Process for producing polysaccharide ron substance with a synthetase

• US 5,565,342 A
• Filed: 09/15/1993
• Issued: 10/15/1996
• Est. Priority Date: 02/08/1989
• Status: Expired due to Fees

• Current Assignee: Sapporo Breweries Limited (Sapporo Holdings Limited)
• Original Assignee: Sapporo Breweries Limited (Sapporo Holdings Limited)
• Inventors: Watanabe, Nobuhiro, Yoneta, Yasuo, Takeo, Suguru, Mitani, Yutaka, Kado, Hisao
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): LANKFORD JR, LEON B
• Application Number:
  US08/122,138
• Time in Patent Office: 1,126 Days
• Field of Search: 435/101, 435/183, 435/822, 435/252.1, 536/55.1, 536/124
• US Class Current: 435/101
• CPC Class Codes: C08B 37/0009 alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12P 19/08 Dextran
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales

Abstract

A polysaccharide RON substance which has antitumor activity can be produced by fermenting sucrose with a synthetase. The synthetase can be isolated from Leuconostoc strains. Preferred strains of the microorganism are Leuconostoc mesenteroides FERM BP-2242, 2711, 2712, 2713, 2714, and 2670. The substance exhibits antitumor activity against syngenic tumor Meth A, a transplantable tumor in mice.

Claims

1. A process for producing a polysaccharide RON substance which comprises

(a) contacting sucrose with a polysaccharide RON substance synthetase derived from a microorganism having a capacity of producing the polysaccharide RON substance, the microorganism belonging to the genus Leuconostoc and being at least one strain of Leuconostoc mesenteroides subsp. dextranicum, said strain selected from the group consisting of strain BL-75 (FERM BP-2242), strain NCFB 517 (FERM BP-2711), strain NCFB 531 (FERM BP-2712), strain NCFB 861 (FERM BP-2713), strain NCBF 864 (FERM BP-2714), strain 46-1 (FERM BP-2670), and mutants thereof, to form the polysaccharide RON substance having the following properties:

(1) form;

the substance is a white amorphous powder;

(2) solubility;

the substance is soluble in water, but the solution becomes milky white and viscous when the concentration is elevated;

the substance is soluble in formamide and dimethylsulfoxide, but the substance is insoluble in alcohols, acetone, benzene, ethyl acetate, hexane, chloroform and carbon tetrachloride;

(3) pH of aqueous solution;

the aqueous solution is neutral or weakly acidic;

(4) constituent saccharide;

the substance comprises solely glucose;

(5) elementary analysis values;

the substance comprises 44.0 to 45.0% C and 6.1 to 6.3% of H;

(6) structure;

the substance is an α

-1,6-glucan and comprises a small amount of 3,6-branched side chains;

(7) proteins;

the substance contains almost no proteins;

(8) molecular weight;

the substance does not permeate through a dialysis membrane and the molecular weight is considered to be higher than 10,000 daltons;

(9) specific rotation;

formamide);

(10) color reaction;

the substance is positive to an anthrone sulfuric acid reaction and a phenol sulfuric acid reaction, but negative to a biurette reaction, the Lowry-Folin reaction, the Elson-Morgan reaction and an iodine reaction;

(11) ultraviolet absorption spectrum;

the substance has no characteristic absorption;

(12) infrared absorption spectrum;

the substance shows a characteristic absorption of α

-glucan;

(13) ¹³ C-NMR spectrum;

the substance shows an absorption having characteristic chemical shift values of α

-1, 6-glucan in the main signals; and

(14) biological activity;

the substance has an antitumor activity against a transplantable tumor in mice, said transplantable tumor being syngeneic tumor Meth A;

and (b) collecting said substance.

2. The process for producing the polysaccharide RON substance according to claim 1, wherein the microorganism having a capacity of producing the polysaccharide RON substance is Leuconostoc mesenteroides subsp. dextranicum, strain BL-75 (FERM BP-2242) or strain 46-1 (FERM BP-2670).

3. The process for producing the polysaccharide RON substance according to claim 1, wherein the sucrose is in a form of white refined sugar, molasses or reagent grade sucrose.

4. The process for producing the polysaccharide RON substance according to claim 3, wherein the synthetase is in a concentration of 0.2 to 2 U/ml.

5. The process for producing the polysaccharide RON substance according to claim 3, wherein the synthetase is in a concentration of 0.5 to 1 U/ml.

6. The process for producing the polysaccharide RON substance according to claim 5, wherein the sucrose is in a concentration of 5 to 30%.

7. A process for producing a polysaccharide RON substance which comprises

(a) contacting sucrose with a polysaccharide RON substance synthetase to form the polysaccharide RON substance having the following properties:

(1) form;

the substance is a white amorphous powder;

(2) solubility;

the substance is soluble in water, the substance is soluble in formamide and dimethylsulfoxide, but the substance is insoluble in alcohols, acetone, benzene, ethyl acetate, hexane, chloroform and carbon tetrachloride;

(3) pH of aqueous solution;

the aqueous solution is neutral or weakly acidic;

(4) constituent saccharide;

the substance comprises solely glucose;

(5) elementary analysis values;

the substance comprises 44.0 to 45.0% of C and 6.1 to 6.3% of H;

(6) structure;

the substance is an α

-1,6-glucan and comprises a small amount of 3,6-branched side chains;

(7) proteins;

the substance comprises almost no proteins;

(8) molecular weight;

the substance does not permeate through a dialysis membrane and the molecular weight is considered to be higher than 10,000 daltons;

(9) specific rotation;

(α

)_D²⁵ =+190°

˜

+220°

(C=0.5, formamide);

(10) color reaction;

the substance is positive to an anthrone sulfuric acid reaction and a phenol sulfuric acid reaction, but negative to a biurette reaction, the Lowry-Folin reaction, the Elson-Morgan reaction and an iodine reaction;

(11) ultraviolet absorption spectrum;

the substance has no characteristic absorption;

(12) infrared absorption spectrum;

the substance shows a characteristic absorption of α

-glucan;

(13) ¹³ C-NMR spectrum;

the substance shows an absorption characteristic of chemical shift values of α

-1,6-glucan in the main signals; and

(14) biological activity;

the substance has an antitumor activity against a transplantable tumor in mice, said tumor being syngeneic tumor Meth A;

and (b) collecting said substance.

8. The process for producing a polysaccharide RON substance according to claim 10, wherein the RON substance synthetase is a polysaccharide RON substance synthetase having the following properties:

(a) action;

the enzyme decomposes 1 mol of sucrose to form 1 mol of fructose, and simultaneously, the enzyme transfers the glucose portion to the polysaccharide RON substance acting as the receptor;

(b) optimum pH;

the optimum pH value is about 5.5;

(c) stable pH range;

the enzyme is stable in the pH range of from 4.5 to 7.0;

(d) substrate specificity;

formation of the biologically active polysaccharide RON substance from glucose, fructose, maltose and isomaltose is not observed, but the enzyme reacts only with sucrose to form the biologically active polysaccharide RON substance;

(e) optimum temperature;

the optimum temperature is about 40°

C.; and

(f) stable temperature range;

the enzyme is stable at a temperature lower than 40°

C.