Field of invention:
A method of producing Isomaltulose or Trehalulose from Sucrose.
Technical field:
The present invention relates to a cell surface layer binding protein having a sugar chain binding area such as glucosyl phosphotidyl inositol attached to at least to the N-terminus or the C-terminus of the sugar chain binding domain.
Background of the invention:
Sucrose is the most common sugar produced/accumulated by plants. Large quantities of this disaccharide accumulate in the edible parts of some plants, making it the most abundant natural sweetener in foods. Sucrose is the most common form of sugar utilized by the world population for routine use in food and beverages. However, there is a growing demand for alternate sugars which are non-cariogenic and reducing the obesity and cardiac related problems.
Isomaltulose [a-D-glucopyranosyl-(l, 6)-D-fructofuranose] and trehalulose [a-D-glucopyranosyl-(l, l)-D-fructofuranose] are nutritive sweetener. Like sucrose, isomaltulose is a disaccharide made up of glucose and fructose. However, in contrast to sucrose, isomaltulose is joined by a a-1, 6 glycosidic bond in isomaltulose compared to a a-1, 2 glycosidic link in sucrose while trehalulose is joined by a -1, 1 glycosidic bonds.
Commercial isomaltulose is produced from food grade sucrose through enzymatic isomerisation with sucrose-6-glucosylmutase (EC 5.4.99.11). It is approximately half as sweet as sucrose, and has a similar sweetness quality. It is found naturally in very small quantities in honey (0.1 - 0.7%) and sugar cane juice. The biocatalyst used to convert the sucrose is obtained from non-viable, non-pathogenic P. rubrum (CBS 574.44) cells. The cells are killed using formaldehyde before they are added to the sucrose. Thus, isomaltulose is not directly obtained from plant, animal, or microorganism. P. rubrum cells are used as the source of the biocatalyst. However, there is no commercial production of trehalulose and in isomaltulose biosynthesis, some amount of trehalulose produced.

Sucrose isomerase (SI) is widely used in industries for production of lsomaltulose. A number of organisms reported to utilized for SI such as Serratia plymuthica, Erwinia rhaponiciti, Klebsiella planticola, Pseudomonas mesoacidophila, protaminobacter rubrum, Pantoea dispera, Enterobacter sp. Etc.
US patent 4898820 describes the enzymatic process for continuous synthesis of isomaltulose from glucose and fructose. US patent 4857461 discloses the process of extraction of sucrose mutase from periplasmic membranes of Protaminobacter rubrum or Serratia plymuthica and utilization of same in a radial type bioreactor for the conversion of sucrose to isomaltulose. Of these organisms, predominantly isomaltulose producing organisms are Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti whereas trehalulose producing organisms are Pseudomonas mesoacidophila and Agrobacterium raadiobacter.
US patent 5336617 discloses two newly isolated strain Pseudomonas mesoacidophilia (Fix-45) and Agrobacterium radiobacter (MX-232) which are capable of converting sucrose to mostly trehalulose and less amount of isomaltulose.
Cell surface layer-associated proteins that are present and immobilized in a cell surface layer such as alpha agglutin or Cwplp, which is a flocculation protein of yeast. These proteins are similar to the secretory proteins, however these proteins are different in nature because they are transported while immobilized in a cell membrane via a GPI-anchor. The cell surface display of protein is well documented in art. EP 1380592 discloses the anchoring of lipase on the cell surface of yeast cell. US 669658 disclose the method of displaying antibody on yeast cell surface.
As described in the preceding section there is no available economical method for conversion of sucrose either into isomaltulose or trehaluloseonly. Therefore there is a need for a better process for bioconversion of sugars exclusively from sucrose.
Objective of the present invention:
The primary objective of the present invention is to develop a process for isomerization/bioconversion of sucrose through enzyme namely Sucrose isomerase and /or trehalulose synthase displayed on the outer surface of the organism either singly or in combination to produce isomaltulose or trehalulose or combination thereof.

Another objective of the present invention is to express sucrose isomerase on the cell surface to convert sucrose to Isomaltulose only.
Yet another objective of the present invention is to express trehalulose synthase on the cell surface to convert sucrose to trehalulose only.
Another objective of the present invention is to express sucrose isomerase and trehalulose synthase on the cell surface to convert sucrose to a combination of Isomaltulose and trehalulose.
Sucrose Yeast armor with sucrose isomerase Isomaltulose
Yeast Armor with trehalulose synthase from B.argentifolii > Trehalulose
Fig1
Summary of the invention
The present invention discloses a novel bio-production process, which utilizes modified cell surfaces for producing altered disaccharides which can be obtained from different sources such as sucrose, sweet sorghum juice, sugar beet juice or sugarcane juice, wherein the modification of the organisms are made through expressing enzymes, proteins or peptides on the surface of host cell. The surface displayed organism used for biosynthesis of altered sugars.
Detailed description of the invention:
According to the invention, the enzyme(s) is displayed on the cell surface by the help of cell surface displaying properties of the organism. However, in the present invention S. cerevisiae strain lacking invertase with the high affinity sucrose H+ symporter encoded by AGT1 gene deleted organism used as this will prevent the organism to use it as primary carbon source.

The selection of organism is cntical for the process. It is well known in the art that the majority of microorganisms utilize natural sugars for their growth and metabolism. Therefore displaying the enzyme on the invertase deleted or repressed organism restricts the organism from metabolizing the disaccharide sugars such as sucrose etc., and still can grow on C6 carbon sugars such as glucose for the growth of the organism till the suitable biomass attain for the desired sugar production.
As mentioned, the said yeast is further modified for the expression of the sucrose isomerase on the cell surface.
The enzyme displayed on the yeast cell surface is isolated polynucleotide that encodes an isomaltulose producing sucrose isomerase enzyme. The polynucleotide is isolated from an isomaltulose producing organism using a probe specific for sucrose isomerase encoding polynucleotides. The probe hybridizes with sucrose isomerase polynucleotides but, in the same condition it does not bind to glucosidase encoding polynucleotides. The polynucleotide is isolated using a probe that essentially consists of a nucleic acid sequence encoding a sucrose isomerase which preferably has at least 80% sequence homology.
In another embodiment of the invention, method of producing isomaltulose from sucrose by expressing the active polynucleotide which may be isolated from any sucrose metabolizing organisms, preferably from sucrose metabolizing microorganisms of the genera Agrobacterium, Enterobacter, Erwinia, Klebsiella, Protaminobater, Serratia, Pseudomonas capable of converting sucrose to isomaltulose.
The said polynucleotide operably linked to promoter and other regulatory sequence which control the transcription and expression of the polynucleotide.
The full length nucleotide sequence of Eiwirnia rhaontici is 1899 base pair which is used for the cell wall display either with N-terminal or C-terminal for the expression with a sugar binding domain such as GPI attached to a cell wall protein such as alpha agglutinin or Flop-1.
As detailed in Fig.l the sugar converted to either Isomaltulose or According to the present invention the sucrose isomerase (Pall, mutB, smuA) displayed on the invertase repressed or deleted cell wall for the conversion of sucrose to isomaltulose. Another inventive process is expressing the trehalulose synthase from Bemisia argentifolii in the hxt null organism for the production of trehalulose only.

According to the present method of invention, isomerization of sucrose can be done to produce altered non-natural sugars such as Isomaltulose and trehaulose from disaccharides such as sucrose by the host cell selected from the group of fungi, bacteria. A method of producing Isomaltulose or Trehalulose or a combination thereof, is described as herein under. The said method comprising:
a) Contacting the transformed biocatalyst with sucrose and in presence of an inducible promoter such as herein described.
b) Allowing the said biocatalyst to isomerize sucrose by incubating the transformed biocatalyst and sucrose at 25-40°C and pH 5-7 thereby obtaining isomaltulose or trehalulose wherein, the Gene capable of producing invertase is deleted or repressed in the transformed biocatalyst.
The transformed biocatalyst is selected from the group comprising Saccharomyces, Pichia, Debrayomyces, Schizosaccharomyces genus, Pseudomonas sp. or Comybacteria sp. (deposited at the International depository).
And the polynucleotide is isolated from group comprising Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti, or Klebsiella sp. and/or group comprising Pseudomonas mesoacidophila, Agrobacterium raadiobacter or Bemisia argentifolia.. and is operably linked to promoter and other regulatory sequences which control the transcription and expression of the polynucleotide and the inducible promoter is GAL 1-10.
The organisms selected are microbial cells and the organisms can be bacteria or fungi.
The group of organisms consists either of Saccharomyces, Pichia, Debrayomyces,
Schizosaccharomyces genus, Pseudomonas sp or Comybacteria sp etc.
The organisms are modified by displaying specific enzymes on the cell surface of the host
organisms.
The protein displayed on host cell surface is sucrose isomerase.
The displayed protein on host cell surface is trehalulose synthase.
Sucrose isomerase gene may be isolated either from Protaminobacter rubrum, Serratia
plymuthica or Erwinia rhaponiciti.

The trehalulose synthase gene may be isolated either from Pseudomonas mesoacidophila ,
Agrobacterium raadiobacter or Bemisia argentifolia..
The Sucrose isomerase or Trehalulose synthase expressed on the cell surface using a single or
multiple promoters.
In accordance with the invention, the enzyme(s) can be displayed on the cell surface with the
help of specific anchor proteins, promoters, vectors.
By expressing either single or combination of enzymes on the surface, a desired product or a combination of desired products can be obtained.
The selection of the organism is critical for the present mode of invention. It is known in the art that majority of the microorganisms utilizing common sugars for their growth and metabolism. Therefore cell surfaces can be modified based on their natural or induced ability and non-preference to utilize the sugar which is used as substrate for bio-conversion. The present process of invention also utilized the process of immobilization of the transformed cells for the continuous production of desired products as such Isomaltulose and/or trehalulose.
Example:
The conversion of sucrose to isomaltulose carried out in 250 ml Erlenmeyer flaks to confirm the best conditions for isomaltulose production, with transformed yeast cells SSI1. Erlenmeyer flasks containing 50 ml of sucrose solution and SSI1 were maintained in an orbital shaker at 150 rpm for 15 min in presence of galactose. At the end of the incubation of batch, samples were collected and the enzyme activity was analyzed. The reaction medium was centrifuged for 20 min at and the free-cells of SSI 1 used for the next batch conversion of sucrose into isomaltulose with fresh substrate.

Result:
Advantages of the invention:
In the present mode of invention the conversion process allows to selective isomerization of disaccharide i.e. either Isomaltulose or Trehalulose which are still hitherto unknown and thereby making the process economical.
Rate and yield of the conversion is superior as isomerization/bioconversion process is simple and enzyme life cycle is longer compare to existing immobilized enzymes system.
We Claim:
1. A method of producing isomaltulose or trehalulose or a combination thereof from
sucrose, the said method comprising:
a) contacting the transformed whole cell biocatalyst with sucrose.
b) allowing the said biocatalyst to isomerize sucrose by incubating the transformed biocatalyst and sucrose at temperature range of 25-40°C and pH 5-8, in the presence of an inducible promoter such as herein described thereby obtaining isomaltulose or trehalulose
wherein, the gene capable of producing invertase is deleted or repressed in the transformed biocatalyst and wherein, the polynucleotide encoding Sucrose isomerase and /or trehalulose synthase is expressed in the transformed whole cell biocatalyst.
2. A method as claimed in claim 1, wherein the transformed biocatalyst is selected from the
group comprising Saccharomyces, Pichia, Debrayomyces, Schizosaccharomyces genus,
Pseudomonas sp. and Cornybacteria sp.
3. A method as claimed in claim 1 wherein, the polynucleotide is operably linked to
promoter and regulatory sequences which control the transcription and expression of the
polynucleotide.
4. A method as claimed in claim 1 wherein, inducible promoter is GAL 1-10.
5. A method as claimed in claim 1, utilized for the continuous production of the desired
products such as Isomaltulose and/or trehalulose by immobilization of the transformed
cells.
6. A transformed invertase negative biocatalyst capable of displaying the sucrose isomerase
on the cell surface for the conversion of sucrose to isomaltulose and/or trehalulose
continuously deposited at the International depository.