Process for the enzymatic isomerization of dextrose to levulose

Abstract

Process for the production of xylose (dextrose) isomerase by means of a mutant strain of Streptomyces that proliferates in a culture medium that may be free of xylose. The xylose (dextrose) isomerase enzyme is therefore constitutive rather than induced.

What is claimed is: 
     
       1. A process for the production of levulose comprising: .[.(a) subjecting a microorganism of the Streptomyces genus to a mutagenic agent;   (b) isolating the mutant strain of the microorganism that has the capacity to form an appreciable quantity of xylose isomerase when cultivated in a nutrient medium that is free of xylose and xylose-supplying materials;   (c) cultivating the isolated mutant strain in a nutrient medium to produce xylose isomerase; and   (d).]. isomerizing dextrose to levulose with .[.said.]. .Iadd.a .Iaddend.xylose isomerase.[...]. .Iadd.enzyme preparation derived from a mutant strain of a Streptomyces microorganism, said mutant strain being characterized in that when cultivated in a nutrient culture medium that is free of xylose and xylose-supplying materials, it produces at least 50% more xylose isomerase activity than Streptomyces olivochromogenes ATCC No. 21,114 under identical conditions of cultivation. .Iaddend.   
     
     
       2. A process in accordance with claim 1, wherein the microorganism is .[.S..]. .Iadd.Streptomyces .Iaddend.olivochromogenes. 
     
     
       3. A process in accordance with claim 2, wherein the .[.isolated.]. mutant strain is selected from the group consisting of .[.S..]. .Iadd.Streptomyces .Iaddend.olivochromogenes ATCC No. 21,713, .[.S..]. .Iadd.Streptomyces .Iaddend.olivochromogenes ATCC No. 21,714 and .[.S..]. .Iadd.Streptomyces .Iaddend.olivochromogenes ATCC No. 21,715.[...]., .Iadd.variants and sub-mutants of said mutant strains. .Iaddend. 
     
     
       4. The process of claim 1, wherein said .[.isolated.]. mutant strain is .[.cultivated in a medium containing xylose or a xylose-supplying material, to thereby produce a greater quantity of xylose isomerase than untreated microorganisms cultured under the same conditions..]. .Iadd.further characterized in that when cultivated in a medium that is free of xylose and xylose-supplying materials, it produces twice as much xylose isomerase activity than Streptomyces olivochromogenes ATCC 21,114, under identical conditions of cultivation. .Iaddend. 
     
     
       5. The process of claim 1, wherein said .[.isolated mutant strain is further characterized by its capacity to form an appreciable quantity of xylose isomerase when cultivated in a nutrient medium that is also free.]. .Iadd.isomerization of dextrose to levulose is conducted in the absence .Iaddend.of added cobalt. 
     
     
       6. The process of claim 1, wherein said xylose isomerase is in its soluble form. 
     
     
       7. A process for the isomerization of dextrose to levulose with a xylose isomerase enzyme preparation that comprises subjecting a solution containing dextrose to the action of a xylose isomerase enzyme preparation, said xylose isomerase enzyme preparation having been prepared by .[.cultivating, in a nutrient medium, an isolated mutant strain of microorganism of the Streptomyces genus, said mutant strain produced by subjecting said microorganism to the action of a mutagenic agent, said isolated strain being capable of reproducing itself to a strain having the characteristics of its parent mutant strain, said mutant strain being further characterized by its capacity to form an appreciable quantity of xylose isomerase when cultivated in a nutrient medium that is free from xylose and xylose-supplying material..]. .Iadd. a) inoculating a nutrient medium that is free from xylose and xylose-supplying materials with cells derived from a mutant strain of a microorganism of the Streptomyces genus to produce cells containing xylose isomerase, said mutant strain being characterized in that when culitvated in a nutrient medium that is free of xylose and xylose-supplying materials, it produces twice as much xylose isomerase activity than Streptomyces olivochromogenes ATCC No. 21,114 under identical conditions of cultivation;   b) fermenting the cells in a nutrient medium which, in at least the final stages of fermentation, contains xylose; and   c) recovering the xylose isomerase enzyme produced. .Iaddend.   
     
     
       8. The process of claim 7, wherein said solution containing dextrose is a starch hydrolysate having a D.E. of at least about 95, said solution being maintained at a temperature of from about 60° C. to about 70° C. and a pH of from about 6 to about 7. .Iadd.9. The process of claim 1, wherein said xylose isomerase enzyme preparation is further characterized as having the following characteristics: when the xylose isomerase is contained within the whole cells of the microorganism, it is capable of converting a 95 D.E. corn starch hydrolysate having a dry substance level of about 600 mg/ml to about 800 mg/ml to a levulose-bearing syrup containing at least about 40% ketose level when the conversion is conducted at 70°  C., at a pH of 6.25 in the presence of about 0.01 molar magnesium sulfate with an enzyme dosage of about 1.2 to about 1.4 units per gram for about 40 to about 48 hours in the absence of added cobalt and under a nitrogen blanket. .Iaddend. .Iadd.10. A process for the production of levulose comprising: isomerizing dextrose to levulose with a xylose isomerase enzyme preparation derived from a mutant strain of a Streptomyces microorganism, said mutant strain being a member selected from the group consisting of Streptomyces olivochromogenes ATCC No. 21,713, Streptomyces olivochromogenes ATCC No. 21,714 and Streptomyces olivochromogenes ATCC No. 21,715, variants and sub-mutants thereof. .Iaddend..Iadd.11. A process for the enzymatic isomerization of dextrose to levulose, with a xylose isomerase enzyme preparation that comprises subjecting a solution containing dextrose to the action of a xylose isomerase enzyme preparation, said xylose isomerase enzyme preparation having been prepared by inoculating in at least one development stage a nutrient medium that is free of added cobalt and xylose, a culture of a microorganism derived from Streptomyces olivochromogenes ATCC No. 21,715, which is a single colony isolate of the mutant strain Streptomyces olivochromogenes ATCC No. 21,713; fermenting the inoculated nutrient medium wherein the culture medium in the final stage of fermentation is characterized as containing xylose and recovering the xylose isomerase enzyme produced therefrom. .Iaddend..Iadd.12. The process of claim 11, wherein said solution containing dextrose is a starch hydrolysate having a D.E. of at least about 95, said solution being maintained at a temperature of from about 60° C. to about 70° C. .Iaddend.