Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel encapsulated lipase in the presence of sporopollenin

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida rugosa lipase (CRL) was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solution/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase particularly had higher conversion and enantioselectivity compared to the sol–gel free lipase. In this study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the free enzyme) with an ee value ～98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the reaction mixture with 98% optical purity.     

Enantioselective enzymatic hydrolysis of racemic drugs by encapsulation in sol–gel magnetic sporopollenin

Candida rugosa lipase was encapsulated within a sol–gel procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of octyltriethoxysilane and tetraethoxysilane in the presence of magnetic sporopollenin. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e., the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of racemic naproxen methyl ester, mandelic acid methyl ester or 2-phenoxypropionic acid methyl ester that were studied in aqueous buffer solution/isooctane reaction system. The encapsulated magnetic sporopollenin (Spo-M-E) was found to give 319 U/g of support with 342% activity yield. It has been observed that the percent activity yields and enantioselectivity of the magnetic sporopollenin encapsulated lipase were higher than that of the encapsulated lipase without support. The substrate specificity of the encapsulated lipase revealed more efficient hydrolysis of the racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester than racemic mandelic acid methyl ester. It was observed that excellent enantioselectivity (E > 400) was obtained for encapsulated lipase with magnetic sporopollenin with an ee value of S-Naproxen and R-2 phenoxypropionic acid about 98%.     

Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles

In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6–11% of the enzyme’s activity after five batches.     

A magnetically separable biocatalyst for resolution of racemic naproxen methyl ester

Candida rugosa lipase (CRL) was encapsulated via the sol–gel method, using 5, 11, 17, 23-tetra-tert-butyl-25,27-bis(2-aminopyridine)carbonylmethoxy-26, 28-dihydroxy-calix[4]arene-grafted magnetic Fe₃O₄ nanoparticles (Calix-M-E). The catalytic activity of encapsulated lipase (Calix-M-E) was tested both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic naproxen methyl ester. The present study demonstrated that the calixarene-based compound has the potential to enhance both reaction rate and enantioselectivity of the lipase-catalyzed hydrolysis of racemic naproxen methyl ester. The encapsulated lipase (Calix-M-E) had great catalytic activity and enantioselectivity (E > 400), as well as remarkable reusability as compared to the encapsulated lipase without supports (E = 137) for S-Naproxen.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

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.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

Abstract

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe₃O₄), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe₃O₄ nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60?°C, free lipase lost its initial activity within 80?min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120?min of heat treatment at 60?°C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).     

Improvement of the enantioselectivity of lipase-catalyzed naproxen ester hydrolysis in organic solvent

A method is presented to improve the enantioselectivity of lipase-catalyzed hydrolysis of naproxen methyl ester in water-saturated isooctane. It is shown that coupling of the enantioselective hydrolysis of Naproxen methyl ester with the photo-dissociation methanol leads to the photocatalytic conversion of methanol into water, by which the equilibrium constant (K) of the lipase-catalyzed hydrolysis was changed. The equilibrium yield and enantiomeric excess are increased. Because the lipase would not dissolve in the organic solvent, it was adsorbed on photocatalyst particles, which may facilitate the isolation of enzyme from reaction system.     

A spectrophotometric transesterification-based assay for lipases in organic solvent

A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30 °C or in decane at 50 °C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification (R² = 0.999) and to results from p-nitrophenyl palmitate (R² = 0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 ± 350 U g⁻¹) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 ± 0.04 U g⁻¹). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.     

Efficient microbial elimination of methanol inhibition for Naproxen resolution by a lipase

To eliminate methanol inhibition of the activity of a lipase, methanotrophic bacteria, which can convert methanol into water and CO₂, were introduced to the reaction of enantioselective hydrolysis of Naproxen methyl ester catalysed by lipase from Candida rugosa. Both the activity and stability of lipase were improved by the removal of methanol by the bacteria.     

Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: significantly enhanced enantioselectivity and stability

The lipase selective hydrolysis of Naproxen methyl ester was explored in both water-saturated isooctane and water-saturated ionic liquid 1-butyl-3-methylimidazolium hexafluoro-phoshate ([bmim]PF₆) to see any significant differences in terms of enantioselectivity and stability between two different classes of reaction media. It is shown that polar and hydrophobic of [bmim]PF₆ made it an unearthly reaction medium for hydrolysis of Naproxen methyl ester. It not only decreases the equilibrium constant (K) and enhances the enantiomeric ratio (E), consequently improves the equilibrium conversion (C_Eq) of the hydrolysis reaction and enantiomeric excess of product (ee_p), but also maintains the lipase activity. Because the lipase would not dissolve in the 1-butyl-3-methylimidazolium hexafluoro-phoshate, it can be filtrated up from 1-butyl-3-methylimidazolium hexafluoro-phoshate and recycled for several runs. The stability of lipase was improved due to the higher solubility of methanol in 1-butyl-3-methylimidazolium hexafluoro-phoshate than in isooctane.     

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35–60 °C and pH 4.0–6.0, with a maximum activity at 50 °C and pH 4.0–5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 °C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0–C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.     

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81 kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35 °C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35 °C for 1 h. Higher activity was observed in the presence of surfactants, Na⁺, NH₄⁺ ions, NaN₃ and ethylenediaminetetraacetic acid (EDTA), while Co²⁺ and Cu²⁺ ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10 days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.     

Maximization of bioconversion of castor oil into ricinoleic acid by response surface methodology

In this study, response surface methodology was applied to optimize process variables like temperature, pH, enzyme concentration (mg/g oil), and buffer concentration (g/g oil) for hydrolysis of castor oil using Candida rugosa lipase. A 2⁴ full factorial central composite design was used to develop the quadratic model that was subsequently optimized and the optimal conditions were as follows: temperature 40 °C, pH 7.72, enzyme concentration 5.28 mg/g oil, buffer concentration 1 g/g oil and there was 65.5% conversion in 6 h. These predicted optimal conditions agreed well with the experimental results. This is the first report on the application of response surface methodology in castor oil hydrolysis using C. rugosa lipase with higher percentage conversion in 6 h.     

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

Abstract

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E?10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E?40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L^-1/h^-1 space-time yield, which decreased to 1.0 g/L^-1/h^-1 after four repetitive batches.     

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E > 200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E > 200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75 Å and 2.04 Å, respectively, showed that both proteins have a canonical α/β hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/β hydrolase fold esterases.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Lipase-catalyzed enantioselective synthesis of (R,R)-lactide from alkyl lactate to produce PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid)

R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5 A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method.