Activity and Adsorption of Lipase from Humicola lanuginosa on

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Langmuir 1997, 13, 784-790

Activity and Adsorption of Lipase from Humicola lanuginosa on Surfaces with Different Wettabilities Kristin Wannerberger,*,† Stefan Welin-Klintstro¨m,‡ and Thomas Arnebrant† Department of Food Technology, University of Lund, Box 124, S-221 00 Lund, Sweden, and Interface Biology Group, Laboratory of Applied Physics, Department of Physics and Measurement Technology, Linko¨ ping Institute of Technology, S-581 83 Linko¨ ping, Sweden Received June 10, 1996. In Final Form: October 29, 1996X The adsorption of Humicola lanuginosa lipase and the activity of the adsorbed lipase were studied as a function of surface wettability. The adsorption was measured by in situ ellipsometry, and the surfaces used were methylated silica surfaces. The activity of the adsorbed lipase was measured after rinsing of the cuvette, i.e., with no lipase in the bulk solution, in situ by the hydrolysis of p-nitrophenyl acetate. The lipase adsorption and activity measurements were made at concentrations of the lipase in the range 63-1050 nM, and from the surface concentrations the specific activity was calculated. The study was carried out using fully methylated surfaces (hydrophobic; 90° water contact angle) and surfaces with a higher wettability (80°, 75°, and 62° water contact angle). All experiments were performed in 3-(Nmorpholino)propanesulphonic acid (MOPS) buffer at pH 7.5. The adsorbed amount was found to be highest at lipase concentrations in the range 200-300 nM and decreased with increasing wettability of the surfaces. The fraction desorbable upon dilution of the adsorbed lipase was found to decrease with the concentration. The specific activity of the lipase was found to increase with increasing wettability of the surface, probably due to changes in the orientation/conformation of the adsorbed lipase. Additional experiments were performed where lipase was adorbed to surfaces with a water contact angle of 90°, in the presence of 0.03 M CaCl2. The lipase concentrations were then 63 and 1050 nM. The amount adsorbed was significantly increased in the presence of CaCl2, but no value for the specific activity of the adsorbed lipase could be calculated due to the high activity originating from lipase adsorbed to the background surfaces (cuvette walls, Teflon tubing, and magnetic stirrer).

Introduction Lipases are enzymes which hydrolyze triacylglycerols. They are found in different body liquids, e.g. pancreatic juice, blood plasma, and saliva, in milk, in triglycerideproducing plants, in molds, and in bacteria.1 Properties of lipases, in general, have been reviewed by different authors.1-3 Adsorption studies of lipases to solid surfaces have recently been reported.4,5 Of particular relevance to this work are the studies by Wannerberger and Arnebrant6,7 and Duinhoven et al.8 regarding the adsorption of lipase from Humicola lanuginosa (HLL) to solid surfaces, together with a study by Martinelle et al.9 concerning the interfacial activation of this lipase. The lipase activity is known to be markedly increased at the oil/water interface, and this so-called interfacial activation has been discussed in several papers. Various explanations to this have been given, e.g. structural changes in the lipase molecule10-12 or increased substrate concentration at the interface.13 * Author to whom correspondence should be addressed. † University of Lund. ‡ Linkoping Institute of Technology. X Abstract published in Advance ACS Abstracts, January 1, 1997. (1) Borgstro¨m, B.; Brockman, H. L. Lipases; Elsevier: Amsterdam, 1984. (2) Brockerhoff, H.; Jensen, R. G. Lipolytic Enzymes; Academic Press: New York, 1974. (3) Woolley, P.; Petersen, S. B. Lipases, Their Structure, Biochemistry and Application; University Press: Cambridge, 1994. (4) Geluk, M. A.; Norde, W.; Van Kalsbeek, H. K. A. I.; Van’t Riet, K. Enzyme Microb. Technol. 1992, 14, 748. (5) Duinhoven, S. Enzyme Adsorption at Solid/liquid Interfaces; Agricultural University: Wageningen, The Netherlands, 1992. (6) Wannerberger, K.; Arnebrant, T. J. Colloid Interface Sci. 1996, 177, 316. (7) Wannerberger, K.; Wahlgren, M.; Arnebrant, T. Colloids Surf., B 1996, 6, 27. (8) Duinhoven, S.; Poort, R.; Van der Voet, G.; Agterof, W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170, 351. (9) Martinelle, M.; Holmquist, M.; Hult, K. Biochim. Biophys. Acta 1995, 1258, 272.

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The first lipase structures to be solved were those of human pancreatic lipase14 and Rhizomucor miehei lipase (RML).15 In none of the lipases was the active site center exposed to the solvent, and therefore a theory arose that the lipase changed its conformation when it was adsorbed to the oil/water interface. This was confirmed by X-ray chrystallographic studies of RML inhibited by n-hexanephosphonate ethyl ester11 and later also with diethyl p-nitrophenyl phosphate.12 Up to date several more structures are known, and recently the structure was presented of the lipase used in this work (from the fungus Humicola lanuginosa).16,17 The lipase from Humicola lanuginosa is mainly used as a component in detergent formulations and is structurally very similar to the lipase from Rhizomucor miehei. One of the most common methods for studying lipase/ substrate interactions is the monolayer technique,18,19 where the water soluble enzyme is acting on the substrate at the lipid/water interface. Another way to measure the lipase activity is by a potentiometric method where the (10) Desnuelle, P.; Sarda, L.; Aihaud, G. Biochim. Biophys. Acta 1960, 37, 570. (11) Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; Bjo¨rkling, F.; Huge-Jensen, B.; Patkar, S.; Thim, L. Nature 1991, 351, 491. (12) Derewenda, U.; Brzozowski, A. M.; Lawson, D. M.; Derewenda, Z. S. Biochemistry 1992, 31, 1532. (13) Brockman, H. L.; Law, J. H.; Ke´zdy, F. J. Biol. Chem.1973, 248, 4965. (14) Winkler, F. K.; D’Arcy, A.; Hunziker, W. Nature 1990, 343, 771. (15) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen, B.; Norskov, L.; Thim, L.; Menge, U. Nature 1990, 343, 767. (16) Lawson, D. M.; Brzozowski, A. M.; Dodson, G. G.; Hubbard, R. E.; Huge-Jensen, B.; Boel, E.; Derewenda, Z. S. In Lipases, Their Structure, Biochemistry and Application; Woolley, P.; Petersen, S. B., Eds.; University Press: Cambridge, 1994; p 77. (17) Derewenda, U.; Swenson, L.; Green, R.; Wei, Y.; Yamaguchi, S.; Joerger, R.; Haas, M. J.; Derewenda, Z. S. Protein Eng. 1994, 7, 551. (18) Verger, R.; de Haas, G. H. Chem. Phys. Lipids 1973, 10, 127. (19) Pie´roni, G.; Gargouri, Y.; Sarda, L.; Verger, R. Adv. Colloid Interface Sci. 1990, 32, 341.

© 1997 American Chemical Society

Activity and Adsorption of Lipase on Solid Surfaces

substrate is emulsified to increase the oil/water interface.20 Methods based on this principle are usually the standard assay for lipase activity. In this work we have studied the adsorption of lipase to solid surfaces by ellipsometry, and after the cuvette was rinsed, the activity of the adsorbed lipase was determined spectrophotometrically by measuring the hydrolysis of a water soluble substrate, p-nitrophenyl acetate (pnpa),9 which was added to the ellipsometer cuvette. The combination of these techniques made it possible to calculate the specific activity from the ellipsometrically determined adsorbed amount of lipase. The natural substrate for lipase is insoluble in water, but there is a reaction also with water soluble substrates although the activity is much lower.2,21 Ellipsometry was used to determine the amounts adsorbed and the kinetics of adsorption/desorption.22-28 This technique facilitates in situ measurements, and the adsorbed amount can be determined29,30 with short time intervals. The surfaces used were methylated silica surfaces similar to those used in previous adsorption studies of the lipase6,7 and a number of other proteins.31-34 To investigate the effect of surface wettability on the adsorption and the activity of the lipase, adsorption measurements were performed onto surfaces with a gradient in wettability,35 and on the basis of the adsorbed amount versus wettability curves achieved, some discrete wettabilities were selected for further investigation. Surfaces with the desired wettability were prepared, and adsorption and activity measurements were then performed on the different surfaces at different concentrations. An attempt was also made to measure the activity of lipase adsorbed in the presence of CaCl2, since it is known that the adsorbed amount of lipase increases in the presence of a divalent salt,6,8 and it would therefore be interesting to determine the effect on the activity of the adsorbed lipase. The information obtained from this study could be directly applied to lipase immobilization for the selection and development of carrier materials. Immobilized lipases are commonly used in industrial processes, e.g. esterification reactions. Furthermore, the results would be of (20) Sarda, L.; Desnuelle, P. Biochim. Biophys. Acta 1958, 30, 513. (21) Martinelle, M.; Hult, K. In Lipases, Their Structure, Biochemistry and Application; Woolley, P.; Petersen, S. B., Eds.; University Press: Cambridge, 1994; p 159. (22) Arnebrant, T. Proteins at the Metal/Water InterfacesAdsorption in Relation to Interfacial Structure. Thesis, Lund University, Lund, Sweden, 1987. (23) Azzam, R. M. A. Proc. Soc. Photo-Opt. Instrum. Eng. 1976, 88, 84. (24) Trurnit, H. J. Arch. Biochem. Biophys. 1953, 47, 251. (25) Trurnit, H. J. Arch. Biochem. Biophys. 1954, 51, 176. (26) Poste, G.; Moss, C. In Progress in Surface Science; Davidson, S. G., Eds.; Pergamon Press: Oxford, 1972; Vol. 2, p 139. (27) Nylander, T. Proteins at the Metal/Water InterfacesAdsorption and Solution Behaviour. Thesis, Lunds University, Lund, Sweden, 1987. (28) Lundstro¨m, I.; Ivarsson, B.; Jo¨nsson, U.; Elwing, H. Polymer Surfaces and Interfaces; John Wiley & Sons Ltd.; Chichester, U.K., 1987. (29) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1977. (30) Cuypers, P. A. Dynamic Ellipsometry: Biochemical and Biomedical Applications; Rijksuniversiteit Limburg: 1976. (31) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1990, 136, 259. (32) Luey, J. K.; McGuire, J.; Sproull, R. D. J. Colloid Interface Sci. 1991, 143, 489. (33) Wahlgren, M. C.; Paulsson, M. A.; Arnebrant, T. Colloids Surf., A 1993, 70, 139. (34) Wahlgren, M.; Arnebrant, T.; Askendal, A.; Welin-Klintstro¨m, S. Colloids Surf., A 1993, 70, 151. (35) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstro¨m, I. J. Colloid Interface Sci. 1987, 119, 203.

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general relevance for the field of detergency due to the application in this field for HLL. Materials and Methods Humicola lanuginosa (HLL) is a 1,3-specific lipase with increasing activity in the pH range 7-11. It has its optimal activity at temperatures around 35-40 °C. Highly purified HLL was kindly provided by NOVO-Nordisk, Bagsvaerd, Denmark. The molecular weight is 31 700 g/mol (glycosylated form), and the isoelectric point (Ip) is at pH 4.4 (personal communication, NOVO-Nordisk). The experiments were carried out in 20 mM 3-(N-morpholino)propanesulphonic acid (MOPS) buffer (Sigma) at pH 7.5. pH was adjusted by the addition of 0.1 M NaOH. p-Nitrophenyl acetate (pnpa) (Sigma) was used as substrate and was dissolved in acetonitrile at a concentration of 0.1 M. The water used was distilled, passed through an ion exchanger and activated charcoal, and finally doubly distilled in a glass still. All chemicals used were of analytical grade. Silicon surfaces (Okmetic OY, Finland) with a thermally grown oxide (≈300 Å) were used and prepared as in ref 31. A cleaning process was carried out as described in ref 36 to give hydrophilic surfaces with a water contact angle of less than 10°.36 The highly hydrophobic surfaces were prepared according to ref 36 and slightly modified as described in ref 31 by the reaction with dichlorodimethylsilane (DDS) in trichloroethylene. The preparation of the gradient surfaces is based on the diffusion of DDS from a trichloroethylene phase into a xylene solution and was carried out as described in ref 35. The surfaces with an intermediate hydrophobicity were obtained as described in ref 7 where the solvent used was xylene. Different reaction times with DDS gave surfaces with different hydrophobicity. The reaction times were 30, 22, and 17 min, giving wettabilities of the surfaces as measured by the water contact angle method37 of 80° ( 2 (n ) 12), 75° ( 2 (n ) 27), and 62° ( 2 (n ) 26), respectively. The highly hydrophobic surfaces are from now on denoted 90° surfaces. The surfaces were stored in ethanol. Prior to use, the surfaces were rinsed with doubly distilled water and ethanol and blown dry with nitrogen. All glassware was cleaned in a 1:1 (v/v) mixture of concentrated sulfuric and nitric acid and then thoroughly rinsed in doubly distilled water. Ellipsometry is an optical method which makes it possible to measure the thickness and refractive index of a thin film on a surface. With an ellipsometer the changes are recorded in the ellipticity of polarized light upon reflection at an interface. The changes are influenced by, among other things, the presence of a thin film of adsorbed molecules. From the ellipsometric angles ∆ and Y the thickness and the refractive index of this film can be calculated.29 In addition, the amount per unit area of the film could be determined if the partial specific volume and the ratio of the molar weight to the molar refractivity38 or the refractive index increment with concentration dn/dc39 of the adsorbed molecules are known. The adsorption/desorption experiments were made with an automated Rudolph thin film ellipsometer, type 43603-200E, equipped with a thermostated cuvette. The experimental setup was described in detail by Nylander.27 The adsorbed amount was calculated according to Cuypers et al.38 using 0.75 mL/g and 4.1 g/mL as the values for the partial specific volume and the ratio between molar mass and the molar refractivity, respectively. The ellipsometer cuvette was carefully cleaned before measurement in detergent solution (Deconex, Borer Chemie AG, Switzerland) and rinsed in doubly distilled water. Finally, it was plasma cleaned in low-pressure (25-40 N/m2) residual air for 5 min, using a radio frequency glow discharge unit (Harrick PDC 3XG, Harrick Scientific Corp., Ossining, NY). The surfaces (36) Jo¨nsson, U.; Ivarsson, B.; Lundstro¨m, I.; Berghem, L. J. Colloid Interface Sci. 1982, 90, 148. (37) Gekas, V.; Persson, K.; Wahlgren, M.; Sivik, B., J. Membr. Sci. 1992, 72, 293. (38) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. C. J. Biol. Chem. 1983, 258, 2426. (39) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759.

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were immersed in the cuvette containing MOPS buffer (4.5 mL), and the ellipsometric angles ∆ and Y for the clean surface were recorded. A stock solution of lipase, freshly prepared each day, was added (0.5 mL) to the cuvette, and the optical changes were recorded continuously. Adsorption measurements were carried out for 30 min (1800 s) before rinsing. Rinsing was then performed with MOPS buffer for 5 min at a flow rate of 20 mL/min, and after another 5 min (2400 s, in total) the substrate was added as described below. Measurement of the optical changes was continued also during the activity measurements. The stirring rate in the cuvette was 325 rpm, and the temperature was 25 °C. The adsorption of a 231 nM lipase solution in MOPS buffer, pH 7.5, to surfaces with a gradient in wettability was performed with a similar ellipsometer setup as described above. The stepping motors controlling the lateral movement of the gradient surface and the procedure for the gradient measurements are described in refs 40 and 41, respectively. Scanning of the gradient was first made to obtain the ∆ and Y values for the clean surface. The lipase was then allowed to adsorb for 25 min prior to the measurement. The cuvette was rinsed after 30 min at a flow rate of 24 mL/minute for 10 min. After an additional 30 min, a measurement was again performed. All experiments were made at least in duplicate. Measurement of the lipase activity was made according to ref 9 and was carried out as follows: 0.1 M pnpa in acetonitrile was added to a final concentration in the cuvette of 1.0 × 10-3 M. Samples (200 µL) were taken from the cuvette with intervals of 5 min for measurement of the absorbance at 405 nM. The activity was obtained as the amount of phenolate ions (in nmol) produced per minute, calulated by using an extinction coefficient of 13 000 M-1 cm-1.42 The total area of the methylated silica surface in contact with the solution was 3.5 cm2. The absorbance measurements were performed with a Beckman spectrophotometer, and quartz cuvettes (1 cm × 1 cm) were used. Part of the total activity measured in this way arises from lipase molecules adsorbed to the ellipsometer cuvette (hydrophilic; total area ) 18.7 cm2), the Teflon tubing, and the magnetic stirrer (hydrophobic; total area ) 1.8 cm2). In order to correct for this, all experiments were performed also without a methylated silica surface, and the reference activity thus achieved could then be subtracted from the total to give the activity originating from the lipase molecules adsorbed to the silica surfaces only. The specific activity was then calculated as the activity for the lipase molecules adsorbed to the methylated silica surface divided by the amount adsorbed after rinsing. From experiments with varying stirring rates, the adsorption and activity measurements were found not to be diffusion controlled under any of the experimental conditions. The lipase concentrations in the cuvette during the adsorption were 63, 115, 231, 345, 460, and 1050 nM for hydrophobic surfaces. For the surfaces with intermediate hydrophobicity the concentrations were 63, 231, 345, and 1050 nM. The adsorption and activity measurements of lipase in 0.03 M CaCl2 solution were carried out at 63 and 1050 nM and to hydrophobic surfaces.

Results The results from the measurements of adsorption to a surface with a gradient in wettability are presented in Figure 1. It should be noted that the water contact angles indicated in the figure are used for reference only and do not correspond to the ones used in the activity study. It is shown from the figure that the adsorbed amount decreased and the desorbable fraction upon rinsing increased with increasing wettability of the surface. Three positions at the gradient surface were chosen for further (40) Welin-Klintstro¨m, S.; Jansson, R.; Elwing, H. J. Colloid Interface Sci. 1993, 157 (2), 498. (41) Welin-Klintstro¨m, S.; Askendal, A.; Elwing, H. J. Colloid Interface Sci. 1993, 158, 188. (42) Dahlborg, J. Action of Lipases on Water Soluble Substrates, Catalysis at the Interface? Graduate Thesis, Department of Biochemistry and Biotechnology, The Royal Institute of Technology, Stockholm, Sweden, 1993.

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Figure 1. Amount of lipase adsorbed onto a gradient surface before (0) and after (4) rinsing. The concentration of lipase was 231 nM in MOPS buffer pH 7.5, 25 °C. Rinsing was performed after 30 min at a rate of 24 mL/min for 10 min. The water contact angle of the surface at different positions is indicated in the figure.

Figure 2. Plateau values for the amount of lipase adsorbed onto surfaces with different wettabilities after rinsing versus concentration. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The experiments were performed in MOPS buffer at pH 7.5. Rinsing was performed after 1800 s at a rate of 20 mL/min.

investigations: one where the amount adsorbed started to decrease, another where the amount adsorbed was very small, and one point in between these. The three positions correspond to water contact angles of ≈80°, ≈75°, and ≈62° (4, 5, and 7 mm from the hydrophobic end), respectively. For the two last positions shown in the figure (6.7 and 7.3 mm), the amount after rinsing was zero. The total distance from the hydrophobic to the hydrophilic end is 18 mm, corresponding to water contact angles of 90° and 7.3 mm. The plateau values for the adsorbed amounts are presented in Figure 2 for the different lipase concentrations and surfaces. For the 90° surface the highest amount adsorbed was found at a lipase concentration of 231 nM. The plateau value after 1800 s of adsorption followed by rinsing varied between 1.83 and 2.09 mg/m2 for lipase concentrations of 63-460 nM. For the highest lipase concentration (1050 nM) the adsorbed amount was 0.81 mg/m2. Measurements for surfaces with varying degrees of wettability were made at lipase concentrations of 63, 231, 345, and 1050 nM. The surfaces with water contact angles of 80° had similar plateau values for the adsorbed amounts as the 90° surfaces. A lowering of the water contact angle

Activity and Adsorption of Lipase on Solid Surfaces

Figure 3. Adsorption of lipase versus time onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The concentration of lipase was 63 nM in MOPS buffer, 25 °C. Rinsing was performed after 1800 s at a rate of 20 mL/min for 300 s. After an additional 300 s (2400 s in total) pnpa was added and the activity was measured during 1500 s.

Figure 4. Adsorption of lipase versus time onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The concentration of lipase was 345 nM. The experimental conditions were the same as in Figure 3.

to 75° led to a reduction of the adsorbed amount for all concentrations, most pronounced at 345 nM. The surface with highest wettability showed about the same adsorbed amount, 0.25-0.31 mg/m2, for all the lipase concentrations. Figures 3-5 show the adsorption curves for the adsorption and activity measurements at the different surfaces for lipase concentrations of 63, 345, and 1050 nM, respectively. The results from these lipase concentrations, as well as those not shown in figures, are presented together with standard deviations/deviations from the mean in Table 1. It should be noted that great effort has been made to perform the experiments as accurately as possible even though the variation sometimes is quite high, especially at the 62° surface. The results from the measurements of the lipase solution with lowest concentration are shown in Figure 3, and it is clear that no desorption during rinsing occurred at the three surfaces with lowest wettability and that for the 62° surface the desorbable fraction was 17%. Figure 4 shows the results from the 345 nM solution, and at this higher concentration there was a minor desorption (5%) from the 90° and 80° surfaces. For the 75° surface the desorbable fraction during rinsing was 25%, and for the 62° surface, it was 40%. The results from the measurements for the highest lipase concentration (1050 nM) are shown in Figure 5.

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Figure 5. Adsorption of lipase versus time onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The concentration of lipase was 1050 nM. The experimental conditions were the same as in Figure 3.

Figure 6. Adsorption of lipase versus time onto 90° surfaces. The concentrations of lipase were 63 and 1050 nM (0, 4), in MOPS-CaCl2 buffer, 25 °C. The concentration of CaCl2 was 0.03 M. The experimental conditions were the same as in Figure 3.

Adsorption to the 90°, 80°, and 75° surfaces resulted in an initial peak which also was found during other conditions, as described in the introductory part of this paper.6 The peak values were 1.8, 2.1, and 2.3 mg/m2, for the 75°, 80°, and 90° surfaces, respectively, and the plateau values after the peak were 1.3 mg/m2 (75° and 80°) and 0.9 mg/m2 (90° surface). The desorbable fraction during rinsing was highest at this concentration for all surfaces (33%, 46%, 46% and 33% for 90°, 80°, 75°, and 62°, respectively) except on the most hydrophilic one, and the plateau values after rinsing were 0.68-0.81 mg/m2 for the 90°, 80°, and 75° surfaces and 0.25 mg/m2 for the 62° surface. The adsorption of lipase in the presence of calcium (0.03 M CaCl2) is shown in Figure 6. The adsorbed amount increased in the presence of CaCl2 in particular for 1050 nM, and the plateau value after rinsing was for this concentration 2.6 mg/m2. Only minor desorption occurred under these conditions. In Table 1 the specific activities are summarized for the different measurements described above (Figures 3-5). It should be noted that the activity values for the 90° and 62° surfaces, at a lipase concentration of 1050 nM, are uncertain due to the low amounts adsorbed. The total activities at the different surfaces together with the reference activities as a function of the lipase concentration are shown in Figure 7. The low difference

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Table 1. Adsorbed Amount after Rinsing, the Total Activity, the Reference Activity, and the Specific Activity of Lipase Adsorbed to Methylated Silica Surfaces with Different Wettabilitiesa lipase conc (nM)

water contact angle (deg)

adsorbed amount (mg/m2)

tot act. (nmol/min)

90 80 75 62

1.83 ( 0.00 1.85 ( 0.05 1.68 ( 0.02 0.25 ( 0.01

79 ( 6 97 ( 1 89 ( 1 47 ( 2

90

2.05 ( 0.05

87 ( 2

63

ref act.b (nmol/min)

sp. act.c (nmol m2/(min mg))

41 ( 12

115

21 ( 3 29 ( 2 29 ( 0 25 ( 8 38 ( 11

231

24 ( 0 20 ( 2

90 80 75 62

2.09 ( 0.30 2.10 ( 0.00 1.78 ( 0.12 0.31 ( 0.01

69 ( 5 86 ( 2 85 ( 5 35 ( 3

90 80 75 62

1.83 ( 0.15 1.68 ( 0.26 1.20 ( 0.00 0.28 ( 0.00

76 ( 7 73 ( 2 68 ( 2 42 ( 3

90

1.84 ( 0.34

60 ( 3

345

24 ( 6 31 ( 1 37 ( 0 47 ( 12 23 ( 2

460

29 ( 5 30 ( 3 38 ( 2 64 ( 13 28 ( 4

1050

18 ( 2 23 ( 11

90 80 75 62

0.81 ( 0.19 0.80 ( 0.00 0.68 ( 0.02 0.25 ( 0.01

25 ( 8 36 ( 3 40 ( 5 24 ( 2

90

2.68 ( 0.02

104 ( 7

63 with CaCl2

d 16 ( 4 25 ( 7 d 101 ( 4

1050 with CaCl2

d 101 ( 1

90

2.65 ( 0.05

96 ( 0

d

n values 4 2 2 2 2 4 2 2 3 2 2 2 2 3 2 2 2 2 2 3 4 2 2 2 2 2 2 2

a

All experiments were performed in MOPS buffer pH 7.5, 25 °C. Two were carried out in the presence of 0.03 M CaCl2. Standard deviations are given for mean values where n > 2. For measurements where n ) 2, the deviations from the mean are given. b The reference activity is the contribution from the cuvette walls, Teflon tubings, and magnetic stirrer. c The specific activity is the total activity with the reference activity subtracted, divided by the adsorbed amount after rinsing. d The specific activity is uncertain due to the proximity of the reference activity to the total activity.

Figure 7. Total activity for lipase versus concentration onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The reference activity (+) for the different concentrations is also shown in the figure. The experimental conditions were the same as in Figure 3.

between the total and the reference activities for 1050 nM (90° and 62°) can be seen in this figure, and it is also shown that the lowest ratio between the reference and total activity is found at lipase concentrations of 231 and 345 nM. Figure 8 shows the specific activity of the lipase adsorbed to the different surfaces as a function of the lipase concentration. Starting with the 90° surface, the specific activity was highest at 345 nM,29 and for 80° the maximum was shifted toward 231 nM. For the 75° surface and especially the 62° surface, the maximum in specific activity was most pronounced at 345 nM. Under these conditions (62°, 345 nM) the highest specific activity in the study was achieved.

Figure 8. Specific activity for lipase versus concentration onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The experimental conditions were the same as in Figure 3.

It was clearly shown that, especially for 231 and 345 nM, the specific activity increased with increasing wettability of the surface. In Figure 9 the adsorbed amount and the specific activity of the adsorbed lipase are given as a function of the surface wettabilities. It is shown that the amount adsorbed to the 62° surface was low but increased with increasing hydrophobicity of the surface for all concentrations except 1050 nM, which had a maximum in the adsorbed amount at a water contact angle of 75°. The specific activity increased with increasing wettability of the surface for 231 and 345 nM while it was highest at 75° for the other concentrations. At this surface the specific activity was less concentration dependent, and the specific activity values were also higher at this surface in comparison with the 80° and 90° surfaces.

Activity and Adsorption of Lipase on Solid Surfaces

Figure 9. Specific activity and the adsorbed amount of lipase after rinsing versus the water contact angles of the surfaces. The lipase concentrations were 63, 115, 231, 345, 460, and 1050 nM (2, 9, b, [, 1, ×) for the specific activity and (4, 0, O, ], 3, +) for the adsorbed amount, respectively. The experimental conditions were the same as in Figure 3.

Figure 10. Specific activity for lipase versus surface concentration after rinsing onto surfaces with different wettabilities. The water contact angles of the surfaces were 90°, 80°, 75°, and 62° (0, 4, O, ]). The experimental conditions were the same as in Figure 3.

The specific activity at the 62° surface was shown to be very concentration dependent. Figure 10 presents the specific activity as a function of the adsorbed amount. It is shown that the specific activity increases with increasing wettability for different values of the adsorbed amount (≈1 and ≈2 mg/m2). It should be noted that the values for the 62° surface were obtained at different solution concentrations and that the high variation is due to the low amounts adsorbed, resulting in low activities, in combination with the high reference activities at low concentrations (63 nM). The total activity of the lipase adsorbed in a mixture with CaCl2 appeared to increase to some extent (Table 1), but the very high value of the reference activity made it difficult to safely conclude anything about the specific activity in these experiments. Discussion The influence of surface wettability on the adsorption behavior of HLL is obvious from the gradient measurements (Figure 1). The decrease in adsorbed amount and increase in desorbability with surface wettability is in line with the observations for several other proteins from studies concerning gradient surfaces and discrete hydrophobic and hydrophilic surfaces.35,43-45 The findings indicate structural changes of the adsorbed lipase with the surface wettability, as discussed below.

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For the discrete surfaces the highest amounts adsorbed were found at 90° surfaces for lipase concentrations in the range 200-300 nM (Figure 2) with a plateau value after rinsing of ≈2 mg/m2. From the dimensions of Humicola lanuginosa lipase used by Martinelle et al.9 (35 × 45 × 50 Å3) the values for close-packed side-on and end-on layers were calculated to be 2.3-3.0 mg/m2, depending on orientation, and 3.3 mg/m2 , respectively. At these experimental conditions the obtained plateau values for adsorbed HLL approximately correspond to a monolayer adsorbed side-on. At the highest lipase concentration (1050 nM) a peak in the adsorbed amount was found (Figure 5), as observed in an earlier work.6 The initial peak corresponded to 2.3 mg/m2, and at the plateau (Figure 2), the adsorbed amount was significantly lower (0.81 mg/m2), indicating a more loosely packed protein layer. The peak might indicate initial orientational/conformational changes of the lipase. To summarize the adsorption data presented in Table 1, the adsorbed amount decreased with increasing wettability of the surfaces, as found from the gradient measurements. The amount adsorbed increased with the concentration for 63, 115, and 231 nM for all surfaces and was for the 90°, 80°, 75°, and 62° surfaces 1.83-2.09 mg/ m2, 1.85-2.10 mg/m2, 1.68-1.78 mg/m2, and 0.25-0.31 mg/m2, respectively. Further increase in the concentration lowered the amounts adsorbed, and at 345 nM, for the 75° surface, the amount adsorbed was significantly lower (1.20 mg/m2). The adsorbed amount to a 80° surface, 345 nM, is in agreement with that found by Martinelle and coworkers for HLL adsorbed to polystyrene surfaces.9 The water contact angle of these surfaces is approximately 80° (Wannerberger, unpublished data). As previously observed for totally methylated surfaces, the desorbability of the lipase decreased with decreasing concentration.6 This was found for the 90°, 80°, and 75° surfaces (Figures 3-5). For each concentration the fraction desorbed upon rinsing increased with the wettability of the surfaces, which was in agreement with the results from the gradient measurements. The increasing amount of HLL adsorbed in the presence of CaCl2 (Figure 6) was previously suggested to be due to interactions with negatively charged amino acid residues in the protein.6 Increased amounts of adsorbed lipase were observed also by Duinhoven and co-workers in an adsorption study of HLL to negatively charged polystyrene surfaces from both CaCl2 and MgCl2 solutions.8 No specific effect of calcium was found, however, pointing to an effect related to the valency and general screening of electrostatics rather than a specific interaction. As discussed above, previous investigations6 revealed initial peaks in the amount adsorbed versus time curves at high lipase concentrations, close to the Ip and in the presence of monovalent electrolyte, indicating the existence of a transient, more densely packed, state. The peak at 1050 nM, observed in the present study, did not occur in the presence of CaCl2, which indicates that calcium inhibits the orientational/conformational changes which most likely occur at 1050 nM. These findings together indicate that a high packing density at the surface is favored at conditions of reduced (screened) electrostatic interactions. The results from the activity measurements show that the specific activity of the lipase increased with decreasing (43) Elwing, H.; Go¨lander, C. G. Adv. Colloid Interface Sci. 1990, 32, 317. (44) Wahlgren, M. Adsorption of Proteins and Interactions with Surfactants at the Solid/liquid Interface; Lund University: Lund, Sweden, 1992. (45) Welin-Klintstro¨m, S. Ellipsometry and Wettability Gradient Surfaces; Linko¨ping University: 1992.

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hydrophobicity (increasing wettability) of the surface (Table 1). For example, the specific activity of the adsorbed lipase from a 345 nM solution increased from 29 (nmol m2/(min mg)) for the 90° surface to 64 (nmol m2/(min mg)) for the 62° surface. This could be interpreted as changes in the orientation of the adsorbed lipase (its hydrophobic active site region) with the surface hydrophobicity. It might be speculated that this can be an effect of increased accessibility for the substrate due to exposure of the active site to solution. Furthermore, since the increase in activity with surface wettability is observed at different amounts adsorbed (Figure 9) this effect of wettability on activity is not due to differences in the supply of the substrate at the different lipase packing densities. From the adsorption experiments performed in the presence of CaCl2 the high values of the reference activities (Table 1) indicate that calcium/divalent ions significantly enhance the lipase adsorption to the 'background' surfaces. The adsorption and activity data presented in this study indicate a possibility for optimization of conditions for reactions involving immobilized lipase. This includes for example choice of carrier material with respect to its hydrophobicity as well as the concentration of lipase used.

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The latter may be of significant economical importance due to the cost of the enzyme. The effect of surface chemistry on enzyme efficiency has also been shown by others, e.g. Norin et al.46 and Bosley and Clayton,47 in both cases for nonaqueous conditions. Future investigations of the influence of other crucial parameters on amount/activity relations for lipase in the adsorbed state might save substantial effort and time in the development of various technical applications. Acknowledgment. We are indebted to Birgitte HugeJensen and Kim Borch, NOVO-Nordisk, Bagsvaerd, Denmark, for the gift of lipase and for valuable discussions, to Professor Karl Hult, Drs. Mats Martinelle and Martin Norin, Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden, and Johanna Dahlborg for valuable help and discussions, and to Agneta Askendahl, University of Linko¨ping, Sweden, for preparing the gradient surfaces. LA9605652 (46) Norin, M.; Boutelje, J.; Holmberg, E.; Hult, K. Appl. Microbiol. Biotechnol. 1988, 28, 527. (47) Bosley, J. A.; Clayton, J. C. Biotechnol. Bioeng.1994, 43, 934.