Improved catalytic properties of immobilized lipases by the presence of very low concentrations of detergents in the reaction medium

The addition of a very small concentration of a detergent (in many instances under the critical micellar concentration (cmc)) has been found to greatly increase the activity of immobilized lipases, using those from Pseudomonas fluorescens (PFL) and Candida antarctica (isoform B) as model enzymes. However, the detergents may also have a negative effect on enzyme activity; in fact, for all enzyme preparations and substrates the activity/detergent concentration curve reached a maximum value and started to decrease, in many instances even under the initial value. The concentration and nature of the detergent (SDS, CTAB, Triton X-100, or X-45) that permitted the maximum hyperactivation was different depending on the substrate. The best hyperactivation values promoted by the presence of detergent were over a 20-fold factor. The presence of detergents permitted the inhibition of lipases by irreversible covalent inhibitors (e.g., 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) (AEBSF) while the enzyme, in the absence of detergent, is not inhibited by these irreversible inhibitors. This suggested that the main effect of the detergents is to shift the conformational equilibrium of lipases toward the open form. Moreover, the presence of detergents also permitted to improve the enantioselectivity exhibited by the immobilized lipases in some cases. For example, the enantioselectivity of PFL-glyoxyl agarose increased from 40 to more than 100 in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester by using 0.1% CTAB.     

General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality

Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration. The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents. However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent. It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization. For example, with use of the lipase from H. lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used. It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica

Glutaraldehyde chemistry has been used to immobilize lipase B from Candida antarctica (CALB) under different situations. Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support via interfacial activation. Using non-ionic detergents (e.g., Triton X-100), the enzyme becomes ionically adsorbed on the activated support. If detergent and salt are simultaneously present during immobilization, a covalent attachment to the support is first produced. In absence of detergent or high ionic strength, a mixture of all of the previous immobilization reasons should coexist. Thus, 5 different CALB biocatalysts were prepared following the previous described protocols, and its stability and activity, pH/activity profile and specificity versus R and S methyl mandelate were analyzed. The existence of covalent attachment of more than 95% of the enzyme molecules was confirmed by washing the biocatalysts in salt and detergent solutions. The glutaraldehyde treatment of the enzyme adsorbed on aminated supports did not produce a significant improvement on the activity of the enzyme versus p-nitrophenylpropinate (pNPB) nor a high stabilization of the enzyme. This differed from the effects of a similar treatment of CAL adsorbed on octyl agarose. However, they were similar to the effects of this treatment on covalently immobilized CALB, suggesting that the immobilization protocol may greatly affect the final effect of a chemical modification on the enzyme properties.Dramatic changes in the enzyme features were observed comparing the different preparations, mainly in the specificity of CALB versus p-NPB and R-methyl mandelate (from 2.5 to 20), or in the enantiospecificity versus R/S methyl mandelate (from 1.8 to 16), confirming that these different immobilization protocols produced biocatalysts with different features. Moreover, changes in experimental conditions produced very different effects on the properties of the different CALB preparations.     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

Template enhanced activity of lipase accommodated in siliceous mesocellular foams

Lipases were adsorbed in siliceous mesocellular foams containing different amounts of residual template in the nanopores. It is found that the hydrolytic activities of the adsorbed lipases are increased with increasing the contents of template in the mesopores. The triacetin hydrolytic activity of the lipase adsorbed in the foam containing 46% of template can be 13 times higher than that of the lipase adsorbed in the foam without template in the nanopores, and its specific activity is about three times higher than that of the free lipase, showing the hyperactivation effect on lipase resulting from the interaction between the lipase and the surfactant in the nanopores. The immobilized lipase cross-linked with glutaraldehyde can retain up to 88% of its original activity after six hydrolysis reaction test. This work provides a new strategy to enhance the activity of immobilized lipase in mesoporous materials.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Lipolytic enzymes with improved activity and selectivity upon adsorption on polymeric nanoparticles

Nanostructured polystyrene (PS) and polymethylmethacrylate (PMMA) were used as carriers for the preparation of bioconjugates with lipolytic enzymes, such as Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL). Simple addition of the lipase solution to the polymeric nanoparticles under protein-friendly conditions (pH 7.6) led to the formation of polymer-enzyme bioconjugates. Energy filtered-transmission electron microscopy (EF-TEM) performed on immuno-gold labeled samples revealed that the enzyme preferentially binds to the polymer nanoparticles and that the binding does not affect the nanostructured features of the carriers. The studies performed on the activity of the bioconjugates pointed out that the lipases adsorbed onto polymeric nanoparticles show an improved performance in terms of activity and selectivity with respect to those shown by lipases adsorbed on the same non-nanostructured carriers. The residual activities of CRL and PCL immobilized on nanostructured PMMA and PS reached 60% and 74%, respectively. Moreover, we found that enantioselectivity and pH and thermal stability increase upon immobilization. These results highlight the fact that new protein conformers with improved enantioselectivity stabilized after adsorption on nanoparticles are obtained. On the basis of the chemical structures of the selected polymers and the slopes of the adsorption isotherms, a hydrophobic binding model for lipase/nanostructured polymers is suggested.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations

Adsorption onto solid supports has proven to be an easy and effective way to improve the mechanical and catalytic properties of lipases. Covalent binding of lipases onto the support surface enhances the active lifetime of the immobilized biocatalysts. Our study indicates that mesoporous silica gels grafted with various functions are ideal supports for both adsorptive and covalent binding for lipase B from Candida antarctica (CaLB). Adsorption of CaLB on phenyl-functionalized silica gels improved in particular its specific activity, whereas adsorption on aminoalkyl-modified silica gels enabling covalent binding with the proper reagents resulted in only moderate specific activity. In addition, adsorption on silica gels modified by mixtures of phenyl- and aminoalkyl silanes significantly increased the productivity of CaLB. Furthermore, CaLB adsorbed onto a phenyl/aminoalkyl-modified surface and then treated with glutardialdehyde (GDA) as cross-linking agent provided a biocatalyst of enhanced durability. Adsorbed and cross-linked CaLB was resistant to detergent washing that would otherwise physically deactivate adsorbed CaLB preparations. The catalytic properties of our best immobilized CaLB variants, including temperature-dependent behavior were compared between 0 and 70 °C with those of two commercial CaLB biocatalysts in the continuous-flow kinetic resolutions of racemic 1-phenylethanol rac-1a and 1-phenylethanamine rac-1b.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Enzymic interesterification of fats: Laboratory and pilot-scale studies with immobilized lipase from Rhizopus arrhizus

An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth. As observed previously with a less active enzyme from Aspergillus sp., the interesterification activity was enhanced by addition of purified lipase or by high loadings of commercial enzyme. The interesterification activities reached maximum values in both cases. For immobilized preparations with purified enzyme, interesterification activity was also enhanced by the presence of a precoat of glutaraldehyde cross-linked commercial lipase. A 2.9-L column of immobilized lipase was used to interesterify batches of shea oleine (67 kg) and shea oil (40 kg). Little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.     

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.     

Studies on the mechanism of the lipase reaction. II. Comparative studies on the adsorption of lipases and various proteins at the air-water interface.

Adsorption of lipases (EC 3.1.1.3) and various proteins at the air-water interface has been investigated in relation to the mechanism of lipase reaction. Aqueous solutions of lipases and denaturated proteins show surface activity as strong as that of synthetic detergents. However, ths surface activity of esterases and various other proteins is little or none. By foam fractionation it was shown that lipases were adsorbed at the air-water interface and the adsorption followed the equation of Langmuir's adsorption isotherm. The properties of lipase at the interface are discussed in relation to the mechanism of lipase reaction and the differences from the esterase reaction.