Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1997-04-18
1998-12-01
Weber, Jon P.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
4352521, C12N 912, C12P 2104
Patent
active
058437509
DESCRIPTION:
BRIEF SUMMARY
This application is a 331 of PCT/JP95/01997 filed Sep. 29, 1995 and claims priority under 35 U.S.C. 119 of Japanese application Ser. No. 6/243539 filed Oct. 7, 1994 and 6/247,419 filed Oct. 13, 1994.
FIELD OF THE INVENTION
This invention relates to a sorbitol kinase, which has properties utilizing mainly ATP, but not substantially phosphoenolpyruvate, as the phosphate donor, having N-terminal amino acid sequence (SEQ ID NO: 10) of the formula ##STR2## with stability up to 60.degree. C. DNA expressing the said sorbitol kinase, a process for producing sorbitol kinase comprising sorbitol kinase producing microorganism belonging to genus Erwinia in a medium and isolating sorbitol kinase therefrom, a substantially pure microorganism which produces said sorbitol kinase and process for producing sorbitol kinase using the said substantially pure microorganism.
PRIOR ARTS
Sorbitol is a representative substance in polyol, and is generated from glucose by aldose reductase.
In diabetes mellitus, when hyperglycemic condition occurred and intracellular hypergeneration of sorbitol continues, sorbitol is accumulated excessively in the cells due to less extracellular difusion of sorbitol, then various failure will occurres caused by accumulation of sorbitol. Accordingly, exact determination of sorbitol is important to find out disease condition of patients with diabetes mellitus.
Sorbitol is known to exist, especially in the crystalline lens, nerves and erythrocytes. Assay of sorbitol has been attempted for various organs. An amount of sorbitol in the erythrocytes converted into whole blood is the levels of approximately 10 .mu.M-50 .mu.M even in the patients with diabetes mellitus. (J. Malone et al.: Red cell sorbitol: An indicator of diabetic control. Diabetes: 861-864, 1980) Accordingly, no precise determination could be made.
Assay methods of sorbitol were knwon including, for example, gas chromatography, HPLC and enzymatic method using sorbitol dehydrogenase. Among them, gas chromatography and HPLC methods for determination of sorbitol are quite complicated. Enzymatic method using sorbitol dehydrogenase is quite disadvantage due to low substrate specificity of sorbitol dehydrogenase for sorbitol and sorbitol could not be assayed precisely.
We have searched in the references an enzyme having activity for sorbitol, and found 2 types of enzyme; i.e. sorbitol kinase from silk worm eggs having optimum pH approx. 9.5 and utilizing ATP and phosphoenol pyruvate as a phosphate donor (T. Yaginuma and O. Yamashita, Comp. Biochem. Physiol. 98B(1), 135-141, 1991) and hexokinase, from bovine and human eye lens, having sorbitol activity which utilize ATP as a phosphate donor and utilizing substrates of sorbitol and glucose (S. Srivastava et al. Biochim. Biophys. Acta, 717(2), 210-214, 1982). ATP is utilized mainly as a phosphate donor. Application of sorbitol kinase for practical use by extracting from silk worm eggs and bovine and human eyes is almost impossible. Futhermore, superior heat stability of the enzyme is not disclosed in the prior references.
Sorbitol kinase from microorganisms origin of Aerobacter aerogenes (N. E. Kelker and R. L. Anderson, J. Bacteriol., 160-164, 1971) has known. However, optimum pH of 7.6 and phosphoenolpyruvate as a phosphate donor have only disclosed in the reference, and no description on superior heat stability.
PROBLEMS TO BE SOLVED BY THE INVENTION
Production of sorbitol kinase from the origin except for microorganisms is known to be trace amount which can only be measured by radioisotope, consequently substantial isolation of the enzyme is impossible and no practical use has known. Sorbitol kinase from microorganism origin has property for only utilize phosphoenolpyruvate as a phosphate donor and does not utilize ATP, consequently practical use for diagnostic enzyme is doubtful.
We have extensively made search sorbitol kinase of microorganism origin having superior substrate specificity for sorbitol with utilizing ATP as a phosphate donor, and found sorbitol kinase, which utilize ATP,
REFERENCES:
Neubaur et al. (1967) Z. Physiol. Chem., 348(7), "Decomposition of Sorbitol by Bacteria in Rat Feces". pp. 871-876.
Srivastava et al. (1982) Biochim. Biophys. Acta, 717(2), "Formation of Sorbitol 6-phosphate by Bovine and Human Lens Aldose Reductase, Sorbitol Dehydrogenase and Sorbitol Kinase", pp. 210-214.
T. Yaginuma, et al., "Does an enzyme activity capable of phosphorylating sorbitol control utilization of sorbitol at the termination of diapause in eggs of the silkworm, Bombyx Mori?", 1991, pp. 135-141, Comp. Biochem. Physial, vol. 98B, No. 1.
S. K. Srivastava, et al., "Formation of sorbitol 6-phosphata by bovine and human lens aldose reductase, sorbitol dehydrogenase and sorbitol kinase", 1982, pp. 210-213, Biochimica et Biophysica Acta, 717.
N. E. Kelker, et al., "Sorbitol Metabolism in aerobacter aerogenes", Jan., 1971, pp. 160-164, Journal of Bacteriology.
Koga Shinji
Suzuki Kouji
Takahashi Mamoru
Asahi Kasei Kogyo Kabushiki Kaisha
Weber Jon P.
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