Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase), Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20-50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20-68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Disubstituted dialkoxysilane precursors in binary and ternary sol–gel systems for lipase immobilization

The disubstituted dimethyldiethoxysilane (DMDEOS), methyl(phenyl)diethoxysilane (MPDEOS) and diphenyldiethoxysilane (DPDEOS) were used in binary silane precursor systems in combination with tetraethoxysilane (TEOS) for the immobilization of lipase from Pseudomonas fluorescens (Lipase AK). In addition, ternary silane precursor systems with TEOS and octyltriethoxysilane (OTEOS) or phenyltriethoxysilane (PTEOS) were also studied for encapsulation. The best performing ternary sol–gel preparations (418–736% activity yields of the immobilized enzyme with 1-phenylethanol rac-1a as compared to the native form) were tested as biocatalysts for kinetic resolutions of rac-1a, 1-phenylpropan-2-ol rac-1b and 4-phenylbutan-2-ol rac-1c. Because the catalytic properties and the operational stability of the DMDEOS-containing preparations proved to be superior to all the tested free and sol–gel entrapped Lipase AK biocatalysts in batch mode, the kinetic resolutions of rac-1a and rac-1b were performed with the TEOS/PTEOS/DMDEOS 4:1:1 Lipase AK in a continuous-flow reactor as well.     

Immobilization of lipases onto the SBA-15 mesoporous silica

Lipase is an important enzyme which can catalyse the hydrolysis of lipids and has several applications and industrial potentials. In addition, different types of lipases are used as efficient catalysts in the enantioselective esterification and/or hydrolysis reactions and produce products in high yields and enantio excess as well. However, immobilization of lipases on the surface of a heterogeneous substrate is necessary to improve its specific catalytic activities as it can be isolated from the reaction mixture easily. Mesoporous silica materials are the best option for this aim due to their high specific surface area, ordered structure, and large pore volume. Hence, in this article, the role of SBA-15 and the modified SBA-15 mesoporous materials as support for different lipases and their catalytic activities are reviewed.     

Lipase-catalyzed enantioselective acylation of 3-benzyloxypropane-1,2-diol in supercritical carbon dioxide

Lipase-catalyzed acylation of 3-benzyloxypropane-1,2-diol with vinyl acetate as acyl donor using different lipases [porcine pancreas lipase (PPL), Lipase AK “Amano”, Lipase PS “Amano”, and crude enzymes from Trichoderma reesei RUT-C30, Thermoascus thermophilus (NRRL5208), Talaromyces emersonii (NRLL3221)] was studied in supercritical carbon dioxide (scCO₂). In the reactions catalyzed by different lipases different amounts of monoacetate and diacetate products along with minor amounts of cyclic acetals forming from the diol and acetaldehyde were obtained.Application of Lipase AK led to the highest conversion (84.7%) and the highest enantiomeric excess values (ee_monoacetates = 38%, ee_diacetate = 85%). Effect of water content of scCO₂ on the productivity and the enantiomer selectivity of the reactions with Lipase AK was also investigated.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

Phyllosilicate sol-gel immobilized lipases for the formation of partial acylglycerides**

Lipase PS-30 (pseudomonas cepacia) and Lipase F (Rhizopus oryzae), immobilized within a phyllosilicate sol-gel matrix, catalyzed the esterification of glycerol with short, medium and long-chain fatty acids to produce mono (MAG), di (DAG) and tri (TAG) acylglycerols. The results from the above esterification reactions were compared to reactions using a commercially available immobilized lipase, Lipozyme IM-60. Time course studies showed that free Lipase PS-30 or Lipase F enhanced esterification reactions with the use of silica-supported glycerol. In contrast, immobilized Lipase PS-30-catalyzed reactions occurred at the same conversion rate when using either free or silica-supported glycerol. For immobilized Lipase F and Lipozyme IM-60 reactions, the use of silica-supported glycerol favored the production of DAG and TAG over MAG. All three immobilized lipases could be reused for acylglycerol production.     

Comparison of differently modified Pseudomonascepacia lipases in enantioselective preparation of a chiral alcohol for agrochemical use

Three methods for enzyme modification/immobilization were compared to enhance the catalytic performance of a commercially available lipase, Lipase PS from Pseudomonascepacia, in highly enantioselective transesterification of an agrochemically useful sec-alcohol, (R,?S)-HMPC [=(R,?S)-4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopenten-1-one], with vinyl acetate as both acyl donor and reaction medium. The stearic acid-coated lipase showed the highest catalytic activity, with a specific activity improved by 54 times over the native lipase. The microcrystal salt-supported lipase and celite-adsorbed lipase also displayed much better performance as compared with the native lipase. All the three modified lipase preparations showed a similar thermal stability to that of the native enzyme. The enantioselectivity (E-value) was also quite satisfactory in all the cases (E>100 at 30°C), though a trend of slight decline was also observed with the temperature increase in the range of 25–60°C. The optimum aqueous pH, from which the modified lipases were prepared, was 6.0–7.0. A low water activity (a_w) of ca. 0.1 was favorable for all the three modified lipases. The stearic acid-coated lipase displayed prominent advantages in catalyzing the transesterification reaction at a very high (R,?S)-HMPC concentration up to 1.0?M.     

Initial water content and lipase-mediated ester formation in hexane

Lipase biocatalysis was investigated as a tool for the production of esters by two model reactions, esterification of 1-butanol with 2-methyl-1-pentanoic acid and irreversible transesterification between 2-methyl-1-pentanol and vinyl acetate. The reactions were carried out in hexane using lipases from Candida cylindracea and porcine pancreas. The initial water content influenced both the yield of the ester and the enantioselectivity of the reaction (esterifica-tions) or the ester formation only (transesterifications).     

Single-step purification of lipase from Burkholderia multivorans using polypropylene matrix

Lipase from Burkholderia multivorans was purified with high yields directly from fermentation broth by a single-step purification protocol involving adsorption and desorption. The crude enzyme (lyophilized powder) from B. multivorans was loaded on Accurel (Membrana, Germany), a polypropylene matrix, using butanol as the solvent in a buffer at pH 9.0 and ambient temperature for a period of 12 h. The enzyme adsorbed onto the matrix with high specific activity (33 units mg^–1 protein). This was followed by desorption of the enzyme from the matrix using Triton X-100 as the eluent. The enzyme was finally recovered by precipitation with acetone (50%, v/v). Thus, an overall enzyme yield of 66% with a 3.0-fold purification was obtained. The purity of the enzyme was ascertained by SDS-PAGE. The phenomenon of adsorption and desorption on Accurel was studied for three more lipases, viz. Mucor meihei lipase (Sigma–Aldrich Co.), Lipolase (Novo Nordisk, Denmark) and Pseudomonas aeruginosa lipase (laboratory isolate).     

Resolution of (±)– Cytallene — A Highly Active Anti-HIV Agent with Axial Dissymmetry

Abstract

Anti-HIV agent (±)-cytallene (1b + 2b) was resolved by enantioselective acylation of (±)-N⁴-benzoylcytallene (1d + 2d) with vinyl butyrate in tetrahydrofuran catalyzed by lipase AK from Pseudomonas sp. and subsequent deacylation of 4d and 1d with ammonia in methanol. Optically pure enantiomers 1b and 2b were obtained.     

Meetings & Symposia

Abstract

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.     

Selective enzymic esterification of free fatty acids with n-butanol under microwave irradiation and under classical heating

Selective enzymic esterification of free fatty acids, obtained from blackcurrant oil by chemical saponification, with n-butanol using four immobilized lipases under microwave irradiation and under classical heating was studied. A positive effect of microwave irradiation on chemical yields of the products of the enzymic reactions and specificity of lipases were observed in comparison with a controlled heating in an incubator equipped with shaking (classical heating) applied during the identical enzyme-mediated processes. The maximum quantity of -linolenic acid (30%) was obtained with Lipozyme used as biocatalyst of the reaction under microwave irradiation. The maximum quantity of butyl -linolenate (20%) was obtained by a Pseudomonas cepacia lipase catalyzed esterification under classical heating.     

Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions 1 and 3 of the molecule of glycerol and docosahexaenoic acid (DHA) in position 2, by acidolysis of tuna oil and CA, catalyzed by several lipases. To this end several lipases and immobilization supports were tested with the aim of avoiding the acyl-migration observed in previous works. The determination of the best catalyst (i.e. the lipase and the immobilization support as a whole) was carried out by experiments of acidolysis of cod liver oil and CA in a bath reactor. The best results were obtained with the lipases from Rhizopus oryzae (Lipase D) and Rhizopus delemar (Lipase Rd), immobilized on Accurel MP1000 (a microporous polypropylene) with a lipase/support ratio 1:1.5 (w/w). The activity of these immobilized lipases was stable for a minimum of 5 days in the operational conditions (up to 40 °C).Lipase Rd was selected for the next step in which it was immobilized on Acurrel MP1000 to obtain STAG enriched in DHA by acidolysis of tuna oil (20% DHA) with CA. The experiments were carried out by recirculating the reaction mixture through an immobilized lipase packed bed reactor at different substrate/hexane ratios, as well as in absence of solvent. In the latter case, STAG with 51% CA and 13% DHA were obtained at 73 h. This result indicates that with this catalyst an acceptable reaction rate was attained in absence of solvent. A structural analysis by the pancreatic lipase method carried out to STAG with 45% CA and 16% DHA indicated that 91% of the CA incorporated is located in positions 1 and 3, and that 51% of the DHA is located in position 2 (MLM structure). This position is also rich in palmitic, eicosapentaenoic and oleic acids.After the acidolysis reaction a mixture of STAG and free fatty acids was obtained. The recovery of STAG from this reaction mixture is difficult because of the high content of free fatty acids. A separation method based on the neutralization of the free fatty acids with a KOH hydroalcoholic solution has been developed. By this procedure pure (100%) STAG were obtained with a recovery yield of 80%.     

Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials

In this work, lipase from Arthrobacter sp. was immobilized by sol–gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, γ-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS = 1/1 (mol/mol), water/silane molar ratio (R value) = 20 and lipase loading = 0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form.     

Terpene ester synthesis by lipase-catalyzed transesterification

Summary Five lipases were screened for their ability to synthesize terpene esters by transesterification. The nature of terpene alcohol and enzyme, as well as the chain length of the acyl donor used affected the product yields. Lipase AY from Candida rugosa gave the best overall yield (96.2%). Geraniol and tributyrin were also found to be the best reactants.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Lipase-Catalyzed Synthesis of Capsaicin Analogs by Amidation of Vanillylamine with Fatty Acid Derivatives

Various lipases catalyzed the synthesis of capsaicin analogs by amidation of vanillylamine with fatty acid derivatives in a two-phase system. When methyl myristate was employed as an acyl donor, moderate yields, 40-59%, of capsaicin analog (3) were obtained using Novozym 435, Lipase AK or Lipase PS. Several capsaicin analogs having 4-18 carboxyl carbons were also synthesized from the corresponding fatty acid or its methyl or ethyl ester in 2-44% yields.     

Application of C-13 NMR Spectrometry to the Structural Investigation of the Novel Bicyclic Anhydrooctose Uronic Acid Nucleosides,Constituents of Ezomycins

Seven kinds of lipase were immobilized to Sepharose 4 B, porous glass beads and ion-exchange resins. Lipase immobilized to porous glass beads was most suitable for the treatment of yolk-contaminated egg white. Optimum pH and temperature were not varied by the immobilization. The immobilized lipoprotein lipase from Pseudomonas fluorescens and lipase from Rhizopus delemar were applied to the treatment of yolk-contaminated egg white. The foam stability of the spray-dried egg white solution containing 0.05 % yolk was completely recovered by the 30 min’s treatment with both immobilized lipases. This demonstrated the feasibility of improving the foaming properties of yolk-contaminated egg white by the immobilized lipase treatment.