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With psicose 3-epimerase; manganese(II) at 60℃; for 2h; Enzymatic reaction; |
6 D-psicose production by Psicose 3-epimerase
D-psicose production by Psicose 3-epimerase [00159] The reaction of the psicose 3-epimerase is performed in a 50 mM PIPES buffer solution containing 14 units of psicose 3-epimerase/ml, 1 mM of Mn2+ ions, and 700 g/L D- fructose at pH 7.0 and at temperature to 60° C. at various times to allow the reaction to proceed sufficiently. The reaction is terminated by heating the reaction solution at 100° C. for 5 minutes, and the enzyme activity is measured by analyzing the reaction mixture for the content of D- psicose and D-fructose. D-psicose production in 120 minutes reaction time is determined. In general the rare sugars are not metabolized by the biological systems and therefore to separates the rare sugars from natural sugars, a fermentation based procedure can be followed to purify the rare sugar from natural sugars. For example, in this example, to purify D-psicose from D- fructose, the mixture containing both these sugars can be mixed with certain microbial strains which can ferment D-fructose but no D-psicose. Upon complete consumption of fructose in the mixture by the microbial cells, the microbial cells are removed by centrifugation or filtration methods and the D-psicose is recovered in an aqueous or organic solution. Although this method is explained with the example of D-psicose, the approach can be used in purifying any other non-fermentable sugars including D-tagatose and L-fructose. |
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With D-tagatose 3-epimerase from Pseudomonas cichorii, expressed with E. coli JM109 In water at 55 - 60℃; Enzymatic reaction; |
1 EXAMPLE 1-FULL PROCESS, MEMBRANE REACTOR
Crystalline fructose is employed as starting material for allulose production. The fructose is dissolved in water and the concentration is adjusted to 40 wt.-%, dry matter, relative to the total weight of the composition. The added water may be tap water, demineralized water, condensed water as provided in a subsequent step of the process, or a mixture of any of the foregoing. The pH value and electrolyte content is adjusted by adding appropriate buffers and salts. (0310) The enzymatic conversion is performed in a membrane reactor (cut off 10 kDa) that is coupled to an ultrafiltration device. The enzymes in the reactor are freely dissolved, i.e. neither immobilized nor contained in microorganisms. (0311) Purified lyophilized enzyme (D-tagatose 3-epimerase from Pseudomonas cichorii, expressed with E. coli JM109) or crude extract (cell free fermentation broth) is added to an aqueous solution of fructose at a concentration within the range of from 50 g/L to 500 g/L in 50 mM TRIS/HCl buffer and 1 mM MnCl2. The pH value is adjusted to pH 7.5 or pH 9 by means of the required amount of HCl aq. and the stirred solution is incubated at 55° C. or 60° C. Depending upon the concentration of the fructose, after 1 hour a yield of 30% allulose relative to the employed fructose may be achieved: [table-us-00006-en] g/L reaction time initial 1 h 24 h fructose fructose allulose yield fructose allulose yield [g/L] [g/L] [g/L] [%] [g/L] [g/L] [%] 51 35 16 30 37 16 31 101 70 30 30 70 30 30 229 165 65 28 168 72 31 420 326 95 23 291 124 30 (0312) The composition containing the fructose is filtered through a filter (0.2 micrometer) and supplied to the membrane reactor. Fructose is converted to allulose by enzymatic catalysis for 36 hours at 30° C. The product is removed from the reactor by ultrafiltration thereby separating the carbohydrates (essentially allulose and residual fructose) from the enzymes which in turn are recycled to the membrane reactor for reuse. (0313) The composition is pre-purified. Decoloring is achieved by means of a decoloring column or by means of active charcoal, in either case at a temperature within the range of from 30° C. to 70° C. Desalting is achieved by means of ion exchange resins, commencing with cations exchangers, followed by anions exchangers, followed by mixed bed exchangers. (0314) The thus provided composition is concentrated by means of an evaporator at a temperature of below 60° C. and the concentration of dry matter is adjusted to a concentration within the range of from 40 wt.-% to 70 wt.-%, relative to the total weight of the composition. The evaporator is selected from rising film plate evaporator (or vertical long tube evaporator), falling film evaporator, Robert evaporator and circulation evaporator, wherein in either case single step or multiple step evaporations are possible. Allulose and residual fructose are separated from one another by chromatography. The chromatography is selected from batch chromatography, continuous chromatography, simulated moving bed (SMB) chromatography and sequential simulated moving bed (SMB) chromatography (SSMB). (0315) The thus provided composition is again concentrated by means of an evaporator at a temperature of below 60° C. and the concentration of dry matter is adjusted to a concentration within the range of from 70 wt.-% to 95 wt.-%, relative to the total weight of the composition. The evaporator is selected from rising film plate evaporator (or vertical long tube evaporator), falling film evaporator, Robert evaporator and circulation evaporator, wherein in either case single step or multiple step evaporations are possible. (0316) From the thus provided composition allulose is provided as a solid material by cooling crystallization and subsequent centrifugation, or by evaporation crystallization and subsequent centrifugation, or by high shear blending and subsequent grinding and sieving, or by spray drying, or by spray granulation, or by spray crystallization, or by means or a belt dryer, or by means of an infrared dryer. The allulose is then (further) dried by means of a drum dryer, or by means of a fluidized bed dryer, or by means of a vibration fluidized bed dryer, or by means of a revolver dryer. The solid allulose is them packaged in bags and palletized. |
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