Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1997-10-16
2001-01-09
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S068100, C435S182000, C435S183000, C435S195000, C435S213000
Reexamination Certificate
active
06171813
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to enzyme-surfactant ion pairs with high catalytic activity and solubility in organic solution. More specifically, the invention relates to the solubilization of hydrolases in organic solvents without reversed micelles, and to enzyme-catalyzed reactions such as peptide synthesis with aqueous-like activity.
2. Related Art
The longstanding desire to achieve catalysis in organic liquids has led to several approaches. Chymotrypsin can be extracted from acidic aqueous solutions into organic solvents at very low surfactant concentrations (e.g., <2 mM) via ion-pairing of a surfactant with the protein. Paradkar and Dordick,
Biotechnol. Bioeng.
1994, 43, 529 (“Paradkar & Dordick 1994”). It was shown in Paradkar & Dordick 1994 that an ion-paired complex of chymotrypsin and an anionic surfactant can be extracted into isooctane in the absence of reversed micelles. This led to the general suggestion that the CT-surfactant complexes may have direct application for preparing biocatalysts active and stable in homogeneous organic solutions.
SUMMARY OF THE INVENTION
It is an object of the invention to provide improved catalysis with stable catalytic enzyme-surfactant complexes that can be dissolved in an organic solvent, maintain their native structure, and exhibit high catalytic activities approaching those of aqueous solutions, and exceeding the activity of a catalyst obtained by optimizing extraction variables and the water content of the reaction medium.
It is another object of the invention to provide methods for enzyme catalysis in organic solutions containing water.
According to the invention, catalytic enzyme-surfactant ion pair complexes can be obtained by extraction from aqueous solution into organic solution, in substantially dehydrated form. These catalytic complexes can be obtained at maximal activity and can be used in a wide variety of catalytic reactions requiring homogeneous organic solutions. These complexes remain dissolved, maintain their native structure, and exhibit high catalytic activities which approach that in aqueous solutions.
An inventive method for improving catalysis by an enzyme-surfactant ion pair complex in an organic reaction solvent comprises providing greater than about 0.03% water in the organic reaction solvent, sufficient to extend the half life of the catalyst and to increase the catalytic efficiency of the catalyst.
A method of catalysis in an organic reaction solvent according to the invention comprises the steps of:
(a) obtaining a catalyst comprising a pre-activated enzyme-surfactant ion pair complex comprising an enzyme and an ionic surfactant capable of forming an ion pair complex with the enzyme, the complex having catalytic efficiency at least an order of magnitude greater than a suspension of an equal amount of the enzyme in the organic reaction solvent without surfactant, and produced by a process comprising dissolving the enzyme in an aqueous solution at a pH of maximal enzyme activity and agitating the enzyme-containing aqueous solution with an aqueous-immiscible organic extraction solvent and the dissolved ionic surfactant so as to extract the enzyme into the organic extraction solvent as a surfactant-enzyme ion pair complex, the ratio of surfactant to enzyme being less than that necessary to form reversed micelles,
(b) combining the catalyst with an organic reaction solvent, comprising an organic solvent and water in an amount sufficient to maximize the rate of catalysis and stabilize the activated enzyme without substantially increasing hydrolysis, and ranging from about 0.03% to about 2.5% v/v,
(c) adding to the organic reaction solvent at least one substrate for the enzyme, and
(d) allowing a sufficient time for the enzyme to catalyze conversion of the substrate to a product.
The water content of the reaction solvent preferably does not exceed the water saturation point for the organic solvent containing the surfactant. The molar ratio of water to enzyme in the organic reaction solvent is preferably less than about 75:1.
The invention contemplates adding up to 2.5% v/v water to the reaction solvent, which may comprise a water miscible hydrophilic organic solvent. Preferably, the amount of water in the reaction solvent is between about 0.1% and about 0.3%, and the half life of the enzyme activity of the catalyst is at least about 0.25 hours, more preferably at least about one hour. The number of turnovers catalyzed by the enzyme-surfactant ion pair complex in the organic reaction solvent during one half life is preferably greater than that of the enzyme dissolved in water.
Preferably, the substrate is added to the organic reaction solvent continuously during conversion of the at least one substrate to a product, and the product is removed from the organic reaction solvent continuously during conversion of the at least one substrate to a product. Or the conversion is carried out as a batch process, in which the reaction is substantially completed before recovering the product, preferably by precipitating the enzyme from the organic reaction solvent by removal of the surfactant from the enzyme-surfactant complex after adding the substrate.
The substrate preferably comprises a blocked or unblocked acyl donor and a nucleophile, and may be an amino acid, an amino acid ester, an N-blocked amino acid, an N-blocked amino acid ester, an amino acid amide, an N-blocked amino acid amide, a polypeptide, a polypeptide ester, an N-blocked polypeptide, an N-blocked polypeptide ester, a polypeptide amide, an N-blocked polypeptide amide, an acyl donor is a methyl or ethyl ester of tyrosine, tryptophan, alanine, or phenylalanine, a methyl or ethyl ester of a dipeptide or tripeptide containing any of tyrosine, tryptophan, alanine, or phenylalanine, or an N-benzoyl, N-acetyl, or N-carbobenzoxy derivative thereof. The acyl donor and nucleophile are preferably added in a ratio between about 2:1 and 1:2.
The enzyme may be a catalytic antibody, an oxidoreductase, a transferase, a lyase, an isomerase, or a ligase, a hydrolase with acyl transferase activity in organic solvents, a peroxidase catalyzing phenolic polymerizations, a tyrosinase catalyzing aromatic hydroxylations, an alcohol dehydrogenase catalyzing stereoselective oxidation and reduction, a lipase, a nuclease, an aldolase, a phosphatase, a sulfatase, subtilisin, papain, pepsin, thermolysin, or thrombin.
The first step of the inventive method may involve
(1) forming a two-phase aqueous/organic system comprising (i) a pre-activated enzyme solution produced by dissolving the enzyme in the aqueous solution at a pH of maximal enzyme activity, the aqueous solution having an enzyme concentration of from about 1 &mgr;g/ml to about 10 mg/ml, and ionic strength less than about 80 mM, (ii) an organic extraction solvent that is immiscible with water, and (iii) an ionic surfactant, the molar ratio of surfactant to enzyme in the two-phase system being sufficient to form an enzyme-surfactant ion pair complex, and substantially less than that necessary to form reversed micelles in the organic solvent;
(2) then agitating the two-phase system for a period sufficient to produce an ion pair complex of the pre-activated enzyme and the ionic surfactant and to extract the ion pair complex directly from the aqueous solution into solution in the organic extraction solvent, without substantial formation of reversed micelles;
(3) then separating the organic phase from the aqueous phase, whereby a homogeneous organic enzyme solution is obtained containing dissolved pre-activated enzyme-surfactant ion pair complex and essentially no reversed micelles.
The organic extraction solvent may be dried after extraction by evaporating the organic extraction solvent after extraction. In one embodiment, the enzyme is &agr;-chymotrypsin, the surfactant is Aerosol OT, AOT, the pH of the aqueous activating solution is from about 7.0 to about 8.5, the ionic strength is less than about 20 mM, the enzyme:surfactant ratio is about 1:30 and the enzyme surfactant
Dordick Jonathan S.
Paradkar Vikram M.
Sergeeva Maria V.
BioTechnology Research & Develop. Corp.
Gollin Michael A.
Venable
Weber Jon P.
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