Enzymatic synthesis of d-allulose and its derivatives from fusion enzymes
OwnerRahul Raju Kanumuruinventor
Life of this patent
Estimating the term…
Abstract
The present invention belongs to the field for enzymatic catalysis, particularly relates to the usage of multienzyme system/fusion proteins for producing of D-allulose, methods for production of the fusion enzyme and their use thereof for the production of D-allulose and its derivatives from carbohydrate sources. More particularly, the present invention provides simple, improved and cost-effective process for the production of D-allulose and its derivatives from carbohydrate sources from such fusion enzymes.
Claims15
A process for the production of D-allulose and its derivatives from carbohydrate source, wherein the method comprises the steps of:
- a. Converting the carbohydrate source to a mixture of glucose and fructose in the presence of extracellular enzymes selected from invertase, amylase and/or pullulanase;
- b. Optionally converting the glucose in the mixture of step (a) to fructose through enzymatic conversion in the presence of isomerase,
- c. Converting the fructose from step (a) and/or step (b) to fructose 6 phosphate in the presence of fructokinase,
- d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in the presence of allulose 6-phosphate 3-epimerase;
- e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence of enzyme allulose 6 phosphate phosphatase;
- wherein the enzymes fructokinase, allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase are expressed by a single fusion protein.
A process for the production of D-allulose and its derivatives from carbohydrate source, wherein the method comprises the steps of:
- a. Converting the carbohydrate source to glucose using extracellular enzymes selected from invertase, amylase and/or pullulanase,
- b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate by the enzyme glucokinase,
- c. Converting the glucose 6-phosphate produced in step (b) to fructose 6 phosphate in the presence of phosphoglucoisomerase,
- d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in the presence of allulose 6-phosphate 3-epimerase;
- e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence of enzyme allulose 6 phosphate phosphatase;
- wherein the allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase are expressed by a single fusion protein.
A process for the production of D-allulose and its derivatives from carbohydrate source, wherein the method comprises the steps of:
- a. Converting the carbohydrate source to glucose-1 phosphate in the presence of glycogen/glucan phosphorylase;
- b. converting the glucose-1 phosphate obtained in step (a) to glucose 6-phosphate in the presence of the enzyme phosphoglucomutase;
- c. converting the glucose 6-phosphate produced in step (b) to fructose 6 phosphate in the presence of phosphoglucoisomerase,
- d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in the presence of allulose 6-phosphate 3-epimerase;
- e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence of enzyme allulose 6 phosphate phosphatase;
- wherein the allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase are expressed by a single fusion protein.
A process for the preparation of the fusion enzymes, the method comprising the steps of:
- a. preparing recombinant cells for expressing the recombinant fusion proteins encoding the enzymes fructokinase, allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase;
- b. culturing the recombinant cells at 37° C. followed by inducing fusion protein expression under different concentration of IPTG and at a temperature of 20-30° C.
- c. extracting and isolating the fusion proteins from culture by causing cell lysis and after separation of soluble and insoluble fractions.
Dependencies are inferred from the claim text.