Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-18
2003-01-14
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007400, C435S007600, C435S184000, C435S239000
Reexamination Certificate
active
06506566
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of identifying enzymes suitable for use in detergents, especially the selection of specific enzyme variants among a large number of such variants created through random mutagenesis.
The invention further relates to specific materials used in said method, the enzymes selected by use of the method, and to detergent compositions comprising aid enzymes.
BACKGROUND OF THE INVENTION
An increasing number of polypeptides, including enzymes and non-enzymatic proteins, are being produced industrially, for use in various industries, household, food/feed, cosmetics, medicine etc. The major source for these proteins is and have been microorganism found in nature. However, since science have developed many new techniques for creating variants of polypeptides through protein engineering, it is becoming increasingly important to be able to make huge populations of protein variants, to screen and select for new properties.
The classic approach for finding polypeptides with new and special properties, have been to screen wild type organism present in nature. This has been a very successful way of generating polypeptides to be used in such diverse areas as the above mentioned industries. Furthermore it is possible to generate new variants of a protein by classical mutation of the microorganism. However, since this approach is a very labour and time consuming process, researchers have in the last two decades been developing improvements on existing polypeptides by creating artificial diversity, using more specific and therefor faster techniques, such as protein and genetic engineering.
Such artificial diversity can be generated by using recombinant DNA techniques such as site-directed or random mutagenesis. This approach generates a population of polypeptide variants which can be selected according to the newly acquired phenotype of the given organism used for the experiments. Such phenotypic screen has its limitations according to the number of mutants it would be possible to screen. First of all one would have to set up a screening assay where a given new property of a polypeptide can be detected and the given mutant can be isolated. Then the DNA encoding the polypeptide is to be isolated and characterized.
These techniques have been used successfully to create new important polypeptides exhibiting improved properties, such as higher specific activity, higher stability under high or low pH, temperature stability, stability under oxidizing conditions etc.
However successful this approach has been, there is still a limitation in the number of polypeptides it is possible to screen and test by using these techniques. Therefore it is of interest to create novel systems, whereby it is possible to combine the screening and the selection in one procedure.
Furthermore such a procedure should make it possible to pick out only those variants with the best properties under given conditions, and these variants should be picked out of a variant population consisting of more than 10
8
members.
One promising approach for making such artificial diversity and selecting for a new or improved specific polypeptide feature is the technique known as “phage display”. This technique couples the genotype with the phenotype making it possible to select the two characteristics together. Hereby eliminating a couple of steps present in the above mentioned approaches.
Phage display is a fairly new technique first used and described by George P. Smith in 1988 (a first attempt was made in 1985). The earliest patents were granted to R. C. Ladner (U.S. Pat. Nos. 5,096,815 and 5,223,409) relating to “Generation and selection of novel DNA-binding proteins and polypeptides” and “Directed evolution of novel binding proteins”, respectively. These two patents contain a wealth of references giving the background for the techniques.
Bacteriophage display systems have been developed that link a polypeptide or peptide of interest to the DNA that encodes it. Such display systems have been used to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties (see references 12 to 15).
Recently, improvements of the display approach have made it possible to express enzymes as well as antibody fragments on the bacteriophage surface thus allowing for selection of specific properties by selecting with specific ligands (see references 2-6, and 16-18).
For display of antibodies there is a large amount of references describing this successful approach. When it comes to the display of enzymes this is a less developed area, however there are some examples of this approach (see references 1, 8, and 10).
Especially the selection principles for enzyme phages are under evaluation and development. It has been shown that the enzymatic mechanism of a given enzyme can be used as means of binding principal by the use of suicidal inhibitors (see references 1 and 9).
SUMMARY OF THE INVENTION
The present invention relates to a method of selecting enzymes or enzyme variants suitable for use in detergents, wherein said enzyme variants to be selected are in a mixture of enzyme variants which are each displayed on the surface of cells or phage particles, comprising the following steps
i) introducing said mixture into a detergent composition in fluid form under conditions that will have a negative impact on the activity of or inactivate most of said enzyme variants,
ii) reacting said mixture with a catcher molecule that will bind specifically only to enzyme variants that exhibit the property sought for, to form a complex between said cell or phage displayed enzyme variant exhibiting the property sought for and said catcher,
iii) separating said complex from the remaining parts of said mixture,
iv) dissociate said complex to isolate such cell(s) or phage(s) that displayed said enzyme variant to be selected without the catcher molecule,
v) introducing said phage into a host wherein it will multiply, or
va) cultivating said cell under conditions conducive to its multiplication, and
vi) isolating a DNA molecule coding for said enzyme variant from the genome of said cell or phage.
Another aspect of the invention relates to such enzymes that have been selected by use of the above method.
A further aspect of the invention relates to the use of detergents in the above method.
The invention also relates to a number of catcher molecules, such as suicide inhibitors and transition state analogs useful in the method of the first aspect of the invention.
Finally the invention relates to the enzymes produced by the method of the invention and detergent compositions comprising such enzymes.
REFERENCES:
patent: 4663071 (1987-05-01), Bush et al.
patent: 5096815 (1992-03-01), Ladner et al.
patent: 5223409 (1993-06-01), Ladner et al.
patent: 5837517 (1998-11-01), Sierkstra et al.
patent: 5914306 (1999-06-01), Svendsen et al.
patent: 6190900 (2001-02-01), Sierkstra et al.
patent: 6194183 (2001-02-01), Markvardsen et al.
patent: 6303340 (2001-10-01), Pollitt et al.
patent: 0 396 608 (1996-03-01), None
patent: WO 92/05249 (1992-02-01), None
patent: WO 93/17124 (1993-09-01), None
patent: WO 95/22615 (1995-08-01), None
McCafferty J. et al , Phage-enzymes: expression and affinity chromatography of functional alkaline phosphates on the surface of bacteriophage, Protein Eng. 1991, 4/8, 955-961.*
Clackson T. et al, Making antibody fragments using phage display libraries, Nature, 1991, 352, 642-628.*
Fuchs P. et al, Targeting recombinant antibodies to the surface ofEscherichia coli: fusion to the peptidoglycan associated lipoprotein, Biotechnology, 1991, 9, 1369-1372.*
Spee J. et al, Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dlTP, Nucleic Acid Research, 1993, 21/3, 777-778.*
Hawkins R. E. et al, Selection of phage antibodies by binding affinity mimicking affinity maturation, J. Mol. Biol., 1992, 226, 889-896.*
Hoogenboom H. R. et al, Building antibodies from their genes, Immu
Bjornvad Mads Eskelund
Diderichsen Borge
Markvardsen Peter
Mikkelsen Frank
Achutamurthy Ponnathapu
Garbell Jason I.
Novozymes A/S
Walicka Malgorzata A.
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