Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Recent advances and future prospects for biolubricant base stocks production using lipases as environmentally friendly catalysts: a mini-review.

In recent years, fluctuating global fossil fuel market prices and growing concern about environmental pollution have increased efforts to obtain novel value-added products from renewable agricultural biomass. To this end, a wide variety of triacylglycerols (edible and non-edible oils and fats) and their derivatives (free fatty acids or monoalkyl esters) stand out as promising feedstocks for the production of biolubricant base stocks, due to their biodegradability, excellent physicochemical properties, and sustainable nature. These raw materials can be transformed into biolubricants using chemical or biochemical (lipases) catalysts, with the enzymatic production of biolubricants using lipases as catalysts being recognized as an environmentally friendly approach. The present mini-review highlights recent advances in this field, published in the last three years. The different chemical modification processes used to develop a wide variety of industrial biolubricant base stocks are comprehensively reviewed, with exploration of future prospects for industrial production via the enzymatic route. This study contributes to the current state-of-the-art, identifying relevant research questions and providing important technical information for new applications of lipases in oleochemical manufacturing industries.

The immobilization protocol greatly alters the effects of metal phosphate modification on the activity/stability of immobilized lipases.

Mineralization of immobilized enzymes has showed to couple the advantages of both processes. Here, the influence of the immobilization protocol on the effects of mineralization has been investigated. The lipases from Thermomyces lanuginosus and Candida rugosa were immobilized on octyl-, vinyl sulfone (VS) octyl (blocked with different nucleophiles) and glutaraldehyde- (at different pH values) agarose beads. The stability, activity and specificity of the biocatalysts were very different, both the differently blocked VS-biocatalysts and the glutaraldehyde biocatalysts prepared at different pH. All biocatalysts were submitted to mineralization using different metals. The activity, specificity and stability effects of the mineralization strongly depended on the enzyme and on the immobilization protocol. For the same enzyme, a mineralization protocol could be negative, positive or present no effect depending on the enzyme immobilization procedure and substrate. In the best cases, activity could be increased by a two-fold factor, while stability was significantly improved in many instances. These results highlight the great potential of mineralization of immobilized enzymes to improve their properties, as well as the great interactions that immobilization protocol and mineralization can exhibit. The combination of both methodologies greatly increases the possibilities to find a biocatalyst that can be suitable for a specific process.

Improvement of functional properties of cow's milk peptides through partial proteins hydrolysis.

Allergy by cow's milk proteins is among the major food allergies and could be reduced by the partial hydrolysis of these proteins by proteases, without significantly affecting its physicochemical properties. In addition, the peptides generated through enzymatic hydrolysis of the cow's milk can present prebiotic and bioactive properties. In this work, the cow's milk proteins were submitted to a controlled hydrolysis by Novo-Pro D® and the influence of the degree of hydrolysis (DH) on peptide size distribution was evaluated, as well as the prebiotic and antimicrobial properties of milk hydrolysates. It was shown that for DH-10%, all the peptides have sizes lower than 12 kDa which is the size of the most allergenic proteins, without apparent changes in the milk, as long as heating of the hydrolysate is avoided. The protein hydrolysis promoted a great improvement in the milk functional properties. In addition, the obtained milk peptides presented great prebiotic activities, as indicated by the significant improvement of the growth of prebiotic L. acidophilus and L. reuteri and by the production of bacteriocins indicated by the inhibition halos in the growth of a pathogenic microorganism. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05533-x.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Stabilization of immobilized lipases by treatment with metallic phosphate salts.

Lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida rugosa (CRL), forms A and B of lipase from Candida antarctica (CALA and CALB) and Eversa Transform 2.0 have been immobilized on octyl-agarose beads at two different loads (1 mg/g and saturated support) and treated with phosphate and/or some metallic salts (Zn2+, Co2+, Cu2+). They have been also immobilized on the support modified by the metallic phosphate, usually driving to biocatalyst with lower stability or marginal improvements. The effects of the phosphate/metal modification on enzyme features depended on the loading of the support. Some enzymes (TLL, CRL or CALA), mainly using the highly loaded biocatalysts, showed very significant improvement on enzyme stability after the treatment with some of the metal phosphates (next to a 20-fold factor), improvements that were not justified by the presence of metallic or phosphate ions in solution, as they had negative effects on enzyme stabilities. In some other cases, a significant increase in enzyme activity was detected (e.g., CALB). This could be explained by the modification of the nucleation places of the enzymes by the metallic phosphate, and this could help to explain the good results obtained in the nanoflower immobilization of many enzymes.

High stabilization and hyperactivation of a Recombinant ß-Xylosidase through Immobilization Strategies.

Attainment of a stable and highly active ß-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant ß-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. ß-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L-1 h-1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized ß-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Design for preparation of more active cross-linked enzyme aggregates of Burkholderia cepacia lipase using palm fiber residue.

A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications.

High stabilization and hyperactivation of a recombinant β-Xylosidase through immobilization strategies

Attainment of a stable and highly active β-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant β-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. β-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L−1 h−1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized β-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization.

Hydroxyapatite (HA) nanoparticles are promising materials for enzyme immobilization, since they provide a high specific surface area for enzyme loading and can also be modified with metal ions (HA-Me2+) to enable interaction with proteins. The adsorption of proteins on HA-Me2+ has been explored for purification purposes, while the use of this material as a support for the immobilization of enzymes remains to be further investigated. Xylanase is an enzyme of considerable industrial interest, being used in the biofuel, pharmaceutical, pulp, and food & beverage sectors, among others. The immobilization of xylanase can enable recovery of the enzyme after biocatalysis, so that it can be reused several times, hence reducing the costs of industrial processes. Here, a systematic study was performed of the immobilization of xylanase on HA nanoparticles modified with metal ions (Cu2+ and Ni2+). A simple, fast, and efficient immobilization protocol was established using statistical experimental design as a tool, generating derivatives by interactions involving complexation of metals of the support with electron donor groups of the enzyme. The affinity towards xylanase was higher for the HA-Cu2+ support, compared to HA and HA-Ni2+. The pH and temperature profiles for the immobilized enzyme activity remained the same as for the soluble enzyme, indicating that the xylanase did not undergo major changes in its conformational state after immobilization. The HA-Cu2+ support was the most effective in reuse assays, retaining up to 80% activity in the second cycle. The results showed that xylanase could be immobilized on HA nanoparticles modified with Cu2+ and Ni2+ metal ions, using a simple and effective method, indicating the promising potential of the system for applications in different industrial processes.

Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

The enzyme phytase has important applications in animal feed, because it favors the bioavailability of phosphorus present in phytate, an antinutritional compound widely found associated with plant proteins. However, for feed applications, the phytase must withstand high temperatures during the feed pelleting process, as well as the gastrointestinal conditions of the animal. This work evaluates the feasibility of immobilizing phytase on hydroxyapatite (HA) nanoparticles, in order to improve its properties. HA is a material with excellent physicochemical characteristics for enzyme immobilization, and it can also act as an inorganic source of phosphorus and calcium in animal feed. The strong affinity of the phytase for the support resulted in rapid adsorption, with total immobilization yield and recovered activity greater than 100%. After immobilization, the phytase showed a broader activity profile in terms of pH and temperature, together with considerably higher thermoresistance at 80 and 90 °C. As a proof of concept, it was shown that the phytase immobilized on HA presented good resistance to acidic conditions and resistance to proteolysis when passing through simulated gastrointestinal conditions of fish. The findings showed that phytase immobilized onto HA presents suitable properties and has great potential for use in animal feed.

Correction to: Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

In the original version of this article, under Calculation of Immobilization Parameters heading, the presentation of the equations are incorrect. The correct presentation of the equations are given below.

Preparation, functionalization and characterization of rice husk silica for lipase immobilization via adsorption.

Silica has been extracted from rice husks via a simple hydrothermal process and functionalized with triethoxy(octyl)silane -OCTES (Octyl-SiO2) and (3-aminopropyl)triethoxysilane - 3-APTES (Amino-SiO2), with the aim of using it as support to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. The supports have been characterized by particle size distribution and elemental analyses, XRD, TGA, SEM, AFM and N2 physisorption so as to confirm their functionalization. Effect of pH, temperature, initial protein loading and contact time on the adsorption process has been systematically evaluated. Maximum immobilized protein loading of 12.3 ± 0.1 mg/g for Amino-SiO2 (5 mM buffer sodium acetate at pH 4.0, 25 °C and initial protein loading of 20 mg/g) and 21.9 ± 0.1 mg/g for Octyl-SiO2 (5 mM buffer sodium acetate at pH 5.0, 25 °C and initial protein loading of 30 mg/g) was observed. However, these biocatalysts presented similar catalytic activity in olive oil emulsion hydrolysis (between 630 and 645 U/g). TLL adsorption was a spontaneous process involving physisorption. Experimental data on Octyl-SiO2 and Amino-SiO2 adsorption were well-fitted to the Langmuir isotherm model. It was also investigated whether these biocatalysts could synthesize cetyl esters via esterification reaction. Thus, it was found that cetyl stearate synthesis required 100-110 min of reaction time to attain maximum conversion percentage (around 94%). Ester productivity of immobilized TLL on Amino-SiO2 was 1.3-3.1 times higher than Octyl-SiO2.

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems.

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100-200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.

Preparation of ion-exchange supports via activation of epoxy-SiO2 with glycine to immobilize microbial lipase - Use of biocatalysts in hydrolysis and esterification reactions.

Ion-exchange supports have been prepared via sequential functionalization of silica-based materials with (3­Glycidyloxypropyl)trimethoxysilane (GPTMS) (Epx-SiO2) and activation with glycine (Gly-Epx-SiO2) in order to immobilize lipase from Thermomyces lanuginosus (TLL) via adsorption. Rice husk silica (RHS) was selected as support with the aim of comparing its performance with commercial silica (Immobead S60S). Sequential functionalization/activation of SiO2-based supports has been confirmed by AFM, SEM and N2 adsorption-desorption analyses. Maximum TLL adsorption capacities of 14.8 ±â€¯0.1 mg/g and 16.1 ±â€¯0.6 mg/g using RHS and Immobead S60S as supports, respectively, have been reached. The Sips isotherm model has been used which was well fitted to experimental data on TLL adsorption. Catalytic activities of immobilized TLL were assayed by olive oil emulsion hydrolysis and butyl stearate synthesis via an esterification reaction. Hydrolytic activity of the biocatalyst prepared with a commercial support (357.6 ±â€¯11.2 U/g) was slightly higher than that of Gly-Epx-SiO2 prepared with RHS (307.4 ±â€¯7.2 U/g). On the other hand, both biocatalysts presented similar activity (around 90% conversion within 9-10 h of reaction) and reusability after 6 consecutive cycles of butyl stearate synthesis in batch systems.

Nanoimmobilization of ß-glucosidase onto hydroxyapatite.

ß-Glucosidase is an enzyme of great industrial interest that is used in biorefineries and in the pharmaceutical, food, and beverage sectors, among others. These industrial processes would benefit from the use of immobilized enzyme systems that allow several reuses of the enzyme. A promising inorganic and nontoxic material for such application is hydroxyapatite (HA), which can be synthesized at nanometric scale, hence providing good accessibility of the substrate to the catalyst. Here, we carried out a systematic study to evaluate the feasibility of immobilizing ß-glucosidase on HA nanoparticles. The immobilization process was highly effective over wide ranges of pH and ionic strength, resulting in immobilization yields and recovered activities up to 90%. Investigation of the type of interaction between ß-glucosidase and HA (using FT-IR, zeta potential measurements, and desorption tests with different salts) indicated the formation of coordination bonds between Ca2+ sites of HA and COO- of amino acids. Even after 10 cycles of reuse, the immobilized ß-glucosidase retained about 70% of its initial activity, demonstrating the operational stability of the immobilized enzyme. The results showed that ß-glucosidase could be efficiently immobilized on HA nanoparticles by means of a very simple adsorption protocol, offering a promising strategy for performing repeated enzymatic hydrolysis reactions.

Eucalyptus xylan: An in-house-produced substrate for xylanase evaluation to substitute birchwood xylan.

Over the past decades, most studies with xylanases have used Birchwood xylan as the standard substrate for activity assays. However, recently, Birchwood xylan production was discontinued by major suppliers, creating an important demand for a substitute. Ongoing and future studies require a substrate with characteristics equivalent to the discontinued xylan, in order to enable the comparison of results. In this context, a protocol for the production of a substrate similar to the discontinued commercial Birchwood xylan is reported. Obtained from bleached Eucalyptus cellulose pulp, xylan was extracted using 4% w/v NaOH solution at 25 °C, precipitated with glacial acetic acid (HOAc), and freeze-dried. A thermal pretreatment in an autoclave for 15 min increased its solubility. The resulting xylan was characterized by infrared spectroscopy, thermogravimetry, and NMR. When assessing the activity of xylanases, the results were the same as those for commercial Birchwood xylan.

1,3-Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates.

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 µmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium.

BACKGROUND: Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions. RESULTS: Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 µmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone. CONCLUSIONS: Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.

Immobilized Lipases on Functionalized Silica Particles as Potential Biocatalysts for the Synthesis of Fructose Oleate in an Organic Solvent/Water System.

Lipases from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) wereimmobilized on functionalized silica particles aiming their use in the synthesis of fructose oleate in a tert-butyl alcohol/water system. Silica particles were chemically modified with octyl (OS), octyl plus glutaraldehyde (OSGlu), octyl plus glyoxyl(OSGlx), and octyl plus epoxy groups(OSEpx). PFL was hyperactivated on all functionalized supports (more than 100% recovered activity) using low protein loading (1 mg/g), however, for TLL, this phenomenon was observed only using octyl-silica (OS). All prepared biocatalysts exhibited high stability by incubating in tert-butyl alcohol (half-lives around 50 h at 65 °C). The biocatalysts prepared using OS and OSGlu as supports showed excellent performance in the synthesis of fructose oleate. High estersynthesis was observed when a small amount of water (1%, v/v) was added to the organic phase, allowing an ester productivity until five times (0.88-0.96 g/L.h) higher than in the absence of water (0.18-0.34 g/L.h) under fixed enzyme concentration (0.51 IU/g of solvent). Maximum ester productivity (16.1-18.1 g/L.h) was achieved for 30 min of reaction catalyzed by immobilized lipases on OS and OSGlu at 8.4 IU/mL of solvent. Operational stability tests showed satisfactory stability after four consecutive cycles of reaction.