Enhancement of Protease Activity in Transesterification of Glycidol with Vinyl n-butyrate by Entrapment into Alkyl-substituted Silicates and Pretreatment with a Substrate

Proteases originating from Aspergillus melleus (Protease P) and Bacillus subtillis (Prolether FG-F) were entrapped into organic-inorganic hybrid silicates on Celite 545 by the sol-gel method, and their activities measured at 35°C for transesterification of chiral glycidol with vinyl n-butyrate in isooctane. n-Butyl- and dimethyl-substituted silicates provided 12.6 times higher activities with Protease P and 5.5 times with Prolether FG-F, respectively, than those deposited on Celite 545. Although pretreatment of those immobilized proteases with the chiral glycidol affected transesterification activities of both enantiomers, the ratio of the initial transesterification rate of (S)-(-)-glycidol to that of (R)-(+)-glycidol, remained unchanged.     

Activation of protease by sol-gel entrapment into organically modified hybrid silicates

To prepare an immobilized protease with a high activity for transesterification of vinyl n-butyrate with 3-methyl-1-butanol (isoamyl alcohol) in organic media, a protease was entrapped into organic–inorganic hybrid silica gel on Celite 545 by the sol-gel method. When propyltrimethoxysilane was used as the organic silane precursor mixed with tetramethoxysilane at a molar ratio of 16:1, the hybrid gel-entrapped protease on Celite 545 had 8 times the activity of the protease deposited on Celite 545 from 35 to 85°C.     

Enhancement of thermostability of lipase by the sol-gel entrapment into methyl-substituted organic silicates formed on diatomaceous earth

Summary Candida cylindracea lipase was entrapped in methyl-substituted organic silicates formed on kieselguhr such as Celite 545 and Hyflo Super-Cel. Various preparation conditions were optimized to obtain a more thermostable immobilized lipase. The hybrid gel-entrapped lipase derived from an equimolar mixture of trimethylmethoxysilane and tetramethoxysilane, retained its full activity for ester synthesis up to the temperatures of 65°C.     

Studies of reaction parameters on synthesis of Citronellyl laurate ester via immobilized Candida rugosa lipase in organic media

Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.     

Modification of Phospholipids with Lipases and Phospholipases

Different commercial lipases and phosphoiipases were studied in the hydrolysis and transesterification of synthetic phosphatidylcholine and soybean lecithin. Wide variations in the lipase and phospholipase activities and in the protein contents of the preparations were observed. The substrate specificity varied between different enzymes. A high degree of hydrolysis of synthetic and soybean phospholipids was achieved with both types of enzymes.

Enzymes immobilized on Celite were used in the transesterification of dimyristoyl phosphatidylcholine and oleic acid. The conversions were carried out both without solvent and in the presence of toluene. The amount of modified phosphatidylcholine was measured using HPLC. The highest amount of modified phosphatidylcholine was obtained in solvent-free transesterification. The best results were obtained with Aspergillus niyer lipase.     

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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.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Immobilization of Pseudomonas cepacia lipase onto electrospun polyacrylonitrile fibers through physical adsorption and application to transesterification in nonaqueous solvent

The lipase of Pseudomonas cepacia was immobilized onto electrospun polyacrylonitrile (PAN) fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane. The rate of reaction with the adsorbed lipase was 23-fold higher than the initial material. After 10 recyclings, the initial reaction rate was 80% of the original rate. This system of enzyme immobilization is therefore suitable for carrying out transesterification reactions in nonaqueous solvents.     

Efficient production of diltiazem chiral intermediate using immobilized lipase from <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.     

Immobilization of Lipases on Clay, Celite 545, Diethylaminoethyl-, and Carboxymethyl-Sephadex and their Interesterification Activity

Lipases AK, PS (both from Pseudomonas sp), and AY (from Candida rugosa) were immobilized on clay, Celite 545, DEAE-Sephadex, or CM-Sephadex and used to synthesize structured lipids from tricaprylin and trilinolein. Lipases AK and AY immobilized on Celite 545 had the highest interesterification activity (0.11 and 0.35 units, respectively) while lipase PS had the highest activity (0.18 units) when immobilized on CM-Sephadex. During 72 h reaction, the highest product yields were obtained in Celite AK (224 µmol), and CM PS (290 µmol) after 48 h and Celite AY (501 µmol) after 24 h. Lipase AK immobilized on DEAE-Sephadex was the most stable for repeated (5 times) operation.     

Stabilization of Candida lipase against acetaldehyde by adsorption onto celite

Summary Immobilization of Candida rugosa lipase (Amano AY-30) by adsorption onto Celite 545 led to a markedly improved performance of the enzyme: Firstly, the enzyme was almost completely resistant to deactivation by acetaldehyde, which is liberated as unavoidable by-product in acyl-transfer reactions with vinyl esters, and secondly, the enantioselectivity was enhanced up to three-fold.     

Lipase-catalyzed resolution of primary alcohol containing quaternary chiral carbon

Summary 2-Cyano-2-phenyl-1-hexanol containing chiral quaternary carbon was resolved using a lipase. Among various reaction conditions, transesterification reaction (solvent; n-hexane, lipase; Candida cylindracea) gave high selectivity(20/80). Additionally, the selectivity could be improved(13/87) when pyridine(1%) was used as an additive. The ratios of resolved alcohols were determined by GC and ¹H-NMR analysis using Mosher's derivative.     

Removal of dibenzothiophene and its oxidized product in anhydrous water-immiscible organic solvents by immobilized cytochrome c

In various anhydrous water-immiscible organic solvents, dibenzothiophene (DBT) was removed by more than 80% by cumene hydroperoxide (,-dimethylbenzyl hydroperoxide) catalyzed by the immobilized cytochrome c on Celite. DBT-sulfone, the oxidation product of DBT, strongly adsorbed on Celite in highly hydrophobic organic solvents such as hexadecane, decane and n-octane. The adsorbed DBT-sulfone was desorbed from Celite by a simple washing of the Celite with a less hydrophobic solvent such as octanol.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Synthesis and biological evaluation of new simple indolic non peptidic HIV Protease inhibitors: The effect of different substitution patterns

New structurally simple indolic non peptidic HIV Protease inhibitors were synthesized from (S)-glycidol by regioselective methods. Following the concept of targeting the protein backbone, different substitution patterns were introduced onto the common stereodefined isopropanolamine core modifying the type of functional group on the indole, the position of the functional group on the indole and the type of the nitrogen containing group (sulfonamides or perhydroisoquinoline), alternatively. The systematic study on in vitro inhibition activity of such compounds confirmed the general beneficial effect of the 5-indolyl substituents in presence of arylsulfonamide moieties, which furnished activities in the micromolar range. Preliminary docking analysis allowed to identify several key features of the binding mode of such compounds to the protease.     

Total enzymatic synthesis of cholecystokinin CCK-5

Summary. This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH₂ of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt·HCl by -chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester.The other fragment H-Asp(OMe)-Phe-NH₂ resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH₂·HCl and thermolysin as catalyst, followed by catalytic hydrogenation.Finally PhAc-Gly-Trp-Met-Asp-Phe-NH₂ was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH₂ with -chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the -methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.     

Coprecipitation of Pseudomonas fluorescens Lipase with Hydrophobic Compounds As an Approach to Its Immobilization for Catalysis in Nonaqueous Media

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.     

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.

     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.