Crude Lipase Research Articles

Enantioselective hydrolysis of ( R, S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of ( R, S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by ( S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for ( R, S)-fenoprofen 2,2,2-trifluoroethyl thioester.

Effect of several reaction parameters in the solvent-free ethyl oleate synthesis using Candida rugosa lipase immobilised on polypropylene

Lipase from Candida rugosa was immobilised onto polypropylene powder by physical adsorption. The immobilised catalyst (CR/PP) was used in the enzymatic synthesis of ethyl oleate in solvent-free medium. The influence of the initial water content, acidity of the aqueous media added, mass of catalyst, reaction temperature, substrate ratio, etc., on enzymatic activity, has been studied. Comparison of specific enzymatic activities achieved using the prepared catalyst and the crude lipase demonstrated that C. rugosa lipase was interfacially activated upon its adsorption on polypropylene. Besides, immobilisation of the lipase led to enhanced thermal stability. Experimental data reported in this manuscript do not belong to equilibrium data but to enzymatic activity attained in the first 2 h of reaction. After this period, it was shown that, in the current synthesis, deactivation/agglomeration/inhibition of the catalyst prevented further CRL activity. However, 2 h measurements allowed fulfilling the aim of this work: the determination of the best conditions for ethyl oleate production in short periods of time, using an immobilised derivative of a relatively cheap lipase as it is C. rugosa lipase. Best results were achieved in the reaction performed at 45 °C and 350 rpm, with an initial stoichiometric ratio of substrates, 20% of aqueous content, and mediated by 50 mg of CR/PP (0.0585 mmol/mg of CR-h). The deleterious effect of ethanol excess and agglomeration of the native and immobilised catalyst have been analysed.

Glucose stimulates production of the alkaline-thermostable lipase of the edible Basidiomycete Antrodia cinnamomea

This paper describes a study of the extracellular lipase synthesised by a submerged culture of Antrodia cinnamomea in two different media (modified ME and modified YM). Lipase production increased substantially upon the addition of glucose; when it comprised 8% of the modified ME and YM media, the lipase production increased remarkably from 0 to 3.35 mU/ml and from 0.59 to 6.17 mU/ml, respectively. The lipase activity decreased, however, beyond certain concentrations of glucose. The lipase synthesis in the modified YM medium was superior to that which occurred in the modified ME medium. In addition, the optimum values of pH and temperature for the crude lipase were 11.0 and 60 °C, respectively. The enzyme was stable at values of pH between 7.0 and 12.0 and at temperatures up to 80 °C. Thus, this novel lipase from A. cinnamomea is particularly alkaline-resistant and thermostable.

Rational strategy for the production of new crude lipases from Candida rugosa

Candida rugosa was cultured using different inducers (oleic acid, olive oil, sunflower oil, n-dodecanol and glycerol) as the only carbon source in batch conditions, as well as in several fed-batch fermentations (oleic acid as inducer) at variable feed rate conditions. The N-terminal analysis of each crude lipase revealed that, while the isoenzymes Lip2 and Lip3 are always secreted (at different proportions depending on the inducer), Lip1 was produced only using n-dodecanol (batch conditions) or oleic acid (fed-batch at high feed rate). The nature of the inducer controls the isoenzyme percentage; when this is fixed, as well as the feed rate in fed-batch fermentation, the isoenzymatic profile remained unaltered and the samples differed only in the activity of the lipases, as determined by heptyl oleate synthesis.

Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 °C and pH at 7.0–8.0. It was active up to 90 °C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca 2+, Mg 2+, Zn 2+, Fe 2+ or Fe 3+, but inhibited by Cu 2+, Mn 2+, and Li +. By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.

Enhancement of enantioselectivity and reaction rate on the synthesis of (S)-ketoprofen hydroxyalkyl ester in organic solvents via isopropanol-dried immobilized lipase

Lipase-catalyzed enantioselective esterification between (R,S)-ketoprofen and alkanediol in organic solvents was developed to produce (S)-ketoprofen hydroxyalkyl esters. The acyl acceptor of 1,6-hexanediol for the resolution of (R,S)-ketoprofen yielded only the enantioselectivity (the enantiomeric ratio of initial rate for (S)-ketoprofen to that of (R)-ketoprofen) VS/VR = 8, when crude Lipase MY originating from Candida rugosa was used. However, isopropanol-dried immobilized lipases (IPA-dried IM-lipase) effectively enhanced the enantioselectivity to greater than 20 in the esterification of (R,S)-ketoprofen when 1,4-butanediol, 1,5-pentanediol or 1,6-hexanediol was employed. IPA-dried IM-lipase and isooctane were selected to use for optimally immobilized lipase and reaction medium, respectively. The IPA-dried IM-lipase exhibited the highest enantioselectivity, E = 26.7, to the (S)-enantiomer with 1,5-pentanediol and the best enzyme activity to the (S)-enantiomer with 1,4-butanediol. The finding indicates that the carbon chain length of the alkanediol strongly affected the enzyme activity and enantioselectivity of lipase-catalyzed esterification. A maximum enantioselectivity of 37 at 27 °C was generated by IPA-dried IM-lipase for the enantioselective esterification of racemic ketoprofen with 1,4-butanediol. IPA-dried IM-lipase can effectively increase the enantioselectivity of lipase. Copyright © 2005 Society of Chemical Industry

Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts

Ultrafine cellulose fiber (diameter 200-400 nm) surfaces were grafted with polyacrylic acid (PAA) via either ceric ion initiated polymerization or methacrylation of cellulose with methacrylate chloride (MACl) and subsequent free-radical polymerization of acrylic acid. PAA grafts by ceric ion initiated polymerization increased with increasing reaction time (2-24 h), monomer (0.3-2.4 M), and initiator (1-10 mM) concentrations, and spanned a broad range from 5.5-850%. PAA grafts on the methacrylated cellulose fibers also increased with increasing molar ratios of MACl to cellulosic hydroxyl groups (MACl/OH, 2-6.4) and monomer acrylic acid (AA) to initiator potassium persulfate (KPS) ratios ([AA]/[KPS], 1.5-6), and were in a much narrower range between 12.8% and 29.4%. The adsorption of lipase (at 1 mg/ml lipase and pH 7) and the activity of adsorbed lipase (pH 8.5, 30 degrees C), in both cases decreased with increasing PAA grafts. The highest adsorption and activity of the lipase on the ceric ion initiated grafted fibers were 1.28 g/g PAA and 4.3 U/mg lipase, respectively, at the lowest grafting level of 5.5% PAA, whereas they were 0.33 g/g PAA and 7.1 U/mg lipase, respectively, at 12.8% PAA grafts on the methacrylated and grafted fibers. The properties of the grafted fibers and the absorption behavior and activity of lipase suggest that the PAA grafts are gel-like by ceric-initiated reaction and brush-like by methacrylation and polymerization. The adsorbed lipase on the ceric ion-initiated grafted surface possessed greatly improved organic solvent stability over the crude lipase. The adsorbed lipases exhibited 0.5 and 0.3 of the initial activity in the second and third assay cycles, respectively.

Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10 mM (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10 U ml(-1) and, at 30 degrees C and pH 7 over 48 h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.

Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis.

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

Lipase factor (LF) as a characterization parameter to explain the catalytic activity of crude lipases from Candida rugosa, free or immobilized in microemulsion-based organogels

Several lipases of Candida rugosa ATCC 14830, obtained in different fermentation conditions and with different proportion and number of isoenzymes, were immobilized in microemulsion-based organogels (MBGs), gelled with hydroxypropylmethyl cellulose (HPMC) as biopolymer, and lecithin or AOT as surfactants. Lipase factor (LF) obtained from initial reaction rate profile is useful parameter to explain the catalytic activity of these crude enzymes in esterification reactions. This parameter is valid for the characterization of native or immobilized enzymes, due to the catalytic behaviour of the lipases immobilized in the MBGs is similar to that described for native enzymes. Lipases hosted in MBGs obtained from microemulsions with lecithin as surfactant are more active than those obtained with AOT in esterification reactions. Esterification of fatty acids in organic media and LF values can be used to evaluate the amount of lipase in different crude enzymes.

Protein engineering and applications of Candida rugosa lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

Abatement of concentration polarization in ultrafiltration using n-hexadecane/water two-phase flow

A novel method using n-hexadecane/water two-phase flow to abate concentration polarization in ultrafiltration was proposed. Compared with a corresponding air-sparged ultrafiltration, the n-hexadecane/water ultrafiltration was shown to have a higher permeate flux when concentrating a crude lipase solution. This was attributable to the much higher shear force induced by n-hexadecane droplets, and the reduction in concentration polarization was thus more pronounced. The range of injection factor resulting in bubble flow was broader in the n-hexadecane/water ultrafiltration than in the air-sparged ultrafiltration. In addition to the permeate flux, the recovery of lipase activity was also enhanced, and enzyme inactivation due to the use of the two-phase flow was not concerned. The benefits from the n-hexadecane/water two-phase flow were found to be common in various ultrafiltration practices.

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation ( Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity ( E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.

Lipase activity in dormant seeds of the African oil bean ( Pentaclethra macrophylla Benth)

Acetone powders were obtained from freshly harvested dormant seeds of African oil bean ( Pentaclethra macrophylla Benth) and the activity as well as substrate specificity of the isolated crude lipase (glycerol ester hydrolase E.C. 3.1.1.3) were examined. The enzyme was more active on lauric oils (containing short-chain fatty acids), especially coconut oil. Hexane at 1:2 v/w solvent:substrate ratio was optimal in enhancing lipolysis of the oils. Optimum substrate concentration was 5 g in 2.5 ml of solvent, above which enzyme inhibition resulted. Lipolysis increased with enzyme concentration (non-linearly), with optimum rate at between 0.5 and 0.75 g and 60 min incubation. Optimum temperature for the activity of the oil bean lipase was 30 °C, although substantial lipolysis was still evident at 80 °C indicating the fairly high thermostability of the enzyme. pH optimum for enzyme activity was near neutrality. The effects of different ions on the activity of the isolated lipase were also investigated. The presence of Ca 2+ increased activity by 64% while sodium chloride and mercuric chloride inhibited activity by 36% and 28.5%, respectively. On addition of EDTA, an inhibition in activity of 28% was observed. The results of the present study show that the oil bean seed will deteriorate with storage.

Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels.

Although a variety of approaches exist for the immobilization of enzymes, the "science" of enzyme immobilization is still in its infancy. In recent years, considerable interest has developed regarding the use of xerogels for enzyme immobilization. There are several advantages to xerogels for enzyme immobilization, including the opportunity to produce them in defined shapes or thin films and the ability to manipulate their physical characteristics (e.g., porosity, hydrophobicity, and optical properties). In this study we examined the effect of xerogel hydrophobicity on the activity of lipase (EC 3.2.2.3) from Rhizomucor miehei. The hydrophobicity of the xerogels was manipulated by generating xerogels with various molar ratios of propyltrimethoxysilane (PTMS) to tetramethoxysilane (TMOS), from 1:1 to 10:1. The belief was that, by increasing the proportion of propyl groups, the hydrophobicity of the resulting xerogel would be increased. Differences in the hydrophobicity of the resulting xerogels were confirmed using water-affinity studies. Two approaches were taken for water-affinity determinations by examining the ability of the xerogels to remove water from air (controlled humidity) and from water-saturated isopropyl ether. Xerogels with higher propyl content showed a reduced affinity for water. A crude lipase preparation from Rhizomucor miehei was then contacted with sized xerogel particulates and the effect of the xerogel on lipase activity was determined. The presence of the xerogel resulted in hyperactivation of the lipase. Analysis of the protein adsorption revealed changes in the profile of proteins adsorbed to the xerogel based on the hydrophobicity of the xerogel. Based on estimations of the specific activity of the hyperactivated lipase, a minimum hyperactivation of 207% was observed. Part of the hyperactivation may be attributable to xerogel-lipase interactions, but also to the adsorption of a component from the crude lipase preparation that may complex with the lipase and the xerogel producing a stabilizing effect. Further improvements in hyperactivation and selectivity of the xerogels is likely possible by working at lower PTMS:TMOS ratios than those investigated in this study.

Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents

The effects of various environmental conditions and chemical compounds on the activity and stability of the lipolytic preparation obtained from a wild strain of Penicillium aurantiogriseum were characterized during a preliminary evaluation of its potential for use in biocatalysis. In aqueous solution, the optimum pH for activity was 8.0 and the enzyme was stable between pH 6.0 and pH 9.0. In assays of 1 min duration carried out at pH 8.0, enzyme activities were quite high from 37 to 70 °C, with a maximum at 60 °C. However, thermal stability was rather low at temperatures higher than 28 °C. Hydrolytic activity was enhanced by Mg 2+, Zn 2+, Co 2+ and Mn 2+, but was inhibited by Cu 2+, Ba 2+ and Hg 2+, while Ca 2+ had no effect. Sodium azide activated the enzyme. Triton X-100 caused an inhibition of 52%, while Tween 80 and SDS had negligible effects on enzymatic activity. Despite the low ratio of the activity towards p-nitrophenyl palmitate (pNPP) in organic medium to that in aqueous medium ( R O/A=4.3×10 −2), the enzyme showed a good stability in organic solvents with high log P values, the best result being in n-heptane (114% residual activity). These promising results with the crude preparation justify the undertaking of purification studies and the use of the pure enzyme in a more in-depth investigation for its potential in biocatalysis in organic solvents.

Characterisation of Accurel MP1004 polypropylene powder and its use as a support for lipase immobilisation

Accurel MP1004, a porous polypropylene powder, was characterised for lipase immobilisation. The particle size ranges between 40–80 mesh, corresponding to particle diameters of 177–420 μm. The pore distribution falls between macroporous and mesoporous domains. A crude lipase preparation from Mucor javanicus was immobilised on Accurel MP1004 by adsorption. During the adsorption process, by measuring the variation of pore volume and of pore size distribution of mesopores as a function of enzyme loading, a significant penetration of the enzyme molecules into the pores was found to occur. The various proteins in the crude lipase preparation are quickly adsorbed by the Accurel MP1004. However, they are progressively displaced by the lipase which shows a greater affinity for the support. A transesterification reaction, between glycerol tricaprylate and 1-butanol in solvent-free conditions, was catalysed by the lipase before and after the immobilisation process. The immobilisation on Accurel MP1004 improves the lipase performance both in terms of activity and of substrate conversion.

Effect of immobilization site and membrane materials on multiphasic enantiocatalytic enzyme membrane reactors.

In the experimental work reported here the optical resolution of racemic naproxen methyl ester with crude lipase immobilized in a membrane reactor was studied. The multiphasic enantiocatalytic enzyme membrane reactor consisted of an organic phase that dissolved the naproxen methyl ester, a lipase-loaded membrane, and an aqueous phase that extracted the reaction product. Lipase preferentially converted the (S)-naproxen methyl ester to (S)-naproxen acid that was simultaneously separated by a membrane. The effect of the immobilization site and membrane type on the performance of the enzyme membrane reactor was studied. Capillary polyamide membrane with 10 kDa nominal molecular weight cutoff (NMWCO) and polysulfone membrane with 30 kDa NMWCO were applied, with lipase loaded in the sponge layer or on a membrane thin layer. With various immobilization sites and membrane types, the enzyme membrane reactors showed different productivity and enantioselectivity resulting from the varying amounts of immobilized enzyme and varying microenvironment of the hydrolysis reaction. Higher amounts of immobilized enzyme led to increased productivity and generally higher enantioslectivity of the membrane reactor. It seems that the location of organic/aqueous interface on membrane, which plays an important role in a multiphasic enzyme membrane reactor, was influenced by the immobilization site and membrane type and this affected the productivity and enantioselectivity. As much as 90% enantioexcess was obtained with a polyamide membrane, and lower values with a polysulfone membrane. A sponge layer of polyamide membrane is the preferred immobilization site for its higher productivity than a thin membrane layer, and for its higher enantioselectivity than a polysulfone membrane. In comparison with the unstable hydrolysis activity of free lipase in stirred tank reactor, a stable lipase activity can be obtained with the lipase-immobilized membrane reactor, irrespective of whether a polyamide or polysulfone membrane is used.

Purification and Partial Characterization of Psychrotrophic Serratia marcescens Lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6h at 30°C and after 12h at 6°C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24h at 4°C in buffer of pH 6.6. The optimum temperature was observed at 37°C and exhibited high activity at 5°C. The thermal inactivation of S. marcescens lipase was more obvious at 80°C; it retained about 15% of its original activity at 80°C and was completely inactivated after heating at 90°C for 5min. Under optimum conditions, activity of the enzyme was maximum after 6min. The Michaelis-Menten constant was 1.35mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5°C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6°C after 12h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.

Preparation of lipases for use in organic solvents

The efficiency of different preparations of lipases was evaluated in organic solvents. Lipases from Humicola lanuginosa, Candida rugosa, Rhizomucor miehei and Pseudomonas cepacia were adsorbed onto the surfactant sorbitan monostearate (Span 60) and the specific activities were compared in hexane to crude powder (used straight from the bottle) and lipase freeze-dried from buffer solution. Lipases adsorbed on the surfactant were “activated” 1.9- to 150-fold compared to the crude lipase. The solubility of the lipase-surfactant preparation in the reaction media was extremely low and the preparation contained aggregates of micrometer size. In further comparisons lipase from H. lanuginosa was freeze-dried in the presence of KCl, crown ethers, immobilised by entrapment into a sol–gel and immobilised on porous polypropylene support (Accurel EP-100). Addition of potassium chloride before freeze-drying of the lipase increased the activity up to 46-fold compared to crude powder. The additive probably worked as an immobilisation matrix for the lipase. When 18-crown-6 was added to the lipase before freeze-drying a 40-fold increase in activity was achieved. In this case a low amount of additive (0.4 mg crown ether/g protein) was needed for activation indicating that specific interactions were involved in the activation. In order to obtain maximal activity, immobilisation on Accurel EP-100 and entrapment into a sol–gel were the best methods to use. The activities were 400- and 320-fold better than that of crude powder. The high activities obtained were due to an improved dispersion of the catalyst in the organic media. The protein loading that could be used when the lipase was adsorbed onto Accurel EP-100 was much higher than what could be used when the lipase was entrapped into a sol–gel. This makes the adsorption technique the best for practical applications.