1. A method of sequential enzymatic conversions for efficient production of Allulose from
substrates such as and not limited to sucrose, starch, cellulose using novel enzymes in
single or in combinations as fusion proteins.
2. The method as claimed in claim 1, wherein the enzymes involved in the sequential
conversion steps are either single or in fusion with subsequent enzyme(s) catalysing two
or multiple enzyme conversions using single fusion enzymes, thereby reducing the number
of steps.
3. The method as claimed in claim 1, where in the production of D-allulose from carbohydrate
source such as and not limited to sucrose or juice, includes combination of the steps:
a. Converting the carbohydrate source to a mixture of glucose and fructose in the presence of
extracellular enzymes selected from invertase;
b. Converting the glucose in the mixture of step (a) to fructose through enzymatic
conversion in the presence of Glucose isomerase (GI) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 1;
c. Converting the fructose obtained in step (b) to fructose 6 phosphate in the presence of
phosphor fructokinase (PPFK) comprising the amino acid sequence which has atleast
85% sequence similarity to SEQ ID: 3;
d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in
the presence of allulose 6-phosphate 3-epimerase (A6PE) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 4; and
e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence
of enzyme allulose 6 phosphate phosphatase (A6PP) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 5
wherein the enzymes fructokinase, allulose 6-phosphate 3-epimerase and allulose 6
phosphate phosphatase are expressed as fusion protein.
4. The method as claimed in claim 3, wherein production of D-allulose from the said
carbohydrate substrate is also achieved through combination of steps:
a. Converting the carbohydrate source to glucose using extracellular enzymes selected
from invertase;
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b. Converting the glucose produced in the step (a) in the presence of polyphosphate to
glucose 6-phosphate (G6P) by the enzyme glucokinase (PPGK) comprising the amino
acid sequence which has atleast 85% sequence similarity to SEQ ID: 3;
c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate
(F6P) in the presence of phosphogluco isomerase (PGI) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 11;
d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in
the presence of allulose 6-phosphate 3-epimerase (A6PE) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 4;
e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence
of enzyme allulose 6 phosphate phosphatase (A6PP) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 5; and
wherein the allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase are
expressed by a part a single fusion protein.
5. The method as claimed in claim 1, where in production of D-allulose from carbohydrate
source such as and not limited to Amylodextrin, starch, glucan, is achieved by combination
of the steps:
a. Converting the carbohydrate source to glucose-1 phosphate in the presence of
glycogen/glucan phosphorylase (GP) comprising the amino acid sequence which has
atleast 85% sequence similarity to SEQ ID: 9;
b. converting the glucose-1 phosphate obtained in step (a) to glucose 6-phosphate in the
presence of the enzyme phosphoglucomutase (PGM) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 10;
c. converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate
(F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 11;
d. Converting the fructose 6 phosphate produced in the step (c) to allulose 6-phosphate in
the presence of allulose 6-phosphate 3-epimerase (A6PE) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 3; and
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e. Converting the allulose 6-phosphate obtained in step (d) to D-allulose in the presence
of enzyme allulose 6 phosphate phosphatase A6PP) comprising the amino acid
sequence which has atleast 85% sequence similarity to SEQ ID: 4;
wherein the allulose 6-phosphate 3-epimerase and allulose 6 phosphate phosphatase are
expressed by a part a single fusion protein.
6. The method as claimed in claim 1, where in the enzymes used in sequential steps of
bioconversion are generated as fusion protein using linkers such as and not limited to
flexible linker, rigid linker, etc.
7. The method as claimed in claim 1, wherein the fusion enzymes are introduced in the
reaction mixture in either free or immobilized form or combination thereof.
8. The method as claimed in claim 1, wherein the enzymes reaction is carried out in the
presence of inorganic polyphosphate or ATP.
9. The method as claimed in claim 1, wherein the said enzymes are used in free or
immobilized form or combination thereof.
10. The method as claimed in claim 1, where in the process for the preparation of the fusion
enzymes includes combination of the steps:
a. preparing recombinant cells for expressing the recombinant fusion proteins
encoding the enzymes fructokinase and/or allulose 6-phosphate 3-epimerase and
allulose 6 phosphate phosphatase;
b. culturing the recombinant cells at 370C followed by inducing fusion protein
expression under different concentration of IPTG and at a temperature of 20-300C;
and
c. extracting and isolating the fusion proteins from culture by causing cell lysis and
after separation of soluble and insoluble fractions.