Application of Goat and Lamb Lipases on the Development of New Immobilized Biocatalysts Aiming at Fish Oil Hydrolysis.

The use of lipases from animal sources for the synthesis of new biocatalysts is barely studied in the literature. The present work focused on the immobilization of lipases from kid goat's and lamb's epiglottis in different ionic supports. For this, anionic supports (monoaminoethyl-N-aminoethyl-agarose (MANAE) and diethylaminoethyl-agarose (DEAE)) and cationic supports (carboxymethyl-agarose and sulfopropyl-agarose) were used. The immobilization parameters were evaluated, as well as the thermal stability of the immobilized enzymes and their stability at different values of pH. Then, the performance of the biocatalysts was evaluated in hydrolysis reactions for obtaining omega-3 fatty acids from fish oil (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)). Values of 100% of recovered activity were obtained for lipase from goats, indicating that it was possible to maintain all the enzymatic activities of the immobilized enzymes on the supports. The immobilized enzymes were more stable in different pH conditions and at a temperature of 50 °C, reaching values of stabilization factor of 12.17 and t1/2 of 9.86 h-1, for lamb lipase immobilized in sulfopropyl agarose. In general, the anionic supports led to lower Km values and the cationic ones to a higher Vmax. Lamb lipase showed the highest selectivity values for EPA/DHA, reaching values of 6.43 using MANAE. Thus, the high potential for using such biocatalysts from animal sources in the food or pharmaceutical industries is observed.

Functionalization of Porous Cellulose with Glyoxyl Groups as a Carrier for Enzyme Immobilization and Stabilization.

The functionalization of the internal surface of macroporous carriers with glyoxyl groups has proven to highly stabilize a large variety of enzymes through multipoint covalent immobilization. In this work, we have translated the surface chemistry developed for the fabrication of glyoxyl-agarose carriers to macroporous cellulose (CEL). To that aim, CEL-based microbeads were functionalized with glyoxyl groups through a stepwise alkoxylation (or alkylation)/oxidation synthetic scheme. This functionalization sequence was analyzed by solid-state NMR, while the scanning electron miscroscopy of CEL microbeads reveals that the mild oxidation conditions negligibly affect the morphological properties of the material. Through the optimal functionalization protocol using rac-glycidol, we introduce up to 200 µmols of aldehyde groups per gram of wet CEL, a similar density to the one obtained for the benchmarked agarose-glyoxyl carrier. This novel CEL-based carrier succeeds to immobilize and stabilize industrially relevant enzymes such as d-amino acid oxidase from Trigonopsis variabilis and xylanases from Trichoderma reseei. Remarkably, the xylanases immobilized on the optimal CEL-based materials present a half-life time of 51 h at 60 °C and convert up to 90% of the xylan after four operation cycles for the synthesis of xylooligosaccharides.

Ethyl esters production catalyzed by immobilized lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents.

Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase's activity and stability in unusual reaction medium is its immobilization on inert supports. Here, lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568-Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568-Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica lipases fraction B (CALB) and phospholipase Lecitase® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized lipases, decreasing the total cost of the process.

Synthesis of omega-3 ethyl esters from chia oil catalyzed by polyethylene glycol-modified lipases with improved stability.

Enzymatic synthesis of fatty acid ethyl esters (FAEE) from chia (Salvia hispanica L.) oil has been performed with different modified derivatives and compared with commercial immobilized lipases to produce omega-3 rich FAEE. Therefore, the main objective was to synthesize omega-3 esters from chia oil catalysed by polyethylene glycol-modified lipases using a biocatalyst with higher stability than commercial derivatives. Lipase from Thermomyces lanuginosus (TLL) was immobilized by hydrophobic adsorption on Sepabeads C-18 followed by a physicochemical coating of lipase surface with a dense layer of PEG. Ethanolysis reactions were carried out using pressurized liquid extracted chia seed oil and with different lipase derivatives to compare the omega-3 FAEE yield and ratio of reaction products, which were analysed by HPLC-ELSD. Furthermore, reutilization of lipase derivatives was studied to evaluate the stability after several reaction cycles to minimize decreasing of catalytic activity and develop a feasible enzymatic process for food industrial applications.

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.

Designing continuous flow reaction of xylan hydrolysis for xylooligosaccharides production in packed-bed reactors using xylanase immobilized on methacrylic polymer-based supports.

The present study focuses on the development and optimization of a packed-bed reactor (PBR) for continuous production of xylooligosaccharides (XOS) from xylan. For this purpose, three different methacrylic polymer-based supports (Relizyme R403/S, Purolite P8204F and Purolite P8215F) activated with glyoxyl groups were morphologically characterized and screened for the multipoint covalent immobilization of a xylanase. Based on its physical and mechanical properties, maximum protein loading and thermal stability, Relizyme R403/S was selected to set up a PRB for continuous production of XOS from corncob xylan. The specific productivity for XOS at 10 mL/min flow rate was 3277 gXOS genzyme-1 h-1 with a PBR. This PBR conserved >90% of its initial activity after 120 h of continuous operation.

Preparation of a robust immobilized biocatalyst of ß-1,4-endoxylanase by surface coating with polymers for production of xylooligosaccharides from different xylan sources.

Xylooligosaccharides display interesting prebiotic effects on human health. The endoxylanase Xys1Δ, from Streptomyces halstedii JM8, was immobilized and stabilized on glyoxyl-agarose beads by multipoint covalent attachment using a novel strategy based on surface coating with a multilayer of polymers. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymers and a layer of polyethylenimine. The optimized biocatalyst was 550-fold more stable than one-point covalent immobilized Xys1Δ (at 70 °C, pH 7). This biocatalyst was tested for the production of xylooligosaccharides from soluble xylans from various sources. Hydrolysis of beechwood, wheat straw and corncob xylans was 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. Maximum values of xylooligosaccharides were found for beechwood at 20.6 mg/mL, wheat at 12.5 mg/mL and corncob at 30.4 mg/mL. The optimized biocatalyst was reused for 15 reaction cycles without affecting its catalytic activity.

Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers.

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases-from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)-were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose.

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

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.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

Immobilization of Moniliella spathulata R25L270 Lipase on Ionic, Hydrophobic and Covalent Supports: Functional Properties and Hydrolysis of Sardine Oil.

The oleaginous yeast Moniliella spathulata R25L270 was the first yeast able to grow and produce extracellular lipase using Macaúba (Acrocomia aculeate) cake as substrate. The novel lipase was recently identified, and presented promising features for biotechnological applications. The M. spathulata R25L270 lipase efficiently hydrolyzed vegetable and animal oils, and showed selectivity for generating cis-5,8,11,15,17-eicosapentaenoic acid from sardine oil. The enzyme can act in a wide range of temperatures (25-48 °C) and pH (6.5-8.4). The present study deals with the immobilization of M. spathulata R25L270 lipase on hydrophobic, covalent and ionic supports to select the most active biocatalyst capable to obtain omega-3 fatty acids (PUFA) from sardine oil. Nine immobilized agarose derivatives were prepared and biochemically characterized for thermostability, pH stability and catalytic properties (KM and Vmax). Ionic supports improved the enzyme-substrate affinity; however, it was not an effective strategy to increase the M. spathulata R25L270 lipase stability against pH and temperature. Covalent support resulted in a biocatalyst with decreased activity, but high thermostability. The enzyme was most stabilized when immobilized on hydrophobic supports, especially Octyl-Sepharose. Compared with the free enzyme, the half-life of the Octyl-Sepharose derivative at 60 °C increased 10-fold, and lipase stability under acidic conditions was achieved. The Octyl-Sepharose derivative was selected to obtain omega-3 fatty acids from sardine oil, and the maximal enzyme selectivity was achieved at pH 5.0.

Immobilization of Lipase from Penicillium sp. Section Gracilenta (CBMAI 1583) on Different Hydrophobic Supports: Modulation of Functional Properties.

Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties.

Critical Role of Different Immobilized Biocatalysts of a Given Lipase in the Selective Ethanolysis of Sardine Oil.

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLL-Sepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLL-Duolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA.

Synthesis of ascorbyl oleate by transesterification of olive oil with ascorbic acid in polar organic media catalyzed by immobilized lipases.

The reaction of transesterification between oils (e.g., olive oil) and ascorbic acid in polar anhydrous media (e.g., tert-amyl alcohol) catalyzed by immobilized lipases for the preparation of natural liposoluble antioxidants (e.g., ascorbyl oleate) was studied. Three commercial lipases were tested: Candida antarctica B lipase (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML). Each lipase was immobilized by three different protocols: hydrophobic adsorption, anionic exchange and multipoint covalent attachment. The highest synthetic yields were obtained with CALB adsorbed on hydrophobic supports (e.g., the commercial derivative Novozym 435). The rates and yields of the synthesis of ascorbyl oleate were higher when using the solvent dried with molecular sieves, at high temperatures (e.g. 45°C) and with a small excess of oil (2 mol of oil per mol of ascorbic acid). The coating of CALB derivatives with polyethyleneimine (PEI) improved its catalytic behavior and allowed the achievement of yields of up to 80% of ascorbyl oleate in less than 24h. CALB adsorbed on a hydrophobic support and coated with PEI was 2-fold more stable than a non-coated derivative and one hundred-fold more stable than the best TLL derivative. The best CALB derivative exhibited a half-life of 3 days at 75°C in fully anhydrous media, and this derivative maintained full activity after 28 days at 45°C in dried tert-amyl alcohol.