Magnetic lipase active in organic solvents

Magnetic lipase (magnetite particles coated with polyethylene glycol-modified lipase) was prepared in two steps: Lipase was coupled with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, activated PEG2, to obtain polyethylene glycol-modified lipase, PEG-lipase. The PEG-lipase was added to the solution of ferrous (Fe2+)- and ferric(Fe3+)-ions with the pH value adjusted to 8.0-8.5 to obtain magnetic lipase. The magnetic lipase was dispersed in organic solvents such as benzene and 1,1,1-trichloroethane with the particle size of 120 +/- 60 nm. The colloidal solution was very stable and no aggregation occurred even after 5 days. A high enzymic activity (11.6 mumol/min/mg protein) for lauryl laurate synthesis was observed in 1,1,1-trichloroethane. The magnetic lipase was readily recovered from the organic solvents in a magnetic field of 6000 Oe without loss of the enzymic activity.     

Application of PEG-enzyme and magnetite-PEG-enzyme conjugates for biotechnological processes

Enzymes can be made soluble and active in organic solvents by chemical modification with an amphipathic macromolecule, polyethylene glycol (PEG). The PEG-enzyme conjugates can also be conjugated to magnetite (Fe₃O₄). The magnetic enzymes stably disperse in both organic solvents and aqueous solutions. When lipase is prepared as such a conjugate, it catalyses ester synthesis in organic solvents, and can be readily recovered by magnetic force without loss of enzymic activity. This approach could have a great practical potential.     

Magnetic lipase adsorbed to a magnetic fluid

A magnetic fluid was synthesized by oxidation of ferrous ions (Fe^2&+) in the presence of a synthetic alternating copolymer of polyethylene glycol (PEG) and maleic acid (MA), poly(PEG-MA). The magnetic fluid dispersed stably both in aqueous solution and in organic solvents. Its particle size was approximately 10 nm. The magnetic fluid was mixed with lipase in water, followed by lyophilization. Although the enzyme and the magnetic fluid were dissociated in aqueous solution, they remained associated in organic solvents such as benzene. The magnetic fluid-adsorbed lipase dispersed in benzene and exerted high enzymic activity (2.9 μmol min⁻¹ mg⁻¹ lyophilized powder) for lauryl laurate synthesis from lauric acid and lauryl alcohol, and was readily recovered from the reaction mixture in a magnetic field (6000 Oe) without loss of enzymic activity.     

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.     

Sugar ester synthesis by a mycelium-bound Mucor circinelloides lipase in a micro-reactor equipped with water activity sensor

The mycelium-bound Mucor circinelloides lipase was used for the synthesis of esters of saccharides and fatty acids in 37 ml reactor equipped with magnetic stirrer and water activity sensor. Either di-n-pentyl ether or the mixture of di-n-pentyl and petroleum ethers were applied as reaction media. Water activity sensor provided on-line monitoring of this parameter and control of continuous processes of ester synthesis. It was found that two natural antioxidants, i.e. carotene and astaxanthin activated this lipase in organic solvents that could be beneficial for the synthesis of esters of compounds sensitive to oxidation, e.g. polyunsaturated fatty acids.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase

Lipase from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase (PEG-lipase) was soluble and active in organic solvents such as benzene and 1,1,1-trichloroethane. PEG-lipase catalyzed esterification of chiral secondary alcohols with fatty acids in benzene and exhibited preference for R isomers over S isomers. Km and Vmax values for each isomer of various alcohols were obtained by kinetic study of the esterification in benzene. PEG-lipase-catalyzed esterification leads to optical resolution of a racemic alcohol.     

Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: Partial purification,characterization and application in non-aqueous environment

The biosynthesis of esters is currently of much commercial interest because of the increasing popularity and demand for natural products among consumers. Biotransformation and enzymatic methods of ester synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ∼32 kDa. The partially purified lipase was optimally active at 37 °C and pH 8.5. The enzyme showed greater stability toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its initial activity. The metal ions such as Ca²⁺, Ba²⁺ and Mg²⁺ had stimulatory effects on lipase activity, whereas Co²⁺ and Zn²⁺ strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application in biocatalysis under non-aqueous environment.     

Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Chemical modification of enzymes with activated magnetic modifier

An activated magnetic modifier, which could render biological materials magnetic property, was synthesized in following two steps: oxidation of ferrous ions (Fe2+) with hydrogen peroxide in the presence of alpha, omega-dicarboxymethylpoly(oxyethylene) (DCPEG) to obtain DCPEG-magnetite (Fe3O4); free carboxyl groups in the DCPEG-magnetite were activated with N-hydroxysuccinimide. By coupling the activated magnetic modifier to amino groups of lipase or L-asparaginase, magnetic enzymes were prepared. They dispersed stably not only in aqueous solution but also in organic solvents with high enzymic activities. Magnetic enzymes were readily recovered from reaction mixture in a magnetic field of 6000 Oe without loss of enzymic activity.     

Chemical modification of lipase with a comb-shaped synthetic copolymer of polyoxyethylene allyl methyl diether and maleic anhydride

Summary Lipase fromPseudomonas fluorescens was coupled with a copolymer of polyoxyethylene allyl methyl diether and maleic anhydride, activated PM. The PM-lipase became soluble and active in organic solvents, and also heat stable. It catalyzed the ester synthesis in benzene and ester hydrolysis in an aqueous system with high enzymic activity.     

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

Refolding,purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca²⁺ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.     

Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene

The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine. The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene. The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C. The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.