Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-10
2001-05-15
Achutamurthy, Ponnathapu (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S192000, C435S320100, C435S252300
Reexamination Certificate
active
06232457
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cloning and recombinant expression of proteins. In particular, it relates to expression of vanadium bromoperoxidase polypeptides.
BACKGROUND OF THE INVENTION
Vanadium haloperoxidase enzymes are useful in industrial catalysis in a variety of contexts (Sheffield, et al.,
Biotechnology Techniques,
8:579-582 (1994)). For instance, they catalyze a variety of halogenation, oxidation and epoxidation reactions (Itoh, et al.,
Eur. J. Biochem.,
172:477 (1988); Itoh, et. al.
Biochimica et Biophysica Acta.,
1994 (1993); Itoh, et al.,
Appl. Microbiol.
&
Biotechnol.,
43:394-401 (1995)). Although a halide ion is a required cofactor for enzyme activity, products may not be halogenated. Numerous uses in synthetic organic chemistry include reactions with diverse substrates such as aliphatic and aromatic hydrocarbons, phenols, &bgr;-diketones and nitrogen- and sulfur-containing heterocycles (Itoh, et al.,
Eur. J. Biochem.
172:477 (1988); Neidleman et al.,
Biohalogenation: Principles, Basic Roles and Applications,
Ellis Horwood, John Wiley & Sons, New York (1986)). Bromoperoxidases can also be used in place of synthetic organic chemistry reactions to make activated intermediates or products such as pesticides. In addition, these enzymes have an advantage over chemical synthesis in producing stereospecific products (Itoh, et al.,
Eur. J. Biochem.,
172:477 (1988)). Moreover, haloperoxidases have unusual stability (both temporal and thermal) and are active in solvents including methanol, ethanol and acetone.
Recent medical applications of bromoperoxidase have been described. Lovqvist, et al.,
Nuclear Medicine and Biology,
22:125-131 (1995) described the enzymatic bromination of a monoclonal antibody with BR-radionuclide for imaging of antibody localization by PET scanning. There is current interest in enzymatic production of antibiotics including fosfomycin and pyrrolnitrin (Itoh, et. al.
Biochimica et Biophysica Acta.
1994 (1993); Itoh, et al.,
Appl. Microbiol.
&
Biotechnol.
43:394-401 (1995)) and 7-chlorotetracycline (van Pée, K. H.,
J. Bacteriol.,
170:5890-5894 (1988)) via haloperoxidase-catalyzed reactions in bacteria.
Known haloperoxidases include bromoperoxidases from brown and red algae including Fucus and Ascophyllum (Butler, et al.,
Chem. Rev.,
93:1937-1994 (1993)), iodoperoxidase from green algae (Mehrtens, G.,
Polar Biol.
14:351-354 (1994)), and chloroperoxidase from the fungus
Curvularia inaequalis
(Van Schijndel, et al.,
Eur. J. Biochem.,
225:151-157 (1994)). A vanadate requirement for algal haloperoxidase was first described by Vilter (Vilter, H.,
Biological Systems,
31, Vanadium and its role in life, Sigel, et al. (Eds.), Marcel Dekker, New York, N.Y., pp. 325-362 (1995)).
The specific bromoperoxidase activity of the native Fucus enzyme is several fold higher (Butler, et al.) than the other algal enzymes for which at least partial sequences have been reported, Ascophyllum (Vilter 1995) and Corallina (Shimonishi, et al. FEBS Letters, 428, 105-110 (1998)), and higher specific activity than the Curvularia fungal chloroperoxidase (van Schijndel et al. BBA 1161:249-256 (1993)).
Extracted and partially purified bromoperoxidase from the red alga
Corallina officinalis
is commercially available from Sigma Chemical Company. Sigma has also investigated immobilization of enzyme on agarose beads (Sheffield, et al.,
Phytochemistry,
38:1103-1107 (1995)) and on cellulose acetate membrane (Sheffield, et al.,
Biotechnology Techniques,
8:579-582 (1994)) for repetitive catalysis of bromination reactions in flow-through reactors in enzyme-driven preparative organic chemistry. Many industrial uses for stable soybean peroxidase are envisioned by A. Pokora of Enzymol International, Inc. as described by Wick (Wick, C. B.,
Genetic Engineering News,
16(3):1, 18-19). Recombinant enzyme biotechnology is of current industrial interest because enzymes are safe, low-polluting alternatives to chemicals in many applications, and can be modified by protein engineering to fit the requirements of specific applications (Kelly, E. B.
Genetic Engineering News,
16(5):1, 30, 32 (1996) Lovqvist, et al.,
Nuclear Medicine and Biology,
22:125-131 (1995)). Peroxidases can also be incorporated into moldable plastics (Service, R. F.,
Science
272:196-197 (1996)).
Multiple representatives of other classes of peroxidases have been produced in recombinant form. A heme peroxidase, manganese peroxidase from the fungus
Phanerochaete chrysosporidium,
was expressed in recombinant form and refolded for activity (Whitwam, R. E.,
Biochem. Biophys. Research. Communications,
216:1013-1017 (1995)). Recombinant horseradish peroxidase isozyme C (a heme peroxidase) for use in chemiluminescent labeling in molecular biology and biotechnology applications has been described (EP 0299682, WO 89/03424). Recombinant non-heme haloperoxidases have been prepared from the bacteria,
Pseudomonas pyrrocinia
(Wolfframm, et al.,
Gene
130:131-135 (1993)) and two related
Streptomyces aureofaciens
enzymes (van Pée, K. H.,
J. Bacteriol.,
170:5890-5894 (1988); Pfeifer, et al.,
J. Gen. Microbiol.
138:1123-1131 (1992)).
Despite the interest in vanadium haloperoxidases, there are relatively few reports in the literature of the cloning and recombinant expression of a vanadium haloperoxidases. Shimonshi et al.
FEBS Lett.
428:105-110 (1998) described cloning of the enzyme from
Corallina pilulifera.
Cloning of the Curvularia gene is described by Hemrika, et al.
PNAS
94,2145-2149 (1997) and 95/27046. A partial sequence of the Ascophyllum gene is described in Vitel (1995). There is a need in the art for efficient means for producing vanadium haloperoxidases using techniques such as recombinant expression. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acids comprising a polynucleotide sequence encoding a vanadium bromoperoxidase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence from residue 441 to residue 676 as set forth in SEQ ID NO:2. The polypeptides of the invention catalyze the oxidation of o-dianisidine (ODA) when complexed with a vanadium ion. The polynucleotide sequence usually has at least 60% sequence identity to a sequence as set forth in SEQ ID NO:1.
The polypeptides of the invention can be of various sizes. Typically, the polypeptides have molecular weight of about 73.4 kD, usually the polypeptide has a molecular weight of about 58 kD, or sometimes at least about 40 kD.
The invention also provides expression cassettes comprising a heterologous promoter operably linked to the polynucleotide sequences of the invention. The promoter can be active in a prokaryotic cell, such as a bacterium. Also provided are cells the expression cassette of the invention.
The invention further provides methods for enzymatically halogenating or oxidizing a compound using the polypeptides of the invention.
Definitions
A “vanadium bromoperoxidase polypeptide” of the invention is an isolated protein capable of catalyzing the oxidation of o-dianisidine (ODA) when complexed with a vanadium ion. Vanadium bromoperoxidases of the invention can also be identified by the presence of a conserved C terminal region located from residue 441 to residue 676 in SEQ ID NO:2. Polypeptides of the invention typically have a sequence at least about 90% identical (as determined below), usually at least about 95% identical to the sequence from residue 441 to residue 676 in SEQ ID NO:2. One of skill will recognize that the sequence of the polypeptide can be altered without substantially altering activity of the polypeptide (e.g., by conservative substitutions). In addition, as explained below, less conservative modifications (e.g., substitutions, additions, and deletions) can be made to facilitate proper refolding, purification, and the like, as desired.
Full length vanadium bromoperoxidase polypeptides of the invention typically have a m
Ng Kwan L.
Vreeland Valerie
Achutamurthy Ponnathapu
Mayhew Bradley S.
The Regents of the University of California
Townsend & Townsend and Crew LLP
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