Role of methoxypolyethylene glycol on the hydration, activity, conformation and dynamic properties of a lipase in a dry film

A combined approach based on the use of ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformation and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molecule when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.4- and 4.7-fold higher than in the absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG acts by hindering intermolecular protein-protein interactions and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for the preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology.     

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.     

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity,enantioselectivity, and conformation

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.     

A lipid-coated lipase as an efficient hydrolytic catalyst in the two-phase aqueous-organic system

The activity of different formulations of Candida antarctica lipase B (CALB), such as crude CALB, purified CALB, purified CALB lyophilized with PEG (CALB + PEG) or oleic acid (CALB + OA), and the commercial formulation Novozym 435, was determined in toluene, carbon tetrachloride, and 1,4-dioxane at various water activities (a(w)). The reaction between vinylacetate and 1-octanol was used as the model reaction and both transesterification (formation of 1-octylacetate) and hydrolytic (formation of acetic acid from vinylacetate) activities were determined. For equal amounts of lipase protein, CALB + PEG (and to a lesser extent CALB + OA) displayed higher activity than that of the other formulations; for instance, in toluene (a(w) < 0.1), it was 260-, 13-, and 1.8-fold more active than crude CALB, purified CALB, and Novozym 435, respectively. Moreover, the transesterification activity of CALB + PEG was of the same order of magnitude (51%) of the activity shown by the enzyme in the hydrolysis of vinylacetate in aqueous buffer. These results suggest that PEG and oleic acid could act as lyoprotectants, preventing the formation of intermolecular interactions during the lyophilization process that might be responsible for protein denaturation. No diffusional limitation was observed for CALB + PEG-catalyzed reactions. Purified CALB, in contrast to the other formulations, showed a marked activity increase (2.1 to 7.8-fold) as a function of a(w) and, in 1,4-dioxane, it was 3.5-fold more active when it was added to the solvent after previous dissolution of the lyophilized powder in water.     

Sol-gel powders and supported sol-gel polymers for immobilization of lipase in ester synthesis

Candida rugosa lipase was entrapped in hybrid organic–inorganic sol-gel powder prepared by acid-catalyzed polymerization of tetramethoxysilane (TMOS) and alkyltrimethoxysilanes, and used in catalyzing esterification reactions between ethanol and butyric acid in hexane. Optimum preparation conditions were studied, which are gels made from propyltrimethoxysilane (PTMS)/TMOS molar ratio=4:1, hydrolysis time of silane precursor=30 min, water/silane molar ratio=24, enzyme loading=6.25% (w/w) of gel, and 1 mg PVA/mg lipase. The percentage of protein immobilization was 95% and the resulting lipase specific activity was 59 times higher than that of a non-immobilized lyophilized lipase. To prepare magnetic lipase-immobilized sol-gel powder (MLSP) for easier recovery of the biocatalyst, Fe₃O₄ nanoparticles were prepared and co-entrapped with lipase during gel formation. This procedure induced surface morphological change of the sol-gel powder and showed adverse effect on enzyme activity. Hence, although only 9% decrease in protein immobilization efficiency was observed, the corresponding reduction in enzyme activity could be up to 45% when sol-gel powder was doped with 25% (v/v) Fe₃O₄ magnetic nanoparticles solution. Lipase-immobilized sol-gel polymer was also formed within the pores of different porous supports to improve its mechanical stability. Non-woven fabric, with a medium pore size of all the supports tested, was found to be the best support for this purpose. The thermal stability of lipase increased 55-fold upon entrapment in sol-gel materials. The half-lives of all forms of sol-gel-immobilized lipase were 4 months at 40 °C in hexane.     

Modulation of lipase hydrolysis and synthesis reactions using carbohydrates.

A novel method for modulation of lipase hydrolysis and synthesis lipase was investigated by using carbohydrates in the microenvironment of the Candida rugosa enzyme. The influence of the addition of different sugars to the previously dialysed enzyme was tested on the two reactions. Rates of hydrolysis were lowered by using dialysed enzyme but were increased after sugar addition, regardless of the identity of the added sugar. In contrast, synthesis reaction rates depended on the nature of the carbohydrate. Rates were increased by adding lactose, which is not a water activity depressor, but were lowered by adding fructose, glucose, sucrose or sorbitol, which are all water activity depressors.     

Extraordinary enantiospecificity of lipase catalysis in organic media induced by purification and catalyst engineering

A purified lipase preparation from Candida rugosa was compared to its crude counterpart in anhydrous and slightly hydrated hydrophobic organic solvents. The purified lipase preparation was less active than the crude enzyme in dry n-heptane, whereas the presence of small concentrations of added water dramatically activated the purified enzyme but not the crude enzyme. Thus, in the presence of as little as 0.25 muL/mL of added water in n-heptane, the purified enzyme is over 230-fold more active and 6-fold more enantioselective than the dry enzyme suspension in the esterification of racemic 2-(4-chlorophenoxy)propionic acid with n-butanol. The reactivity and selectivity of this biocatalyst, however, was affected by coalescence of the enzyme preparation suspended in the wet organic solvent. Engineering the biocatalyst environment by dissolving the purified lipase in aqueous buffer and then adding this solution to n-heptane resulted in a precipitated enzyme preparation with smaller particle sizes that did not coalesce severely. In the presence of 5 muL/mL of water added with the enzyme, this pretreatment resulted in an activation over the dry, purified enzyme preparation of over 1800-fold and nearly enantiospecific catalysis (E > 100). Hence, by simply modifying the way enzymes are hydrated, dramatic activation of catalytic competency can be achieved. (c) 1996 John Wiley & Sons, Inc.     

Coprecipitation of Pseudomonas fluorescens Lipase with Hydrophobic Compounds As an Approach to Its Immobilization for Catalysis in Nonaqueous Media

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.     

A glycerol-inducible thermostable lipase from Bacillus sp.: medium optimization by a Plackett-Burman design and by response surface methodology

The production of a neutral lipase from a Bacillus sp. was improved tremendously (193-fold) following media optimization involving both the "one-at-a-time" and the statistical designing approaches. The present lipase was poorly induced by oils, instead its production was induced in the presence of sugars and sugar alcohols, mainly galactose, lactose, glycerol, and mannitol. A high inoculum density of 15% v/v (A550 = 0.8) led to maximum lipase production. Interestingly, the enzyme induction was growth independent, a property very different from most of the lipases investigated to date. The optimal composition of the growth medium to achieve maximum lipase production was determined to be as follows: NH4Cl, 35 g x L(-1); glycerol, 10 mL x L(-1); K2HPO4, 3 g x L(-1); KH2PO4, 1 g x L(-1); MgSO4.7H2O, 0.1 g x L(-1); glucose, 2 g x L(-1); MgCl2, 0.6 mmol x L(-1), with 15% inoculum density and an incubation period of 24 h. About 62 U x mL(-1) of enzyme production was achieved in the optimized medium.     

Analysis of the products from enzymatic scouring of cotton

This article discusses the analysis of the hydrolysis products from one-step scouring of cotton using pectinase and two-step scouring of cotton using lipase then cellulase, protease then cellulase, or lipase/protease then cellulase, to improve water absorbency of cotton. UV spectrophotometric analysis indicated that the pectinase scouring process produced approximately 18-fold higher amounts of reducing sugars and galacturonic acid than any of the two-step scouring processes. The production rate of reducing sugars and galacturonic acid from most of the scouring processes showed a decrease with an increase in time. HPLC analysis revealed that the lipase/protease/cellulase scouring processes produced approximately 5-fold higher amounts of 17 amino acids than the pectinase scouring process. GC analysis for 18 fatty acids (C(8)-C(24)) revealed that three major fatty acids, palmitic acid, stearic acid, and behenic acid, were found on both the scoured and the unscoured fabrics. Scoured fabrics were tested for content of proteins, extractable components, waxes, and anionic components including pectins, and some differences among the fabric scoured with different enzyme combinations were found.     

Coprecipitation of the Pseudomonas fluorescens lipase with hydrophobic compounds as an approach to its immobilization for catalysis in nonaqueous media

The precipitation of N-cetylamine, N-cetylacetamide, hexan-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone-water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. The immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme. The English version of the paper.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents

The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 degrees C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 degrees C, the activation effect on the lyophilized protease is even higher, reaching 1000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, alpha-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous millieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts. (c) 1996 John Wiley & Sons, Inc.     

Fourier‐transform infrared spectroscopy study of dehydrated lipases from Candida antarctica B and Pseudomonas cepacia

Fourier‐transform infrared (FT‐IR) spectroscopy was employed to investigate potential lyophilization‐induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the α‐helix and increased the β‐sheet content. However, FT‐IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the β‐sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT‐IR for analysis of the structure of solid proteins, at least as far as the β‐sheet content is concerned, because it is possible that the method overestimates the β‐sheets by measuring other hydrogen‐bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 545–551, 1999.     

Role of solvent on protein-matrix coupling in MbCO embedded in water-saccharide systems: a Fourier transform infrared spectroscopy study

Embedding protein in sugar systems of low water content enables one to investigate the protein dynamic-structure function in matrixes whose rigidity is modulated by varying the content of residual water. Accordingly, studying the dynamics and structure thermal evolution of a protein in sugar systems of different hydration constitutes a tool for disentangling solvent rigidity from temperature effects. Furthermore, studies performed using different sugars may give information on how the detailed composition of the surrounding solvent affects the internal protein dynamics and structural evolution. In this work, we compare Fourier transform infrared spectroscopy measurements (300-20 K) on MbCO embedded in trehalose, sucrose, maltose, raffinose, and glucose matrixes of different water content. At all the water contents investigated, the protein-solvent coupling was tighter in trehalose than in the other sugars, thus suggesting a molecular basis for the trehalose peculiarity. These results are in line with the observation that protein-matrix phase separation takes place in lysozyme-lactose, whereas it is absent in lysozyme-trehalose systems; indeed, these behaviors may respectively be due to the lack or presence of suitable water-mediated hydrogen-bond networks, which match the protein surface to the surroundings. The above processes might be at the basis of pattern recognition in crowded living systems; indeed, hydration shells structural and dynamic matching is first needed for successful come together of interacting biomolecules.     

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.     

Isolation of a Aspergillus niger lipase from a solid culture medium with aqueous two-phase systems

The aim of this work is to find the best conditions to isolate lipase from a solid culture medium of Aspergillus niger NRRL3 strains using aqueous two-phase systems formed with polyethylene glycol and potassium phosphate or polyethylene glycol and sodium citrate. We studied the partitioning of a commercial lyophilizate from A. niger. Also, the lipase enzymatic activity was studied in all the phases of the systems and the results indicate that citrate anion increases lipase activity. An analysis by fluorescence spectroscopy of the interaction between lipase and the bottom and top phases of the systems shows that the protein tryptophan-environments are modified by the presence of PEG and salts. Separation of the enzyme from the rest of the proteins that make up the lyophilized was achieved with good yield and separation factor by ATPS formed by PEG 1000/Pi at pH 7, PEG 2000/Ci at pH 5.2 and PEG 4000/Ci at pH 5.2. The above mentioned systems were used in order to isolate extracellular lipase from a strain of A. niger in submerged culture and solid culture. The best system for solid culture, with high purification factor (30.50), is the PEG 4000/Ci at pH 5.2. The enzyme was produced in a solid culture medium whose production is simple and recovered in a phase poor in polymer, bottom phase. An additional advantage is that the citrate produces less pollution than the phosphate. This methodology could be used as a first step for the isolation of the extracellular lipase from A. niger.     

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.     

Regulation of Leaf Senescence by Cytokinin, Sugars, and Light : Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.