AU731229B2

AU731229B2 - Substitutes for modified starch in paper manufacture

Info

Publication number: AU731229B2
Application number: AU62767/96A
Authority: AU
Inventors: Scott E Nichols
Original assignee: Pioneer Hi Bred International Inc
Current assignee: Pioneer Hi Bred International Inc
Priority date: 1996-06-12
Filing date: 1996-06-12
Publication date: 2001-03-29
Anticipated expiration: 2016-06-12
Also published as: JP2001503607A; WO1997047806A1; EP0904452A1; CA2257621A1; CA2257621C; AU6276796A

Description

WO 97/47806 PCT/US96/10190 SUBSTITUTES FOR MODIFIED STARCH IN PAPER MANUFACTURE Field of the Invention The present invention involves the field of paper manufacture. Specifically, the present invention provides sources alternative to modified starch in paper manufacture.

Background of the Invention There are three major phases in paper manufacture where starch is used as an ingredient. The first is the "wet end" where cellulose fibers are mixed with starch in a slurry, and the slurry is forced through a narrow opening onto a wire belt. Water is rapidly removed as the forming sheet travels the length of the belt. After a distance of typically five to fifteen meters on the belt, the sheet has had enough water removed from it so that it can support its own weight. The sheet travels through a number of foils and rolls wherein more water is removed. It is dried to about 11% moisture.

The second phase in paper manufacturing involving starch is the "sizing step". Here, the paper goes through a sizing press where a starch slurry is applied to the sheet.

The sheet again goes through a series of foils and rolls.

It is dried on rollers and can be taken off the press as a finished product.

The third step involves coating the paper with a mixture of starch and a thermoplastic molecule. On certain lines, this occurs after the sizing step. The nascent roll can also be removed and reinstalled onto a different press for coating. A typical coating device has two blades that run the width of the paper. The blades apply the coating material onto two rolling drums. The paper passes between the drums and the coating material, comprising starch and the thermoplastic moiety, comes off the drums onto the paper. After the paper leaves the drums, it goes through a WO 97/47806 PCT/US96/10190 number of dryers. When the paper is dry, it goes onto a "soft calendar" comprising two drums, one made of a hard density fabric and the other a heated steel drum. The paper passes between the two drums and the heated steel drum is sufficiently hot to melt thermoplastic components of the coating mix providing a hard gloss finish on the paper.

The cellulosic wood pulp fibers, typically used in the above process, are anionic in nature. The addition of a cationic starch to the "wet end" slurry acts as an adhesive by cross linking the pulp fibers through salt linkages.

Thus a cross linked polymeric network is made, comprising the starch and cellulose fibers. Typically, the cationic starches used in the "wet end" are tertiary or quaternary amines. These amino groups are added to the starch by wet millers.

Surface sizing starches are used to impart both strength and smooth finish to the sheet after it leaves the "wet end". Such starches also prepare the sheet to receive the various coatings. In cheaper grades of paper and in fiberboard manufacture, sizing starches are used simply as unmodified corn starch. For high grades of paper, chemically-modified starches are used. This is important for the application of a smooth, uniform high quality surface to the paper.

There is a tendency for starches to retrograde i.e. reform high ordered structures (both helices and crystallites) in an otherwise gelatinous starch slurry. Deposition of retrograded starch onto high quality paper causes regional inconsistencies on the paper and is unacceptable.

Furthermore, retrograded starch in the sizing press may necessitate shutting the line down to clear the apparatus.

The starch most often used for sizing applications is a starch having a covalently attached neutral adduct, for instance hydroxyethyl starch. This is prepared by the reaction of ethylene oxide with starch after it is isolated at the wet milling plant. The function of the hydroxyethyl (or similar) adduct is independent of its chemical nature; WO 97/47806 PCT/US96/10190 rather, it serves to provide steric hindrance, inhibiting the formation of high ordered structures. This steric hindrance is critical to decrease retrogradation. The periodic protuberance afforded by the adduct disrupts the formation of higher ordered structures that leads to retrogradation.

Speed is of paramount importance in paper manufacturing. Limiting in press speed is starch consistency. Presses often run below their full capacity speeds. Depending on the application, starch slurries are between 3-15% (usually solids. An increase in solids would necessarily result in a decrease in the amount of water that would have to be removed from a paper sheet being manufactured. This would allow the press to work at higher speeds.

Hydroxethylated starch also forms higher ordered structures as the temperature decreases or the concentration increases. The formation of the higher ordered structures on the surface of the paper is required. -After application to the sheet the starch reforms some of these higher ordered structures and creates a uniform surface that imparts structural strength and facilitates the acceptance of inks and dyes. However, the higher ordered structures should not form in the slurry nor on the application device because this necessitates shutting down the production line to clear off retrograded starch.

The function of the hydroxyethyl group is to lower the temperature and/or raise the concentration of starch at which retrogradation occurs. As the processing lines have already been optimized for a particular temperature of the starch slurry, a decrease in the tendency to retrograde would allow for a higher carbohydrate content in the slurry.

The mixture applied to the paper sheet in the coating process contains hydroxethylated starch and thermoplastic molecules. The most prevalent thermoplastic molecules used are latexes, such as styrene butadiene. The function of the hydroxethyl starch is as indicated above. The function of WO 97/47806 PCT/US96/10190 the thermoplastic molecule is to form a high gloss finish on the paper. This causes an increased ability to take inks and dyes and improves the resolution, in general, on the printed sheet.

Based on the foregoing, there exists a need, in paper manufacturing, for modified starch substitutes which are functionally similar to modified starch. There is a further need to provide substitutes for modified starch which are less prone to retrogradation. There is a further need to provide methods of manufacturing paper which are faster than current methods and allow presses to run closer to their full capacity speed. There is a further need to provide methods of manufacturing paper that are environmentallyfriendly and do not involve input materials that require chemical processing.

It is therefore an object of the present invention to provide substitutes for modified starch which are less prone to retrogradation when used in paper manufacture.

It is a further object of the present invention to provide methods of manufacturing paper which are faster and more efficient than existing methods.

It is a further object of the present invention to provide substitutes for starch in paper manufacturing that do not require costly chemical modification as does starch.

It is a further object of the present invention to provide methods for manufacturing paper that are more environmentally-friendly than existing methods.

It is a further object of the present invention to provide substitutes for thermoplastic molecules currently used in the coating step during paper manufacture.

SUMMARY OF THE INVENTION The present invention provides glucans which can be used as substitutes for modified starch and/or latex in paper manufacturing. The present glucans are produced by glucosyltransferase B ("GTF enzymes of the species Streptococcus mutans, and are functionally similar to the modified starch currently used in paper manufacturing. The present glucans also exhibit similar physical properties to the thermoplastic molecules currently used in the coating step of paper manufacturing.

The present invention also provides methods of manufacturing paper utilizing the present glucans, input materials that are produced biologically. Thus, the present methods are more cost-effective and environmentally-friendly than current methods which require input materials producing chemical effluents.

According to a first embodiment of the invention there is provided a method of manufacturing paper comprising adding a glucan to one or more steps in paper in manufacturing, wherein the glucan is synthesized by a glucosyltransferase B enzyme, wild-type or mutant.

According to a second embodiment of the invention there is provided a method of imparting gloss on paper during the manufacturing process comprising adding a glucan to a coating step, wherein the glucan is synthesized by a glucosyltransferase B enzyme, i 5 wild-type or mutant.

According to a third embodiment of the invention there is provided an isolated nucleic acid sequence comprising a member selected from the group consisting of: i o a polynucleotide which encodes a glucosyltransferase B polypeptide having changes at positions selected from the group consisting of 1448V; D457N; D567T; i1 K1014T: D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/KI014T; 1448 V/D457N/D567T/D571K/K779Q/K1014T; Y169A/Y170A/Y171A; and K779Q, a polynucleotide complementary to a polynucleotide of According to a fourth embodiment of the invention there is provided an expression .cassette comprising at least one gi/b nucleic acid operably linked to a promoter.

According to a fifth embodiment of the invention there is provided a method for producing a glucan in a plant comprising: transforming a plant cell with the expression cassette of the invention, growing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to produce the glucan in the regenerated plant.

According to a sixth embodiment of the invention there is provided an isolated protein comprising a polypeptide encoded by a gtfb nucleic acid having changes at [I:\DAYLIB\LIBFF]8652spec.doc:gcc positions selected from the group consisting of; 1448V; D457N; D567T; K1OI4T; 1D457N/D567T; D457N/D571K; D567T/D571K; D567T/D57IKI14T; 1448 V/D457N/D567T/D571K1K779Q/KlI4T; YI 69A/YI7OA/YI71A; and K779Q.

According to a seventh embodiment of the invention, there is provided a transgenic llnt cell containing a DNA molecule, encoding a transit sequence and a Strept7ococcus Inl/tns glucosyltransferase B enzyme, wild type or mutant, wherein the mutant is 1448V; D457N: D567T; Ki1l 4T; D457N/D567T; D457N/D571IK; D567T/D57 I K; li567T/D57 I K/K 10 1 4T; 1448 V/D457N/D567T/ D571I K/K779Q/ K 101 4T; or Y1I69A-/Y I70A/Y 171 A, wherein the transit sequence directs the enzyme to the amnyloplast or vacuole and wherein the plant cell is derived from a plant selected from the g'roup consisting of potato, cassava, sweet potato and sugar cane.

According to an eighth embodiment of the invention, there is provided a transgenic plant -seed containing a DNA molecule, encoding a transit sequence and a Strep tococcus ':'tan gluCOSyl transferase B enzyme, wild type or mutant, wherein the mi-utant is 1448V; :::D457N: D567T; KIO14T; D457N/D567T; D457N/D571K; D567T/D571K; )567Tr/D571K/KI0l4T; 1448V/D457N/D567T/D571 K/ K779Q/KIOI4T; or Y1I69A/Y]I70A/Y 171 A, wherein the transit sequence directs the enzyme to the ainyloplast or vacuole and wherein the plant seed is derived from a plant selected from thIIe grou consisting of maize, rye, barley, wheat, sorghum, oats, millet, triticale and rice.

2 -ccor dinc to a ninth embodiment of the invention, there is provided a m-aize line in starch biosynthesis containing, a DNA molecule, encoding a transit sequence nd a Streptococcuts nitans gILucosyltr-ansferase B enzyme. wild type or mutant, wherein OIC mILltL is 1448V; D457N, D567T; KlOI4T; D457N/D567T; D457N/D571K; D567'17D571 K: D567T/D571 KJKlI4T; 1448V/D457N/D567T/D571 K! i 779QO/IOI4T1: or Y169A/YI7OA/YI7lA, wherein the transit sequence directs the enzyme to the -im-yloplast or vacuole.

D~etailed Description of the Invention A-S Used herein, "glucan" mreans a g',lucose polymer having, linkages that are (xL( I and branching A-S used herein, amnylop last" m-eans starch accumulating, organelle in plant storage tISSueC A-S used here in, 'vacuole" mneans the cellular compartment bounded by the tonoplast membrane.

[I:\DAYLIB\LII3FF]08652spec.doc:gcc Streptococcus mulans is a species that is endogenous to the oral cavity and colonizes tooth enamel. See e.g. Kararnitsu, "Characterization of Extracellular Glucosyl 'Fransferase Activity of Streptococcus-mutans". lnfrcl Immun.; vol. 12(4); pp. 738-749; 1 I975): and Yam-ashita, ei ali., "Role of the StreptococcuIs-Mutanis-gtf Genes in Caries ndn1cLItiOn in the Specific-Pathogen Free Rat M~odel," Infeci Immun.: Vol. 61(9); pp.

'XI 11-381 7 (1993); both incorporated herein their entirety by reference. Strepto31coccusll Imltuo species secrete glucosyltransferase B ("GTF enzymes Which Utilize dietary sutcrose to make a variety of extracellular glUcans. See e.g. Kainetaka, el ali., "Purification aind Characterization of Glucosyltransferase from Streptococcus-mu1Ltans OMZI176 with I Chromnatofocusing," Mlicrobios; Vol. 5 1(206); pp. 29-36; (1978); incorporation herein by rc ference.

B~oth Soluble and insoluble glucans are synthesized, and the proteins responsible have been isolated and characterized. See Aoki, el ali., "Cloning of a Pi:\DAY LI B\L I B IF]08652spec.doc: gcc WO 97/47806 PCT/US96/10190 Streptococcus-mutans Glucosyltransferase' Gene Coding for Insoluble Glucan Synthesis," Infect. Immun.; Vol. 53(3); pp. 587-594; (1986); Shimamura, et al., "Identification of Amino Acid Residues in Streptococcus Mutans Glucosyltransferases Influencing the Structure of the Glucan Produced," J. Bacteriol.; Vol. 176(16); pp. 4845-50; (1994); and Kametaka, et al., "Purification and Characterization of Glucosyltransferase from Streptococcus-mutans OMZ176 with Chromatofocusing," Microbios; Vol. 51(206); pp. 29-36; (1987); all incorporated herein their entirety by reference.

The proteins involved are large (-155 kDa) and catalyze the group transfer of the glucosyl portion of sucrose to an acceptor glucan via oc and oc linkages. See Wenham, et al., "Regulation of Glucosyl Transferase and Fructosyl Transferase Synthesis by Continuous Cultures of Streptococcus-mutans," J. Gen. Microbiol.; Vol. 114 (Part pp. 117-124; (1979); Fu, et al., "Maltodextrin Acceptor Reactions of Streptococcus-mutans 6715 glucosyltransferases," Carbohydr. Res.; Vol. 217; pp. 210- 211; (1991); and Bhattacharjee, et al., "Formation of Alpha Alpha and Alpha (1-+2)Glycosidic Linkages by Dextransucrase from Streptococcus Sanguis in Acceptor- Dependent Reactions," Carbohydr. Res.; Vol. 242; pp. 191- 201; (1993); all incorporated herein their entirety by reference.

The genes involved in glucan synthesis have been isolated and sequenced. See Shimamura, et al., cited hereinabove and Russel, et al., "Expression of a Gene for Glucan-binding Protein from Streptococcus-mutans in Escherichia-coli," J. Gen. Microbiol.; Vol. 131(2); pp.

295-300; (1985); Russell et al., "Characterization of Glucosyltransferase Expressed from a Streptococcus-Sobrinus Gene Cloned in Escherichia-coli," J. Gen. Microbiol.; Vol.

133(4); pp. 935-944; (1987); and Shiroza, et al., "Sequence Analysis of the GTF B Gene from Streptococcus mutans," J.

Bacteriol.; Vol. 169(9); pp. 4263-4270; (1987); all incorporated herein in their entirety by reference.

WO 97/47806 PCT/US96/10190 The structures of the various glucans produced by GTF enzymes are quite heterogeneous with respect to the proportions of and cc branches present in any given glucan. Transformation of genes which encode naturally occurring GTF B and GTF B mutant proteins into plants, such as maize, provides amyloplasts and vacuoles with novel compositions.

GTF B enzyme activity incorporated into the amyloplast and/or vacuole leads to the accumulation of starch and glucan in the same amyloplast and/or vacuole.

Retrogradation occurs as portions of starch molecules interact and subsequently form inter- or intra-chain helices. In a mixture of starch and glucans, the frequency of starch-starch interactions, that lead to helix formation, is diminished. A paste made from the mixed polymers is less prone to retrogradation as a result. This is especially true in the starch accumulation mutants envisioned as transformation targets where the relative proportion of starch is reduced.

Glucans produced in maize amyloplasts and/or vacuoles by the transgenic GTF B enzymes can function in paper processing without chemical modification, as required of starch. The polymer solution consequently has altered rheological properties and is less prone to retrogradation compared to starch. The glucans are branched and irregular and able to supplant modified starches with comparable or superior efficacy. They do not require any costly chemical modification as does starch. For coating applications, the present glucans exhibit thermoplastic properties in addition to the above advantages.

The wild type GTF and mutants thereof useful in producing glucans according to the present invention are provided below. The following code is employed: WO 97/47806 PCT/US96/10190 Amino Acid One-letter Symbol Alanine

A

Asparagine

N

Aspartic Acid

D

Glutamine

Q

Glutamic Acid

E

Isoleucine

I

Lysine

K

Threonine

T

Tyrosine Valine

V

The nomenclature used to identify the mutant GTF B enzymes used to produce the present glucans is as follows: the number refers to the amino acid position in the polypeptide chain; the first letter refers to the amino acid in the wild type enzyme; the second letter refers to the amino acid in the mutated enzyme; and enzymes with multiple mutations have each mutation separated by The mutant GTF B enzyme used to produce glucans for paper coating is preferably selected from the group consisting of I448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; I448V/D457N/D567T/D571K/K779Q/ K1014T; and Y169A/Y170A/Y171A.

The mutant GTF B enzyme used to produce glucans for paper coating is more preferably selected from the group consisting of I448V; K1014T;D567T/D571K/K1014T; I448V/D457N/D567T/D571K/K779Q/ K1014T; and Y169A/Y170A/Y171A.

The mutant GTF B enzyme used to produce glucans for paper coating is even more preferably selected from the group consisting of K1014T; I448V/D457N/D567T/D571K/K779Q/K1014T; and Y169A/Y170A/Y171A.

The mutant GTF B enzyme used to produce glucans for paper coating is most preferably I448V/D457N/D567T/ D571K/K779Q/K1014T; or Y169A/Y170A/Y171A.

WO 97/47806 PCT/US96/10190 The mutant GTF B enzyme used to produce glucans for paper sizing is preferably selected from the group consisting ofI448V; D457N; D567T; K779Q; K1014T; D457N/D567T; D457N/D571K; D567T/D571K and D567T/D571K/K1014T.

The mutant GTF B enzyme used to produce glucans for paper sizing is more preferably selected from the group consisting of I448V; D457N: K779Q; D567T/D571K; and D567T/D571K/K1014T.

The mutant GTF B enzyme used to produce glucans for paper sizing is most preferably I448V.

The glucans of the present invention are preferably produced in transgenic maize, potato, cassava, sweet potato, rye, barley, wheat, sorghum, oats, millet, triticale, sugarcane or rice. More preferably, the present glucans are produced in maize, potato, sugarcane, cassava or sweet potato. Even more preferably, the present glucans are produced in maize or potato. Most preferably, the present glucans are produced in maize.

In a highly preferred embodiment of the present invention, maize lines deficient in starch biosynthesis are transformed with mutant GTF B genes. Such lines may be naturally occurring maize mutants sh 2 bt 2 btl) or transgenic maize engineered so as to accumulate low amounts of starch in the endosperm when compared to wild type maize.

See e.g. Muller-Rbber, et al., "Inhibition of the ADPglucose Pyrophosphorylase in Transgenic Potatoes Leads to Sugar-Storing Tubers and Influences Tuber Formation and Expression of Tuber Storage Protein Genes," The EMBO Journal; Vol. 11(4); pp. 1229-1238; (1992); and Creech, "Carbohydrate Synthesis in Maize," Advances in Agronomy; Vol. 20; pp. 275-322; (1968); both incorporated herein in their entirety by reference.

The production of the present glucans is performed according to methods of transformation that are well known in the art, and thus constitute no part of this invention.

The compounds of the present invention are synthesized by WO 97/47806 PCT/US96/10190 insertion of an expression cassette containing a synthetic gene which, when transcribed and translated, yields a GTF enzyme that produces the desired glucan. Such empty expression cassettes, providing appropriate regulatory sequences for plant expression of the desired sequence, are also well-known, and the nucleotide sequence for the synthetic gene, either RNA or DNA, can readily be derived from the amino acid sequence for the protein using standard texts and the references provided. The above-mentioned synthetic genes preferably employ plant-preferred codons to enhance expression of the desired protein.

The following description further exemplifies the compositions of this invention and the methods of making and using them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.

The genes which code for the present enzyme or mutants can be inserted into an appropriate expression cassette and introduced into cells of a plant species. Thus, an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for a mutant or wild type gene in proper reading frame, together with transcription promoter and initiator sequences active in the plant. Transcription and translation of the DNA sequence under control of the regulatory sequences causes expression of the protein sequence at levels which provide an elevated amount of the protein in the tissues of the plant.

Synthetic DNA sequences can then be prepared which code for the appropriate sequence of amino acids of a GTF B protein, and this synthetic DNA sequence can be inserted into an appropriate plant expression cassette.

Likewise, numerous plant expression cassettes and vectors are well known in the art. By the term "expression cassette" is meant a complete set of control sequences including promoter, initiation, and termination sequences which function in a plant cell when they flank a structural WO 97/47806 PCT/US96/10190 gene in the proper reading frame. Expression cassettes frequently and preferably contain an assortment of restriction sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence.

By the term "vector" herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell. Typically, the vector has one or more restriction endonuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as markers to identify and separate transformed cells. Preferred markers/selection agents include kanamycin, chlorosulfuron, phosphonothricin, hygromycin and methotrexate. A cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant".

A particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule which is not a part of the chromosomes of the cell.

As mentioned above, both genomic DNA and cDNA encoding the gene of interest may be used in this invention. The gene of interest may also be constructed partially from a cDNA clone and partially from a genomic clone. When the gene of interest has been isolated, genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene in the host cell.

According to this invention, the genetic construct will contain a genetic sequence coding for the protein or trait of interest and one or more regulatory sequences operably linked on either side of the structural gene of interest. Typically, the regulatory sequences will be selected from the group comprising of promoters and WO 97/47806 PCT/US96/10190 terminators. The regulatory sequences may be from autologous or heterologous sources.

The expression cassette comprising the structural gene for a mutant of this invention operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells.

Typically, genes conferring resistance to antibiotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.

Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and Serratia marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. Since these hosts are also microorganisms, it will be essential to ensure that plant promoters which do not cause expression of the protein in bacteria are used in the vector.

The isolated cloning vector will then be introduced into the plant cell using any convenient technique, including electroporation (in protoplasts), retroviruses, bombardment, and microinjection into cells from monocotyledonous or dicotyledonous plants in cell or tissue culture to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette. Using known techniques, WO 97/47806 PCT/US96/10190 protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the gene for a protein according to this invention. Accordingly, a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette of a GTF B mutant.

It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species. Thus, this invention provides a method for introducing GTF B in Agrobacterium tumefacienssusceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infecting the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include a plant expression cassette of this invention.

For example, the potato plant can be transformed via Agrobacterium tumefaciens to produce the present glucans.

The transformation cassette comprises a patatin promoter, followed by the relevant GTF B coding sequence and the neomycin phosphotransferase polyadenylation site/terminator.

See e.g. Utsumi, et al., "Expression and Accumulation for Normal and Modified Soybean Glycinins in Potato Tubers," Plant Science; Vol. 102(2); pp. 181-188; (1994); (Limerick); incorporated herein in its entirety by reference. The transgenic cassette is placed into a transformation vector.

For example, BIN19, or derivatives thereof, are useful when transforming via Agrobacterium tumefaciens. See e.g.

Visser, et al., "Transformation of Homozygous Diploid Potato with an Agrobacterium-tumefaciens Binary Vector System by Adventitious Shoot Regeneration on Leaf and Stem Segments," Plant Mol. Biol.; Vol. 12(3); pp. 329-338; (1989); incorporated herein in its entirety by reference.

For maize transformation vectors, the promoters include any promoter whose expression is specific and limited to WO 97/47806 PCT/US96/10190 endosperm cells. Included are those encoding either 22 kDa zein, opaque2, gamma zein and waxy. These lead into the GTF B gene and are followed by the endogenous terminator or the heterogeneous PINII terminator. The GTF B protein are directed to the maize endosperm amyloplast using a suitable transit sequence. Transit sequences useful in directing the enzyme into the amyloplast for accumulation within the amyloplast include but are not limited to ribulose biophosphate carboxylase small subunit, waxy, brittle-l, and chlorophyll AB binding protein. The transit sequences are juxtaposed between the promoter and the GTF B coding sequence and fused in translational reading frame with the GTF B moiety.

Transit sequences useful in directing the enzyme into the vacuole for accumulation within the vacuole are well known in the art. For vacuolar targeting, see e.g. Ebskamp, et al., "Accumulation of Fructose Polymers in Transgenic Tobacco," Bio/technology; Vol. 12; pp. 272-275; (1994); incorporated herein in its entirety by reference.

For maize transformation and regeneration see e.g.

Armstrong, "Regeneration of Plants from Somatic Cell Cultures: Applications for in vitro Genetic Manipulation," The Maize Handbook; Freeling, et al., eds, pp. 663-671; (1994); incorporated herein in its entirety by reference.

Once a given plant is transformed, the glucans synthesized can be isolated, by standard methods, known to one skilled in the art. The glucan thus obtained in the transgenic plant can be substituted for modified starches and utilized in the sizing and/or coating steps. For formulations useful in the coating step, see e.g. Heiser, et al., "Starch Formations," Starch and Starch Products in Paper Coating; Kearney, et al., eds., pp. 147-162; (1990); Tappi Press; incorporated herein in its entirety by reference.

In both sizing and coating, the present glucans are utilized in an amount of from about 4 to about 15 weight percent, more preferably from about 5 to about 12 weight WO 97/47806 PCT/US96/10190 percent, also preferably from about 6 to about 8 weight percent. Weight percent is defined as grams of molecule per 100 ml solution.

The present glucans are used to replace the starch and/or latex molecules completely, or a starch-glucan or a latex-glucan mixture is used in the slurry. In the sizing application, the glucan:starch ratio ranges from about 10:90 to about 100:0; more preferably from about 40:60 to about 100:0; more preferably still from about 60:40 to about 100:0; most preferably about 100:0.

In the coating application, the glucan:starch ratio ranges from about 10:90 to about 100:0; more preferably from about 40:60 to about 100:0; more preferably still from about 60:40 to about 100:0; most preferably about 100:0. In the coating application, the glucan:latex ratio ranges from about 10:90 to about 100:0; more preferably from about 40:60 to about 100:0; more preferably still from about 60:40 to about 100:0; most preferably about 100:0.

All publications cited in this application are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Variations on the above embodiments are within the ability of one of ordinary skill in the art, and such variations do not depart from the scope of the present invention as described in the following claims.

Claims

1. A method of manufacturing paper comprising adding a glucan to one or more steps in paper manufacturing, wherein the glucan is synthesized by a glucosyltransferase B enzyme, wild-type or mutant.

2. The method of claim 1 wherein the glucan is added to a wet end, sizing or coating step.

3. The method of claim 2 wherein the glucan is added to a coating step.

4. The method of claim 3 wherein the amount of glucan utilized is from about 4 to about 15 weight percent of the slurry used in the coating application.

5. The method of claim 4 wherein the amount of glucan utilized is from about to about 12 weight percent of the slurry used in the coating application.

6. The method of any one of claims 1 to 5 wherein the glucan is produced by a glucosyltransferase B mutant having changes at positions selected from the group consisting of 1448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; 1448V/D457N/D567T/D571K/K779Q/Kl014T; Y169A/Y170A/Y171A; and K779Q.

7. The method of claim 6 wherein the glucosyltransferase B mutant has changes at positions 1448V; 1448V/D457N/D567T/D571K/K779Q/K1014T or Y169A/Y170A/Y171A. 20 8. The method of claim 7 wherein the glucosyltransferase B mutant has a change at position 1448V.

9. A method of imparting gloss on paper during the manufacturing process comprising adding a glucan to a coating step, wherein the glucan is synthesized by a glucosyltransferase B enzyme, wild-type or mutant.

10. The method of claim 9 wherein the amount of glucan utilized is from about 4 to about 15 weight percent of the slurry used in the coating application.

11. The method of claim 10 wherein the amount of glucan utilized is from about to about 12 weight percent of the slurry used in the coating application.

12. An isolated nucleic acid sequence comprising a member selected from the group consisting of: a polynucleotide which encodes a glucosyltransferase B polypeptide having changes at positions selected from the group consisting of 1448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; 1448V/D457N/D567T/D571K/K779Q/K1014T; 35 Y169A/Y170A/Y171A; and K779Q, a polynucleotide complementary to a polynucleotide of

13. An expression cassette comprising at least one gtfb nucleic acid operably linked to a promoter.

14. The expression cassette of claim 13, wherein the promoter is a 22Kda zein, paque 2, gamma zein or waxy promoter. S S. S 9e [n:\libh]00263:KWW A vector comprising the expression cassette of claim 13 or claim 14.

16. A host cell introduced with at least one expression cassette of claim 13 or claim 14.

17. The host cell of claim 16 that is a plant cell.

18. A transgenic plant comprising at least one expression cassette of claim 13 or claim 14.

19. The plant of claim 18, wherein the plant is maize, potato, sugar cane, cassava or sweet potato. The plant of claim 19 wherein the plant is maize.

21. The plant of claim 20 wherein the maize is deficient in starch biosynthesis.

22. The plant of claim 21 which is selected from the group consisting of sh-2, bt-1 and io bt-2.

23. A seed or tuber from the plant of any one of claims 18 to 21, wherein said seed or tuber comprises at least one expression cassette of claim 13 or 14.

24. The seed or tuber of claim 23 that is from maize, potato, sugar cane, cassava or sweet potato. i 25. A method for producing a glucan in a plant comprising: transforming a plant cell with the expression cassette of claim 13 or claim 14. growing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to produce the *20 glucan in the regenerated plant.

26. The method of claim 25 wherein the plant is maize, potato, sugar cane, cassava or sweet potato.

27. The method of claim 26 wherein the plant is a maize plant.

28. The method of claim 27 wherein the maize plant is deficient in starch biosynthesis.

29. The method of claim 28 wherein the plant is sh-2, bt-1 or bt-2. The method of any one of claims 25 to 29 wherein the promoter is selected from the group consisting of 22kDa zein, opaque 2, gamma zein and waxy.

31. The method of any one of claims 25 to 30 wherein the expression cassette contains a transit sequence selected from the group consisting of ribulose biphosphate carboxylase small .3 subunit, waxy, brittle-1 and chlorophyll AB binding protein to produce a transgenic plant.

32. The method of any one of claims 25 to 31 wherein the glucan is produced in the amyloplast or vacuole of the plant cell.

33. The method of claim 32 wherein the glucan is produced in the amyloplast of maize or R potato. II :\DAYLIIIALIBI1108652spec.doc:-cc 18

34. An isolated protein comprising a polypeptide encoded by a gtfb nucleic acid having changes at positions selected from the group consisting of; 1448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; 1448V/D457N/D567T/D571K/K779Q/K1014T; Y169A/Y170A/Y171A; and K779Q. A glucan produced by the protein of claim 34.

36. A ribonucleic acid sequence encoding the protein of claim 34.

37. A transgenic plant comprising at least one protein of claim 34.

38. A method of manufacturing paper of claim 1, substantially as hereinbefore described.

39. A method of imparting gloss on paper during the manufacturing process of claim 9, io substantially as hereinbefore described. An isolated nucleic acid sequence of claim 12, substantially as hereinbefore described.

41. An expression cassette of claim 13, substantially as hereinbefore described.

42. A method for producing a glucan of claim 25, substantially as hereinbefore described.

43. An isolated protein of claim 34, substantially as hereinbefore described.

44. Paper produced by the method of any one of claims 1 to 11 and 38.

45. A transgenic plant cell containing a DNA molecule, encoding a transit sequence and a Streptococcus mutans glucosyltransferase B enzyme, wild type or mutant, wherein the mutant is 448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; 1448V/D457N/D567T/ D571K/K779Q/ K1014T; or Y169A/Y170A/Y171A, wherein the transit sequence directs the enzyme to the amyloplast or vacuole and wherein the plant cell is derived from a plant selected from the group consisting of potato, cassava, sweet potato and sugarcane.

46. A transgenic plant seed containing a DNA molecule, encoding a transit sequence and a Streptococcus mutans glucosyltransferase B enzyme, wild type or mutant, wherein the mutant is 1448V; D457N; D567T; K1014T; D457N/D567T; D457N/D571K; D567T/D571K; D567T/D571K/K1014T; 1448V/D457N/D567T/D571K/ K779Q/K1014T; or Y169A/Y170A/Y171A, wherein the transit sequence directs the enzyme to the amyloplast or vacuole and wherein the plant seed is derived from a plant selected from the group consisting of maize, rye, barley, wheat, sorghum, oats, millet, triticale and rice.

47. A maize line deficient in starch biosynthesis containing a DNA molecule, encoding a transit sequence and a Streptococcus mutans glucosyltransferase B enzyme, [I:\DAYLI B\IJBFI-F]08652spcc.doc:gcc 19 wVildl type or- Mutant, wherein the mutant is 1448V; D457N; D567T; KlO14T; 1D457N/D567T; D457N/D57 1K; D567T/D57 1K; D567T/D571 K/K1 01 4T; 448 V/D457N/D567T/D57 1K! K779Q/K 101 4T; or Y 169A/Y 170A/Y 171 A, wherein the transit sequence directs the enzym~e to the amyloplast or vacuole.

48. The plant cell of claim 45 which is transformed by Agrobacter-im minefticiens, electroporation, retroviruses, bombardment or' microinj ection.

49. A transgenic plant regenerated from the plant cell of claim 45 or 48. 'The plant of clbaim- 49 wherein the plan-t is maize of genotype sib), bt-) or bti. 51I. 'The plant seed of claim- 46 wherein the enzyme produces anl insoluble I MrflILUCI

52. The plant seed of claim 46 whereini the DNA molecule contains a promoter selected fromn the group consisting of 22 kDa zein, opaque2, gamnma zein and waxy gene pro-nRoters. Dated 31 August, 2000 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON tIA:DAY LlB\1-1BF F]08652spec.doc:gcc