Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1995-01-06
1998-05-12
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
435 89, 435 90, 435 911, 435183, 435195, C12N 910
Patent
active
057503893
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Sucrose-synthase (Glycosyltransferase EC2.4.1.13 UDPG: D-Fructose-2-glucosyltransferase) is an enzyme which has been long known and is especially widespread in plants (e.g., wheat, rice, corn, sugar beets, etc.) see Y. Milner and others in "Nature" 206 (1965), Page 825; the function of this enzyme as a catalyst in the production of activated sugars in the metabolism of plants has been extensively studied and compendia have been produced (Avigad, G in Loewus, F.A. et al. (eds.) Encyclopedia of Plant Physiology New Series Vol. 13A, Carbohydrates I, Intracellular Carbohydrates, Springer-Verlag, Berlin 1983, pages 217-347). The enzyme catalyzes in vivo the splitting of sucrose according to the following equation: guanosine and adenosine, and NDP is nucleoside diphosphate.
Purification and characteristics of the enzyme have been described inter alia by T. Nomura et al in Arch. Biochem. Biophysics 156 (1973), pages 644-652, which gives a yield of 8.8% at 11.4 fold purification by ammonium sulfate precipitation and column chromatography on DEAE-Cellulose and Neusilin (MgO+Al.sub.2 O.sub.3 +2SiO.sub.2) and K.sub.m -values for synthase reaction and splitting reaction are given.
Recently, S.L. Haynie and G.M. Whitesides have reported in Appl. Biochem. & Biotechnol. 23 (1990) about a sucrose-synthase purified by an ammonium sulfate precipitation based process and its use for the sucrose synthesis by reaction of UDP-Glucose and Fructose.
That describes the limited stability of the enzyme (page 158), especially of highly purified enzyme preparations (page 160) as well as the drawback of highly stabilized enzyme preparations of reduced purity based upon byproduct activity, especially of phosphoglucomutase and the consequent low activity requirement of the use of larger gel volume (of the gel immobilized enzyme). R.H. Juang and others describe in J. Chinese Biochem. Soc. 17 (1988) 42-51. A sucrose-synthase purification by column chromatography and electrophoresis is disclosed with 38-times purification which is carried out depending upon the protein composition.
In spite of the long known manner functioning of sucrose-synthase and the purification process, the production of activated sugars according to the above equation has not hitherto been economically utilized although the sucrose-synthase is commercially available and activated sugars as well as disaccharides and oligosaccharides are of considerable significance in sugar chemistry.
Mono-,. Oligo- and Polysaccharides have multiple functions as antigen determinants, in cell-cell recognition, in cell differentiation and as binding cites for toxins, bacteria and viruses.
A compilation of the production and use can be found in S. David and others in Advances in Carbohydrate Chem. A. Biochem. 49 (1991), 175-237. Y. Ichikawa and others present in ANal. Biochem. 202 (1992) 215-238 different reaction mechanisms. As "large scale synthesis", however, especially the reaction of sugar-1-phosphate (especially glucose-1-phosphate) with nucleoside triphosphate, especially with UTP in the presence of pyrophosphorylase, reference may be had to C.H. Wong and others (J. Org. Chem. 47 (1982) 5416-18) which describes a multistage enzymatic synthesis of nucleotide sugars.
This synthesis according to C.H. Wong is also described as the method of choice by Toone and Whiteside in Am. Chem. Soc. Sympos. Sr. 466 (1991) 1-22.
SUMMARY OF THE INVENTION
It has been surprisingly found that sucrose synthase isolate (especially of commercially available enzyme contained generally more or less high proportions of nucleotidephosphatases and that the presence of them, even in small amounts, is so greatly detrimental to the synthesis of NDP Glucose and homologous compounds that the confirmed synthesis suitability of sucrose synthesis could not be recognized heretofore.
By appropriate purification methods and sensitive phosphatase determinations, a sucrose synthetase has been developed which is surprisingly stable and enables a clean single stage synthesis reaction accordin
REFERENCES:
Journal of the Chinese Biochemical Society, vol. 17, No. 1, 1988, pp. 42-51, Rong-Huay Juang et al "Purification of Rice Grain Sucrose Synthetase by Preparative Electrophoresis".
Applied Biochemistry and Biotechnology, vol. 23, No. 2, Feb. 1990, pp. 155-170, Sharon L. Haynie et al, "Enzyme Catalyzed Organic Synthesis of Sucrose and Trehalose With in Situ Regeneration of UDP-Glucose".
Plant Physiology, vol. 45, 1970, pp. 782-786, Deborah P. Delmer et al, "The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaselous aureus".
Wong et al, J. Org. Chem., vol. 47, (1982), pp. 5416-5418.
J. Org. Chem., 1990, 55, 1834-1841, Simon et al, Convenient Synthesis of Cytidine 5'-Triphosphate, Guanosine, 5'-Triphosphate, and uridine 5'-Triphosphate and Their Use in the Preparation of UDP-Glucose, UDP-Glucoronic Acid, and GDP-Mannose.
Journal of Biological Chemistry, vol. 245, No. 1, pp. 188 to 197, Jan. 10, 1970, Grimes, W.J. et al, "Sucrose Synthetase from Phaselous aureus Seedlings".
Analytical Biochemistry 202, 215 to 238 (1992), Ichikawa, Yoshitaka et al, "Enzyme-Catalyzed Oligosaccharide Synthesis";.
J. Org. Chem., 1992, 57, 152 to 157, Heidlas, Jurgen E. et al, "Practical Enzyme-Based Syntheses of Uridine 5'-Diphosphogalactose and Uridine 5'-Diphospho-N-acetyl-galactosamine on a Gram Scale".
Elling Lothar
Kula Maria-Regina
Dubno Herbert
Forschungszentrum Julich GmbH
Lankford , Jr. Leon B.
Myers Jonathan
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