Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Isomerase
Patent
1995-11-22
1998-08-18
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Isomerase
4351721, 4353201, 435325, 536 232, 935 22, C07H 2104, C12N 500, C12N 990, C12N 1500
Patent
active
057957673
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to acylglucosamine 2-epimerase and derivatives thereof, a DNA molecule encoding the enzyme, a recombinant vector integrated thereinto the DNA molecule, a transformant containing the vector and a method for producing the epimerase.
The invention relates to a novel polypeptide, acylglucosamine 2-epimerase and derivatives thereof having renin binding activities, a DNA molecule encoding the enzyme, a recombinant vector integrating thereinto the DNA molecule, a transformant containing the vector, a method for producing the epimerase, an antihypertensive agent containing the enzyme or derivative thereof as an essential component, an epimerization agent and methods for producing N-acetylmannosamine and N-acetylneuraminic acid.
BACKGROUND ART
In recent years, N-acetylneuraminic acid is noted as raw materials of drugs. It is known that said N-acetylneuraminic acid may be enzymatically synthesized from N-acetylmannosamine and pyruvic acid using N-acetylneuraminic acid lyase. However, because of expensiveness and difficulty of large-scale preparation of N-acetylmannosamine, a method for preparing N-acetylneuraminic acid by reacting inexpensive N-acetylglucosamine and pyruvic acid in the presence of N-acetylneuraminic acid lyase is proposed (Udo Kragl et al., Angewandte Chemi-International Edition in English, 30, 827-828 (1991)). This method utilizes that acylglucosamine 2-epimerase epimerizes N-acetyl-glucosamine to N-acetylmannosamine. However, acylglucosamine 2-epimerase employed in this method exists only in a trace amount in animal tissues and techniques of large-scale production thereof has not been developed. Accordingly, above-mentioned method may not be employed practically.
On the other hand, Teshima et al. (Clinical Chemistry, 34, 2291-2294 (1988)) disclose that acylglucosamine 2-epimerase is useful for determination of N-acetylhexosamine.
As shown above, acylglucosamine 2-epimerase is a very important enzyme and establishment of an efficient method for production thereof is earnestly desired.
It is known that acylglucosamine 2-epimerase exists in animal tissues. For example, Asis Datta (Methods in Enzymology, 41, 407-412 (1975)) reported that acylglucosamine 2-epimerase existed in porcine kidney. It also widely exists in kidney, liver, mucosal cell, submandibular gland, intestinal mucosa, colon, salivary gland, etc.
Purification of acylglucosamine 2-epimerase from animal tissues is, however, very difficult, and only crude acylglucosamine 2-epimerase is obtained up to the present. For example, Ghosh et al (Methods in Enzymology, 8, 191-195 (1966)) and Asis Datta (Methods in Enzymology, 41, 407-412 (1975)) tried to isolate and purify acylglucosamine 2-epimerase. However, degree of purity is low according to the report of Ghosh. According to Asis Datta, specific activity thereof is about as low as 6 unit/mg protein.
These reports demonstrate that purification of enzyme from crude extract of porcine kidney cortex prepared by homogenizer followed by a combination of conventional purification means, such as protamine concentration, bentonite treatment, DEAE-cellulose column chromatography, adsorption on calcium phosphate gel, etc. is difficult.
The inventors further conduct gel filtration, hydroxyapatite, hydrophobic gel and like a variety of chromatographies and chromatofocusing in addition to said purification means, which do not lead to recovering said enzyme in a purified form due to dilution of enzymatic activities and loss of enzymatic activities caused by inactivation of the enzyme. A trace amount of existence of the enzyme in kidney is one of reasons for difficulties of purification thereof.
Recently, preparation of heterologous proteins using microorganisms becomes relatively easy with the progress of gene recombination techniques. However, because of necessity of isolation of protein for utilizing said means, materials to specify said enzyme, such as DNA probes and antibodies may not be prepared with respect to acylglucosamine 2-epimerase which is obtain
REFERENCES:
patent: 5071750 (1991-12-01), Kragl et al.
The Journal of Biological Chemistry, vol. 265, No. 12, Apr. 1990, Hiroyasu Inoue et al., "Molecular Cloning and Sequence Analysis of a cDNA encoding a porcine Kidney Renin-binding Protein", pp. 6556-6561.
The Journal of Biological Chemistry, vol. 110, No. 4, Oct. 1991, Hiroyasu Inoue et al., "Genetic and Molecular Properties of Human and Rat Renin-Binding Proteins with Reference to the Function of the Leucine Zipper Motif", pp. 493-500.
Datta (Jan. 1975) N-Acetylglucosamine 2-Epimerase from Hog Kidney, Methods in Enzymology 41 (Part B): 407-412.
Kragl (Jun. 1992) Reaction technology for biocatalytic processes exemplified by the continuous enzymic synthesis of N-acetylneuraminic acid, Ber. Forschungszent, Juelich, Juel 2583, 186 pp.
Ngo et al. (Feb. 1994) In The Protein Folding Problem and Tertiary Structure Prediction, Eds. Merz et al., Birkhauser, Bostaon, MA, pp. 433 and 492-495.
Maru Isafumi
Ohta Yasuhiro
Tsukada Yoji
Marukin Shoyu Co., Ltd.
Stole Einar
Wax Robert A.
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