A PROCESS TO SYNTHESIZE FALLING OLIGOS ACÁRIDOS
Technical Field of the Invention The invention relates to a process for synthesizing oligosaccharide compositions. More specifically, the invention pertains to an economical method for synthesizing ohgosacapdos using unpurified crude sugar nucleotides and / or glycosyltransferases. Background of the Invention The term "carbohydrate" covers a wide variety of chemical compounds having the general formula (CH20) n and encompasses such compounds as monosaccharides, disacapdos, polysaccharide ohgosacapdos and their aminated, sulfonated acetylated forms and other derivatives (US Patent 5,288 637, this patent as well as other patents and publications described herein are incorporated herein by reference) The ohgosacapdos are chains composed of sugar units that are also known as monosacapdos. The sugar units can be arranged in any order and joined by their sugar units in any number of different ways. Id Therefore, the num However, different chains of possible stereoisophores are extremely large. Many classic chemical techniques have been developed for the synthesis of carbohydrates but these techniques require selective protection and deprotection. The organic synthesis of oligosaccharides is further complicated by the lability of many glycosidic linkages. achieve regioselective sugar coupling and generally with low synthetic yield Therefore, unlike peptide synthesis, traditional synthetic organic chemistry can not provide quantitative, reliable synthesis of even simpler oligosaccharides. Recent advances in the synthesis of oligosaccharides have occurred with the characterization, cloning and isolation of ghcosiltransferasas These enzymes can be used m vitro to prepare polysaccharide oligosaccharides and other glycoconjugates The advantage of biosynthesis with glycosyltransferases is that the ligatures g The enzymes formed by the enzymes are highly stereo- and regiospecific. Each enzyme catalyzes the ligation of specific sugar residues to other specific acceptor moieties such as a hybrid oligosaccharide or protein. For example, US Pat. No. 5,288,637 describes the synthesis of oligosaccharides using potassium portions. purified sugar nucleotide acceptors and ghcosyltransferases The problem with this process however is that it is not commercially feasible due to the extremely high cost of purified sugar nucleotides. Therefore, there is a need for an economical process to synthesize ohgosacapdos using nucleotides and ghcosyltransferases. of the Invention The present invention relates to a process for synthesizing oligosaccharides in wine. The process involves adding an acceptor portion and a catalytic amount of a glycosyltransferase to a culture of microorganisms that produces a sugar nucleotide or combination thereof. nucleotide sugar and nucleotide groups The mixture is maintained under conditions and for a period sufficient for the formation of ohgosacapdos. Alternatively, the present invention also involves a process for synthesizing ohgosacapdos by adding an acceptor moiety and intact host cells transformed with a polynucleotide encoding a catalytic amount of a glycosyltransferase to sugar nucleotides having a sugar unit to form a mixture and maintaining the mixture under conditions and for a sufficient period of time for the formation of oligosaccharides. Detailed Description of the Invention The present invention relates to a process to synthesize ohgosacapdos m vitro The process involves adding an acceptor molecule and a glycosyltransferase to sugar nucleotides under sufficient conditions and time to allow the formation of oligosaccharides The sugar nucleotide and / or the glycosyltransferase used in the process of this invention. tion are present in an unpurified crude form The use of the unpurified sugar nucleotide and / or the unpurified glycosyltransferase makes the process of the present invention more economical than the known processes employed in the prior art using purified sugar nucleotides and purified ghcosyltransferases or Semi-pupils (See US Pat. No. 5,288,637) The process of the present invention can be used in a single-batch or multiple-batch operation and the batch-fed products produced, according to the process of the present invention, they can be synthesized in a single reaction vessel or multiple reaction vessels. As is known in the art, a monosaccase is a sugar molecule that contains a sugar unit. present the term 'sugar unit' means a monosacapdo Also in the art it is known that a disacapdo is a sugar molecule that contains 2 units of sugar, a tsaczade is a sugar molecule that contains 3 units of sugar, an ohgosacapdo is a sugar molecule that contains between 2-10 units of sugar and a polysaccharide is a sugar molecule that contains more than 10 units of sugar. The units of sugar in a di- and ohgosacapdo are connected by glycosidic ligatures. However, as it is used In the present invention, the term "oligosaccharide" means a sugar molecule that contains at least two sugar units. The acceptor portion used in the present invention can be any molecule that is capable of being covalently bound to the sugar unit. Suitable acceptor moieties that can also be used in this invention include for example, glycoprotein proteins, lipid lipids, carbohydrates or any molecule having a sugar unit contained in its structure The preferred acceptor portion is a carbohydrate The preferred acceptor portion is a mono- or t-oligosaccharide When the acceptor portion is terminated by a sugar unit, the subsequent added sugar units will normally be covalently attached to the sugar unit. the molecule in the non-reduced sugar terminal end of the molecule The sugar unit that will be transferred to the acceptor portion is provided by a sugar nucleotide In mammals the sugar nucleotides are the building blocks for most oligosaccharides The nucleotides of sugar are considered to be donor molecules as they provide sugar units to the acceptor moieties during oligosaccharide synthesis Sugar nucleotides that can be used in the process of the present invention include, for example, mono- or di-phosphates of updina terminated is mono- or di-saccharides. -guanosine phosphates terminated in saccharides and cytidine mono- or di-phosphates terminated in sacapdos Examples of sugar nucleotides that can be used in this invention include UDP-glucose (UDP-Glc) UDP-N-acetylglucosamine (UDP-GIcNAc) , UDP-Galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc) GDP-Mannose (GDP-Man), GDP-Fucose (GDP-Fuc) and CM PN-acetylneuraminic acid (CMP-Neu-Ac) The semi-purified or unpurified purified sugar nucleotides can be used in the present invention. As used herein, the term "purified and semi-purified sugar nucleotides refers to sugar nucleotides that have been processed in the same manner such as by exchange chromatography or ionic or ultrafiltration, to increase its concentration and to separate nucleotides of sugar from any other chemical compounds produced during the synthesis of sugar nucleotides A preparation of purified sugar nucleotides is a preparation in which, at least approximately 80 % of the total weight of the preparation are sugar nucleotides and not more than about 95% of the total weight of the sugar nucleotide preparation, counter-ions and water. For example, purified sugar nucleotides can be obtained by stirring sugar nucleotides in a solution by precipitation and then filtering the solution and applying the solution to a selective ion exchange chromatography. The resulting solution is then hardened or in an alternative, lyophilized and subjected to evaporation of ethanol or methanol to obtain purified sugar nucleotides. The term semi -pupf icado "as used in the present, refers to the preparation of sugar nucleotides in which the actual residual content of sugar nucleotides in the preparation does not exceed 80% by weight of the preparation. For example, Semi-pupficados sugar nucleotides can be obtained by filtering a yeast solution containing sugar nucleotides through a 10 000 PM filter and then drying the filtrate. The term "unpurified" as used herein refers to sugar nucleotides. which have not been processed in any way such as ion exchange chromatography or affinity chromatography, to increase their concentration and to separate sugar nucleotides from any chemical compound produced during the biological or chemical synthesis of sugar nucleotides Sugar nucleotides they can be purified using any technique known in the art, such as ion exchange chromatography and ultrafiltration. See Smith, D, and others. The purified sugar nucleotides are well known in the art and will often be used in oligosaccharide synthesis (Blake D, and others, Meth Enzy 83 (1982) 127, Smith DF, Meth Enzy 83 (1982) 2 41) Additionally the sugar nucleotides produced by a culture of microorganisms can also be used in the present invention. The culture of microorganisms produces the nucleotides of sugar in an unpurified crude form. Preferably the sugar nucleotides used in the process of this invention are in an unpurified form and is produced by a culture of microorganisms or by permeabilized or previously dried microorganisms. The production of sugar using microorganisms, particularly yeast, is well known in the art (see Tochikura T and others J Feíment Technol Vol 46 No 12 page 957 -969 (1968) and Tochikura, T et al., Agr Biol Chem Vol 35 No 2 pages 163-176 (1971) ^ Generally the culture of microorganisms can be prepared by stirring sugars at room temperature or in a refrigerator with microorganisms and nucleotides or precursors of nucleotides such as updine mono- or di-phosphate, guanosine mono- or di-phosphate, or mono- or cytidine di-phosphate, orotate in the presence of a phosphate source (such as phosphate or pH buffer), an energy source (such as glucose, fructose or maltose) and magnesium ions, for a sufficient amount of time to allow that the microorganism begins to produce sugar nucleotides As used herein, the term "nucleotide precursor" refers to molecules that are used as intermediates in the synthesis of nucleotides via metabolic pathways, such as nucleotides, pupils or pipmidines. Examples of precursors of sugar nucleotides are updina orotato cytidine, adenosm, inosine, guanine guanidine, guanosma, updina, mono-, di- and tp-phosphates of updina, mono-, di- and tp-cytidine phosphates, mono- di- and tp-guanosine phosphates The precursors of pupna synthesis, such as UMP and UDP sugar nucleotides, are carbamoyl phosphate, aspartate, N-carbamoylaspartate dihydro-orotate and orotidylate, other precursors are pbonucleotide glycinamide pbonucleotide of formylglycinamide pbonucleotide of formylglycine, pbonucleotide of 5-ammonium-m? dazole pbonucleotide of 4-amino? m? dazol-4-carbox? lato pbonucleotide of 5-amino? m? dazol-4 -N-succ-nocarboxamide 5-ammono-5-m-dazole-4-carboxamide-dazole-4-carboxamide, phenoxyacetone, adenosu-canate and xantilate In the present invention, any microorganism which is capable of producing sugar nucleotides For example, microorganisms of the genus Saccharomyces, Zygosaccharomyces torulopsis, Candida can be usedCrypococcus, Brettanomyces Mucor Hansenula and Debaryomyces Examples of strains of microorganisms that can be used to produce sugar nucleotides are listed in Table 1 Table 1 Saccharomyces S cerevisiae Baker yeast (UDP-GIcNAc, UDP-Glc, GDP-Man) Brewer yeast (UDP-GIcNAc, UDP-Glc, GDP-Man)
S fragilis (UDP-Gal) S lactis (UDP-Glc, UDP-Gal) S ludwigí '(UDP-GIcNAc) Zygosaccharomyces Z rouxn (UDP-GIcNAc) Torulopsis T candida (UDP-GIcNAc UDP-Glc UDP-Gal) T spaenca (UDP-Glc UDP-Gal) T xlinus (GDP-Man) T versatile (UDP-GIcNAc Candida C famata .UDP-GIcNAc UDP-Glc GDP-Gal) C intermediate (UDP-Gal C kiuse, JDP-Glc) C paraps osus (UDP-G c) C. utilis (UDP-Man) C. mycoderma (UDP-Glc) C. pseudostopicalis (UDP-Glc) C. tropicalis (UDP-GIcNAc) Crypococcus albidus (UDP-Glc) Brettanomyces B. anomalus (UDP-Glc, UDP-GIcNAc)
B. clausenií (UDP-Glc. UDP-Gal) Mucor M. javanicus (UDP-Glc) M. racemosus (UDP-Glc) M. circinelloides (UDP-Glc) Hensenula H. jadinii (GDP-Man) H. saturnus ( GDP-Man) H. suaveolens (GDP-Man) H. capsulata (UDP-Glc) Debaryomyces D. subglobosus (UDP-Glc, UDP-GIcNAc) D. globosus (UDP-GIcNAc) D. cantavellii (UDP-GIcNAc) D japonicus (UDP-GIcNAc) D. hansemi (UDP-GIcNAc) Preferably, the microorganisms used in this invention have undergone some type of process such as drying, sound treatment or exposure to solvents or detergents. The preferred microorganism for use in This invention is S cerevisiae, particularly, dry Baker's yeast and dry Brewer's yeast, dry Candida famata or Zygosaccharomyces rouxp. In addition to sugar nucleotides, the culture of microorganisms or the glycosyltransferase preparation may also contain one or more epimerases. An epimerase is an enzyme that changes the stereospecificity of hydroxyl groups to ca specific rbones in a saccharin For example, an epimerase can be used to convert glucose to galactose and galactose glucose In the present invention an epimerase can be used to convert UDP-Gic to UDP-Gal The crude unpurified sugar nucleotides, described above are capable of providing component sugar units to an acceptor portion when placed in contact with an acceptor portion in the presence of at least one glycosyltransferase. As used herein, "giicosyltransferase" refers to an enzyme that facilitates the transfer of a unit. of sugar from one chemical entity (the donor molecule) to another (the acceptor portion) and is named phenomenologically according to the sugar unit it transfers For example, galactosyl transferases transfer galactose and fucosytransferases transfer fucose The glycosyltransferases that can be used In this invention they include, for example, ucosyrtransferases, sialiitransferases N-acetyl-ucosa-amyltransferases, galactosyltransferases, N-acetylgalactosamyltransferases, glucosyl-transferases and mannosyltransferases. Glycosyltransferases are known to have three domains that correspond to three different areas of the gene encoding the enzyme. The area of the gene found at the 3-end is known by encoding the catalytic functionality domain (Lowe, Seminars m Cell Biology, (1991) 2 289-307) The glycosyltransferases used in the process of this invention contain at least this catalytic domain but can contain up to the entire protein sequence
The ghcosyltransferases used in this invention can be obtained from any source and be in a purified or curd or unpurified form. As used herein, a "purified glycosyltransferase" refers to a glycosyltransferase that has been processed in some way, such as by affinity chromatography. , to increase the specific activity of the enzyme. The term specific activity refers to units per milligram of protein. The methods for purifying glycosyltransferases are bine known in the art. An "unpurified glycosyltransferase" as used herein refers to a glycosyltransferase that does not it has been processed in no way such as affinity chromatography to increase the specific activity of the enzyme. Preferably the glycosyltransferases used in the process of the present invention are in non-purified form. The genes encoding glycosyltransferases and methods for producing recombinant molecules that express glyc Osyltransferases are well known in the art For example, genes encoding glycosyltransferases of Neissena gonorrhoeae and recombinant molecules expressing these genes are described in WO 96/10086 and U.S. Patent No. 5,545,553 Any gene encoding glycosyltransferase can be inserted into a recombinant molecule. The pohnucleotides constituting the gene can be obtained by normal procedures known in the art, such as cloned DNA (such as a "bank" of DNA), chemical synthesis, cloning of cDNA or by cloning of genomic DNA or fragments thereof from a desired cell as described in Sambrook , J and others Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Sppng Harbor Laboratoy Press (1989) In molecular cloning of the genomic DNA gene, DNA fragments are generated some of which encode the desired gene DNA can be separated into specific sites using various restriction enzymes Alternatively DNAse can be used in the presence of manganese for the DNA fragment or DNA can be physically shared, for example by sound treatment. Linear DNA fragments can be separated according to size by standard techniques, such as agarose and polyacrylamide gel electrophoresis and column chromatography. DNA fragments are generated the identification of the specific DNA fragment containing the desired glycosyltransferase gene can be achieved in a number of ways that are well known in the art, such as by hybridizing nucleic acid with one or more labeled probes as described in Sambrook, J, and others Molecular Cloning A Laboratory Manual 2nd Edition, Cold Sppng Harbor Laboratory Press (1989) The presence of the desired gene can then be detected using analyzes based on the physical, chemical or immunological properties of the expressed product. that the gene encoding a glycosyltransferase has isolated can be inserted into a vector of cl Suitable vectors A large number of vector-host systems as used in the art can be used. Possible vectors include, but are not limited to, modified plasmids or viruses, as long as the vector system is compatible with the host cell used. which may be used include, for example, a cloning vector of E coli bacteriophages such as lambda derivatives, plasmids such as pBR322 derivatives or pUC plasmid derivatives. Insertion of the gene into the cloning vector can be achieved by any known process in the material such as by ligand the DNA fragment in a cloning vector having complementary cohesive terminations Sambrook, J, and others Moleci ar Clanmg a Laboratory Manual 2nd Edition, Cold Sprmg Harbor Laboratory Press (1989) However, if the sites of complementary restriction used to fragment the DNA are not present in the cloning vector, then the ends of DNA molecules may have to be modified enzymatically. Alternatively, any desired site may be produced by ligating the nucleotide 'binding' sequences at the DNA endings. These linked linkers may comprise chemically specific synthesized oligonucleotides that encode the restriction endonuclease recognition sequences. Cloning vector can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequences are generated. Transformation of the host cells with a cloning vector incorporating the glycosyltransferase gene allows Generation of multiple copies of the gene Therefore, the gene can be obtained in large quantities by growth transformants by isolating the cloning vector of the transformants and, when needed, recovering the inserted gene from the isolated cloning vector. or cloning may contain genes encoding truncated forms of the enzyme (fragments) and gene derivatives that have the same functional activity as the full-length gene A fragment or derivative is functionally active if it is capable of measuring the transfer of a unit of sugar to an acceptor portion For example, a cloning vector may contain a gene encoding the catalytically functional domain of a glycosyltransferase Once sufficient copies of the gene sequence have been generated, the gene encoding a glycosyltransferase, or a functionally active fragment or other derivative thereof, can be inserted into a suitable recombinant molecule for use in the process of the present invention. The recombinant molecule is an expression vector of polynucleotides that contain the necessary elements for the transcription and translation of the sequence encoding the inserted protein Preferably the expression vector also includes an origin of replication The necessary trans-transcriptional and translational signals can also be delivered by the native glycosyltransferase gene and / or its flanking regions A Once a recombinant molecule has been prepared, it is inserted into an acceptable host cell that will grow and divide to produce clones. To express the gene a variety of host-vector cell systems can be generated. The host-vector cell systems include example bacterial expression systems, mammalian cell systems infected with viruses such as vaccinia virus or adenovirus insect cell systems with a virus such as a baculovirus microorganisms such as yeast-containing yeast and transfo bacteria. Bacteria with bacteriophage DNA plasmid DNA or cosmide DNA The preferred host-vector cell system for use in this invention is the bacterial cell expression system. The most preferred host cells can be used in this invention are E coli cells. The methods previously described for the insertion of DNA fragments into a vector can be used to produce expression vectors containing a chimeric gene consisting of appropriate transcription / translation control signals and protein coding sequences. The expression of the polynucleotide which encoding a glycosyltransferase fragment or peptide thereof can be regulated by a second nucleic acid sequence such that the glycosyltransferase or peptide fragment is expressed in a host transformed with the recombinant DNA molecule. For example the expression of a glycosyltransferase can be controlled by a promoter / incr element eminently known in the art but these regulatory elements must be functional in the host selected for expression For bacterial expression, bacterial promoters are required Promoters that can be used to control the expression of glycosyltransferase genes include for example the early promoter region SV40, the promoter contained in the long terminal repeat 3 of Roux sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metaotionin gene, procapotin expression vectors such as lactamase promoter or tac promoter Recombinant molecules containing the glycosyltransferase gene can be identified by PCR amplification of the desired plasmid DNA or specific mRNA, nucleic acid hybridization, presence or absence of marker gene functions and expression of the inserted sequences Once an appropriate host system and growth conditions are established l The recombinant molecules containing the glycosyltransferase gene can be introduced into the host cells of any procedure known in the art such as transformation, infection transfection, electroporation, etc. Once a source of glycosyltransferases, the source can be added directly to a reaction vessel. For synthesis of oligosaccharides For example, host cells, such as E coli cells transformed with a polynucleotide encoding a crude unpurified glycosyltransferase to be added directly to a reaction vessel for use in the process of this invention. If the host cells express the glycosyltransferase in a culture medium, then the culture medium can be added to the reaction vessel Additionally the glycosyltransferase source can be homogenized and the homogenate can be added directly to the reaction vessel If a purified glycosyltransferase is employed, the purified enzyme can be added directly to the reaction vessel. For example, if the glycosyltransferase source is homogenized the glycosyltransferase can be purified from the homogenate by affinity chromatography using the acceptor moiety as the affinity ligand, using techniques well known in the art. In view of adding the unpurified or purified glycosyltransferase directly to the reaction vessel, the glycosyltransferase can instead be placed in a dialysis bag that can be inserted into a reaction vessel. The dialysis shell can be removed periodically from the reaction vessel and can be added additional glycosyltransferases when required. When the glycosytransferase is contained in a dialysis bag, the acceptor portion and the sugar nucleotides migrate to the pocket and react with the glycosyltransferase to synthesize an oligosacchad. When the synthesis of the oligosacchado is complete, the oligosaccharide migrates out of the pocket and into the surrounding medium. The glycosyltransferases used in the present invention catalyze the transfer of sugar units from the sugar nucleotides to an acceptor portion. The glycosyltransferases are preferably specific to a saccades unit to at least some significant portion, active or exposed thereof. Specifically, a glycosyltransferase is manifested by its tendency to bind with a particular sequenced portion of the acceptor portion when placed in close contact or proximity to it to effect the transfer of a particular sugar unit to the acceptor portion. A catalytic amount of a glycosyltransferase is used in the process of the present invention. As used herein a "catalytic amount" refers to the amount of glycosyltransferase that must be present to sufficiently catalyze the transfer of a sugar unit from a sugar nucleotide to a sugar unit. acceptor portion The catalytic amount of glycosyltransferase employed in the process of the present invention can be determined by one of ordinary skill in the art through routine experimentation. In the process of the present invention, an oligosaccharide can be synthesized by adding an acceptor portion and a glycosyltransfera Sa to a sugar nucleotide to form a mixture and then maintain the mixture under conditions and a sufficient time to allow the formation of oligosaccharides. The conditions and type required for the formation of the ohgosacapdo using the process of the present invention can be determined by someone with ordinary experience in the matter through routine experimentation In general, however, the mixture is incubated from about 4 to about 48 hours at a temperature of about 4 to about 35 ° C and a pH of about 4 to about 9 0 in the process of the present invention can synthesize a oligosacapdo using a nucleotide raw sugar unpurified microorganisms and an unpurified glycosyltransferase curda host cell transformed with polmucleotido expressing a glycosyltransferase for example can synthesize a ohgosacapdo adding a acceptor portion and E coli cells transformed with A polynucleotide encoding a catalytic amount of a glycosyltransferase to a culture of S cerevisiae that produce sugar nucleotides to produce a mixture The mixture is then maintained under conditions and for a time sufficient to allow formation of oligosacapdos Alternatively, one can synthesize a oligosacapdo adding an acceptor portion and a catalytic amount of a purified glycosyltransferase to a culture of microorganisms producing sugar nucleotides to form a mixture and incubate the mixture under conditions and for a sufficient time to allow the formation of oligosaccharides. synthesizing an ohgosacapdo by adding an acceptor moiety and host cells transformed with a polynucleotide that encodes a catalytic amount of a glycosyltransferase to purified sugar nucleotides to form a mixture and incubate the mixture under conditions and for a sufficient time to allow formation The oligosaccharide formed by the process of this invention can serve as an acceptor portion for the further synthesis of oligosaccharides. If additional synthesis is required the oligosacchad and a catalytic amount of a glycosyltransferase are added to sugar nucleotides to form another mixture. it is maintained under conditions and for a sufficient time for the formation of oligosaccharides This process is repeated until a sufficient number of sugar units are transferred to form the desired oligosaccharide. The synthesis of oligosaccharides according to the process of the present invention can take place in or no or a number of reaction vessels If only one reaction vessel is used, the ingredients required for the synthesis of oligosaccharides can be added sequentially, one at a time. For example, the ingredients required for the formation of a culture of microorganisms that produce the sugar nucleotides can be added first followed by the acceptor portion and then transformed host cells as a polynucleotide encoding a glycosyltransferase. Depending on the ohgosacapdo that will be synthesized additional sugar nucieotides and glycosyltransferases can be added to the reaction vessel if necessary. Alternatively, all the ingredients required for the synthesis of a particular oligosape can be added to the reaction vessel at the same time. A number of reaction vessels can also be used to The synthesis of oligosaccharides For example the ingredients required for the formation of a culture of microorganisms that produce sugar nucleotides an acceptor portion and host cells transformed with a polynucleotide that encodes a glycosyltransferase can be added or the same time in a reaction vessel The resultant coughing is then removed from the reaction vessel to a second reaction vessel for further synthesis. The oligosaccharide and host cells transformed with a polynucleotide which expresses a glycosyltransferase are added to nucleotides of sugar produced by a culture of microorganisms as required until the desired oligosaccharide is synthesized. Once the desired oligosacchado has been synthesized, it is removed from the reaction vessel and exposed to further processing such as centrifugation or decantation, ion exchange chromatography, tangential flow, filtration, reverse osmosis or spray drying and hofilization to obtain a pure ohgosacapdo The process of the present invention can be used to synthesize any oligosacchar For example, the process of the present invention can be used to synthesize oligosaccharides such as Lacto-N -neo T Lacto-N-fucopentase (LNF-V) 2-phenylactose II (LNF-II), difyucosylactose (DFL), lacto-N-fucopentaose (LNF-I) and Lacto-N-tetraose (LNT) Oligosaccharides produced according to the process of this invention are used as a wide variety of surplus applications and can be used in the same manner as the sacchapod compositions available from known sources. The present invention provides pharmaceutical and other oligosaccharide pharmaceutical compositions containing prepared compositions. in accordance with the present invention The following examples illustrate the preferred embodiments of the present invention and do not limit the specific claims in any way. Example 1 Production of Ohgosacapdos LNnT in a Single Yeast UDP-GIcNAc Production System
Reaction vessel Dry yeast cells (S cerevisiae) are fed with
mM of glucosamm, 170 mM of KH2P04, 5 mM of MgSO4, 70 mM of fructose and 20 mM of UMP This yeast culture is incubated at room temperature The yeast culture is monitored for the nucleotide formation of UDP-GlcNAc sugar When the amount of UDP-GIcNAc produced is approximately 6.0 mM and the yeast production system of sugar nucleotides is added to a reaction vessel Synthesis of Tr? saca? do-LNT-2 (GicN Ac 1-3Gal 1-4 Glc ) E coli cells expressing GIcNAc transferase (100 Units / liter of Reaction) are homogenized, and, without any further purification, they are added to the reaction vessel containing the UDP-GIcNAc LA GIcNAc transferase production system and the Yeast production UDP-GIcNAc are incubated at room temperature until the production of residual GIcNAc UDP is approximately 1.0 mM 30 mM lactose are added to the reaction vessel as a substrate to produce material containing the inte tpsacapdo LNT-2 mediator (GIcNAcBI 3Gal 1-4Glc) The amount of LNT-2- that contains the material produced should be greater than 6 mM E coli whole cell homogenates can be substituted with intact E coli +/- cells Triton X-100 Synthesis of LNnT (Gal 1-4GlcNAc 1-3Glc) E-cells coh that express Gal transferase (350
Units / Liter of Reaction) and eventually an epimerase is homogenized and added to the reaction vessel without any purification. The Gal transferase, the material containing LNT-2 and the solution of U DP-Glc / yeast are incubated at room temperature until the residual production of GIcNAc 1-3 Gal 1-4Glc is about 0 1 mM UDP-Glc is produced by feeding dry yeast cells (Candida famata) 200-400 mM glucose (Glc), 180 mM KH2PO. ,, 12 mM MgSO4 and 30-100 mM UMP Yeast is incubated at room temperature for 10-48 hours and monitored for the production of UDP-Glc When the amount of UDP-Glc produced exceeds 6 mM the mixture of UDP-Glc is added to the reaction vessel to produce Lacto-N-neoTetraose (LNnT) The amount of LNnT containing the material produced should be greater than 6 mM The material containing LNnT is then removed from the reaction vessel and moved downward for further processing such as by centrifugation or dec Antagonization, ion exchange chromatography tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 2 Production of LNnT in a Single Reaction Canister Using a Two Yeast System System Synthesis LNT-2- (GIcNAc 1 -3Gal 1-4Glc) All the reagents necessary for the synthesis of the intermediate LNT-2 are added simultaneously to a reaction vessel More specifically, a mixture of Candida famata and dry S cerevisiae were fed with 20 mM of glucosamide, 20 mM of UMP 170 mM KH2P04, 5 mM MgSO4, 70-175 mM fructose to produce UDP-GIcNAc from sugar nucleotide At the same time, 30 mM lactose and non-purified, homogenized E coli cells expressing GIcNAc transferase (100 Units / Liter of Reaction mixture) All the reaction mixture was incubated at room temperature until the levels of the material containing LNT-2 produced are greater than 6 00 mM. esis of LNnT (Gal 1-4GlcNAc 1-3Gal 1-4Glc) Additional homogenized non-purified coli E cells expressing Gal transferase (350 units / liter of Reaction) and an epimerase are added to the reaction vessel More specifically dry Candida famata is feed with 200.-400 mM of glucose, 180 mM of 12 mM KH2P04 of MgSO4 and 20-100 mM of UMP The yeast is incubated at room temperature until the amount of UDP-Glc exceeds 6 mM The yeast portion system of sugar-nucleotide is then added to the reaction vessel containing the material containing LNnT and the coli cells expressing Gal transferases to produce the material containing LNnT The material containing LNnT is then removed from the reaction container and moved to down for additional process such as by centrifugation or decanting, ion exchange chromatography, tangential flow filtration, spray-dried reverse osmosis or lyophilization to obtain pure LNnT Example 3 Production of LNnT in a Single Reaction Canister Using a Two Yeast System by Addition of
Simultaneous Reagents All reagents necessary for the synthesis of LNnT are added simultaneously to a reaction vessel More specifically, a mixture of dry Baker's yeast (S cerevisiae) and Candida famata are fed with 30 mM of ina glucose, 40 mM of UMP 170 mM of 12 mM KH2P04 of MgSO4 and 200 mM of maltose to produce UDP-GIcNAc from sugar nucleotides and UDP-Glc At the same time, E coli cells, not purified homogenized in 30 M lactose, expressing GIcNAc transferases ( 1900 U / L reaction) and non-purified, homogenized coli E cells expressing Gal transferases (1000-2000 U / L reaction) were added to the same reaction vessel. The reaction mixture is incubated at room temperature and the level of the material containing LNnT is greater than 3 mM The material containing LNnT is then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or chromating ion exchange atography tangential flow filtration reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 4 Production of LNnT of Oligosacapdo in a single
Reaction Vessel Using the Two System
Yeasts by the Exogenous Addition of LNT-2 All reagents necessary for the synthesis of LNnT are added simultaneously to a reaction vessel More specifically, a mixture of dry Baker's yeast and Candida famata are fed with 30 mM of glucosamine, 40 mM of UMP, 170 mM of KH2P04, 12 mM of MgSO4 and 200 mM of maltose to produce
UDP-GIcNAc of sugar nucleotides and UDP-Glc At the same time in 30 mM lactose cells from non-purified E coli, homogenates expressing GIcNAc transferases (1900 U / L reaction) and homozyzed non-purified coli E cells expressing Gal transferases (1000-2000 U / L reaction) were added to the reaction vessel. The reaction mixture was cooled or incubated at room temperature and allowed to proceed until the levels of the material containing LNnT reach at least 15 mM. contains LNnT then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decantation ion exchange chromatography tangential flow filtration reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 5 Production of LNnT of Solo Reaction vessel
Using a Single Yeast Production System All the reagents needed for the synthesis of LNnT are added simultaneously to a reaction vessel. More specifically, dry Candida famata were fed with 30 mM of glucosamine, 40 mM of UMP, 170 mM of KH2P04 , 12 mM MgSO4 and 200 mM maltose to produce UDP-GIcNAc from sugar nucleotides and UDP-Glc At the same time 30 mM lactose, intact E coli cells expressing GIcNAc transferases (1900 U / L mixture) were also added of reaction) and intact E co cells expressing Gal transferases (1000-2000 U / L of reaction mixture) are also added to the reaction vessel. The complete reaction mixture is incubated at room temperature and allowed to proceed until the LNnT-containing material reaches at least 18 mM The material containing LNnT is then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or hofilization to obtain pure LNnT Example 6 Production of LNnT in a Single Reaction Vessel Using a Single Yeast and Orotate Production System All necessary reagents for the synthesis of LNnT were added simultaneously to a reaction vessel More specifically, dry Candida famata was fed with 30 mM of 40 mM glucosamine of UMP, 170 mM of KH: P04, 12 mM of MgSO4 and 200 mM of maltose to produce UDP-GIcNAc of sugar nucleotides and UDP-Glc At the same time 30 mM lactose were added to the uncoated E coit cells expressing GIcNAc transferase (1900 U / L of reaction mixture) and non-purified E coli cells expressing Gal transferases (1000-2000 U / L reaction mixture) were added to the reaction vessel. All the reaction mixture was cooled or incubated at room temperature and allowed to proceed until the levels of the material containing LNnT reached at least 13 mM. The material containing LNnT is then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decanting, ion exchange chromatography, tangential flow filtration reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 7 Production of LNnT in a Single Reaction Canister Using a Single Yeast Production System and LNT-2 A method that is can be used to synthesize LNnT using a single yeast production system and LNT-2 will now be described. All the reagents needed for the synthesis of LNnT can be added simultaneously to a reaction vessel. More specifically, dry Candida famata is fed with 30 mM of glucosamm 40 mM of UMP, 170 mM of KH_P04, 12 mM of MgSO4 and 200 mM of maltose to produce sugar nucleotides UDP-GIcNAc and UDP-Glc At the same time 30 mM lactose 20 mM of LNT-2 cells of E coli not purified GIcNAc transferases (1900 UL reaction mixture) and non-purified E coli cells expressing Gal transferases (2000 U / L reaction mixture) were added to the recipient Reaction time The entire reaction mixture is incubated at room temperature and allowed to proceed until the levels of the material containing LNnT reaches at least 18 mM. The material containing LNnT is then removed from the reaction vessel and moved downstream. for additional process such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 8 Production of LNnT Ogosycapd in Two Reaction Vessels Using a Two Yeast System UDP-GIcNAc Production System Dry Baker's yeast is fed with 20 mM of 170 mM glucosamine of KH2P04, 5 mM MgSO and 70 mM of fructose. 20 mM of UMP was added to the yeast. The yeast was incubated at room temperature and monitoring for the formation of the sugar nucleotide UDP-GIcNAc The yeast is incubated at room temperature until the amount of UDP-GIcNAc is approximately 6.0 mM The yeast production system of sugar nucleotides was added to a first reaction vessel Synthesis of Tr? Saca? Do-LNT-2 (GIcNAc 1-3Gal 1-4Glc) The cells of E coli expressing GIcNAc transferase (350 units / Liter of Reaction Mixture) are homogenized and without any further purification they are added to the reaction vessel containing the yeast production system of UDP-GIcNAc * *. *
32 The production system of GIcNAc transferase and UDP-GIcNAc were incubated at room temperature until the production of residual UDP-GIcNAc is less than 1.0 mM. Lactose was also added to the reaction vessel as a substrate to produce the material containing the trisaccharide intermediate LNT-2 (GlcNAc-3Gal 1- 4Glc). The amount of material containing LNT-2 produced must be greater than 6 mM. The material containing LNT-2 is removed from the first reaction vessel and transported to a second reaction vessel. 10 Synthesis of LNnT (Gal 1-4 GIcNAc 1-3Gal 1-4Gic) Additional homogenized and non-purified E. coli cells expressing Gal transferase (350 units / Liter of Reaction) are added to the second reaction vessel containing the material containing LNT-2 and a source of UDP-15 Glucose This material was incubated at room temperature until the residual production of GIcNAc 1-3Gal 1-4Glc is about 0.1 mM. The source of UDP-Gic was supplied by the incubation of Candida famata, 200-400 mM glucose, 180 mM KP04. 12 mM of MgSO and 20-100 mM of UMP. The microorganisms were incubated
Room temperature until the amount of UDP-Glc produced is greater than 6 mM. The sugar nucleotide yeast production system is then added to the second reaction vessel containing the gIcNAc 1-3Gal 1-4Glc and the Gal transferase to produce LNnT. The mater that contains LNnT
then removed from the reaction vessel > cn and moves downstream for further processing such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 9 Production of Oligosacapdo LNnT in a single Reaction Vessel Using a Single Synthesis Yeast System of Tpsacapdo (LNT-2 GIcNAc 1-3Gal 1-4Glc) in the absence of added nucleotide. To dry Candida famata cells were added 50 mM of 200 mM glucosamine of 12 mM KH2P04 of 200 mM MgSO4. maltose, 100 mM lactose and homogenized non-purified E coli cells expressing GIcNAc transferase (1200 Units / Liter reaction mixture) The whole mixture is incubated at room temperature with aeration After 24 hours of incubation the reaction is supplemented with 40 mM of glucosamine and 200 mM maltose The mixture was further incubated until more than 55 mM of LNT-2 was generated. Synthesis of LNnT (Gal 1-4GlcNAc 1-3 Ga 1-4Glc) To the yeast / LNT-2 mixture was added Candida famata dried with air, 400 mM glucose (or galactose) 180 mM
12 mM MgS0 KH2P0 and homogenized non-purified coli E cells expressing Gal transferase (1000 Units / Liter reaction mixture) The reaction was incubated at room temperature with aeration until at least 50 mM LNnT was produced The material containing LNnT is then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decantation ion exchange chromatography, tangential flow filtration, reverse osmosis spray drying or lyophilization to obtain pure LNnT Example 10 Production of Oligosacapdo LNnT in Two Reaction Vessels Using a Single Yeast System Synthesis of Tpsacapdo (LNT-2, GIcNAc 1-3Gal 1-4Glc) in
Absence of added nucleotide To the dried Candida famata cells were added 50 mM of 200 mM glucosamine of 12 mM KH2P04 of 200 mM MgSO4 of maltose, 100 mM of lactose and homogenized non-purified E coli cells expressing GIcNAc transferase (1200
Units / Liter reaction mixture) All the mixture is incubated at room temperature with aeration After 24 hours of incubation, the reaction is supplemented with 40 mM of glucosamine and
200 mM of maltose The mixture was further incubated until more than 55 mM of LNT-2 was generated. The LNT-2 was semi-puppied by a combination of methods including centrifugation ion exchange chromatography, tangential flow filtration and reverse osmosis. of LNT-2 was transferred to a second reactor. Synthesis of LNnT (Gal 1-4GlcNAc 1-3 Gal 1-4Glc) In the second reactor 400 mM Candida famata of glucose (or galactose) 180 mM KH2PO was added. 12 mM of 100 mM MgSO4 of UMP the material of LNT 2 and non-purified E coli cells *?
homogenates expressing Gal transferase (1000 Units / Liter reaction mixture) The reaction was incubated at room temperature with aeration until at least 50 mM LNnT was produced. The LNnT-containing material was then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 12 Production of Oligosacapdo LNnT in a single
Reaction Vessel Using a Single Yeast Production System in the Absence of Aggregate Nucleotide All reagents necessary for the synthesis of LNnT were simultaneously added to a reaction vessel More
Specifically, dry Candida famata was fed with 50 mM glucosamine, 200 mM KH: P04, 12 mM MgSO4 and 100 mM maltose to produce the sugar nucleotides UDP-GlcNAc and UDP-Glc. At the same time 100 mM were added. lactose and homogenized non-purified E coli cells expressing GIcNAc
transferase (1500 U / L reaction mixture) were added to the reaction vessel. All the reaction mixture was incubated at room temperature with aeration. After 18 hours, the reaction was supplemented with 50 mM of additional glucosamine and 100 mM of maltose. After 23 hours, 200 mM of
galactose and homogenized non-purified coli E cells expressing Gal transferase (1000 Units / Liter reaction mixture) to the reaction vessel The mixture was allowed to proceed until the LNnT levels reached at least 40 mM (approximately 46 hours). LNnT containing material is then removed from the reaction vessel and moved downstream for further processing such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT Example 12 Production of Lacto-N-fucopentase Oligosacapdo III (LNF-III) in a Single Reaction Canister by the Addition of LNnT A method that can be used for synthesis of lacto-N-fucopentaose ohgosacapdo (LNF-III) will now be described More specifically , Dry Torulopsis candida is fed with 30 mM of 40 mM fucose of GMP and 30 mM of fructose to produce the sugar nucleotides of GMP-fucose At the same time, 20 mM of LNnT-2 and non-purified homogenized E coli cells expressing 1,3 fucosyltransferase (1900 Units / Liter of Reaction Mixture) were homogenized and added without any purification to the reaction vessel. The reaction mixture was incubated at room temperature and allowed to proceed until the levels of the material containing LNF-III reached at least 5 M. The material containing LNnT was then removed from the reaction vessel and moved downstream to the process. additional such as by centrifugation or decantation, ion exchange chromatography, tangential flow filtration, reverse osmosis, spray drying or lyophilization to obtain pure LNnT.