Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Patent
1995-02-27
2000-02-22
Grimes, Eric
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
435 697, 435 698, 435 699, 4351721, 4351723, 4352523, 4353201, C12N 1500, C12N 120, C12P 100
Patent
active
060279109
DESCRIPTION:
BRIEF SUMMARY
The present invention is in the field of conversion processes using immobilized enzymes, produced by genetic engineering.
BACKGROUND OF THE INVENTION
In the detergent, personal care and food products industry there is a strong trend towards natural ingredients of these products and to environmentally acceptable production processes. Enzymic conversions are very important for fulfilling these consumer demands, as these processes can be completely natural. Moreover enzymic processes are very specific and consequently will produce minimum amounts of waste products. Such processes can be carried out in water at mild temperatures and atmospheric pressure. However enzymic processes based on free enzymes are either quite expensive due to the loss of enzymes or require expensive equipment, like ultra-membrane systems to entrap the enzyme.
Alternatively enzymes can be immobilized either physically or chemically. The latter method has often the disadvantage that coupling is carried out using non-natural chemicals and in processes that are not attractive from an environmental point of view. Moreover chemical modification of enzymes is nearly always not very specific, which means that coupling can affect the activity of the enzyme negatively. Physical immobilization can comply with consumer demands, however also physical immobilization may affect the activity of the enzyme in a negative way. Moreover, a physically immobilized enzyme is in equilibrium with free enzyme, which means that in continuous reactors, according to the laws of thermodynamics, substantial losses of enzyme are unavoidable.
There are a few publications on immobilization of enzymes to microbial cells (see reference 1). The present invention provides a method for immobilizing enzymes to cell walls of microbial cells in a very precise way. Additionally, the immobilization does not require any chemical or physical coupling step and is very efficient. Some extracellular proteins are known to have special functions which they can perform only if they remain bound to the cell wall of the host cell. Often this type of protein has a long C-terminal part that anchors it in the cell wall. These C-terminal parts have very special amino acid sequences. A typical example is anchoring via C-terminal sequences enriched in proline (see reference 2). Another mechanism to anchor proteins in cell walls is that the protein has a glycosyl-phosphatidyl-inositol (GPI) anchor (see reference 3) and that the C-terminal part of the protein contains a substantial number of potential serine and threonine glycosylation sites. O-Glycosylation of these sites gives a rod-like conformation to the C-terminal part of these proteins. Another feature of these manno-proteins is that they seem to be linked to the glucan in the cell wall of lower eukaryotes, as they cannot be extracted from the cell wall with SDS, but can be liberated by glucanase treatment.
SUMMARY OF THE INVENTION
The present invention provides a method for immobilizing an enzyme, which comprises the use of recombinant DNA techniques for producing an enzyme or a functional part thereof linked to the cell wall of a host cell, preferably a microbial cell, and whereby the enzyme or functional fragment thereof is localized at the exterior of the cell wall. Preferably the enzyme or the functional part thereof is immobilized by linking to the C-terminal part of a protein that ensures anchoring in the cell wall.
In one embodiment of the invention a recombinant polynucleotide is provided comprising a structural gene encoding a protein providing catalytic activity and at least a part of a gene encoding a protein capable of anchoring in a eukaryotic or prokaryotic cell wall, said part encoding at least the C-terminal part of said anchoring protein. Preferably the polynucleotide further comprises a sequence encoding a signal peptide ensuring secretion of the expression product of the polynucleotide. Such signal peptide can be derived from a glycosyl-phosphatidyl-inositol (GPI) anchoring protein, .alpha.-factor, .alpha.-agglut
REFERENCES:
patent: 5348867 (1994-09-01), Geogiou et al.
Pelham, H. R. B. "Control of protein exit from endoplasmic reticulum" Ann. Rev. Cell Biol. 5, 1-23, 1989.
Martineau et al. "A Genetic System to Elicit and Monitor Antipep . . . " Bio/technology 9, 170-172, Feb. 1991.
Lipke et al. AG.alpha.1 is the structural gene for the Saccaromyces cervisiae .alpha.-agglutinin, . . . , Mol. and Cell. Biol. 9 (8), 3155-3165, Aug. 1989.
Francisco, Joseph A.; Proceedings of the National Academy of Sciences of USA; vol. 89; Apr. 1992; Washington, US; pp. 2713-2717.
George et al; Journal of Bacteriology; vol. 171; No. 9; Sep. 1989; pp. 4569-4576.
Hiebert, Scott W. et al; Journal of Cell Biology; vol. 107; Sep. 1988; pp. 865-876.
Chen, Cecil C. et al; Journal of Biological Chemistry; vol. 265; No. 6; Feb. 25, 1990; Baltimore US; pp. 3161-3167.
EMR, Scott D. et al; Chemical Abstracts; vol. 102; No. 3; Jan. 21, 1985; Columbus, Ohio, US; abstract No. 18728c.
S. Vijaya et al; Molecular and Cellular Biology; vol. 8; No. 4; Apr. 1988; Washington, US; pp. 1709-1714.
Klis Franciscus M
Schreuder Maarten P
Toschka Holger Y
Verrips Cornelis T
Grimes Eric
Nashed Nashaat T.
Unilever Patent Holdings B.V.
LandOfFree
Process for immobilizing enzymes to the cell wall of a microbial does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for immobilizing enzymes to the cell wall of a microbial, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for immobilizing enzymes to the cell wall of a microbial will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-519585