Ester synthesis in organic solvent catalyzed by lipases immobilized on hydrophilic supports

Summary Eight microbial lipases and one animal lipase were immobilized on hydrophilic supports either by adsorption or entrapment. All preparations catalyzed the synthesis of geranyl or menthyl butyrate or laurate using heptane as solvent. This is a simple and easy method for ester synthesis.     

Immobilization of lipases on hydrophobic supports: immobilization mechanism,advantages, problems,and solutions

Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic, vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.     

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.     

Modification of hydrophilic and hydrophobic surfaces using an ionic-complementary peptide

Ionic-complementary peptides are novel nano-biomaterials with a variety of biomedical applications including potential biosurface engineering. This study presents evidence that a model ionic-complementary peptide EAK16-II is capable of assembling/coating on hydrophilic mica as well as hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces with different nano-patterns. EAK16-II forms randomly oriented nanofibers or nanofiber networks on mica, while ordered nanofibers parallel or oriented 60 degrees or 120 degrees to each other on HOPG, reflecting the crystallographic symmetry of graphite (0001). The density of coated nanofibers on both surfaces can be controlled by adjusting the peptide concentration and the contact time of the peptide solution with the surface. The coated EAK16-II nanofibers alter the wettability of the two surfaces differently: the water contact angle of bare mica surface is measured to be <10 degrees , while it increases to 20.3+/-2.9 degrees upon 2 h modification of the surface using a 29 microM EAK16-II solution. In contrast, the water contact angle decreases significantly from 71.2+/-11.1 degrees to 39.4+/-4.3 degrees after the HOPG surface is coated with a 29 microM peptide solution for 2 h. The stability of the EAK16-II nanofibers on both surfaces is further evaluated by immersing the surface into acidic and basic solutions and analyzing the changes in the nanofiber surface coverage. The EAK16-II nanofibers on mica remain stable in acidic solution but not in alkaline solution, while they are stable on the HOPG surface regardless of the solution pH. This work demonstrates the possibility of using self-assembling peptides for surface modification applications.     

From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: an atomic force microscopy study

The sequence of events involved in the transition from attached liposomes to bilayer patches on hydrophilic and hydrophobic solid supports were visualized in situ by Tapping Mode atomic force microscopy in liquid. In a smooth manner, the attached liposomes spread and flattened from the outer edges toward the center until the two membrane bilayers were stacked on top of each other. The top bilayer then either rolls or slides over the bottom bilayer, and the adjacent edges join to form a larger membrane patch. This is clearly visible from the apparent height of 6.0-7.5 nm of the single bilayer, measured in situ. The addition of calcium appeared to increase the rate of the processes preventing the visualization of the intermediate stages. The same intermediate steps appeared to be present on hydrophobic surfaces, although the attached liposomes seemed to be distorted and the resultant membrane edges were uneven. This work has provided visual and detailed information on liposome coalescence (fusion) onto solid supports and demonstrated how the atomic force microscope can be used to study the process.     

Lipase immobilized on hydrophobic porous polymer supports prepared by concentrated emulsion polymerization and their activity in the hydrolysis of triacylglycerides

Microporous polymer supports for the immobilization of lipase have been prepared by the polymerization of a concentrated emulsion precursor. The concentrated emulsion consists of a mixture of styrene and divinyl-benzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. The volume fraction of the latter phase was greater than 0.74, which is the volume fraction of the dispersed phase for the most compact arrangement of spheres of equal radius. The lipase from Candida rugosa has been immobilized on the internal surface of the hydrophobic microporous poly(styrene-divinyl benzene) supports and used as biocatalysts for the hydrolysis of triacylglycerides. The effects of the amount of surfactant, of the molar ratio of divinylbenzene/styrene in the continuous phase, and of the aquaphilicity of the supports on the adsorption, activity, and stability of the immobilized lipase have been investigated. The microporous poly(styrene-divinylbenzene) adsorbents constitute excellent supports for lipase because both the amount adsorbed is large and the rate of enzymatic reaction per molecule of lipase is higher for the immobilized enzyme than for the free one. (c) 1993 John Wiley & Sons, Inc.     

Preparation of new lipases derivatives with high activity–stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media.

Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst). Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.     

Drastically lowered protein adsorption on microbicidal hydrophobic/hydrophilic polyelectrolyte multilayers

Polyelectrolyte multilayer films assembled from a hydrophobic N-alkylated polyethylenimine and a hydrophilic polyacrylate were discovered to exhibit strong antifouling, as well as antimicrobial, activities. Surfaces coated with these layer-by-layer (LbL) films, which range from 6 to 10 bilayers (up to 45 nm in thickness), adsorbed up to 20 times less protein from blood plasma than the uncoated controls. The dependence of the antifouling activity on the nature of the polycation, as well as on assembly conditions and the number of layers in the LbL films, was investigated. Changing the hydrophobicity of the polycation altered the surface composition and the resistance to protein adsorption of the LbL films. Importantly, this resistance was greater for coated surfaces with the polyanion on top; for these films, the average zeta potential pointed to a near neutral surface charge, thus, presumably minimizing their electrostatic interactions with the protein. The film surface exhibited a large contact angle hysteresis, indicating a heterogeneous topology likely due to the existence of hydrophobic-hydrophilic regions on the surface. Scanning electron micrographs of the film surface revealed the existence of nanoscale domains. We hypothesize that the existence of hydrophobic/hydrophilic nanodomains, as well as surface charge neutrality, contributes to the LbL film's resistance to protein adsorption.     

Adsorption of fibrinogen on silicon oxide with controlled hydrophobic/hydrophilic properties

The amount of fibrinogen irreversibly adsorbed on silicon dioxide does not exceed 3.6 pmol/cm2 and depends on the protein concentration, solution pH and surface hydrophobic/hydrophilic properties. Electrostatic interactions determine the fibrinogen adsorption rate. Partial denaturation of fibrinogen takes place in its adsorption form diluted solutions with the pH value lower than the protein isoelectric point.     

PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports

Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, β‐lactoglobulin, and lysozyme. All proteins were PEGylated in the N‐terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono‐ and di‐PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of β‐lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702–707, 2016     

Molecular dynamics study of the folding of hydrophobin SC3 at a hydrophilic/hydrophobic interface

Hydrophobins are fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes. These assemblages are extremely stable and posses the remarkable ability to invert the polarity of the surface on which they are adsorbed. Neither the three-dimensional structure of a hydrophobin nor the mechanism by which they function is known. Nevertheless, there are experimental indications that the self-assembled form of the hydrophobins SC3 and EAS at a water/air interface is rich with beta-sheet secondary structure. In this paper we report results from molecular dynamics simulations, showing that fully extended SC3 undergoes fast (approximately 100 ns) folding at a water/hexane interface to an elongated planar structure with extensive beta-sheet secondary elements. Simulations in each of the bulk solvents result in a mainly unstructured globular protein. The dramatic enhancement in secondary structure, whether kinetic or thermodynamic in origin, highlights the role interfaces between phases with large differences in polarity can have on folding. The partitioning of the residue side-chains to one of the two phases can serve as a strong driving force to initiate secondary structure formation. The interactions of the side-chains with the environment at an interface can also stabilize configurations that otherwise would not occur in a homogenous solution.     

Extramatrical structures of hydrophobic and hydrophilic ectomycorrhizal fungi

The extramatrical mycelia of Suillus bovinus, Rhizopogon luteolus and R. vinicolor, all examples of hydrophobic (ho), mat-forming mycorrhizal fungi, were examined while associated with their hosts in the unsterilized rhizoscope, and efforts were made to produce and examine similar structures in vitro. Comparisons were made with four hydrophilic (hi) mycorrhizal fungi, Thelephora terrestris, Cenococcum geophilum, Laccaria laccata and Hebeloma crustuliniforme. The ho fungi formed linear structures (coarse, rhizomorph-like cords, with vessels in the center) and fans, both in the rhizoscope and in vitro. The same was seen in mycorrhizal mycelia in forest soils. These cords did not themselves give rise to the fans peripherally, and were not proper rhizomorphs, but were created continuously from single exploring air hyphae in the preexisting fan. Thus the ho exploring hyphae aggregated into strands, which grew in thickness only when no suitable, exploitable substrate was found. The assembly of hyphae creating ho cords was seen in the air as well as on inert hydrophilic (glass) or hydrophobic (plastic) surfaces, but never in water. It is hypothesized that the ho cell wall surface glues hyphae together while cords are formed. Water disturbed strands and mantles already formed. The ho exploring hyphae could also create ho mycelial patches (as in a mat) at the water-air interface of a number of substrates. The periphery of these patches seemed to be composed of shorter exploiting hyphae penetrating different water-soaked substrates. Exploring, aerial hyphal tips of the ho fungi were shown to excrete water droplets from openings in the ho cell wall surface, both in vitro and in the rhizoscope. In the rhizoscope, droplet excretion was apparently directly governed by photosynthesis in the shoot of the seedling. It is proposed that the drop exudation represents a kidney-like function of the extramatrical hyphae and a bridge to drier soil particles to initiate nutrient uptake by the hyphae. The ecological function of the different extramatrical structures of ho fungi are discussed. The ho cords or hyphae may translocate water only in the vessels or symplastically and not in the cell walls. The ho property may be essential among the S-selected (stress-tolerant) factors in these forest fungi. The transfer from water-repelling exploring structures into more hi exploiting structures in water contact with surrounding soil debris is, therefore, of great importance. The hi fungi did not form rhizomorph-like strands, in most cases, but an extending hyphal mycelium, representing foraging, exploring and exploiting structures at the same time. In the field, short strands may be found. On the hi fungi droplets were also produced but readily fused into a water sheath around the hypha. The hyphae thus tended to wick water via the cell wall.     

Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers.

Saccharification and adsorption characteristics of native and modified cellulases were investigated. Copolymers, containing polyoxyalkylene and maleic anhydride (MA) were used to modify cellulase. Amino groups of the cellulase were covalently coupled with the MA. As the degree of modification (DM) increased, the activity of modified cellulase slightly decreased. At the maximum DM, the modified cellulase activity retained more than 75% of the unmodified native cellulase activity. In saccharification, native cellulase rapidly adsorbed onto the substrate at initial reaction time. Native cellulase adsorbed tightly onto the substrate surface and did not desorb as reaction time proceeded. The strong adsorption of cellulase onto the substrate can, however, be controlled by the modification. As the hydrophilicity of modified cellulase increased, free modified enzyme concentration also increased. As a result, the conversion rate of modified cellulase was higher than the native one.     

Modulation of the properties of immobilized CALB by chemical modification with 2,3,4-trinitrobenzenesulfonate or ethylendiamine. Advantages of using adsorbed lipases on hydrophobic supports

Lipase B from Candida antarctica (CALB) has been adsorbed on octyl-agarose or covalently immobilized on cyanogen bromide agarose. Then, both biocatalysts have been modified with ethylenediamine (EDA) or 2,4,6-trinitrobenzensulfonic acid (TNBS) just using one reactive or using several modifications in a sequential way (the most complex preparation was CALB–TNBS–EDA–TNBS). Covalently immobilized enzyme decreased the activity by 40–60% after chemical modifications, while the adsorbed enzyme improved the activity on p-nitrophenylbutyrate (pNPB) by EDA modification (even by a 2-fold factor). These biocatalysts were further characterized. The results showed that the effects of the chemical modification on the enzyme features were strongly dependent on the immobilization protocol utilized, the experimental conditions where the catalyst will be utilized, and the substrate. Significant changes in the activity/pH profile were observed after the chemical modifications. The effect of the modifications on the enzyme activity depends on the substrate and the reaction conditions: enzyme specificity is strongly altered by the chemical modification. Moreover, enzyme activity versus pNPB (using octyl-CALB–EDA) or versus R methyl mandelate (using octyl-CALB–TNBS) increased by almost a 2-fold factor at pH 5. The stability of the modified enzymes at different pH and in the presence of organic solvents generally decreased after the modifications, usually by no more than a 2-fold factor. However, under some conditions, some stabilization was found. CALB enantioselectivity in the hydrolysis of R/S methyl mandelate could be also improved by these chemical modifications (e.g., E-value went from 11 to 16 using octyl-CALB–TNBS at pH 5). Therefore, solid phase chemical modification of immobilized lipases may become a powerful tool in the design of lipase libraries with very different properties, each immobilized preparation may be used to produce a variety of forms with altered properties.     

Isolation of hydrophobic and hydrophilic variants of Candida albicans

We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.     

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF‐SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF‐SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.