3488
Langmuir 1997, 13, 3488-3493
Comparison of the Adsorption and Activity of Lipases from Humicola lanuginosa and Candida antarctica on Solid Surfaces Kristin Wannerberger* and Thomas Arnebrant Deparment of Food Technology, University of Lund, Box 124, S-221 00 Lund, Sweden Received December 2, 1996. In Final Form: April 16, 1997X The adsorption of lipases from Humicola lanuginosasthe wild type (WT) and a mutant with increased hydrophobicity in the active site regionsand lipase B from Candida antarctica to solid surfaces was studied by in situ ellipsometry. In addition, the activity of the adsorbed lipase was measured in situ and from the different surface concentrations, the specific activity was calculated. Concentration and temperature dependence as well as the influence of surface wettability was studied using silica surfaces with varying degree of methylation. The higher hydrophobicity of the mutant compared to the WT resulted in increased amounts adsorbed, no desorbable fraction during rinsing and absence of an initial maximum in adsorbed amount (as seen for the WT) at higher concentrations. No temperature dependence for the mutant could be observed. This was in contrast to the WT where both the plateau value of the adsorbed amount and the activity decreased with increasing temperature. The influence of surface wettability was similar for both featuring a decreased adsorbed amount and increased specific activity with increasing wettability. The amount adsorbed of the Candida lipase was significantly higher at all concentrations, compared to the other lipases, and the activity was very low, indicating adsorption with the active site region directed toward the surface. The surface wettability did not affect the activity of the Candida lipase.
Introduction According to the Enzyme Commission1 lipases are triglyceride acylhydrolases, i.e. enzymes that hydrolyze triglycerides. They may or may not have positional specificity for the primary ester bonds. Usually the enzymes also hydrolyze di- and monoglycerides.2 Lipases are found in different body liquids, e.g., pancreatic juice, blood plasma, and saliva, in milk, in triglyceride-producing plants, in molds, and in bacteria.3 There are many applications for microbial lipases, e.g., in the food (dairy, bakery, brewing) and the chemical fields (oil processing and detergents).4 The discovery of new microorganisms together with the quite new field of protein engineering5 will lead to an increase in the diversity of lipases and probably also extend the fields of application for these enzymes. The first lipase structures to be solved were those of human pancreatic lipase6 and a lipase from the mold Rhizomucor miehie.7 To date several more structures are known. The lipases studied in this work were the Humicola lanuginosa lipase (HLL) and the Candida antarctica lipase B (CALB). The structures of both lipases were presented recently.8-10 The main use for these * Author to whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, June 1, 1997. (1) Florkin, M., Stotz, E. H., Eds. Report of the Enzyme Commission of the International Union of Biochemistry; Elsevier: Amsterdam, 1964. (2) Brockerhoff, H.; Jensen, R. G. Lipolytic Enzymes; Academic Press: New York, 1974. (3) Whitaker, J. R. Principles of Enzymology for the Food Sciences; Marcel Dekker: New York, 1972. (4) Iwai, M.; Tsujisaka, Y. In Lipases; Borgstro¨m, B., Brockman, H. L., Eds.; Elsevier: New York, 1984; p 443. (5) Bott, R.; Shield, J. W.; Poulose, A. J. In Lipases: Their Structure, Biochemistry and Application; Woolley, P., Petersen, S. B., Eds.; University Press: Cambridge, 1994; p 337. (6) Winkler, F. K.; D’Arcy, A.; Hunziker, W. Nature 1990, 343, 771. (7) 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. (8) 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.
S0743-7463(96)02061-6 CCC: $14.00
lipases is as components in detergent formulations (HLL) and as catalysts in transesterification reactions (CALB). HLL and CALB differ with respect to their activation. HLL is thought to act by the so-called interfacial activation where the enzyme in its inactive form has the active site covered by a lid which, when the enzyme is at an interface, opens and exhibits the active site. The lid is not found in CALB but instead there is a helix that may play a similar role as the lids of other lipases. The entrance to the active site is located in the middle of a large hydrophobic surface.11 Recent studies have been performed concerning lipase adsorption12,13 and of particular interest are those where the adsorption of HLL has been studied.12,14-16 In ref 15 a study of the lipase adsorption and activity on solid surfaces with different wettabilities was performed and it was found that the specific activity of the lipase increased with the surface wettability. Several studies on CALB have also been performed, and as mentioned above the structure was studied by Uppenberg et al.10,11 A comparison of the interfacial activation between CALB and HLL was recently made by Martinelle et al.17 CALB as catalyst in acyl transfer reactions was studied by O ¨ hrner and co-workers.18 (9) Derewenda, U.; Swenson, L.; Green, R.; Wei, Y.; Yamaguchi, S.; Joerger, R.; Haas, M. J.; Derewenda, Z. S. Protein Eng. 1994, 7, 551. (10) Uppenberg, J.; Trier Hansen, M.; Patkar, S.; Jones, T. A. Structure 1994, 2, 293. (11) Uppenberg, J. The Three-Dimensional Structure of Lipase B from Candida antarctica. Thesis, University of Uppsala, Sweden, 1994. (12) Wannerberger, K.; Arnebrant, T. J. Colloid Interface Sci. (in press). (13) Geluk, M. A.; Norde, W.; Van Kalsbeek, H. K. A. I.; Van’t Riet, K. Enzyme Microb. Technol. 1992, 14, 748. (14) Wannerberger, K.; Wahlgren, M.; Arnebrant, T. Colloids Surf. B (in press). (15) Wannerberger, K.; Welin-Klintstro¨m, S.; Arnebrant, T. Langmuir (submitted). (16) Duinhoven, S.; Poort, R.; Van der Voet, G.; Agterof, W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170, 351. (17) Martinelle, M.; Holmquist, M.; Hult, K. Biochim. Biophys. Acta 1995, 1258, 272. (18) O ¨ hrner, N. Lipase B from Candida antarctica as a Stereoselective Biocatalyst in Acyl Transfer Reactions. Thesis, Royal Institute of Technology, Stockholm, Sweden, 1994.
© 1997 American Chemical Society
Lipase Adsorption and Activity on Solid Surfaces
In the present work we expanded the adsorption and activity measurements performed on the wild type (WT) of H. lanuginosa lipase15 to include two other lipases, a mutant of HLL and CALB. The adsorption of lipase to solid surfaces was studied by ellipsometry, the activity was determined during the measurement by adding a water soluble substrate (p-nitrophenylacetate)17 to the ellipsometer cuvette, and then the absorbance of samples from the solution was measured. The aim was to investigate whether structural/functional differences between lipases could be observed by this method, and CALB and the mutant of HLL were chosen because of their lack of interfacial activation and increased hydrophobicity in the active site region, respectively. Ellipsometry was used to determine the amounts adsorbed and the kinetics of adsorption-desorption.19-25 The technique facilitates in situ measurements, and the adsorbed amount can be determined with short intervals.20,26 Methylated silica surfaces were used in the study, similar to those used in previous adsorption studies of lipase12,14,15 and a number of other proteins.27-30 Surfaces with different wettabilities were also used and were similar to those used in the previous investigation.15 The adsorption and activity measurements were performed to hydrophobic surfaces at different concentrations (63, 345, and 1050 nM) for all the lipases. The behavior of the WT and the mutant at increased temperature (45 °C) was studied as well as the influence of surface wettability on the adsorption and activity. Materials and Methods Highly purified lipase from H. lanuginosa (HLL) and from C. antarctica type B (CALB) was kindly provided by Novo-Nordisk, Bagsværd, Denmark. HLL is a 1,3-specific lipase with increasing activity in the pH range 7-11, and has its optimal activity at temperatures around 35-40 °C. Its molar weight is 31700 g/mol (glycosylated form) and it has an isoelectric point (pI) of 4.4 (personal communication, Novo-Nordisk). Both the wild type of the protein (WT) and a mutant which was modified by replacement of D (aspartic acid) 96 with L (leucine) were used in the study. The lipase from C. antarctica has a molecular weight of 33000 g/mol and an pI of 6.11 The experiments were carried out in 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma), at pH 7.5. 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 27. A cleaning process was carried out as described in ref 31 to give hydrophilic surfaces with a water contact angle of less than 10°.31 (19) Arnebrant, T. Proteins at the Metal/Water InterfacesAdsorption in Relation to Interfacial Structure. Thesis, Lund University, Lund, Sweden, 1987. (20) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1977. (21) Trurnit, H. J. Arch. Biochem. Biophys. 1953, 47, 251. (22) Trurnit, H. J. Arch. Biochem. Biophys. 1954, 51, 176. (23) Poste, G.; Moss, C. In Progress in Surface Science; Davidson, S. G., Ed.; Pergamon Press: Oxford, 1972; p 139. (24) Nylander, T. Proteins at the Metal/Water InterfacesAdsorption and Solution Behaviour. Thesis, Lund University, Lund, Sweden, 1987. (25) Lundstro¨m, I.; Ivarsson, B.; Jo¨nsson, U.; Elwing, H. In Polymer Surfaces and Interfaces; Feast, W. J., Munro, H. S., Eds.; John Wiley & Sons Ltd: Chichester, 1987; p 201. (26) Cuypers, P. A. Dynamic Ellipsometry: Biochemical and Biomedical Applications. Thesis, Rijksuniversiteit Limburg, The Netherlands, 1976. (27) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1990, 136, 259. (28) Luey, J. K.; McGuire, J.; Sproull, R. D. J. Colloid Interface Sci. 1991, 143, 489.
Langmuir, Vol. 13, No. 13, 1997 3489 The highly hydrophobic surfaces were prepared according to ref 31 slightly modified as described in ref 27 by the reaction of dichlorodimethylsilane (DDS) in trichloroethylene. The surfaces with an intermediate hydrophobicity were obtained as described in ref 14 where the solvent was xylene. Different reaction times with DDS gave surfaces with different hydrophobicity. The reaction times were 30 and 22 min, giving wettabilities of the surfaces as measured by the water contact angle method32 of 80° ( 2° (n ) 12) and 75° ( 2° (n ) 27). The surfaces were stored in ethanol. Prior to use, the surfaces were rinsed with doubly distilled water and ethanol and blown dry with nitrogen. The hydrophilic surfaces were plasma cleaned prior to use 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). 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 measures the changes in the state of polarization of elliptically polarized light upon reflection at a surface. From the changes, expressed by the ellipsometric angles ∆ and Ψ, the thickness and the refractive index of a thin film on a surface can be calculated.20 In addition the amount per unit area of the film can be determined if the partial specific volume and the ratio of the molar weight to the molar refractivity33 or the refractive index increment with concentration dn/dc34 of the adsorbing molecules are known. The adsorption/desorption experiments were performed with an automated Rudolph thin-film ellipsometer, type 43603-200E, equipped with a thermostated cuvette. The experimental setup was described in detail by Nylander.24 The adsorbed amount was calculated according to Cuypers et al.,33 and 0.75 mL/g and 4.1 g/mL were used as the values for the partial specific volume and the ratio between molar mass and the molar refractivity, respectively. Before each measurement the ellipsometer cuvette was carefully cleaned in detergent solution (Sparkleen, Fischer Scientific Co., U.S.A.) and rinsed in doubly distilled water. The ellipsometric angles ∆ and Ψ for the clean surface were recorded after immersion in the cuvette containing MOPS buffer (4.5 mL). Freshly prepared stock solution of lipase 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 made 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. Measurements of the optical changes were continued also during the activity measurements. The stirring rate in the cuvette was 325 rpm, and the temperature was 25 or 45 °C. All experiments were made at least in duplicate. Measurement of the lipase activity was made according to an assay described by Martinelle et al.17 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 different time intervals for measurement of the absorbance at 405 nm. The activity was obtained as the amount phenolate ions produced per minute. The absorbance measurements were performed with a Beckman spectrophotometer and quartz cuvettes (1 cm × 1 cm) were used in the study. Part of the activity achieved in this way arose from lipase molecules adsorbed to the ellipsometer cuvette, but since all experiments were performed also without surface, this background activity (or reference activity) could be subtracted to give the activity originating from the lipase molecules adsorbed to the methylated silica surfaces only. (29) Wahlgren, M. C.; Paulsson, M. A.; Arnebrant, T. Colloids Surf. A 1993, 70, 139. (30) Wahlgren, M.; Arnebrant, T.; Askendal, A.; Welin-Klintstro¨m, S. Colloids Surfaces A 1993, 70, 151. (31) Jo¨nsson, U.; Ivarsson, B.; Lundstro¨m, I.; Berghem, L. J. Colloid Interface Sci. 1982, 90, 148. (32) Gekas, V.; Persson, K.; Wahlgren, M.; Sivik, B. J. Membr. Sci. 1992, 72, 293. (33) 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. (34) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759.
3490 Langmuir, Vol. 13, No. 13, 1997
Wannerberger and Arnebrant
Table 1. Adsorbed Amount after Rinsing, the Total Activity, the Reference Activity, and the Specific Activity of 345 nM Lipase Solutions Adsorbed to Totally Methylated Silica Surfaces at Different Temperaturesa
conditions HLL WT 25 °C HLL WT 45 °C HLL mutant 25 °C HLL mutant 45 °C CALB 25 °C
adsorbed amount (mg/m2)
activity (nmol/min) total
referenceb
specific activityc [nmol m2/ min mg)]
23 ( 2 1.83 ( 0.15
74 ( 6
29 ( 5 27 ( 8
1.10 ( 0.00
43 ( 1
15 ( 0 56 ( 0
2.18 ( 0.02
75 ( 6
2.22 ( 0.02
95 ( 2
2.80 ( 0.00
15 ( 1
9(3 73 ( 6 10 ( 1 6(1 3(0
no. of values (n) 2 2 2 2 2 2 2 2 2 2
a
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 tubing, and magnetic stirrer. c The specific activity is the total activity, with the reference activity subtracted, divided by the adsorbed amount after rinsing. Table 2. Adsorbed Amount after Rinsing, the Total Activity, the Reference Activity, and the Specific Activity of Lipase of Different Concentrations Adsorbed to Totally Methylated Silica Surfaces at 25 °Ca
conditions HLL WT 63 nM HLL WT 345 nM HLL WT 1050 nM HLL mutant 63 nM HLL mutant 345 nM HLL mutant 1050 nM CALB 63 nM CALB 345 nM CALB 1050 nM
adsorbed amount (mg/m2)
activity (nmol/min) total
referenceb
specific activityc [nmol m2/ (min mg)]
41 ( 12 1.83 ( 0.00
79 ( 6
1.83 ( 0.15
74 ( 7
21 ( 3 23 ( 2 29 ( 5 23 ( 11
0.81 ( 0.19
25 ( 8
d 46 ( 2
2.12 ( 0.02
85 ( 2
18 ( 1 56 ( 0
2.18 ( 0.02
75 ( 6
9(3 51 ( 2
2.35 ( 0.05
84 ( 5
14 ( 2 6(0
2.55 ( 0.05
14 ( 1
3(0 6(1
2.80 ( 0.00
15 ( 1
3(0 9(1
2.85 ( 0.00
20 ( 2
4(1
Table 3. Adsorbed Amount after Rinsing, the Total Activity, the Reference Activity, and the Specific Activity of 345 nM Lipase Solutions Adsorbed to Totally Methylated Silica Surfaces of Different Wettability at 25 °Ca
conditions HLL WT 90° 80° 75° HLL mutant 90° 80° 75° CALB 90° 80° 75°
adsorbed amount (mg/m2)
activity (nmol/min) total
referenceb
specific activityc [nmol m2/ min mg)]
23 ( 2 1.83 ( 0.15 1.68 ( 0.26 1.20 ( 0.00
74 ( 7 73 ( 2 68 ( 2
" " "
29 ( 5 30 ( 3 38 ( 2
56 ( 0 2.18 ( 0.02 1.97 ( 0.08 1.92 ( 0.03
75 ( 6 87 ( 3 87 ( 8
" " "
9(3 16 ( 1 16 ( 4
6(1 2.80 ( 0.00 2.45 ( 0.18 2.45 ( 0.05
15 ( 1 18 ( 2 15 ( 2
" " "
3(0 5(1 4(1
no. of values (n) 2 3 2 2 2 2 3 3 2 2 3 2
a
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 tubing, and magnetic stirrer. c The specific activity is the total activity, with the reference activity subtracted, divided by the adsorbed amount after rinsing.
no. of values (n) 4 2 2 2 3 4 2 2 2 2 2 2 2 2 2 2 2 2
a
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 tubing, and magnetic stirrer. c The specific activity is the total activity, with the reference activity subtracted, divided by the adsorbed amount after rinsing. d No value is given since the difference was very small between the total and the reference activity.
The results from measurements with different stirring rates (not presented) show that the reaction was not diffusion controlled. The lipase concentrations in the cuvette during the adsorption to hydrophobic surfaces [water contact angle (θ) ) 90°] were 63, 345, and 1050 nM (T ) 25 °C). The temperature dependence of the adsorption was measured for the WT and the mutant at 25 and 45 °C (θ ) 90°). For the surfaces with higher wettability (θ ) 80°, 75°, and
