Royer, J. C.; Kretzschmar, T.; Halkier, T.; Vind, J.; Jorgensen, S. T. Lipolytic enzymes. U.S. Patent US6495357, December 17, 2002. (27) Wolff, A. M.; Showell, M. S. Application of lipases in detergents. In Enzymes in Detergency; van Ee, J. H., Misset, O., Baas, E. J., Eds.; Marcel Dekker: New York, 1997; pp 93106. (28) Kawase, T.; Hashimoto, T.; Fujii, T.; Minagawa, M. Studies on the effects of surfactants on lipase activity. Yukagaku 1985, 7, 530. (29) Jurado, E.; Bravo, V.; Nu~ nez-Olea, J.; Bailon, R.; Altmajer-Vaz, D.; García-Roman, M.; Fernandez-Arteaga, A. Enzyme-based detergent formulas for fatty soils and hard surfaces in a continuous-flow device. J. Surfactants Deterg. 2006, 1, 83. (30) Lee, Y. K.; Choo, C. L. The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea. Biotechnol. Bioeng. 1989, 2, 183. (31) Stahmann, K. P.; B€oddecker, T.; Sahm, H. Regulation and properties of a fungal lipase showing interfacial inactivation by gas bubbles, or droplets of lipid or fatty acid. Eur. J. Biochem. 1997, 1, 220. (32) Mohanty, M.; Ghadge, R. S.; Patil, N. S.; Sawant, S. B.; Joshi, J. B.; Deshpande, A. V. Deactivation of lipase at gas-liquid interface in stirred vessel. Chem. Eng. Sci. 2001, 11, 3401. (33) Ghadge, R. S.; Ekambara, K.; Joshi, J. B. Role of hydrodynamic flow parameters in lipase deactivation in bubble column reactor. Chem. Eng. Sci. 2005, 22, 6320. (34) Jurado, E.; Camacho, F.; Luz on, G.; Fernandez-Serrano, M.; García-Roman, M. Kinetics of the enzymatic hydrolysis of triglycerides in o/w emulsions. Study of the initial rates and the reaction time course. Biochem. Eng. J. 2008, 3, 473. (35) Fujii, T.; Tatara, T.; Minogawa, M. Studies on Applications of Lipolytic Enzyme in Detergency I. Effect of Lipase from Candida cylindracea on Removal of Olive Oil from Cotton Fabric. J. Am. Oil Chem. Soc. 1986, 6, 796. (36) Snabe, T.; Petersen, S. B. Lag phase and hydrolysis mechanisms of triacylglycerol film lipolysis. Chem. Phys. Lipids 2003, 1, 69. (37) Snabe, T.; Neves-Petersen, M. T.; Petersen, S. B. Enzymatic lipid removal from surfaces-lipid desorption by a pH-induced “electrostatic explosion. Chem. Phys. Lipids 2005, 1, 37. (38) Arakawa, T.; Timasheff, S. N. The stabilization of proteins by osmolytes. Biophys. J. 1985, 3, 411. (39) Lozano, P.; Combes, D.; Iborra, J. Effect of polyols on αchymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization. J. Biotechnol. 1994, 1, 9. (40) Rosen, M. J. Surfactants and Interfacial Phenomena; John Wiley & Sons, Inc.: Hoboken, NJ, 2004; pp 6264. (41) Jurado, E.; Bravo Rodriguez, V.; Bailon Moreno, R.; Nu~ nez Olea, J.; Altmajer Vaz, D. Bath-Substrate-Flow method for evaluating the detersive and dispersant performance of hard-surface detergents. Ind. Eng. Chem. Res. 2003, 19, 4303. (42) Vilchez, S.; Azemar, N.; Solans, C. Enzyme stability and detergent efficiency study in surfactant mixture aqueous solutions. Comun. Jorn. Com. Esp. Deterg. 2008, 38, 309. 11509



ARTICLE

(43) Helisto, P.; Korpela, T. Effects of detergents on activity of microbial lipases as measured by the nitrophenyl alkanoate esters method. Enzyme Microb. Technol. 1998, 1-2, 113. (44) Otzen, D. E.; Sehgal, P.; Westh, P. α-Lactalbumin is unfolded by all classes of surfactants but by different mechanisms. J. Colloid Interface Sci. 2009, 2, 273. (45) Xia, J.; Chen, X.; Nnanna, I. A. Activity and stability of Penicillium cyclopium lipase in surfactant and detergent solutions. J. Am. Oil Chem. Soc. 1996, 1, 115. (46) Derewenda, U.; Brzozowski, A. M.; Lawson, D. M.; Derewenda, Z. S. Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase. Biochemistry 1992, 5, 1532. (47) Skagerlind, P.; Jansson, M.; Bergenstahl, B.; Hult, K. Binding of Rhizomucor miehei lipase to emulsion interfaces and its interference with surfactants. Colloids Surf., B 1995, 3, 129. (48) Van Deurzen, M. P. J. Liquid detergent trends. Increasing opportunities for enzymes. Household Pers. Care Today 2008, 3, 64. (49) Montgomery, D. C. Design and analysis of experiments; John Wiley & Sons, Inc.: Hoboken, NJ, 2008; pp 162206. (50) Altmajer Vaz, D. Formulaciones Detergentes Biodegradables: Ensayos de Lavado, Ph.D. Thesis, Universidad de Granada, Spain, September 2004. (51) Jurado Alameda, E.; Bravo Rodríguez, V.; Nu~nez Olea, J.; Bailon Moreno, R.; Galvez Borrego, A.; Altmajer Vaz, D. Design of experiments to evaluate the detergency of surfactants on fatty soils in a continuous-flow device. J. Surfactants Deterg. 2009, 3, 191.

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Optimization of Lipase Performance in Detergent Formulations for

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