Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-02-14
2002-12-24
Achutamurthy, Ponnathapua (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S440000, C536S023200
Reexamination Certificate
active
06498026
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of genetics, cellular and molecular biology, and enzymology. More particularly, the invention relates to novel galactose oxidase enzymes, polynucleotides, and polypeptides.
BACKGROUND
The enzyme galactose oxidase (GO), encoded by the galactose oxidase gene (go) of
Dactylium dendroides
and other organisms, catalyzes a reaction in which a primary alcohol such as the C6 hydroxyl group of galactose is oxidized to an aldehyde with concomitant reduction of molecular oxygen to hydrogen peroxide, as shown generally in Formula I.
RCH
2
OH+O
2
→RCHO+H
2
O
2
(I)
GO enzymes may be readily used to oxidize the D-galactose (and other hydroxyl-containing) side-chains of many complex compounds, including, but not limited to molecules comprising a D-galactose moiety that is not sterically hindered or blocked at the C6 hydroxyl, and molecules comprising a moiety such as dihydroxyacetone, glycerol, or similar short-chain alcohols in which a primary hydroxyl functional group is present. Of particular interest is the oxidation of guar gum. When guar gum is oxidized by GO, the resulting compound, called oxidized guar, can be used for many purposes, including use in paper manufacturing to add strength to paper products via the formation of acetal, hemiacetal, and other crosslinks with cellulose fibers (see, e.g., 1996; Aldehyde cationic derivatives of galactose containing polysaccharides used as paper strength additives; U.S. Pat. No. 5,554,745 (Chiu et al.); U.S. Pat. No. 5,502,091 (Dasgupta).
Galactose oxidases have been isolated from several species. For example, U.S. Pat. No. 6,090,604 discloses a genomic DNA sequence and deduced amino acid sequence for the GO enzyme from
Fusarium venenatum.
Wildtype GO enzymes, however, are relatively inefficient oxidizers of guar gum and other compounds. Thus, there exists a need in the art for superior oxidizers of such compounds. Additionally, one obstacle to the development of variant GO's is the high viscosity of guar (e.g., the cationic guar used in this work has a viscosity of 1000 cps in 1% aqueous solution), a high molecular weight polymeric substrate. Indeed, many natural or synthetic polymers are insoluble or highly viscous when in solution, and are consequently difficult to pipette by hand or robotic means. Therefore, various methods of high-throughput screening used to evaluate such variant enzymes useful in adding functionality to viscous or insoluble polymers are needed by the art. Such methods enable those of skill in the art to create mutant GO enzymes capable of more efficient oxidative enzymatic reactions.
SUMMARY OF THE INVENTION
Although GO does display significant activity towards guar, the present inventors improved its specific activity via in vitro evolution of the enzyme. Using selected methods of mutagenesis, the present inventors created mutant galactose oxidase genes (mgo's) which encode variant galactose oxidase enzymes (vGO's), which variants are superior to wildtype GO in terms of efficiency of oxidizing guar and other compounds, as well as in conferring improved thermostability.
Error prone PCR (EPP) was used to generate mutant go genes, encoding variant GO enzymatic proteins. One of skill in the art will appreciate that any method capable of generating mutant genes would be suitable for practicing the present invention. In order to evaluate the efficiency of oxidation of the variants, certain recently developed methods of high throughput screening were used. However, one of skill in the art may choose a method of screening suitable to the substrate of interest. One aspect of the particular screening method used for the evolution of vGO's by the present inventors is the use of a proxy, i.e., a substrate that represents an adequate substitute for a problematic compound. Particularly, in order to evaluate vGO's oxidation of guar, the proxy methyl-&agr;-D-galactose (methyl galactose) was used. The variant GO's of the present invention are demonstrably superior to wildtype GO in terms of their ability to oxidize guar and other complex compounds having hydroxyl-containing sidechains.
In one aspect, the present invention provides polynucleotides comprising mutant go genes. In another aspect, the invention provides polypeptides encoded by such polynucleotides. In another aspect, the invention provides variant GO enzymes having superior enzymatic activity (on methyl galactose or other substrates), and/or thermostability (i.e., resistance to heat inactivation), and which differ from wildtype GO by having at least one substituted amino acid. In another aspect, the present invention provides vectors comprising the polynucleotides. In another aspect, the present invention provides cells transfected or transformed with such vectors. In still another aspect, the present invention provides antibodies specific to the variant GOs. In another aspect, the present invention provides methods of using these molecules and constructs.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding variant GO enzymes which differ from wildtype GO by at least one amino acid, and which exhibit superior enzymatic activity (on methyl galactose or other substrates) and/or thermostability. DNA polynucleotides of the invention include genomic DNA, cDNA, and DNA that has been chemically synthesized in whole or in part. The present invention also provides vectors comprising such polynucleotides, and cells transfected with such vectors. The present invention also provides the proteins encoded by such polynucleotides (i.e., variant GO enzymes), and methods of using the polynucleotides and polypeptides. The present invention also provides antibodies specific to vGOs capable of binding specifically to the variants while remaining unbound to wildtype GO.
Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.
“Synthesized” as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. “Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. By the term “region” is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. The term “domain” is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular re
Bylina Edward J.
Coleman William J.
Delagrave Simon
Maffia, III Anthony M.
Murphy Dennis J.
Achutamurthy Ponnathapua
Hercules Incorporated
Pak Yong
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