Method for enhancing catalytic activity of cells

Details Method for enhancing catalytic activity of cells Method for enhancing catalytic activity of cells

2001-03-12

2003-02-25

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Chemical stimulation of growth or activity by addition of...

C435S251000, C435S174000, C435S041000, C435S130000, C435S134000

Reexamination Certificate

active

06524839

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for producing a desired substance using cells as a catalyst. More specifically, the present invention relates to a method for producing a desired substance using cells treated with lower alcohol as a catalyst.
BACKGROUND OF THE INVENTION
There have been attempts to use cells as a catalyst in biological processes. In order to enhance the catalytic activity of the cells, it is necessary to lower the permeability barrier of cell walls, cell membranes or the like. There have been a large number of reports on attempts to lower the permeability barriers of microorganisms to increase the permeability (Felix et al., Ana. Biochem. 120:211-234 (1982)).
For example, the results of research on enhancing membrane permeability have been reported with respect to yeasts treated with a mixed solution of toluene and ethanol (Serrano Eur. J. Biochem. 34: 479-482 (1973)); cetyltrimethylammonium bromide (Gowda et al., Enzyme Micro. Technol. 13: 154-157 (1991)); ethyl ether (Seip and Cosimo et al., Biotechnol. Bioeng. 40:638-642 (1992)); alcohols (Fenton et al., Enzyme Micro. Technol. 4: 229-232 (1982)); digitonin (Joshi et al., Enzyme Micro. Technol. 11: 439-444 (1989)); Triton X-100 (Vlach et al., J. Mol. Catal. 26:173-185 (1984)); and hexamethylene diamine (Inoue et al., Process biochem. 29: 271-275 (1994)).
However, such research on permeability was conducted for the purpose of determining intracellular enzyme activities in situ and not for the purpose of industrial applications (Felix et al., ibid.).
On the other hand, in recent years, genetic recombination technology has advanced dramatically and excellent gene expression systems have been developed. For example, regarding yeasts, Romanos et al., Yeast 8: 423-488 (1992) describes various gene expression systems. In these gene expression systems, excessive enzymes are produced and accumulated in cells, so that the cells themselves can be an effective catalyst.
Furthermore, for example, if enzymes present in cell cortexes, periplasms or cell membranes, such as lipase which is retained in cell cortices, are used without any treatments or if these enzymes have been expressed by gene manipulation, the cells themselves can be an effective catalyst.
However, in order to utilize the enzymes accumulated in the cells or the enzymes retained in cell cortices, periplasms or cell membranes, it is necessary to lower the barriers of cell walls, cell membranes or the like as described above. However, there is no effective means for lowering the permeability barrier, so that a process of disrupting cells is required. Furthermore, because a process of strict temperature control to prevent inactivation of the enzymes, a purification process, and other processes are required, there are disadvantages that the processes are complicated and lead to high costs.
Therefore, there is a demand for technologies that eliminate disruption of cells, extraction of enzymes and purification, and provide a low-cost catalyst that can be manipulated in a very simple manner, namely, technologies that lower the permeability barrier of cells and use cells themselves as a catalyst.
SUMMARY OF THE INVENTION
The present invention is carried out to solve the above-described problems. The present invention is directed to a cell treated with a lower alcohol, wherein the reaction rate of the treated cell is at least 50 times higher than that of a cell that is not treated with the lower alcohol.
In a preferable embodiment, the cell is yeast or filamentous fungus, and the cell is immobilized onto a carrier.
Furthermore, in a preferable embodiment, the lower alcohol is selected from the group consisting of methanol, ethanol, propanol, and isopropanol.
In a preferable embodiment, the cell is dried.
In a preferable embodiment, the cell contains an enzyme having a resistance to treatment with a lower alcohol.
In a preferable embodiment, the enzyme is glyoxalase I or lipase.
In a preferable embodiment, the enzyme is produced by the expression of a recombinant gene.
In another aspect, the present invention is directed to a catalyst comprising a cell treated with a lower alcohol, wherein the reaction rate of the catalyst is at least 50 times higher than that of a catalyst comprising a cell that is not treated with the lower alcohol.
In a preferable embodiment, the cell is yeast or filamentous fungus.
In another aspect, the present invention is directed to a method for increasing the reaction rate of a cell to at least 50 times higher than that of an untreated cell, comprising treating the cell with a lower alcohol.
In a preferable embodiment, the cell is yeast or filamentous fungus.
In another aspect, the present invention is directed to a method for producing a desired substance by reacting a cell with a substrate, comprising reacting a cell treated with a lower alcohol with the substrate to an enzyme, wherein the reaction rate of the cell treated with the lower alcohol is at least 50 times higher than that of an untreated cell.
In a preferable embodiment, the cell is yeast or filamentous fungus, and preferably the cell is immobilized onto a carrier.
In a preferable embodiment, the cell contains an enzyme having a resistance to treatment with a lower alcohol.
In a preferable embodiment, the enzyme is glyoxalase I, and the desired substance is S-lactoylglutathione.
In a preferable embodiment, the enzyme is lipase and the desired substance is fatty acid ester.
In a preferable embodiment, the glyoxalase I or the lipase is produced by the expression of a recombinant gene.
In a preferable embodiment, the cell is immobilized onto a carrier and filled in a column.
In another aspect, the present invention is directed to a sensor having a cell treated with a lower alcohol, wherein the reaction rate of the treated cell is at least 50 times higher than that of a cell that is not treated with the lower alcohol.
In a preferable embodiment, the cell is yeast or filamentous fungus.
The present invention solves the above-described problems. More specifically, by treating cells with a lower alcohol, the reaction rate of the cells becomes at least 50 times higher than that of untreated cells, and 200 to 600 times higher in preferable cases. This seems to be a result that the treatment with a lower alcohol lowers the permeability barrier of the cell walls or the cell membranes. In the present invention, free cells treated with lower alcohol can be used as a catalyst without any further treatment. Immobilized cells also can be treated with a lower alcohol to be used as a catalyst without any further treatment. The cells or the immobilized cells treated with lower alcohol are further dried for use. Thus, the present invention provides cells having an excellent catalyst activity that can be produced by a very simple method and can be used repeatedly.
Furthermore, the present invention provides a method for producing a desired substance using the cells treated with a lower alcohol as a catalyst.


REFERENCES:
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Liu Y. et al, Production of S-Lactoglutathione by High Activity Whole Cell Biocatalysts Prepared by Permeabilization of Recombinant Saccharomyces cerevisiae with Alcohols, Biotechnology and Bioengeneering, (1999), 64, 54-60.*
Inoue Y. et al, Continuous production of S-lactoylglutatione by immobilized Hansenula mrakii cells, Process. Biochemistry, 1994, 29, 271-275.*
Felix, H., et al. “Permeabilized Cells,” Ana. Biochem. 120, 211-234 1982.
Serrano, R., et al. “Assay of Yeast Enzymesin situA Potential Tool in Regulation Studies,” Eur. J. Biochem. 34, 479-482 1973.
Gowda, Lalitha R., et al. “Permeabilization of Bakers' yeast by cetyltrimethylammonium bromide for intracellular enzyme catalysis,” Enzyme Microb. Technol. 1991, vol. 13, Feb. 154-157.
Seip and Cosimo, “Optimization of Accessible Catalase Activity in Polyacrylamide Gel-Immobilized Saccharomyces cerevisiae,” Biotechnol

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