Adsorption Properties and Activities of Lipase on a Gold Substrate Modified by Self-assembled Monolayers

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm⁻²), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immobilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state.     

Action of a microbial lipase/acyltransferase on phospholipid monolayers

Vibrio species release a lipase which shares many properties with mammalian lecithin-cholesterol acyltransferase. We have studied the action of the enzyme on phospholipid monolayers. At similar surface pressures, reaction velocities were higher with monolayers of dilauroylphosphatidylcholine than with the corresponding phosphatidylglycerol or phosphatidylethanolamine. The dependence of reaction velocity on molecular density was very similar for phosphatidylcholine and phosphatidylethanolamine monolayers. Lag times were shortest with phosphatidylglycerol at low molecular densities, but maximum velocity was reached at considerably lower densities than with the other two lipids. We have found [Hilton, S., McCubbin, N. D., Kay, C., & Buckley, J.T. (1990) Biochemistry 29, 9072-9078] that nicking of the enzyme with trypsin or other proteases results in an increase in its activity against lipids in membranes. Here we show that trypsin treatment results in a large change in the surface activity of the lipase, allowing it to penetrate monolayers at pressures higher than 40 mN.m-1.     

Hydrolysis of mixed monomolecular films of triglyceride/lecithin by pancreatic lipase.

The main purpose of this study was to describe the influence of lecithin upon lipolysis of mixed monomolecular films of trioctanoylglycerol/didodecanoylphosphatidycholine by pancreatic lipase in order to mimic some physiological situations. The quantity of enzyme adsorbed to the interface was simultaneously determined using 5-thio-2-nitro[14C]benzoyl lipase. Lipolytic activity was enhanced 3- to 4-fold in the presence of colipase, an effect which is attributed to increased enzyme turnover number. When a pure triglyceride film was progressively diluted with lecithin, the minimum specific activity of lipase exhibited a bell-shaped curve: a mixed film containing only 20% trioctanoylglycerol was hydrolyzed at the same rate as a monolayer of pure triglyceride.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.

Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipoprotein lipase hydrolysis of trioleoylglycerol in a phospholipid interface. Effect of cholesteryl oleate on catalysis

The effect of cholesteryl oleate on the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol was determined in monolayers of egg phosphatidylcholine at a constant surface pressure of 24 mN m-1. The phospholipid monolayers contained 1.0 to 7.5 mol % trioleoylglycerol and various amounts (0 to 20 mol %) of cholesteryl oleate. The initial rates of trioleoylglycerol hydrolysis were determined with lipoprotein lipase purified from bovine milk. In phospholipid monolayers containing 5.0 or 7.5 mol % trioleoylglycerol, the further addition of cholesteryl oleate caused a decrease in lipoprotein lipase activity. In contrast, addition of cholesteryl oleate to phospholipid monolayers containing 1.0 or 2.5 mol % trioleoylglycerol enhanced enzyme activity; a 3-fold enhancement was observed with 5.0-7.5 mol % cholesteryl oleate. Based on force-area measurements, the cholesteryl ester-mediated decrease in lipoprotein lipase activity observed at high substrate concentrations may be explained by displacement of trioleoylglycerol from the interface, thereby reducing the interfacial trioleoylglycerol concentration available for enzyme catalysis. One explanation for the cholesteryl oleate-mediated enhancement of lipoprotein lipase activity at low trioleoylglycerol concentrations is that the additional spreading of cholesteryl oleate disrupts microemulsions of trioleoylglycerol, thereby increasing the effective monomer substrate concentration available for enzyme catalysis. Based on these monolayer studies with model systems, we suggest that the relative amount of cholesteryl esters in plasma triacylglycerol-rich lipoproteins plays a regulatory role in determining the rate at which triacylglycerols are cleared from the circulation.     

Amphiphilic conetworks as activating carriers for the enhancement of enzymatic activity in supercritical CO2

Enzymatic reactions in supercritical carbon dioxide (scCO2) represent a way of combining the advantages of biocatalysis with the environmental benign nature of scCO2 as a solvent. Here we demonstrate that activities of enzymes in scCO2 can be greatly enhanced by incorporating them into amphiphilic conetworks (APCNs), a novel type of enzyme support. Two sets of hydrophilic/scCO2-philic APCNs, poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) and poly(2-hydroxyethyl acrylate)-linked by-perfluoropolyether (PHEA-l-PFPE), were prepared and loaded with the synthetically relevant lipase from Rhizomucor miehei. The effect of the APCNs' composition on the amount of the absorbed lipase was studied. It is observed that both sets of lipase-loaded APCNs enhance the catalytic activity of the enzyme in scCO2. The chemical nature of the scCO2-philic phase as well as the conetworkscomposition greatly influences the activity of the lipase in the conetworks. Activities obtained with PFPE-basedAPCNS were up to 10-fold higher than those obtained with PDMS-based conetworks. The highest specific activity measured corresponds to a 2,000-fold activation compared to the lyophilized enzyme powder. This activity is 10 times higher than the specific activity of the lipase immobilized on an optimized commercial carrier.     

Enzyme--substrate interaction in lipid monolayers. III. A study of the variation of the surface concentration with lipolysis.

Monolayers of a diacylglycerol were submitted to the action of lipase, keeping the area constant. The variation of lipase, keeping the area constant. The variation of the surface concentration gamma of the substrate with time was derived from the recorded reduction of the surface pressure pi (the isotherm of the monolayer being previously established). The rate -d gamma/dt was determined both as a function of the surface concentration gamma of the substrate and as a function of the bulk concentration C of the enzyme in the underlying solution. The rate depends on the quantity of enzyme ze adsorbed on the monolayer and on the enzymatic specific activity alpha of these adsorbed enzyme molecules. Both ze and alpha vary with gamma. The two variations have been quantitatively dissociated. The curves of ze and of alpha as functions of gamma coincide with those previously established in the study of hydrolysis under constant surface pressure.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.     

Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance

Lipases from Candida rugosa (CRL) and lipase isoforms A and B from Candida antarctica (CAL-A and CAL-B) were adsorbed on aminated supports in the presence of detergents to have individual lipase molecules. Then, one fraction was washed to eliminate the detergent, and both preparations were treated with glutaraldehyde. The presence of detergent during the cross-linking of the lipases to the support permitted an increase in the recovered activity (in some instances, even by a 10-fold factor). This activity was higher even than that exhibited by the just adsorbed lipases, suggesting that it was not a result of some protective effect of the detergent in the enzyme activity during glutaraldehyde chemical modification. Moreover, the enantioselectivity of the different enzyme preparations was very different if the glutaraldehyde was offered in the presence or in the absence of detergent, in some cases increasing the E value (even by a 7-fold factor in the case of CAL-A in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester), in other cases even inverting the enantio preference (e.g., in the case of CRL). The irreversible chemical inhibition of the enzyme that was immobilized and cross-linked with glutaraldehyde in the presence of detergents was more rapid than that in the other preparations (by more than a 10-fold factor). This experiment reveals an exposition degree of the active serine in the preparation cross-linked with the support in the presence of detergent that is higher than that in the other preparations. The results suggested that different enzyme structures were "stabilized" by the glutaraldehyde treatment if performed in the presence or in the absence of detergent, and that, in the presence of detergent, a form of the lipase with the serine residue more exposed to the medium and much more active could be obtained. This strategy seems to be of general use to improve the lipase activity to be used in macroaqueous media.     

Surface density of cellobiohydrolase on crystalline celluloses. A critical parameter to evaluate enzymatic kinetics at a solid-liquid interface

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid-liquid interface was evaluated by applying the novel concept of surface density (rho) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme. When the adsorption levels of Trichoderma viride Cel7A on cellulose I(alpha) from Cladophora and cellulose I(beta) from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose I(beta) was approximately 1.5 times higher than that on cellulose I(alpha), although the rate of cellobiose production from cellulose I(beta) was lower than that from cellulose I(alpha). This indicates that the specific activity (k) of Cel7A adsorbed on cellulose I(alpha) is higher than that of Cel7A adsorbed on cellulose I(beta). When k was plotted versus rho, a dramatic decrease of the specific activity was observed with the increase of surface density (rho-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activity. An apparent difference of the specific activity was observed between crystalline polymorphs, i.e. the specific activity for cellulose I(alpha) was almost twice that for cellulose I(beta). When cellulose I(alpha) was converted to cellulose I(beta) by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose I(beta) at the same rho-value. These results indicate that the hydrolytic activity (rate) of bound Cel7A depends on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid-liquid interface.     

Immobilization of phospholipase A2 from Central Asian cobra venom on polyamide sorbents

The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption.     

Structural organization of DMPC lipid layers on chemically micropatterned self-assembled monolayers as biomimetic systems

The growth structure of DMPC lipid layers on hydrophobic and hydrophilic alkylsilane-based self-assembled monolayers adsorbed on silicon has been investigated by means of X-ray reflectometry and atomic force microscopy. Hydrophilic modification of hydrophobically terminated ODS-SAMs has been achieved by dose-controlled irradiation with DUV light. While island formation of small DMPC bilayer islands is observed on hydrophobic SAM surfaces, closed layers of DMPC monolayers are formed on hydrophilic SAM surfaces. Furthermore, DMPC adsorption on chemically micropatterned substrates with alternating hydrophobic/hydrophilic surface properties has been studied by imaging ellipsometry and photoemission microscopy. Indication for at least partial bridging of hydrophobic areas by an adsorbed DMPC monolayer has been found.     

Enzyme-substrate interaction in lipid monolayers. I. Experimental conditions and fundamental kinetics.

A systematic study has shown the importance of the different factors which are concerned with the action of lipase on a substrate (1,3-didecanoylglycerol). These consist of a) the process of adsorption of lipase to the surface, b) the necessity of limited stirring to reach equilibrium, and c) the persistence during the reaction process of the enzyme molecules adsorbed on the monolayer. On the basis of this preliminary investigation, a technique was established to analyze the mechanism of lipase action with defined quantities of enzyme and lipid segregated in the monolayer. Thus, the process of the reaction itself is separated from the adsorption process, and it is demonstrated that the quantity of substrate hydrolyzed per minute depends only on the quantity of initially adsorbed lipase and not on the quantity of substrate or on the surface concentration of the enzyme. An appropriate new definition of the rate is consequently adopted.