MXPA00007079A - Cosmetic or dermatological use of 7-hydroxylated steroids alone and/or in combination with elastin derived peptides - Google Patents

Abstract

The present invention is directed to a composition for improving tissue texture, wherein the composition includes an elastin peptide which is synthesized by selectively cleaving elastin using thermolysin. The composition further provides the incorporation of a 7-hydroxylated steroid. The composition is capable of modifying the physical characteristics of mammalian tissue to which it is applied, thus increasing elasticity.

Description

PROCESSING PROCESSING OF SUCROSE IN GLUCOSE BACKGROUND OF THE INVENTION FIELD OF THE INVENTION: The present invention relates to a process for preparing commercial quantities of glucose from sucrose, and a reactor for the practice thereof. In particular, the present invention relates to a process for preparing glucose from sucrose by contacting sucrose with a B-2, 1-fructosyltransferase, followed by glucose isolation and a branched fructan, whereby efficiencies are improved of production.

DESCRIPTION OF THE BACKGROUND: Glucose is a saccharide found in nature, either as a monosaccharide or incorporated in polysaccharides. Glucose is used clinically as a fluid and replacement nutrient, as a source of carbon in the culture of microorganisms, and is widely used as a food additive. Glucose has been prepared commercially from starch (Dean, Gottfried, Advan, Carbohyd, Chem 5, 127 (1950) and by acid hydrolysis of sucrose, despite the availability of starting materials to prepare glucose the cost of this material remains high, relative to the cost of starting materials, therefore, commercial glucose syntheses can be improved, fructans are found in nature everywhere.

(Science and Technology of Fructans, 1933 ed. M. Suzuki and N.J. Chatterton, CRC Press, Inc.). In the plants, there are four fructans described: 1) Inulin, a fructan linked in 2, 1- found mainly in dicotyledons such as Jerusalem artichokes and chicory roots; 2) raise a fructan linked in 2,6- found in some monocots such as Phleum pratense; 3) A branched fructan in 2.1- and 2.6- found in monocots such as barley, blue agave and wheat; and 4) A fructan of neoserie, a fructose linked in 2,1- and 2,6- in glucose. Glucose is internal to these molecules, instead of terminal. The fructose residues are then bound in 2.1- and 2.6- to both terminal fructose, creating a complex structure (asparagus). Many plants produce more than one of these fructans. In (Yeast and fungi have been reported fructans linked in 2.1- In bacteria, two fructans have been described: 1) An inulin of 2,1-fructan streptococcus mutans; and 2) a levan of fructan linked in 2, -6 has been described of Bacillus subtilis, Zymomonas mobilis and many others. The inulins are made up of fructose chains linked in β-2,1- to a D-glucoside; they have a linear structure and typically comprise many units of β-O-fructofuranose. The length of the average chain and molecular weight distribution will depend on both the species of the plant, the growth phase, and the method of preparation. Average chain lengths of 10 to 25 are common, in which case the individual units have about 9 to 24 units of fructose. Branched inulins have been reported, which comprise a linear chain of fructose chains linked in β-2,1- linked to a D-glucoside, which has fructose units in β-2,6 branched thereon. It has been reported that such branched inulin material has been isolated from the sap of the blue agave plant (GO Aspinall and PC Das aupta, Proceeding of the Chermical Society 1959 718-722 and MN Satvanaravana, Indian J. of Biochem and Biophys. (1976) 13: 408-412) and barley leaves (Simmen et al. Plant Physiol. (1933) 101: 459-468). The properties of an inulin can vary depending on the length of the chain and the degree of branching. Compositions comprising short straight chain inulins having a degree of polymerization of about 3 to 7 units of fructose have been used as low calorie sugar substitutes (DE 4,003,140). Longer chain inulins have been used as fat mimics and branched fructones can be used as both. Coussement et al. E.U. 5, 659.028 discloses branched fructo-oligosaccharides consisting of a chain which mainly comprises fructose units and have a preferred chain length of 2 to 15 units, in which one or more side chains mainly composed of fructose units are fixed. In the area of glucose production, Naqle et al. E.U. 4,637,835 reports the preparation of glucose and other saccharides from an α-cellulose using a calcium chloride catalyst and hydrogen ions.

Miyawaki et al. E.U. 5,524,075 reports the production of high purity glucose by saccharifying liquefied starch with an enzyme. Venkatasubramanian et al. E.U. 4,299,677 reports the direct separation of fructose and glucose from a mixture of glucose and fructose by ion exchange membranes. Harada et al. E.U. 5,169,679 report the use of fructans composed mainly of ligatures in β-2,1 that have a molecular weight from 2,000 to 20,000,000 as food additives such as, for example, bulking agents or fat substitutes, to produce low calorie foods. Kurtz et al. E.U. 5,478,732 reports a method for obtaining intermediate chain inulins (v. G., A degree of polymerization of 10-12), by treatment of crude inulin suspensions with a hydroiase enzyme. During the enzymatic treatment, the short chain components are degraded to mono- and disaccharides while long chain inulins are separated, then converted to a dry form. Adachi et al., Reports in US 4,681, 771 that when sucrose (GF) is contacted with an enzyme having activity to transfer fructose (hereinafter referred to as a fructosyltransferase), a low calorie sweetening composition is obtained, low in cariogenic which includes glucose, sucrose, trisaccharide (GF2), tetrasaccharide (GF3) as well as minor amounts of fructose; penta saccharide (GF4) and hexasaccharide (GF6). The amount of higher linear inulins falls dramatically, the majority fraction being inulin GF2.3.

Kono et al. U.S. 5,314,810 reports that the half-life of an immobilized fructosyltransferase used in the reaction with sucrose can be improved by supporting it in a granular carrier such as derived from a chitosan or anion exchange resin. It is reported that such supported above allows the industrial production of a low cariogenic sweetening composition. Heady E.U. No. 4,317,880 reports the production of novel fructose polymers and high fructose syrups from sucrose by the combined action of a fructosyltransferase preparation and an isomerase glucose enzyme. A method to produce glucose and / or fructose from sucrose is reported by Catani et al. In the application of E.U. co pending E.U. Serial No. 09 / 019,709 filed on February 6, 1998. Current methods for glucose preparation from sucrose, however, suffer from poor efficiency, so that the production of commercial quantities of glucose can be improved. In addition there remains a need for processes to prepare commercial quantities of polysaccharides such as linear and branched inulins.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the present invention to provide a process for preparing commercial quantities of glucose from sucrose. It is another object to provide a process for preparing commercial quantities of glucose and a branched fructan from sucrose.

The above objectives can also be carried out with a process for preparing glucose by contacting sucrose with a β-2,1-fructosyltransferase and a β-2,6-fructosyltransferase in a reactor to produce reaction products comprising glucose and a fructan. branched, followed by glucose isolation and a branched fructan.

In another embodiment, a process for preparing glucose can be accomplished by sequentially contacting sucrose first with a chain-extending β-2-1-fructosyltransferase and second with a branching β-2,6-fructosyltransferase in a reactor to produce reaction products comprising glucose and a branched fructan, which are almost depleted of sucrose. The present invention is based, in part, on the discovery that a combination of fructosyltransferases can be used to prepare glucose from sucrose (GF) with greater efficiency. In addition branched fructans produced during the formation of glucose by the reaction of sucrose and two fructosyltransferases can be isolated in commercial quantities to further increase the economic value of the present process.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the advantages thereof will be readily obtained as it is better understood by reference to the following detailed description when considered in connection with the accompanying drawings in which: Figure 1 depicts a flowchart in which sucrose is converted to glucose and a branched fructan; Figure 1 a depicts a flow chart in which sucrose is converted to glucose and a branched fructan in a reactor vessel equipped with an external separator for glucose; Figure 2 depicts a flow chart in which sucrose is converted into glucose and branched fructan into two reaction vessels; Figure 2a depicts a flowchart in which sucrose is converted to glucose and branched fructan in two reaction vessels, each equipped with external separators for glucose.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Glucose is a basic article of commerce and is sold for pharmaceutical and food uses. A branched inulin fructan has good performance in organoleptic tests and has similar bulk properties to sucrose for food use, such as unbranched inulins, but without inducing excessive gas to be consumed. A fructan de branched levan also has good bulking properties for food use. As used herein, the term "β-211-fructosyltransferase" refers to any enzyme or enzymes capable of transferring portions of fructose from sucrose as a donor, to sucrose or other saccharide (v, g., A fructan ) as acceptors and forms bonds in β-2, 1. The fructose unit is preferably transferred in the form of furanose. In addition, it is preferable that β-2, 1-fructyltransferase be selective for fructose in the form of furanose as an acceptor. In plants, these enzymatic activities are specifically provided by a sucrose: sucrose 1-fructosyltransferase (1-SST) and fructan: fructan 1-fructosyltransferase (1-FFT) (see Pollock et al., Annu Rev. Plant Physiol. Mol. Bio 42, 77-101 (1991) An isolated B-2,1-fructosyltransferase of S. mutans exhibits activity to transfer fructose to both sucrose and a fructan The result of transferring the fructose portion of sucrose is the production of a glucose unit.The β-2,1-fructosyltransferase can transfer fructose to the Ci position of a terminal fructose (eg, forming straight chain extensions) or to the Ci position of a linear fructan (eg, a bound in ß-2,6-) forming branching points Some fructosyltransferases will have both activities (v, g., chain extension and branched), however, some fructosyltransferases will only have one activity The selection of chain-extending fructosyltransferases and / or provide suitable branching is within the skill level of those of ordinary skill in the art. Non-limiting examples of suitable β-2,1-fructosyltransferases can be obtained from microorganisms of the genus Aspergilus such as A. oryzae ATCC20498; A. sp. ATCC 20524; A. awamorí, A. Sydowi and A. Niger ATCC 20611; of the genus Penicillium, such as P. Jancezewskii ATCC10115 and 26546; P. nigricans, of the genus Fusarium, such as F. Lini IAM 5011; and of the genus Aureobasidium, such as A. Pullulans ACTT 9348; Streptococcus mutans ATCC 25175; and A. Pullulans var. melanigenum A-8 ATCC 20612. Suitable enzymes can also be obtained from yeasts and other microorganisms such as the genus Saccharomyces, such as S. cerevisiae; the genus Rhodotorula, such as R. lutinis; the genus Pichia, such as P. miso; the genus Hansenula, such as H. miso; the genus Candida, such as C. tropicali; and of higher plants, such as asparagus, dahlia tubers, chicory roots, and Jerusalem artichoke; as described in JP-A-56-154967 and JP-B-59-53834. A particularly preferred enzyme is a β-2, Bacterial 1-fructosyltransferase which can be obtained from a gene isolated from Streptococcus mutans. In particular S. mutans ATCC 25175 can be a source of a fructosyltransferase gene. The fructosyltransferase can be obtained as a fusion construct with a heterologous protein sequence. An available fusion protein is, for example, the fructosyltransferase isolated from Streptococcus mutans fused to the C-terminus of glutathione-S-transferase. The coding sequence of the fructosyltransferase of Streptococcus mutans, which lacks the predicted signal sequence can be isolated from ATCC 25175 by PCR of the strain Streptococcus mutans which can be to form a transformant which expresses a fusion protein of fructosyltransferase . Another suitable fructosyltransferase gene sequence of the GS-5 strain of Streptococcus mutans is reported by Shiroza, T. and Kuramitsu, H.K. J. Bacteriol., 170, 810-816 (1988). As used herein, the term "β-2,6-fructosyltransferase" (also known as a levan synthetase) refers to any enzyme or enzymes capable of transferring portions of sucrose fructose as a donor, to sucrose or other saccharide (v, g., a fructan) as an acceptor forming ligatures in β-2,6. The fructose unit is preferably transferred in the form of furanose. Furthermore, it is preferable that ß-2,1-fructosyltransferase be selective for fructose in the form of furanose as an acceptor. This specifically describes a sucrose: fructan 6-fructosyltransferase (6-SFT) (See Sprenger et al., Proc. Nati, Acad. Sci. USA, 92, 11652-11656 (1995)) and a fructan: fructan 6G-fructosyltransferase ( 6G-FFT) (See Vijn et al., The Plant Journal (1997) 11 (3) 387-398). As a result of transferring the fructose portion of sucrose under the action of a β-2,6 fructosyltransferase is the production of a glucose unit. The β-2,6-fructosyltransferase can transfer fructose to the Ce position of a terminal fructose (v, g., Forming straight chain extensions) or to the Ce position of a linear fructan (v, g., An inulin bonded to B). 2, 1-) forming branch points. Some fructosyltransferases will have both activities (v, g., Chain extension and branching), however, some fructosyltransferases will only have one activity. The selection of suitable fructosyltransferases of chain extension and or branching is within skill level of that ordinary skill in the art. A suitable β-2,6-fructositransferase can be obtained from plant sources such as grasses and barley leaves. Such β-2,6-fructosyltransferase is described from barley leaves by Simmen et. al .. Plant Physiol. (193) 101: 459-468; Duchateau et.al .. Plant Physiol. (1995) 107: 1249 ^ -1255. Leosucrose having a β-2,6-fructosyltransferase activity is also available from Bacillus subtils (ATCC 6.051) and Zymomonas mobilis. The purification, cloning and expression of barley sucrose: frutan 6-fructosyltransferase is described by Sprenger et al. Proc. Nati Acad. Sel., USA vol. 92, pp. 1 1, 652-1 1 .656 (1995) and FEBS Lett January 1997 6: 400 (3): 355-8. The present process provides the sucrose reaction with both a β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase, however, it is within the scope of the present invention to use additional glycosyltransferases, including other fructosyltranferases which do not interfere with the reaction of either a β-2, 1 'fructosyltransferase or a β-2,6-fructosyltransferase. The following descriptions of fructosyltransferases are applied independently to both, β-2, 1-fructosyltransferase and β-2,6-fructosyltransferase. The fructolsyltranferases can be immobilized in a carrier having a primary to quaternary amine as described in US Pat. No. 5,314,810. In a preferred embodiment, the fructosyltransferases are at least partially purified. As used herein, the term "purified" means that the enzyme has been purified, at least partially from the host organism from which it was naturally produced Purification is preferably at least the partial removal of degrading enzymes. such as inulinases which would degrade fructan, and proteases which can degrade the fructosyltransferase enzyme.Preferably the enzyme is purified to the degree that there is no degradation enzymes.When the source of the enzyme is a transfected E. coli microorganism, a crude cell lysate can be used when the transfected E. coli does not have native degrading enzymes In a preferred embodiment, each of the purified fructosyltranferases have a synthesis to degradation activity ratio of = 1,000 to 1, preferably = 1500 to 1, and even more preferably = 2,000 to 1 (eg, for each adhesion of a fructan ligature, there is a pref at least 1,000 fructose ligatures formed). When a unit equals one μmol of monosaccharide transferred to an acceptor per minute, a growth of A. niger crude supernatant contains -90 units / mg. of protein, and a preparation of A. niger purified with DEAE has ~ 2,000 units / mg of protein. 10 μg of purified preparation with DEAE has enough activity to completely convert one liter of 50% sucrose to glucose and a linear fructan in about one day at 50 ° C. Alternatively, the same enzyme preparation can operate continuously, and without falling in efficiency, for at least two weeks at 50 ° C, while continuously adding sucrose. Each of the fructosyltransferases can be purified to an activity of from 90 to 3,000 U / mg, preferably from 100 to 2,000 U / mg. In a preferred embodiment, each of the fructosyltransferases will have an activity of > 100 U / mg, preferably > 150 U / mg, even more preferably > 200 U / mg. A third fructosyltransferase is a 2,6-G-fructosyltransferase which transfers fructose to the C6 hydroxyl group of glucose. Such 2,6-G-fructosyltransferase preferably uses sucrose as a fructose donor. A suitable 2,6-G-fructosyltransferase can be isolated from natural sources by conventional methods known to those of ordinary skill in the art. Non-limiting examples of suitable sources include onions, asparagus and all lily plants. When used in conjunction with a fructosyltransferase extending in a linear chain in β-2,1 and a fructosyltransferase extending in a chain in β-2,6, a fructan star can be formed which comprises linear chains in β-2 , 1 and ß-2.6. Such a compound can be useful as a polyvalent support, as an agent to increase volume for food and as an entanglement agent or nucleus for polymers. The starting material for the present process will be sucrose or a composition containing sucrose. Sucrose refers to the disaccharide in refined or crude form, as a solution or dry, from any source of sucrose raw material, e.g. , sugar cane, or sugar beet. Preferably the amount of sucrose contained in the sucrose raw material is >; 10% by weight, more preferably = 20% by weight, even more preferable > 50% by weight, most preferable > 70% by weight. The existence of food may contain other materials as long as they do not interfere significantly with the conversion of sucrose to glucose (v-9-. 1-cestose (GI-2FI-2F). Sucrose can be introduced in any of the ways described above. In order to maintain the overall ionic strength and concentration of the reaction medium, however, sucrose is introduced continuously or intermittently in dry or solution form.The rate and frequency of addition of sucrose to the reaction mixture will be such to maintain a high rate of oligosaccharide production and will depend in part on the nature and specific activity of the fructosyltransferase, the reaction temperature and the glucose and fructan removal regimes.The determination of the optimum rate and frequency of sucrose addition can be achieved by routine experimentation and is within the skill level of those of ordinary skill in the art. that of the present invention is preferably conducted in aqueous solution. The concentration of sucrose in the reaction medium is not particularly limited and can be from 50 mM to saturation. In terms of percent by weight, the amount of sucrose in the reaction solution can be from 1 to 80% by weight, based on the total weight of the reaction mixture, typically from 40 to 80% w / w, of preference of 50 to 70% w / w and more preferably about 60% w / w. The structure of branched fructan can vary depending on the selected reaction conditions. Within the context of the present invention, branched fructans comprising 2 basic ligatures can be formed: 1) fructose: fructose bound in β-2, 1 -; and 2) fructose: fructose bound in β-2,6. The specific branched fructan structures provided include 1) a linear inulin -2.1 - with branches in β-2,6-; 2) a levan in ß-2,6-linear with branches in ß-2, 1-; 3) a linear inulin in β-2, 1- with branches in β-2,6- which contain branches in β-2,1-; and 4) a linear levan in ß-2,6- with branches ß-2,1- which contain branches in ß-2,6-. The addition of branch points to any string line) which by itself is a branch point that can be continued. It will be appreciated by those of ordinary skill in the art that fructan may comprise a glucose unit, as a result of the initial transfer of fructose to sucrose. In one embodiment, a linear inulin fructan is formed comprising ligations in β-2, 1- under the action of a chain-extending β-2, 1-fructosyltransferase, followed by branching with fructose units under the action of a β- Branching 2,6-fructosyltransferase. Sucrose would be the fructose donor for both fructosyltransferases. The sucrose and fructose terminal of a fructan would be acceptors for the β-2, 1-fructosyltransferase, while a linear chain of fructan would be an acceptor for a β-2,6-fructositransferase. The linear inulin fructan comprising ligations in β-2,1 - can be formed in one or more stages, the use of a one-step process that favors the formation of a larger number of low DP inulin chains, the use of a process comprising more than one stage favoring the production of few, but longer chains of inulin (eg, larger DPs). In order to form longer inulin chains, the formation of fructan is induced with a portion of the sucrose to be converted, followed by the addition of the remaining sucrose. Such a procedure for forming extended linear fructans (e.g., major DPs) described by Catani et al. in the copending from U. Serial No. 09 / 019,709, filed February 6, 1998, the entire contents of which are incorporated herein by reference. In another embodiment, a linear levan fructan is formed which comprises ß-2,6- ligations under the action of a β-2, 6-fructosyltransferase, followed by branching with fructose units under the action of a β-2, 1-fructosyltransferase. Sucrose would be the fructose donor for both fructosyltransferases. The sucrose and fructose terminal of a fructan would be an acceptor for the β-2,6-fructosyltransferase while the linear fructal chain would be an acceptor for a β-2,1-fructosyltransferase. The fructan of linear levan comprising ligatures in β-2,6- can be formed in one or more stages, the use of a one-step process that favors the formation of a greater number of chains of low-DP levan, the use of a process that comprises more than one stage favoring the production of few chains, but longer ones (eg, larger DPs). In order to form longer levan chains, the formation of fructan is induced with a portion of the sucrose to be converted, followed by the addition of the remaining sucrose. Such a process for forming extended linear levans fructans is as described for the preparation of extended linear inulin fructans. A linear levan fructan comprising ß-2,6 ligatures containing branching fructose units linked in β-2,1-can be used as a bulking agent for food and food sweeteners. Such fructan will typically have a linear levan fructan structure comprising from 2 to 15 units of fructose, preferably from 3 to 10, even more preferably from 4 to 7, which are linked in β-2.6 to a glucose. Attached to it will be one or more units of fructose joined in ß-2,1-, forming branching points. The branching point can occur randomly in the structure of linear levan fructan. The molecular weight of tai fructan can range from about 700 to 3600 gms / mol. Each branch point can itself be an extended chain. In another embodiment, the simultaneous action of both a β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase in sucrose can give rise to a fructan comprising ß-2, 1 and β-2,6- ligations , both branched and linear. Each fructosyltransferase uses sucrose as the fructose donor and may use sucrose and / or a fructan as an acceptor. In addition, each fructosyltransferase can form linear or branching ligatures. In another embodiment, a linear inulin or linear fructan levan is branched under the action of both, a β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase. Each fructosyltransferase uses sucrose as the fructose donor and a fructan as an acceptor. In addition, each fructosiltranferose can form ligatures either linear or branching. Each of the fructans described above can be further elaborated to comprise one or more glucose units by the action of a glucosyltransferase which uses a fructan as an acceptor and preferably uses sucrose as a glucose donor. An advantage of forming branched fructans is that the efficiency of glucose formation is increased in relation to the formation of only linear fructans since each branch group formed from sucrose will produce one unit of glucose. When a linear fructan is formed of sucrose, the first coupling of fructose to sucrose, a reaction that consumes two units of sucrose, will produce only one unit of glucose. As such, the synthesis of branched fructans from sucrose offers a highly efficient method for synthesizing glucose. The reaction of sucrose with fructosyltransferases can be conducted over a wide temperature range. The reaction temperature can be room temperature, that is, 18-25 ° C, to temperatures just below the temperature where rapid inactivation of the fructosyltransferases occurs. A preferred temperature range is 25 to 60 ° C. More preferably, the reaction is conducted at a temperature of 35 to 55 ° C. Most preferable, the temperature is 30 to 50 ° C. Aqueous reaction solutions can be un-buffered or buffered at the appropriate pH using well-known buffer components, such as citrate, phosphate, and TRIS buffers. The use of a buffer is preferred when the reaction is conducted for an extended period of time, such as 2 weeks. The reaction of sucrose with fructosyltransferases is conducted for a sufficient time to produce commercial quantities of glucose. The reaction time can be from 2 to 48 days, depending on the size of the batch. When conducted in a continuous manner, a volume of 10mL can react at a rate of 2.5 g / hr, without a significant loss of activity, for a period of 2 to 4 weeks. The pH of the sucrose reaction with fructosyltransferases is not particularly limited and the optimum pH of the reaction may vary depending on the specific enzyme used. Typically, the pH will be from 4.0 to 8.0, preferably from 5.0 to 7.5, more preferably around 6.0. The present process can be conducted in any mode, batch or continuous. The continuous reaction can be conducted by circulating a reaction mixture through an ultrafiltration apparatus whereby the product (s) are continuously removed as permeated from the ultrafilters, a transferase enzyme that is retained in the retentate of ultrafilters. Fresh substrate and fresh enzyme can be added, as needed, to replenish those that have become inactive, with the addition to the reaction mixture at the same rate at which the permeate from the ultrafilters is removed. The reaction of sucrose with a β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase can be conducted in a tubular reactor. The tubular reactor will typically comprise a pipe length, a pump for moving the reaction stream through the pipe, an inlet for reagents - and an outlet for reaction products. The tubular reactor can be made of conventional reactor materials known to those skilled in the art. For example, a tubular reactor may be made of stainless steel, glazed stainless steel, or a polymer tai such as high density polyethylene, polypropylene, potyvinyl chloride or a polyester. In a preferred embodiment, the tubular reactor is made of polyvinyl chloride. The tubular reactor will typically have the shape of a tube, the length and diameter of which may vary depending on the specific reaction being conducted. Generally a tube will have an inside diameter from 2.54 to 61.0 c, preferably from 10.16 to 50.8 cm, more preferably from 15.24 to 25.4 cm. A pump is provided to move the contents of the tubular reactor along the length of the reactor. Conventional pumps known to those of ordinary skill in the art can be used. Non-limiting examples of suitable pumps are: Typically, the pump will be provided with sufficient force to provide a flow rate of from 3.05 to 61 cm / sec, preferably from 6.1 to 30.5 cm / sec, more preferably from 9.15 to 21.35 cm / sec. Preferably, the pump will produce a flow which behaves like a solid plug. The inlet for the tubular reactor is not particularly limited and may simply comprise an opening which will allow the introduction of reagents, either as an initial charge or continuously in the course of operation of the reactor. The reagents can be measured to the reactor, either gravimetrically or by a pump. The reagents can be introduced in solid form tat as a powder, or as a solution in a suitable solvent. The reactor may also be equipped with additional inputs, as described above, located along the length of the reactor, downstream of the initial input. These inputs can be used to add additional reagents at various points along the reaction stream. The outlet for the product is not particularly limited and may fear the form of a direct intake of the entire contents of the reactor from the reactor stream., or provide selective removal of a reaction product. The selective removal of product can be by a size exclusion filter. The length of the reactor will vary depending on the specific reaction and the reaction conditions such as the rate of flow through the reactor and the temperature. The reactor may also be equipped with a temperature control system such as a heater or cooler. The ability to adjust the temperature will preferably vary through the length of the reactor, allowing different temperature zones over the length of the reactor. In one embodiment, sucrose is reacted with a β-2,1-fructosyltransferase in a first portion of a tubular reactor, followed by reaction with a β-2,6-fructosyltransferase in a downstream portion. Before the reaction with the β-2,6-fructosyltransferase, the β-2,1-fructosyltransferase can be deactivated by catellation for a sufficient time and temperature, typically around 65 to 95 ° C, preferably around 75 to 90 ° C, to one more preferably around 85 ° C, for about 1 minute. Alternatively, a deactivation zone may comprise the removal of the biocatalyst from the flow of the tube reactor, such as via a size exclusion membrane or the like. The deactivation can also be achieved by the introduction of a suicide substrate for the biocatalyst which deactivates the biocatalytic activity. Preferably, the fructosyltransferase is deactivated by thermal deactivation. Leosucrose having a β-2,6-fructosyltransferase activity using a fructan as an acceptor, which is available by expression of a Bacillus subtilis (ATCC6051), can be especially used, which provides efficient use of sucrose in the formation of ligatures in β-2,6- of fructose to the inulin chain. Such fructosyltransferase can be obtained by conventional means known to those of ordinary skill in the art. When sucrose reacts efficiently, a problem of glucose separation from residual sucrose is simplified. In a preferred embodiment, the concentration of sucrose after the action of a β-2,6-fructosyltransferase, if any, is <; 20% by weight, more preferably < 10% by weight, even more preferably < 5% by weight, even more preferably = 1% by weight. The reaction can also be conducted in a reactor or series of reactors, which can be equipped with reagent inlets and outlets for suitable products. The outputs can be selective for the renewal of a specific product. The selectivity can be obtained by providing suitable separators that allow the removal of product and return of other materials to the reactor. A separator can be in the form of a membrane or a chromatographic column. In some cases, a separator may comprise a plurality of membranes and / or chromatographic columns that provide selective removal of the desired product. After the reaction to produce glucose, the fructosyltransferases can be inactivated by heating a reaction mixture at about 100 ° C for 10 to 15 minutes. If desired, the enzymes can be removed from the reaction mixture either before or after inactivation by heating by means of ultrafiltration through a filter of suitable size. Typically, fructan will have a linear structure of fructose units in the β-O-fructofuranose, a form linked in β-2,1-. The number of units of β-O-fructofuranosa will typically be from 4 to 20, preferably from 4 to 15, more preferably from 4 to 8. Implanted in the linear fructan structure will be one or more units of β-O-fructofuranosa linked in ß-2,6- to the structure. The number and density of fructose units grafted in the structure may vary, however typically, on average there will be at least one grafting fructose for every 5 units of fructose bonded in β-2, 1-linearly, preferably at least 1 for every 4, more preferably 1 for every 3, even more preferably at least 1 for every 2. These relations refer only to the number of branch points found in the linear structure. In another embodiment, the branched fructan may be chain extended fructose branching units of ß-O-fructofuranose linked in beta-2, 1 - a unit fructofuranose branching, providing a structure of branched fructan having a structure like a comb. The number of ß-O-fructofuranose units bound in β-2, 1 - to the branching fructose may vary depending on the reaction conditions and will typically be from 2 to 20, more preferably from 2 to 10, even more preferably from 2 to 8 fructose units will be attached to the individual branching fructose units. It is not necessary for each unit of branching fructose to support the same number of chain extender fructose units. The term "comb polymer" is well known in the field of polymer chemistry, such that the branched fructan structure described herein is clear to those of ordinary skill in the art. Within the scope of the present invention is also to form branches and branches extended in chains in individual comb chains which themselves are grafted in a linear fructan joined in β-2.1-. The number and density of fructose units grafted on the individual comb chains may vary, however, typically, on average there will be at least one grafting fructose for every 5 units of fructose linearly linked in β-2.1-, preferably at least 1 for every 4, more preferably at least 1 for every 3, even more preferably for at least 1 for every 2. These relations refer only to the number of branch points found in the comb linear chain. The number of ß-O-fructofuranose units linked in β-2,1- to the branching fructose in the comb chain may vary depending on the reaction conditions and will typically be from 2 to 20, more preferably from 2 to 10. , even more preferably from 2 to 8 fructose units will be linked to the individual branching fructose units. It is not necessary for each unit of branching fructose to support the same number of chain extender fructose units. For the purposes of illustration, specific details are provided for the preparation of glucose from sucrose using at least one β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase. Sucrose and some β-2,1-fructosyltransferases are reacted in a reactor. The reactor may comprise an inlet for sucrose and an outlet for glucose. As the degree of polymerization increases, the glucose concentration will also increase such that it is possible that the rate of glucose-forming reaction will decrease. Accordingly, in a preferred embodiment, the glucose is removed from the reaction medium during the reaction. Glucose can be removed by conventional methods known to those of ordinary skill in the art such as by membrane filtration or chromatography. Within the context of the present invention, chromatography includes ion exchange and gel exclusion techniques known to those of ordinary skill in the art. A pump can be used to increase the pressure against the membrane or chromatographic column. In a preferred embodiment, the glucose outlet comprises a membrane, which allows the flow of glucose to the reaction medium, without allowing the sucrose, fructan or fructosyltransferase to pass therethrough. A β-2,6-fructosyltransferase is then added, which, by reaction with sucrose, provides branching of the straight chain. Glucose can be removed continuously, batchwise or as a semilote, however, in a preferred embodiment, the glucose is continuously removed from the reaction medium. The glucose can be isolated and purified by conventional methods known to those of ordinary skill in the art such as by filtration which can also be followed by crystallization. In a preferred embodiment, the fructan is also removed from the reaction mixture, more preferably, the fructan is continuously removed from the reaction mixture. A fructan can be removed by conventional methods known to those of ordinary skill in the art such as by membrane filtration or chromatography, such as ion exchange or gel exclusion. In a preferred embodiment, an outlet for fructan comprises a membrane which allows the flow of fructan from the reaction medium, without allowing the sucrose, glucose or fructosyltransferase to pass therethrough. Alternatively, fructan can be separated from the reaction mixture by returning sucrose and glucose to the reaction mixture. In a preferred embodiment the amount of fructan produced, based on the initial weight of sucrose is >; 10% by weight, preferably > 20% by weight, even more preferably - = 30% by weight, even more preferably = 40% by weight, and most preferably = 50% by weight. In a preferred embodiment, the glucose yield produced, based on the reacted weight of sucrose, is from 25 to 50% by weight, preferably > 25% by weight, preferably = 33% by weight, even more preferably > 37% by weight, even more preferably > 40% by weight, and most preferably around 50% by weight. Within the context of the present invention, commercial quantities are defined as a glucose production rate of 103 to 10d kg / day and preferably will be an amount of > 1000 kg / day, preferably > 2000 kg / day, even more preferably > 5000 kg / day. In addition, the rate of production of commercial quantities is relative to the amount of sucrose starting material. Therefore, the production regimes identified above are based on a unit that processes 6000 kg of sucrose. Consequently, the term "commercial quantities" does not refer to an absolute quantity, but rather refers to a commercially acceptable production regime. In order to increase the efficiency of glucose production, in a preferred embodiment for batch chain elongation, each batch will comprise from 20 to 25% by weight of the product of the previous reaction. Accordingly, after an initial batch of chain lengthening is completed, from 75 to 80% by weight of the reactants are removed, the remaining 20-25% by weight remain as a reagent for a second batch of chain lengthening. Therefore, 20-25% by weight of the first reaction product is charged to a reactor with sucrose and β-2,1-tructosyltransferase. Thus, the transfer of fructose from sucrose with the β-2,1-fructosyltransferase will be in the presence of a higher concentration of higher fructans, thus favoring the formation of additional higher fructans, rather than trisaccharides (FFG). Since the production of higher fructans produces more efficient glucose, this provides an even more efficient method for forming glucose. In a further preferred embodiment, the efficiency of the process can be further increased by the recovery of unreacted sucrose, if any, from the chain-lengthening product (the β-2, 1-fructosyltransferase) or branching (the β-2, 6-fructosyltransferase). Typically the action of a fructosyltransferase with sucrose will produce a reaction mixture comprising glucose, unreacted sucrose, fructan and fructose. The removal of unreacted sucrose and recycling it as a feedstock for a fructosyltransferase greatly increases the efficiency of glucose production. For example, sucrose can be removed by conventional chromatographic techniques known to those of ordinary skill in the art. As a specific example, simulated mobile bed techniques can be used to separate glucose, higher fructans (DPS and greater) and sucrose. The sucrose fraction will typically also comprise lower fructans DP3 and DP4 and glucose, all of which can be returned to a reaction with fructosyltransferase, increasing the efficiency of glucose production. Typically, a simulated moving bed technique will use as a support, a salt of an ion exchange resin, such as the sodium salt of a styrene-divinyl benzene sulfonic acid resin which has a high degree of entanglement from 4 to 6% When the degree of entanglement of the resin is below 4%, the mechanical integrity of the resin is undesirable. A suitable resin is available from Dow as DOWEX® ion exchange resin. The glucose separation can also be performed by size exclusion techniques using conventional hollow-type membranes known to those of ordinary skill in the art. The sequential combination of the commercially available size exclusion membranes Gio and Gs provide effective glucose isolation as well as a fraction comprising sucrose and lower fructans which can be recycled to the reactor.

Referring now to Fig. 1, where 1 represents a reactor, 2 represents an inlet for sucrose, 3 represents an outlet for glucose, 4 represents an outlet for a fructan, and 5 represents a separator which is permeable to glucose, but not permeable to sucrose, to fructosyltransferase or to fructan. Sucrose is introduced to the reactor via inlet 2 to a portion of reactor 1 which contains a β-2,1-fructosyltransferase and β-2,6-fructosyltransferase. In such a configuration, a division is created such that the fructans are concentrated on one side of the separator. The reactor is equipped as a glucose outlet 3, located on the glucose side of the separator 5. The outlet 4 for fructan, can be equipped with a separator (not shown) which allows the passage of fructan, but does not allow the passage of sucrose, glucose or fructosyltransferases. If a membrane system is used to isolate fructan, typically the membrane will allow passage of glucose and sucrose, but not of branched fructan. Therefore, fructan has been effectively separated. Referring now to FIG. 1 a, where 1 represents a reactor, 1 represents a separate portion of the reactor, 2 represents an inlet for sucrose, 3 represents an outlet for glucose, 4 represents a satida for a fructan and 5 represents a separator for glucose. . Sucrose is introduced to the reactor via inlet 2 to a portion 1a of reactor 1, which contains a β-2, 1-fructosyltransferase and a β-2,6-fructosyltransferase. In such a configuration, glucose is separated from the reaction medium by separator 5, before being removed via exit 3 for glucose. During the glucose separation, the remaining materials can be recycled to the portion 1a of the reactor. Exit 4 for fructan may be equipped with a separator (not shown) which allows the passage of fructan, but does not allow the passage of sucrose, glucose or fructosiltransfera & as. In another embodiment, a reactor comprising an inlet for sucrose is equipped with an external separator, which separates both glucose and a fructan from the sucrose. Unreacted sucrose, if any, can be returned to the reactor. Referring now to Fig. 2, where 1 represents a first reactor, 2 and 11 represent entries for sucrose, 3 and 8 represent outputs for glucose, 4 represents an output for a linear fructan, 5 and 10 represent separators which are permeable to glucose, but not permeable to sucrose, to fructosyltransferase or an upper fructan, 6 represents a second reactor, 7 represents an inlet for a linear fructan and 9 represents an outlet for a branched fructan. Two reactors are used, each divided with separators 5 and 10 which are permeable to glucose, but impermeable to sucrose; fructosyltransferases or linear or branched fructans. In the first reactor 1, the concentration of sucrose is such as to provide synthesis of fructans of inulin in β-2.1, the product being then transferred to the second reactor 6 via the inlet by a linear fructan. In a preferred embodiment, either the output 4 for linear fructan or the input 7 for linear fructan does not allow the passage of an active fructosyltransferase. This can be done by equipping either the outlet 4 or the inlet 7 with a membrane which does not allow the passage of a fructosyltransferase. Alternatively, one or the other of the salt 4 or the inlet 7 may be equipped with a zone of inactivation of the fructosyltransferase, such as by heating for a sufficient time and temperature, typically around 65 to 95 ° C, preferably around 70 to 90 ° C, even more preferably 85 ° C, for about one minute. In the second reactor 6, a β-2,6-fructosyltransferase is contained in a portion of a second reactor 6 and a linear fructan is reacted with sucrose. Glucose is allowed to pass through separator 10 and is removed via glucose outlet 8. During glucose separation, the remaining materials can be recycled to the reactor portion 6. The branched fructan can be removed via the 9 exit of branched fructan. The outlet 9 for branched fructan can be equipped with a separator (not shown) which allows the passage of branched fructans, but does not allow the passage of sucrose, glucose or fructosyltransferases. Referring now to Fig. 2a where 1 represents a first reactor and 1a represents a separate portion of the reactor, 2 and 11 represent entries for sucrose, 3 and 8 represent outputs for glucose, 4 represents an output for a linear fructan, 5 and 10 represent external separators which are permeable to glucose, but not permeable to sucrose, fructosyltransferase or linear or branched fructans, 6 represents a second reactor and 6a represents a separate reactor portion, 7 represents an inlet for a linear fructan and represents an outlet for a branched fructan. Two reactors are used, equipped with external separators 5 and 10 which are permeable to glucose, but impermeable to sucrose, fructosyltransferases or to linear or branched fructans. In the portion 1 a of the first reactor, the concentration of sucrose is such as to provide the synthesis of linear fructans, bound in β-2, 1-, the product that is transferred to the second portion 6a separated from the reactor via the inlet 7 for a linear fructan. In a preferred embodiment, both the output 4 for fructan linear or the input 7 for fructan linear, does not allow the passage of an active fructosyltransferase. This can be done by equipping either outlet 4 or inlet 7 with a membrane which does not allow the passage of a fructosyltransferase. Alternatively, either, outlet 4 or inlet 7 may be equipped with a fructosyltransferase deactivation zone, such as by heating for a sufficient time and temperature, typically around 65 to 95 ° C, preferably about 70 to 90 °. C, even more preferably around 85 ° C, for about one minute. In the second reactor 6, a β-2,6-fructosyltransferase is contained in the separate portion 6a of the reactor and a linear fructan is reacted with sucrose. The glucose is allowed to pass through the separator 10 and is removed via the glucose outlet 8. The branched fructan can be removed via the 9 exit of branched fructan. The outlet 9 for branched fructan can be equipped with a separator (not shown) which allows the passage of the branched fructan, but does not allow the passage of sucrose, glucose or β-2,6-fructosyltransferase. Both separators 5 and 10 are represented with a recycling line for non-glucose return materials such as sucrose and fructan, if necessary. The process of the present invention is preferably conducted in a suitable reactor to make commercial quantities of branched fructan. Preferably the reactor comprises one or more inlets for introducing sucrose and / or the fructosyltransferase and a means for isolating commercial quantities of branched fructans from the reactor. The reactor may comprise multiple vessels as illustrated in Fig. 2 and 2a, functioning as a reactor system. It is also within the scope of the present invention to conduct further modifications of branched fructans produced enzymatically by either conventional chemical modification or additional enzymatic modifications.Non-limiting examples of chemical modification may include alkylation, esterification, dehydration, cyclization and partial hydrolysis. Non-limiting enzymatic modification may include glycosylation A branched fructan may be used as a bulking agent for food and food sweeteners, which themselves have sweetness. Having generally described this mention, a greater understanding may be obtained by reference to certain specific examples which are given here for purposes of illustration only and is not intended to be limiting unless otherwise specified CLONING AND PROCEDURE OF EXPRESSION: The coding sequence of the ctosyltransferase of streptococcus mutans, which lacks the predicted sequence signal can be isolated from the strain of Streptococcus mutans ATTC 25175 by PCR. Two primaries were designated and synthesized. The first, a 5'-TCTGCGGGATCCCAGGCAGATGAAGCCAATTCAAC-3 \ containing a restriction site BamHl followed by sequence identical to the sequence immediately following the end of the signal sequence predicted in the coding sequence of fructosyltransferase in streptococcus mutans. The second, a 5'- TCTGCGAAGCTTTTATTTAAAACCAATGCTTACACA-3 ', contained a Hindtit restriction site followed by the corresponding reverse complement sequence at the end of the C terminus of the fructosiitransferase coding sequence of streptococcus mutans. Both primaries were combined with genomic DNA isolated from strain ATCC25175 of streptococcus mutans and used in PCR. The resulting DNA fragment was digested with Bamlll and Hind 111 and ligated to the digested pyramide BamHI-Hindlll, pGEX-KT-ext. This ligation resulted in the fructosyltransferase code sequence of streptococcus mutans described above, which is immediately placed downstream, in tag, with the coding sequence of glutathione-S-transferase (GST). The pásmid pGEX-KT-ext-Streptococcus mutans-fructosyltransferase was transformed into E.Coti BL21 cells. Protein expression of the resulting transformant resulted in intracellular accumulation of a GST-ext fusion protein Streptococcus mutans-fructosyltransferase. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings.

Therefore, it should be understood that within the scope of the appended claims, the invention may be practiced in other ways than those specifically described herein.

Claims (17)

1. REVIVAL DICATIONS 1. A process for preparing glucose from sucrose comprising: i) contacting sucrose with a β-2, 1-fructosyltransferase and β-2,6-fructosyltransferase to produce glucose; and ii) Isolate glucose from there. The process of claim 1, wherein said β-2, 1-fructosyltransferase is obtained from an organism selected from the group consisting of Aureobasidium pullulans, Aspergillus oryzae, Aspergillus awamori, Aspergillus sydowi, Aureobasidium sp. , Aspergillus niger, Penicillium roquefortii, Streptococcus mutans, Penicillium jancezewskii and higher plants. The process of claim 1, wherein at least one of said β-2, 1-fructosyltransferase or said β-2,6-fructosyltransferase is obtained by expression in a host of a fructosyltransferase gene which is not native to said guest. 4. The process of claim 1, wherein said glucose is continuously isolated or in the form of semi-batches. The process of claim 1, wherein said reaction product further comprises a branched fructan and said process further comprises isolating said branched fructan. 6. The process of claim 6, wherein said branched fructan is continuously isolated. 7. The process of claim 1, wherein said glucose is isolated by membrane filtration or chromatography. 8. The process of claim 5, wherein said branched fructan is isolated by membrane filtration chromatography. The process of claim 1, wherein said β-2,1-fructosyltransferase is a chain-extending fructosyltransferase. The process of claim 1, wherein said β-2,6-fructosyltransferase is a branching fructosyltransferase. The process of claim 1, wherein said β-2,1-fructosyltransferase is a branching fructosyltransferase. The process of claim 1, wherein said β-2,6-fructosyltransferase is a chain-extending fructosyltransferase. 13. A reactor for preparing commercial quantities of glucose comprising: a) A reactor vessel; b) An entry for sucrose; c) An outlet for glucose; and d) A β-2, 1 -fsyltransferase and a β-2,6-fructostltransferase. 14. The reactor of claim 13, further comprising an outlet for a branched fructan. 15. The reactor of claim 13, wherein said entry for sucrose is a continuous input. 16. The reactor of claim 13, wherein said outlet for said branched fructan is a continuous outlet. 17. The reactor of claim 13, wherein said output for glucose is a continuous output.