Wheat germ lipase catalyzed kinetic resolution of secondary alcohols in non-aqueous media

The lipase from wheat germ was used for the kinetic resolution of secondary alcohols. It has the opposite enantioselectivity against the Kazlauskas rule and acts as an anti-Kazlauskas catalyst. The effect of initial water activity, organic solvent, acyl donor and temperature were investigated. Wheat germ lipase had a high activity and enantioselectivity only in n-hexane with a high initial water activity (α_w = 0.97), especially with 1-phenylethanol (C 32%, E > 200). Its performance changed little with the chain length of acyl donor and temperature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (ee_p 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

     

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.     

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).     

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.     

Synthesis of CuPF6‐(S)‐BINAP loaded resin and its enantioselectivity toward phenylalanine enantiomers

A type of resin‐anchored CuPF₆‐(S )‐BINAP was synthesized and identified. The PS‐CuPF₆‐(S )‐BINAP resin was used to adsorb the phenylalanine enantiomers. The results showed that the adsorption capacity of PS‐CuPF₆‐(S )‐BINAP resin toward L‐phenylalanine was higher than that of resin toward D‐phenylalanine. PS‐CuPF₆‐(S )‐BINAP resin exhibited good enantioselectivity toward L‐phenylalanine and D‐phenylalanine. The influence of phenylalanine concentration, pH, adsorption time, and temperature on the enantioselectivity of the resin were investigated. The results showed that the enantioselectivity of the resin increased with increasing the phenylalanine concentration, pH, and adsorption time, while it decreased with an increase in temperature. The causes for these influences are discussed. The highest enantioselectivity (α = 2.81) was obtained when the condition of phenylalanine concentration was 0.05 mmol/mL, pH was 8, adsorption time was 12 h, and temperature 5°C. The desorption test for removing D/L‐phenylalanine on PS‐CuPF₆‐(S )‐BINAP resin was also investigated. The desorption ratios of D‐phenylalanine and L‐phenylalanine at pH of 1 were 95.7% and 94.3%, respectively. This result indicated that the PS‐CuPF₆‐(S )‐BINAP resin could be regenerated by shaking with an acidic solution. The reusability of the PS‐CuPF₆‐(S )‐BINAP resin was also assessed and the resin exhibited considerable reusability.     

Enantioselective resolution of racemic flurbiprofen methyl ester by lipase encapsulated mercapto calix[4]arenes capped Fe3O4 nanoparticles

This work presents the synthesis of new mercapto calix[4]arenes derivatives (4 and 5). These derivatives were capped on Fe₃O₄ magnetic nanoparticles and subsequently encapsulated with Candida rugosa through sol–gel method to furnish enc-4 and enc-5, respectively, to enhance catalytic activity and enantioselectivity of lipase for hydrolysis reaction of racemic flurbiprofen methyl ester. Catalytic activity and enantioselectivity of enc-4 and enc-5 were assayed at different pH and temperature conditions and it was found that the resultant encapsulated enzyme exhibited higher thermal and operational stabilities compared to the free lipase in which enc-5 showed the excellent rate of enantioselectivity (E = 176) for S-flurbiprofen better than free lipase (E = 137) at pH 7 and 35 °C for 48 h. The time study shows that enantioselectivity reached the maximum value of E = 244 after 72 h. Catalytic activity  of these materials was hardly affected by 20 and 23% after five usages of enc-4 and enc-5, respectively.

     

Water activity does not influence the enantioselectivity of Lipase PS and lipoprotein lipase in organic solvents

Summary The activity and enantioselectivity of Lipase PS from Pseudomonas cepacia and lipoprotein lipase from Pseudomonas sp. were investigated in organic solvents preequilibrated to water activities ranging from <0.1 to 0.53, using as a model reaction the transesterification between (±)-sulcatol and vinyl acetate. Variations of water activity markedly influenced the transesterification rate but did not modify the enantioselectivity of the two enzymes.     

Intranasal Microemulsion of Sildenafil Citrate: In Vitro Evaluation and In Vivo Pharmacokinetic Study in Rabbits

The purpose of the present study was to prepare intranasal delivery system of sildenafil citrate and estimate its relative bioavailability after nasal administration in rabbits to attain rapid onset of action with good efficacy at lower doses. Sildenafil citrate saturated solubility was determined in different solvents, cosolvents, and microemulsion systems. For nasal application, sildenafil citrate was formulated in two different systems: the first was a cosolvent system (S3) of benzyl alcohol/ethanol/water/Transcutol/taurodeoxy cholate/Tween 20 (0.5:16.8:47.7:15.9:1:18.1% w/w). The second was a microemulsion system (ME6) containing Oleic acid: Labrasol/Transcutol/water (8.33:33.3:16.66:41.66% w/w). The prepared systems were characterized in relation to their clarity, particle size, viscosity, pH, and nasal ciliotoxicity. In vivo pharmacokinetic performance of the selected system ME6 (with no nasal ciliotoxicity) was evaluated in a group of six rabbits in a randomized crossover study and compared to the marketed oral tablets. The targeted solubility (>20 mg/ml) of sildenafil citrate was achieved with cosolvent systems S1, S3, and S5 and with microemulsion systems ME3–ME6. The saturated solubility of sildenafil citrate in cosolvent system S3 and microemulsion system ME6 were 22.98 ± 1.26 and 23.79 ± 1.16 mg/ml, respectively. Microemulsion formulation ME6 showed shorter t _max (0.75 h) and higher AUC_(0-∞) (1,412.42 ng h/ml) compared to the oral tablets which showed t _max equals 1.25 h and AUC_(0-∞) of 1,251.14 ng h/ml after administration to rabbits at dose level of 5 mg/kg. The relative bioavailability was 112.89%. In conclusion, the nasal absorption of sildenafil citrate microemulsion was found to be fast, indicating the potential of nasal delivery instead of the conventional oral administration of such drug.     

Improvements of enzyme activity and enantioselectivity in lipase-catalyzed alcoholysis of (R,S)-azolides

With Candida antarctica lipase B (CALB)-catalyzed alcoholysis of (R,S)-naproxenyl 1,2,4-triazolide at the optimal conditions (i.e. anhydrous MTBE as the solvent, and methanol as the acyl acceptor at 45 °C) as the model system, the enzyme enantioselectivity in terms of V_R/V_S = 105.8 and specific activity for the fast-reacting (R)-azolide V_R/(E_t) = 0.979 mmol/(h g) were greatly improved in comparison with V_R/V_S = 8.0 and V_R/(E_t) = 0.113 mmol/(h g) of using (R,S)-naproxenyl 2,2,2-trifluoroethyl ester as the substrate. The resolution strategy was successfully extended to other (R,S)-profenyl 1,2,4-triazolides and lipases from Candida rugosa (Lipase MY) and Carica papaya (CPL) having opposite enantioselectivity to CALB. Moreover, the kinetic constants were estimated, compared with those obtained via hydrolysis, and employed for modeling time-course conversions of (R,S)-naproxenyl 1,2,4-triazolide in anhydrous MTBE. The advantages of easy substrate preparation, high enzyme reactivity and enantioselectivity, as well as easy product separation from the remaining substrate via reactive extraction demonstrate merits of using (R,S)-azolides but not the corresponding esters for the alcoholytic resolution.     

A novel mono- and diacylglycerol lipase highly expressed in Pichia pastoris and its application for food emulsifier preparation

A mono- and diacylglycerol lipase (MDL) was cloned from Penicillium cyclopium and expressed in Pichia pastoris strain GS115. The recombinant enzyme was named Lipase GH1. High cell density fermentation was performed by culture in a 7.5-L fermenter using BSMG medium, in which the phosphate in basal salt medium was replaced by sodium glycerophosphate (Na₂GP). The maximal lipase activity detected was 18,000 U per mL, and total protein content in the fermentation supernatant was 3.94 g per L. The activity of the liquid enzyme remained stable under alkaline conditions at 4 °C for 6 months and was 50% after one year. Lipase GH1 was used for the synthesis of mono- and diacylglycerols (MAGs and DAGs), which are commonly used emulsifiers for industrial applications. A conversion rate of 84% after 24 h of reaction was obtained using glycerol/oleic acid molar ratio 11:1, water content 1.5 wt%, enzyme dosage 80 U per g, and reaction temperature 35 °C. Lipase GH1 was more efficient for the synthesis of MAGs and DAGs than was Lipase G50 (a similar, commercially available lipase derived from Penicillium camemberti) when oleic acid was used as an acyl donor. Lipase GH1 has potential for food emulsifier preparation.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

     

Studies on a novel carbon source and cosolvent for lipase production by<Emphasis Type="Italic"> Candida rugosa</Emphasis>

Oleic acid esters were shown to be the best carbon source for both cell growth and lipase production by Candida rugosa. Use of a cosolvent, dodecane, in fermentations improved the solubility of solid substrates and increased oxygen solubility. This resulted in the highest lipase activity in batch fermentation with glycerol trioleate and dodecane. Lipase activity reached 77.1 units ml^–1.     

Optimization of <Emphasis Type="Italic">Serratia marcescens</Emphasis> lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester

Lipase production and cell growth of Serratia marcescens ECU1010 were optimized in shake flasks, with lipase production being enhanced 9.5-fold (4,780 U/l) compared with the initial activity (500 U/l). Optimal carbon and nitrogen sources were Tween-80 and peptone, and the optimal ratio of Tween-80 to peptone was 1:3. The optimized cultivation conditions were 25°C and pH 6.5. Lipase activity, particularly specific activity, could be improved by decreasing the cultivation temperature from 35 to 25°C. Enzyme stability was significantly improved by simple immobilization with synthetic adsorption resin no. 8244. After five reaction cycles, enzyme activity decreased only very slightly, while enantioselectivity of the preparation remained constant, and the ee_s (enantiomeric excess of the remaining substrate) achieved in all cases was higher than 97%. The resin-8244-lipase preparation can be used for efficient enantioselective hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of Diltiazem.     

Surface display of active lipase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using Cwp2 as an anchor protein

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 ± 0.4 U/g (dry cell) when using (G₄S)₃ sequence as the linker, with an optimal temperature and pH at 40°C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.     

Overexpression of a recombinant amidase in a complex auto-inducing culture: purification,biochemical characterization,and regio- and stereoselectivity

Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V _max and K _m values of the purified enzyme with acetamide (50 mM) were 6.89 μmol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated β-hydroxy amides, and N-benzylated β-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF₆] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.     

Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(?)-mandelic acid production

A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His₆-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V _max and K _m parameters were determined to be 27.9 μmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(−)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(−)-mandelic acid.     

Enantioselective esterification of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-2-methylalkanoic acid with <Emphasis Type="Italic">Carica papaya</Emphasis> lipase in organic solvents

Isooctane was the best reaction medium for the enantioselective esterification of (R,S)-2-methylalkanoic acid with n-butanol using Carica papaya lipase as catalyst. Increasing linear alkyl-chain length of racemic 2-methylalkanoic acids from ethyl to hexyl increased the enantioselectivity (E) from 2.1 to 98.2 for the esterification of racemic 2-methylalkanoic acids with n-butanol at 35°C. Decreasing reaction temperature from 40 to 20°C increased the enantioselectivity (E) from 14 to 33 for the esterification of racemic 2-methylhexanoic acids with n-butanol. We obtained a maximum enantioselectivity, of E = 24.3, for the enantioselective esterification of racemic 2-methylhexanoic acids with n-butanol in isooctane at water activity 0.33, and at 35°C.     

Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304

Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28°C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60°C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60°C. Lipase activity was significantly enhanced by Fe³⁺ and strongly inhibited by Hg²⁺. Li⁺, Mg²⁺ and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.