Patent GB-800634-A (GB)
Title: Improvements in or relating to the preparation of 2-keto-l-gulonic acid
IPC: C12P7/60
Filed: 1955-07-27
Abstract: 2-Keto-l-gulonic acid is obtained by subjecting l-idonic acid or a non-toxic l-iodonate under aerobic conditions to the oxidizing system produced by cultivating a mixture of the two organisms Pseudomonas ATCC 11867 and Pseudomonas ATCC11868 under aerobic conditions on an otherwise sterile nutrient medium.  It is stated that a living culture of each of the organisms has been deposited with the American Type Culture Collection in Washington, D.C., where they have been given the above numbers.  The process may be effected in various ways, thus the mixture may be grown under aerobic conditions on an otherwise sterile nutrient medium containing l-idonic acid or the l-idonate.  Alternatively, to such a medium in which growth has been established l-idonic acid or the l-idonate may be added.  The reaction may also be carried out by subjecting l-idonic acid or a non-toxic l-idonate to a suspension of cells so grown or to a cell-free broth in which the mixture has been grown.  The term non-toxic l-idonate includes salts of metals, e.g. alkali metals, ammonia and amines, and esters of simple alcohols, e.g. of methanol or ethanol, with l-idonic acid. The mixture of organisms may readily be grown on standard nutrient media, thus a liquid nutrient medium containing a source of carbohydrate such as dextrose, inorganic salts such as potassium phosphate and magnesium sulphate, a protein source such as peptone, and growth-promoting substances such as yeast extract, may be used.  The cultivation may also be readily carried out on ordinary nutrient agar slants.  In one method a solution containing nutrients such as dextrose and yeast extract is charged with l-idonic acid or a nontoxic l-idonate and inoculated with a mixed culture of the organisms which has previously been grown under aerobic conditions on either agar slants or a liquid nutrient medium. The fermentation broth is shaken under aerobic conditions at between about 25 DEG  C. and 30 DEG  C. and generally good conversion is produced in about 34 hours by 1 cc. of a 48-hour inoculum in 100 cc. of fermentation mixture and the progress of the reaction may be followed by means of a colour test using orthophenylenediamine dihydrochloride in the form of a 2 1/2 per cent aqueous solution.  In an example a culture of a mixture of ATCC 11867 and ATCC 11868 is grown for 48 hours under aerobic conditions on a nutrient broth containing 0.25 per cent yeast extract.  One cc. of this broth is inoculated into 100 cc. of an aqueous fermentation mixture of pH 7.0 and having the following composition by weight: 2 per cent sodium l-idonate-, 0.1 per cent dextrose and 0.5 per cent yeast extract. The mixture is maintained at 28 DEG  C. under aerobic conditions and after 34 hours 2-keto-gulonic acid is isolated by the following procedure: the broth is concentrated to about one quarter of its original volume by evaporation, acetic acid is added to reduce the pH to about 4 and sodium hydroxide is then added to raise the pH to about 7.5. Methyl alcohol is then added and the sodium 2-keto-l-gulonate which is precipitated is filtered off and treated with a non-oxidizing strong acid such as hydrochloric acid to yield the free acid.  The organisms used are stated to be Gram-negative, straightcelled bacilli and other characteristics of the organisms are described.