Y. Kodera

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.

Ester synthesis from α-substituted carboxylic acid catalyzed by polyethylene glycol-modified lipase fromCandidacylindracea in benzene

SummaryLipase fromCandidacylindracea was coupled with polyethylene glycol(PEG). In contrast to the previously used lipase fromPseudomonasfluorescens, the modified lipase catalyzed the ester synthesis in benzene at 25°C from short-chain alcohols and α- or β-substituted carboxylic acid. Using the modified lipase, following esters were synthesized; pentyl α-methylpentanate, methyl benzoate, and methyl retinoate.

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

Abstract 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 (Fe 3 O 4 ). 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.

Ester-exchange catalyzed by lipase modified with polyethylene glycol.

Lipoprotein lipase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine; forty-six percent out of seven amino groups in the molecule were substituted. The modified lipase catalyzed ester-exchange reactions between an ester and an alcohol, between an ester and an acid, and between two esters. The modified enzyme catalyzed these reactions not only in organic solvents, but also in straight hydrophobic substrates. As the modified enzyme was extremely stable at elevated temperature, for example at 70 degrees C, this can find many practical applications.

Terpene alcohol ester synthesis by polyethylene glycol-modified lipase in benzene

SummaryLipase fromPseudomonasfragi modified with polyethylene glycol was soluble and active in organic solvents such as benzene and chlorinated hydrocarbons. Using the modified lipase, terpene alcohol esters were synthesized with various combinations of terpene alcohols (citronellol, geraniol, farnesol and phytol) and carboxylic acids (acetic-, propionic-, n-butyric-, and valeric acids) in benzene at 25°C. The yield was generally very high.

Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene.

Cholesterol oxidase from Nocardia sp. was modified with a synthetic copolymer of polyoxyethylene allylmethyldiether (PEG) and maleic acid anhydride (MA anhydride), poly(PEG-MA anhydride). The modified cholesterol oxidase, in which 64% of the amino groups in the protein molecule were coupled to poly(PEG-MA), was soluble in organic solvents and catalyzed the oxidation reaction of cholesterol in benzene to form 4-cholesten-3-one with the enzymic activity of 0.6 mumol/min/mg protein. Using the modified cholesterol oxidase together with polyethylene glycol-modified peroxidase, coupled reactions shown below took place in Cholesterol + O2----4-Cholesten-3-one + H2O2 H2O2 + o-Phenylenediamine----H2O + Oxidized o-Phenylenediamine transparent benzene solution, not in an emulsified system. The oxidation of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phenylenediamine at 490 nm.

Chemical modification of lipase with ferromagnetic modifier: a ferromagnetic-modified lipase

SummaryA ferromagnetic modifier was prepared by reacting ferrous(Fe2+)- and ferric(Fe3+)-ions with polyethylene glycol having two carboxyl groups (MW:2000) at pH 8.0–8.5. Lipase fromPseudomonasfragi 22–39B was coupled with the modifier using water-soluble carbodiimide. The modified lipase, which was dispersed into buffered solutions in the size range of 30–70 nm, exerted the hydrolytic activity of 8.0 U/mg. In a magnetic field of 250 Oe, the ferromagnetic-modified lipase was readily recovered from the colloidal solution.

Ester-exchange reaction between triglycerides with polyethylene glycol-modified lipase

SummaryPolyethylene glycol-modified lipase efficiently catalyzed esterexchange reaction between trilaurin and triolein in the straight hydrophobic substrates. Dilauroyl-monooleoyl-glycerol and monolauroyl-dioleoyl-glycerol were formed from two triglyceride-substrates, trilaurin and triolein, in the presence of the modified lipase at 58°C. Consequently, the melting temperature of the mixture of two substrates was decreased from 33–36°C to 11–13°C. Similar ester-exchange reaction took place between fat and oil composed of triglycerides, accompanied by the decrease in the melting temperature of the reaction mixture.

Fibrinolysis by urokinase endowed with magnetic property.

The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.

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

Abstract 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.