Patent Description:
The bio-conversion of D-fructose to D-allulose by D-tagatose-<NUM>-epimerase (DT3E) or by D-psicose-<NUM>-epimerase (<FIG>) has long been recognized, however, the methods of production have typically come with high production cost. The conversion of D-fructose to D-allulose will diversify the traditional sweetener product portfolio associated with corn processing by adding a natural low caloric sweetener and bulking agent to the traditional portfolio of sweeteners derived from corn starch, i.e. corn syrup, high fructose corn syrup (HFCS), glucose and fructose.

<CIT>) discloses an enzyme from the soil microorganism Desmospora sp. that has psicose-<NUM>-epimerase activity. This enzyme is shown to convert D-fructose to D-allulose (also known as D-psicose).

<CIT> and <CIT> disclose an enzyme derived from Agrobacterium tumefaciens that has psicose-<NUM>-epimerase activity.

<NPL>" also discloses an enzyme that can epimerize D-fructose to yield D-psicose.

There remains a need in the art to discover other genes that express psicose-<NUM>-epimerase activity to improve efficiency of converting fructose to allulose in a cost efficient manner. The present invention discloses a novel class of psicose-<NUM>-epimases from the betaproteobacteria Burkholderia which is a symbiont found the posterior midgut of the bean insect Riptortus pedestris. This symbiotic microorganism is thought to be essential for host survival and reproduction and plays pivotal roles in host metabolism by providing essential nutrients, digesting food materials and/ or influencing host plant use, resistance against parasitoids and body color change. Once identified and isolated, an exemplary psicose-<NUM>-epimase gene from a Burkholderia sp. strain RPE64 was expressed in an E. colt production strain and the enzyme was isolated and evaluated in both benchtop and pilot scale production environments. The enzyme was evaluated for commercial allulose production by immobilization of the enzyme on a solid matrix weak base anion exchange resin as described in more detail herein. <CIT> relates to D-psicose-epimerase and methods of its use in the peparation of psicose from fructose. It discloses a D-psicose-<NUM>-epimerase from P. cichorii which is <NUM> % identical to SEQ I D NO: <NUM> disclosed herein. Gen Bank Accession No. BAN26336. <NUM> discloses a sequence from Burkholderia RP64 that is identical to SEQ I D NO: <NUM> disclosed herein.

The present invention describes a method of producing allulose comprising, contacting a solution containing fructose with a psicose-<NUM>-epimerase enzyme from a Burkholderia species having a polypeptide sequence selected from SEQ ID NO:<NUM> and SEQ I D NO:<NUM> for a time and under conditions suitable to convert at least a portion of the fructose to allulose. Certain embodiments include a method wherein the psicose-<NUM>-epimerase is immobilized on a solid matrix resin and used to convert fructose to allulose, preferably at temperatures between <NUM> ° and <NUM>. Preferred embodiments include a method wherein the solid matrix is a weak base anion exchange resin.

Exemplary embodiments of the invention include a method wherein the solid matrix resin is a phenol formaldehyde based condensate resin functionalized to contain tertiary amine free base groups, exemplified by DUOLITE™ A568. Additional exemplary embodiments of the invention include a method wherein the solid matrix resin is a methacrylic acid based resin functionalized to contain C2-C6 amine linkages, such as Lifetech™ ECR8315, Lifetech™ ECR <NUM>, and SEPABEADS™ EC-HA.

Additional embodiments of the invention include a method wherein the fructose solution is selected from solubilized crystalline fructose and high fructose corn syrup (HFCS). Preferred embodiments of the invention include a method of wherein the fructose solution has a dissolved solids content of about <NUM>%w/w, the contacting is done at a pH range of <NUM>-<NUM> in the presence of magnesium at a concentration of <NUM>-<NUM> ppm (corresponding to <NUM> - <NUM><NUM>/ L), and is done with a flow rate of <NUM>-<NUM> volumes of fructose solution per bed volume of the solid matrix resin.

Other embodiments of the invention include a method wherein the psicose-<NUM>-epimirase enzyme is obtained from a microorganism containing a recombinant nucleic acid vector operably configured to express a nucleic acid sequence encoding the protein having a polypeptide sequence selected from SEQ ID NO: <NUM> and SEQ ID NO: <NUM>. Preferred embodiments of the invention include a method wherein the microorganism is E. coli and B.

An additional aspect of the invention is a recombinant nucleic acid sequence operably configured to express a nucleic acid encoding psicose-<NUM>-epimerase enzyme having a polypeptide sequence of SEQ ID NO: <NUM>.

Another aspect of the invention is a microorganism transformed with a recombinant nucleic acid sequence operably configured to express a nucleic acid encoding psicose-<NUM>-epimerase having a polypeptide sequence selected from SEQ ID NO: <NUM> and SEQ ID NO: <NUM>. Preferred embodiments of the invention include a microorganism selected from E. coli and B.

Additional aspects of the invention include a solid matrix resin containing a psicose-<NUM>-epimerase enzyme having a polypeptide sequence selected from SEQ ID NO:<NUM> and SEQ I D NO: <NUM>. Further aspects of the invention include a column containing the solid matrix resin which contains a psicose-<NUM>-epimerase enzyme having a polypeptide sequence selected from SEQ ID NO:<NUM> and SEQ ID NO:<NUM>; and, configured to receive an input flow input flow of a solution to flow over the solid matrix resin and permit exit of an output flow of the solution.

The following description and foregoing background makes citations to certain references that may aid one of ordinary skill in the art to understand the present invention and that may provide material, information, techniques, proteins, vectors and nucleotide sequences that may assist one of ordinary skill in the art to make and use aspects of the present invention in its fullest scope.

One aspect of the present invention is the discovery of a class of psicose-<NUM>-epimerase enzymes encoded by genes from the betaproteobacterial genus Burkholderia, which is exemplified herein by SEQ ID NO: <NUM>, which is the protein sequence of a psicose-<NUM>-epimerase from a Burkholderia sp strain RPE <NUM> which is a symbiont found in the posterior midgut of the bean insect Riptortus pedestris. Other examples of psicose-<NUM>-epimerase genes from this class are shown in Table <NUM>, all of which share at least <NUM>% amino acid sequence identity to SEQ ID NO: <NUM>, all of which substantially differ from other psicose-<NUM>-epimerases disclosed for use in converting fructose to allulose, such as those from Clostridium sp. , Agrobacterium tumefaciens, Desmospora sp. that have previously been described in the art, and all of which, except for SEQ ID NO: <NUM>, are not part of the claimed invention. SEQ ID NO: <NUM> only has <NUM>% identity to the Desmospora sp polypeptide sequence disclosed by Woodyer et al. Likewise, the Clostridium cellulolyticum sequence described by Chan et al. has <NUM>% identity to SEQ ID NO:<NUM> and the Agrobacterium tumefaciens polypeptide described by <CIT> and <CIT> only showed <NUM>% identity to SEQ ID NO <NUM>. An exception to this rule is the epimerase from Pseudomonas cichorii, (SEQ ID NO: <NUM>) shown in <FIG>, which has <NUM>% sequence identity to SEQ ID NO: <NUM> but which is excluded from the present invention.

SEQ ID NO: <NUM>: was identified in a database by the National Center for Biotechnology Information (NCBI) as accession number YP_008039310 and was labeled as being a xylose isomerase gene. Prior to the present invention, it was not known that this enzyme has a psicose-<NUM>-epimerase activity capable of isomerizing fructose to allulose. In order to determine that the exemplary enzyme of the present invention displayed epimerase activity, the present inventors had the gene encoding the protein identified by accession number YP_008039310 chemically synthesized by GenScript (Biscataway, NJ). The synthetic DNA was created to include NdeI and XhoI restriction sites at the <NUM>' and <NUM>' ends of the polynucleotide in order to ensure simple cloning. The sequence of this synthesized polynucleotide is according to SEQ ID NO: <NUM> as shown in <FIG> and is referred to herein as the BRPV3 gene cassette. The BRPV3 cassette was designed with the NdeI and XhoI restriction sites and was cloned into a NdeI/XhoI digested expression vector pET-22b+ obtained from Novagen, Inc. (Madison, WI) so the N terminus would be in frame with a polyhistidine tag sequence contained within the vector. The resulting expression vector is herein referred to as BRPV3_pET22b(+), a map of which is provided in <FIG>.

The expression vector BRPV3_pET22b(+) was transformed into the E. coli strain BL21(DE3) (<NPL>). The strain that resulted from this transformation is herein referred to as BL21(DE3)/BRPV3.

Single colonies were selected and incubated overnight (<NUM>, <NUM> rpm, <NUM>) in <NUM> LB broth (Miller) (BD Biosciences, Inc. ) with <NUM>µg/µL ampicillin (Sigma-Aldrich Corp. ) to maintain the plasmids. After <NUM>, <NUM> of each culture was diluted <NUM>: <NUM> in a baffled shake flask containing <NUM> LB (<NUM>µg/µL ampicillin) and grown until OD<NUM> reached approximately <NUM>(<NUM>, <NUM> rpm). Then the cultures were induced with a final concentration of <NUM> Isopropyl β-D-<NUM>-thiogalactopyranoside (IPTG from Sigma-Aldrich Corp. ) and incubated again for <NUM>. Cells were harvested by <NUM> minute centrifugation at <NUM>,<NUM> rpm, <NUM>. The supernatant was discarded and cell pellets were frozen at - <NUM>.

The frozen cells were thawed on ice, lysed, and centrifuged. The supernatant was used to evaluate if the expressed gene could catalyze epimerization of fructose to allulose, which confirmed that SEQ ID NO: <NUM> in fact encoded an enzyme with the epimerase enzyme activity. Results of this experiment can be seen in <FIG>.

While there was clear demonstration of the plasmid BRPV3_pET-<NUM>(b)+ expressing the encoded enzyme in the E. coli strain BL21(DE3)/BRPV3, a strain free of antibiotic markers was ultimately desired. In order to accomplish this, the E. coli strain BL21(DE3)thrC- was created, which is auxotrophic for threonine biosynthesis. BL21(DE3)thrC- was generated by the integration of an antibiotic cassette deleting thrC and excision of the antibiotic cassette by the following steps. The thrC gene is already disrupted in an E. coli K-<NUM> derivative in the Keio collection (<NPL>). coli K-<NUM> derivative, a kanamycin cassette has replaced the native thrC gene and is flanked by DNA sequences recognized by the FLP recombinase, an enzyme capable of excising the cassette. A P1 phage lysate generated from this thrC::kmR strain was used to transduce kanamycin resistance into BL21 (DE3). The transductants were transformed with a temperature sensitive ampicillin resistant plasmid pCP20 (<NPL>) to transiently introduce the FLP recombinase and isolates that were sensitive to kanamycin and ampicillin were confirmed as being auxotrophic for threonine biosynthesis.

In parallel, the plasmid BRPV3_pET-<NUM>(b)+ was used as the template in a PCR reaction where the primers shown in <FIG> and <FIG> (SEQ ID NO:<NUM> and <NUM>, respectively) were used to amplify the polynucleotide sequence that expresses the epimerase activity. The amplified PCR product and pET-<NUM>(b)+ were digested with the restriction enzymes NdeI and XhoI and ligated together to create the plasmid p-<NUM>-13_ pET-<NUM>(b)+. The <NUM>' primer (SEQ ID NO: <NUM>) contains a stop codon upstream of the XhoI restriction site so, once ligated, the His tag of the pET-<NUM>(b)+ expression vector is no longer in frame.

The plasmid p-<NUM>-13_pET-22b (+) was digested with DraI and DraIII NEB restriction enzymes to remove all ampicillin resistance-encoding DNA. The thrC gene of E. coli K-<NUM> was amplified with primers SEQ ID NO:<NUM> and NO:<NUM> (as seen in <FIG>) that appended extended homology to pET22b at the DraI and DraIII cut sites. The PCR and digested p-<NUM>-13_pET-<NUM>(b)+ plasmid were assembled by means of a Gibson Assembly Master Mix (NEB). The resulting plasmid, p-<NUM>-13_pET-<NUM>(b)+thrC, was transformed into BL21(DE3)thrC- and the colonies that appeared on M9 minimal media constitute the strain designated herein as ASR180. ASR180 is an E. coli strain containing the antibiotic resistant marker free vector as indicated in <FIG>.

In order to construct ASR182, which is an E. coli strain with a copy of the psicose-<NUM>-epimerase gene from Burkholderia RPE64 integrated into the chromosome, a synthetic nucleotide cassette was made by Integrated DNA Technologies (Coralville, IA) according to SEQ ID NO: <NUM>. The cassette functionally encodes a small piece of homologous DNA upstream of the thrC gene, the thrC gene, a T7 promoter, a sequence encoding the enzyme according to SEQ ID NO: <NUM> and a small piece of homologous DNA downstream of thrC. This cassette was amplified with Go Taq (Promega, Inc. , Fitchburg, WI) using IDT primers according to SEQ ID NO: <NUM> and <NUM> (<FIG>, respectively). The amplified cassette and the plasmid pKD46 (<NPL>) was introduced into strain BL21 (DE3)thrC-, previously described herein. As described more fully in Datsenko et al. , the plasmid pKD46 has a recombination mechanism capable of integrating the amplified cassette into the chromosome of BL21(DE3)thrC-. The cassette was integrated into the chromosome of BL21(DE3)thrC- by selecting for prototrophy on M9 minimal plates. The strain was cured of pKD46 by passage at <NUM>. Prototrophic isolates sensitive to ampicillin and expressing the desired enzymatic activity were characterized.

While the enzyme exemplified herein was obtained by expression of a vector encoding the enzyme in E. coli, one of ordinary skill in the art can overexpress any of the psicose-<NUM>-epimerases enzymes in any suitable microorganism, for example iB. subtilis is another preferred bacteria for overexpressing enzymes.

Once the strains ASR180 and ASR182 were constructed, small benchtop fermentations and large pilot scale fermentations were done as shown in the following examples. Isolation of the psicose-<NUM>-epimerase enzyme that was produced from these fermentations was done by immobilization onto solid matrix resins described in further detail in the examples below.

The following examples and embodiments not falling within the scope of the claims are offered for purposes of illustration.

Example <NUM>: Small scale production of BL21DE3/BRPV3: After construction of the BL21DE3/BRPV3 strain, cultivation was done by first obtaining single colonies. Selected single colonies were inoculated into <NUM> of LB medium containing ampicillin (<NUM> ug/mL) held in <NUM> flask. Cultures were incubated with shaking at <NUM> rpm at <NUM> on New Brunswick Scientific G25 Gyratory shakers. A <NUM>% inoculum derived from overnight stage I cultures was used to initiate fresh LB cultures (<NUM>) with antibiotics in a <NUM> Fembach flask. The culture was incubated at <NUM> for <NUM> with shaking at <NUM> rpm on a gyratory shaker. When the optical density was <NUM>, the expression of protein was induced with IPTG to a final concentration of <NUM>. coli cells were harvested by centrifugation at <NUM>,<NUM> × g for <NUM> at <NUM>. The cells were lysed and the psicose-<NUM>-epimerase enzyme was purified using a Ni-NTA column. The eluted protein was dialyzed with tris buffer and evaluated for enzyme activity according to the procedure as described herein in Example <NUM>.

The results of this evaluation can be seen in <FIG>, which is a SDS page gel analyzing different fraction after small scale purification. Lane <NUM> is a Pre-stained molecular weight markers, Lane <NUM> is cell lysate, Lane <NUM> is cell lysate eluate from the nickel column, Lane <NUM> is Load FT, Lane <NUM> is Wash, Lane <NUM> is fraction <NUM>, Lane <NUM> is Fraction <NUM>, Lane <NUM> is Fraction <NUM>, Lane <NUM> is fraction <NUM>, and Lane <NUM> is cell debris.

BL21(DE3)/BRPV3 was cultured in a Biostat ® C-plus (Sartorius) fermenter. Seed cultures were initiated by inoculating <NUM>µl of frozen glycerol stock in a <NUM> flask containing <NUM> of LB Lenox media with ampicillin (<NUM> ug/mL). The flask was shaken at <NUM>, <NUM> rpm for <NUM>. The overnight culture were transferred to Biostat ® C-plus (Sartorius) fermenters containing <NUM> of LB Lennox media with ampicillin (<NUM> ug/mL). The fermenter was maintained at <NUM>, <NUM> % dissolved oxygen and at pH <NUM> ± <NUM>. pH was adjusted with either 2N HCL or 5N NaOH. When the culture attained an OD600 of <NUM>, the culture was induced with β-isopropyl thiogalactoside (IPTG) (final conc <NUM>). After the culture was grown overnight, the fermentation was stopped and cells were harvested by centrifugation. The cells were stored at -<NUM> until further use.

The frozen cells were thawed and suspended in <NUM> lysis buffer (<NUM> Tris Cl, <NUM> imidiazole, <NUM> NaCl, <NUM> MnCl<NUM>, ph7. The cell were lysed using a microfluidizer (<NUM> kbar (<NUM>,<NUM> psi)). The cell lysate was centrifuged at <NUM>,<NUM> g to separate cell debris. The supernatant was used for further purification.

The active enzyme was purified using AKTA explorer FPLC system equipped with <NUM> of Ni-NTA column. The column was equilibrated with the lysis buffer and the supernatant was loaded onto the column at <NUM>/min. After all the cell lysate was loaded, the column was washed with <NUM> bed volumes of wash buffer (<NUM> Tris. Cl, <NUM> imidiazole, <NUM> NaCl, <NUM> MnCl<NUM>, pH <NUM>) to separate out all the other undesired proteins. The Ni-NTA column was then further washed with elution buffer (<NUM> Tris. Cl, <NUM> imidiazole, <NUM> NaCl, <NUM> MnCl<NUM>, pH <NUM>) to elute the His tagged enzyme from the column. The fraction containing active enzyme was pooled, concentrated and dialyzed with <NUM> TrisCl, <NUM> MnCl<NUM>, <NUM>% glycerol pH <NUM>. The stabilized enzyme was stored at <NUM>. The purified enzyme was stable for > <NUM> years when stored under these conditions. The enzyme containing fractions were run on an SDS page gel and the results can be seen in <FIG>. The enzyme activity was evaluated according the methods described herein in Example <NUM>.

Example <NUM>: Purified psicose-<NUM>-epimerase enzyme from the fermentation as described in Example <NUM> and <NUM> was evaluated using an epimerase assay performed at <NUM> for <NUM> in <NUM> TrisCl buffer (pH <NUM>) containing <NUM> fructose and <NUM> ug of purified enzyme. The reaction was stopped by heating at <NUM> for <NUM>. One unit of D-psicose <NUM>-epimerase activity was defined as the amount of the enzyme required to produce <NUM> umol of psicose per min at pH <NUM> and <NUM>.

Purified enzyme was diluted <NUM>-fold into <NUM> tris buffer containing <NUM> MnClz, pH <NUM>. This solution was incubated at different temperature (<NUM>-<NUM>). At appropriate time after incubation at the desired temperature, an aliquot of enzyme sample (<NUM>) was withdrawn and evaluated for residual activity. The purified enzyme showed a similar level of enzyme activity at all temperatures tested. It was observed that the enzyme was catalytically active after incubating at <NUM> for <NUM>. These results can be seen in <FIG>. The bioconversion of fructose to allulose at <NUM> can also be seen in <FIG>.

Example <NUM>: Immobilization of psicose-<NUM>-epimerase on solid matrix resins. The cell lysate from BL21(DE3)/BRPV3 as grown under the condition disclosed in Example <NUM> was screened for epimerase activity on several immobilization support resins. The results comparing the resins can be seen in Table <NUM> showing the activity of the present enzyme measured by GC analysis, which indicate that weak base solid matrix resins are the best support for immobilizing this enzyme. Table <NUM> further suggests that strong anion exchange resins, such as Lifetech™ ECR1504, Lifetech™ ECR1640, and Lifetech™ ECR1604, are not most suitable support resins for immobilizing the enzyme described herein.

Where there are two numbers in a row of Table <NUM>, the bolded bottom number indicates the enzyme activity as measured by HPLC. Most importantly, the last column indicates that the resins ECR8315F/M, ECR8415F/M and DUOLITE™ A568 show a very low % of activity left in the supernatant as it passes through the column, indicating a high enzyme loading capacity of these resins. ECR8315 F/M and ECR8415 F/M (Lifetech™) and DUOLITE™ A568 were identified as optimal immobilization support for this enzyme. SEPABEADS™ EC-HA which is also a methacrylic based amino modified resin (similar to ECR8415) will also work for immobilization. Based on this, it can be hypothesized, this psicose-<NUM>-epimerase has a high affinity for amino modified resin and shows high activity of the psicose-<NUM>-epimerase on these resins once immobilized.

Psicose-<NUM>-epimerase was produced in the lab at a scale of <NUM>- <NUM>. A batch process was designed using E. coli strain ASR180. This experiment was done with ASR <NUM> also, but for exemplary purposes, data and results from ASR180 are shown herein. M9 media was used in the flask: <NUM>/L Dextrose; <NUM>/L Na<NUM>HPO<NUM>- <NUM><NUM>O; <NUM>/L KH<NUM>PO<NUM>; <NUM>/L NH<NUM>Cl; <NUM>/L NaCl; <NUM>/L MgSO<NUM>; <NUM>/L CaCl<NUM>; <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L FeCl<NUM>·<NUM><NUM>O; and <NUM>/L ZnSO<NUM>·<NUM><NUM>O. Thiamine was aseptically added at a concentration of <NUM>/L. <NUM> to <NUM> inoculum from a frozen vial was used to inoculate one <NUM> shake flask. Flasks were incubated at <NUM> and <NUM> RPM for <NUM> to <NUM> or until an OD660 of <NUM>-<NUM> units was reached.

When OD660 reached <NUM> to <NUM> units in the shake flask, the culture was transferred to a seed fermenter at a <NUM>% ratio. M9 media was used in the seed fermenter: <NUM>/L Na<NUM>HPO<NUM>·<NUM><NUM>O; <NUM>/L KH<NUM>PO<NUM>; <NUM>/L NH<NUM>Cl; <NUM>/L NaCl; <NUM>/L MgSO<NUM>; <NUM>/L CaCl<NUM>; <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L FeCl<NUM>·<NUM><NUM>O; <NUM>/L ZnSO<NUM>·<NUM><NUM>O; <NUM> to <NUM>/L citric acid; and <NUM>/L Antifoam. Dextrose and thiamine were aseptically added at a concentration of <NUM>/L and <NUM>/L, respectively. Seed tank conditions at EFT=<NUM>: temperature was <NUM>, pH was <NUM>, agitator was <NUM> RPM, and airflow was <NUM> vvm. During incubation, pH was controlled with <NUM>% aqueous NH<NUM>OH at set point of <NUM>, dissolved oxygen was controlled at <NUM>% using agitation (max <NUM> RPM), and dextrose concentration was maintained between <NUM> and <NUM>/L. The culture incubated for <NUM> to <NUM> or until OD660 was <NUM> to <NUM> units.

When OD660 reached <NUM> to <NUM> units in the seed fermenter, the culture was transferred to a production fermenter at a <NUM>% ratio. Media was used in the production fermenter: <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L K<NUM>HPO<NUM>; <NUM>/L NaCl; <NUM>/L Na<NUM>C<NUM>H<NUM>O<NUM>-<NUM><NUM>O; <NUM>/L MgSO<NUM>-<NUM><NUM>O; <NUM>/L CaCl<NUM>-<NUM><NUM>O; <NUM>/L FeSO<NUM>-<NUM><NUM>O; <NUM> to <NUM>/L citric acid; <NUM>/L Antifoam; and <NUM>/L Neidhardt micronutrients solution. The Neidhardt micronutrients solution consisted of: <NUM>/L (NH<NUM>)<NUM>(MO<NUM>)<NUM>·<NUM><NUM>O; <NUM>/L H<NUM>BO<NUM>; <NUM>/L CoCl<NUM>·<NUM><NUM>O; <NUM>/L CuSO<NUM>·<NUM><NUM>O; <NUM>/L MnCl<NUM>·<NUM><NUM>O; and <NUM>/L ZnSO<NUM>·<NUM><NUM>O. Dextrose and thiamine were aseptically added at a concentration of <NUM>/L and <NUM>/L, respectively. Production tank conditions at EFT=<NUM>: temperature was <NUM>, pH was <NUM>, agitator was <NUM> RPM, and airflow was <NUM> vvm. During the growth phase, pH was controlled with <NUM>% aqueous NH<NUM>OH at set point of <NUM>, dissolved oxygen was controlled at <NUM>% using agitation (max <NUM> RPM), and dextrose concentration was maintained between <NUM> and <NUM>/L. The growth phase lasted until OD660 was <NUM> to <NUM> units.

Once the OD <NUM> reached <NUM> to <NUM> units in the production fermenter, the induction phase began. A bolus of <NUM>/L to <NUM>/L lactose or <NUM> mmol/L IPTG and <NUM> to <NUM>/L yeast extract was added and the temperature was linearly ramped down from <NUM> to <NUM> over the course of <NUM>. Production tank conditions after induction: temperature was <NUM>, pH was <NUM>, agitator was <NUM> to <NUM> RPM, and airflow was <NUM> vvm. During the induction phase, pH was controlled with <NUM>% aqueous NH<NUM>OH at set point of <NUM> and dextrose concentration was maintained less than <NUM>/L.

A fed-batch protocol was developed to increase the production of psicose-<NUM>-epimerase. coli strain ASR180 was used. This experiment was done with ASR182 also, but for exemplary purposes, data and results from ASR180 will be shown herein. M9 media was used in the flask: <NUM>/L Dextrose; <NUM>/L Na2HPO4·7H2O; <NUM>/L KH<NUM>PO<NUM>; <NUM>/L NH<NUM>Cl; <NUM>/L NaCl; <NUM>/L MgSO<NUM>; <NUM>/L CaCl<NUM>; <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L FeCl<NUM>·<NUM><NUM>O; and <NUM>/L ZnSO<NUM>·<NUM><NUM>O. Thiamine was aseptically added at a concentration of <NUM>/L. <NUM> to <NUM> inoculum from a frozen vial was used to inoculate two <NUM> flasks with <NUM> of media. Flasks were incubated at <NUM> and <NUM> RPM for <NUM> to <NUM> or until an OD660 of <NUM>-<NUM> units was reached.

When OD660 reached <NUM> to <NUM> units in the shake flask, the culture was transferred to a seed fermenter at a <NUM>% ratio. M9 media was used in the seed fermenter: <NUM>/L Na<NUM>HPO<NUM>·<NUM><NUM>O; <NUM>/L KH<NUM>PO<NUM>; <NUM>/L NH<NUM>Cl; <NUM>/L NaCl; <NUM>/L MgSO<NUM>; <NUM>/L CaCl<NUM>; <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L FeCl<NUM>·<NUM><NUM>O; <NUM>/L ZnSO<NUM>·<NUM><NUM>O; <NUM> to <NUM>/L citric acid; and <NUM>/L antifoam. Dextrose and thiamine were aseptically added at a concentration of <NUM> to <NUM>/L and <NUM>/L, respectively. Seed tank conditions at EFT=<NUM>: temperature was <NUM>, pH was <NUM>, agitation was <NUM> RPM, airflow was <NUM> vvm, and back pressure was <NUM> bar (<NUM> psig). During incubation, pH was controlled with <NUM>% aqueous NH4OH at set point of <NUM> and dissolved oxygen was controlled at <NUM>% using agitation (max <NUM> RPM). The culture incubated until OD660 was <NUM> to <NUM> units.

When OD660 reached <NUM> to <NUM> units in the seed fermenter, the culture was transferred to a production fermenter at a <NUM>% ratio. Media was used in the production fermenter: <NUM>/L (NH<NUM>)<NUM>SO<NUM>; <NUM>/L K<NUM>HPO<NUM>; <NUM>/L NaCl; <NUM>/L Na<NUM>C<NUM>H<NUM>O<NUM>· <NUM><NUM>O; <NUM>/L MgSO<NUM>·<NUM><NUM>O; <NUM>/L CaCl<NUM>·<NUM><NUM>O; <NUM>/L FeSO<NUM>·<NUM><NUM>O; <NUM> to <NUM>/L citric acid; <NUM>/L antifoam; <NUM> to <NUM>/L yeast extract; and <NUM>/L Neidhardt micronutrients solution. The Neidhardt micronutrients solution consisted of: <NUM>/L (NH<NUM>)<NUM>(MO<NUM>)<NUM>·<NUM><NUM>O; <NUM>/L H<NUM>BO<NUM>; <NUM>/L CoCl<NUM>·<NUM><NUM>O; <NUM>/L CuSO<NUM>·<NUM><NUM>O; <NUM>/L MnCl<NUM>·<NUM><NUM>O; and <NUM>/L ZnSO<NUM>·<NUM><NUM>O. Dextrose and thiamine were aseptically added at a concentration of <NUM>/L and <NUM>/L, respectively. Production tank conditions at EFT=<NUM>: temperature was <NUM>, pH was <NUM>, agitation was <NUM> RPM, airflow was <NUM> vvm, and back pressure was <NUM> bar (<NUM> psig). During growth phase, pH was controlled with <NUM>% aqueous NH<NUM>OH at set point of <NUM>, dissolved oxygen was controlled at <NUM>% using agitation (max <NUM> RPM), and dextrose concentration was maintained between <NUM> and <NUM>/L. The growth phase lasted until OD660 was <NUM> to <NUM> units.

Once the OD <NUM> reached <NUM> to <NUM> units in the production fermenter, induction phase began. A bolus of <NUM>/L lactose or <NUM> mol/ L IPTG was added and the temperature was linearly ramped down from <NUM> to <NUM> over the course of <NUM>. Production tank conditions after induction: temperature was <NUM>, pH was <NUM>, agitation was <NUM> RPM, airflow was <NUM> vvm, and back pressure was <NUM> bar (<NUM> psig). During induction phase, pH was controlled with <NUM>% aqueous NH<NUM>OH at set point of <NUM>, dissolved oxygen was "as is," and dextrose concentration was maintained less than <NUM>/L. The results of the enzyme specific activity over the course of the pilot scale fermentation can be seen in <FIG>. In addition, <FIG> shows the production of psicose-<NUM>-epimerase being produced by ASR180 over the course of this fermentation.

At the end of fermentation, the fermentation broth was processed by sodium phosphate salts being added to a final concentration of <NUM> phosphate and pH of <NUM>. The fermentation broth was passed through a homogenizer (Panda NS1001L <NUM> from Niro-Soavi, Inc. ) at <NUM>-<NUM> bar, <NUM> to disrupt the cells. The crude lysate was clarified by centrifugation followed by filtration with diatomaceous earth to remove any cell debris. The supernatant was used for immobilization.

Dow resin DUOLITE™ A568 was used for immobilization of the enzyme. Prior to immobilization, the resin was regenerated per manufacture's protocol and washed with <NUM> phosphate buffer with <NUM> MgSO<NUM>, pH <NUM>. It was determined that a protein load of <NUM>/ g of dry resin was optimal for getting maximum enzyme immobilization. A jacketed glass column (Ace glass #<NUM><NUM> × <NUM>) was filled with a known volume of resin. A known volume of crude lysate was recirculated through the resin bed at <NUM>-<NUM> BV/h for <NUM>. The resin bed was then washed with <NUM> bed volumes of <NUM> phosphate buffer with <NUM> MgSO<NUM>, pH <NUM> to remove any unbound protein. The resin again was washed with <NUM>:<NUM>(v/v) Glycerol: <NUM> phosphate buffer with <NUM> MgSO<NUM>, pH <NUM> and stored for future use. The results of this can be seen in <FIG>. As seen in the gel data, the binding of the psicose-<NUM>-epimerase enzyme to DUOLITE™ A568 is very specific. The <NUM> kd band for psicose-<NUM>-epimerase is not observed in the eluate fraction (lane <NUM>) or in the subsequent wash fraction. The psicose-<NUM>-epimerase activity is observed in the resin and no activity is observed in the eluate fraction or wash fraction.

The reaction were carried out using feed consisting of solubilized crystalline fructose (<NUM>% w/w) or high fructose corn syrup <NUM>(<NUM>% DS), magnesium Mg+<NUM> concentration of <NUM>-<NUM> ppm, pH range of <NUM> -<NUM>. The flow rate was adjusted to <NUM> bed volume per h and temperature of the column was maintained at <NUM>. Results can be seen in <FIG>. Under these conditions, near equilibrium production of allulose was observed for <NUM> with minimum loss in performance, which was surprising for this resin. As described by Woodyer et al. , an epimerase enzyme that was immobilized on the DUOLITE™ A568 resin showed a decrease in the percentage conversion of fructose to allulose over the course of as little as <NUM> when the reaction was maintained at <NUM>.

The reaction were carried out using feed consisting of solubilized crystalline fructose (<NUM>% w/w) or high fructose corn syrup <NUM>(<NUM>% DS), Mg + <NUM> concentration of <NUM>-<NUM> ppm, pH range of <NUM> -<NUM>. The flow rate was adjusted to <NUM>-<NUM> bed volume per hour and temperature of the column was maintained at <NUM>. Under these conditions, near equilibrium production of allulose was observed for ~<NUM> with minimum loss in performance. Results can be seen in the chart in <FIG>.

Example <NUM>: Enzyme assay for fermentation samples: The fermentation samples were stored at <NUM> until processed. Fermentation samples (<NUM>) was centrifuged and supernatant was discarded. The cell pellet was lysed using Bugbuster® (EMD Millipore), centrifuged and the supernatant was used for the enzyme assay. The psicose-<NUM>-epimerase enzyme assays were performed at <NUM> for <NUM> in <NUM>% (w/w) fructose and cell lysate (<NUM>:<NUM> dilution v/v) of crude lysate. The reactions were stopped by adjusting the pH to <NUM> with <NUM>% (v/v) HCl. The reaction mixtures were analyzed by HPLC. One unit of D-psicose-<NUM>-epimerase activity was defined as the amount of the enzyme required to produce <NUM> umol of psicose per min at pH <NUM> and <NUM>. These results seen in <FIG>.

Claim 1:
A method of producing allulose comprising:
contacting a solution containing fructose with a psicose-<NUM>-epimerase enzyme from a Burkholderia species having a polypeptide sequence selected from SEQ ID NO:<NUM> and SEQ ID NO:<NUM> for a time and under conditions suitable to convert at least a portion of the fructose to allulose.