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.     

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%.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

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.     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Stereoselective synthesis of caffeic acid amides via enzyme-catalyzed asymmetric aminolysis reaction

In this study, a new method was developed to prepare enantiopure caffeic acid amides by enzyme-catalyzed asymmetric aminolysis reaction. Methoxymethyl chloride (MOMCl) was first introduced as a protective and esterified reagent to obtain the MOM-protected caffeic acid MOM ester 1d. Aminolysis reaction occurred between 1d and (R, S)-α-phenylethylamine in the presence of an immobilized lipase (Novozym 435) from Candida antarctica. Compared with the methyl-protected caffeic acid methyl ester 1c, 1d as substrate improved the lipase-catalyzed reaction rate by 5.5-fold. After Novozym 435-catalyzed aminolysis reaction was established, we evaluated the effects of synthesis parameters on the catalytic activity and enantioselectivity of Novozym 435. A reaction conversion rate of 25.5% and an E value of >100 were achieved under the following optimum conditions: reaction solvent, anhydrous isooctane; reaction temperature, 70 °C; reaction time, 24 h; ester-to-amine substrate molar ratio, 1:40; and enzyme additive amount, 40 mg. Kinetic and thermodynamic analyses were conducted to determine the main factors affecting enantiomeric discrimination. Novozym 435 still showed 80% of its initial activity after recycling five times. Highly optically pure caffeic acid amides with an enantiomeric excess of 98.5% were finally obtained by HCl deprotection. The established enzyme-catalyzed asymmetric aminolysis method in this study might be used to prepare other caffeic acid amides.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

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.     

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%.     

Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil

The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g^?1). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.     

Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase

Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35?°C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase.     

Gas phase enantioselective reduction catalyzed by immobilized ketoreductase: Effects of water activity and reaction temperature

The performance (activity, stability, enantioselectivity and productivity) of the commercial ketoreductase immobilized on non-porous glass supports was investigated as functions of the water activity and the reaction temperature in a continuous gas phase reactor. The enantioselective reduction of 2-butanone to (S)-2-butanol with the in situ regeneration of β-nicotinamide adenine dinucleotide phosphate by 2-propanol catalyzed by the immobilized ketoreductase was used as a model reaction. The activity, stability and enantioselectivity were strongly influenced by the water activity and the reaction temperature. The optimal water activity and reaction temperature were obtained at 0.8 and 313–323 K in terms of the productivity, respectively. Successfully, the enantioselectivity for the gas phase system attained the level identical to that for the aqueous phase system.     

Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Abstract

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.     

Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012 This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].     

Enantioselective esterification of ibuprofen in supercritical carbon dioxide by immobilized lipase

The enantioselective esterification of racemic ibuprofen with n-propanol by immobilized Mucor miehel lipase in supercritical carbon dioxide was studied. The enantiomeric excess of the product (ee_p) was 70 % at 15...20 % conversion. The enantioselectivity was faintly affected by temperature and the concentration of ibuprofen and lipase. The optimum temperature was 45 °C. The initial reaction rate increased with pressure, but enantioselectivity was not affected by pressure changes. The reaction rates in supercritical carbon dioxide at optimized conditions and in n-hexane were similar.     

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.     

Enhancement of the activity and enantioselectivity of lipase in organic systems by immobilization onto low-cost support

Lipase from Arthrobacter sp. was immobilized onto low-cost diatomite materials using different protocols for the resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) by asymmetric acylation. The support surface was grafted various functional groups including methacryloxypropyl, vinyl, octyl, dodecyl and γ-(aminopropyl)-glutaraldehyde. These modifications resulted in various mechanisms during the immobilization and thus introduced different characteristics to the prepared lipases. The interfacially adsorbed lipase onto dodecyl-modified support exhibited both higher activity and stability among these immobilized preparations. The modified enzyme-aggregate coating method was performed based on interfacial adsorption in our work, and the characteristics of this immobilized lipase were investigated and compared with those by cross-linking and interfacial adsorption methods. It was shown that the enzyme-aggregate coated lipase yielded the highest activity with a recovered activity of 8.5-fold of the free enzyme, and the highest operational stability with 85% of initial activity remained after 10 recycles. Excellent enantioselectivity (E ≥ 400, with e.e. = 99% of S-HMPC) was obtained for most lipase preparations in our paper (E = 85 for the free enzyme).     

Biodiesel production from pomace oil by using lipase immobilized onto olive pomace

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto olive pomace. Immobilized support material used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy (SEM). The maximum immobilization of T. lanuginosus was obtained as 18.67 mg/g support and the highest specific activity was 10.31 U/mg protein. The properties of immobilized lipase were studied. The effects of protein concentration, pH and buffer concentration on the immobilization and lipase activity were investigated. Biodiesel production using the immobilized lipase was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 93% at 25 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.     

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.