A method for obtaining l-arabinose

Abstract

This invention belongs to the field of functional small molecule sugar separation and preparation, and relates to a method for obtaining L-arabinose, specifically a method for preparing, separating, and purifying L-arabinose using enzyme catalysis. This method uses D-xylose as raw material, and involves a first-step enzyme-catalyzed reaction, a first-step chromatographic separation, a second-step enzyme-catalyzed reaction, and a second-step chromatographic separation, followed by crystallization to obtain a high-quality L-arabinose crystal product with a purity ≥99%. The stepwise enzyme-catalyzed coupling L-arabinose production process developed in this invention, based on simulated moving bed separation, has the advantages of simple separation and purification processes and environmental friendliness. Simultaneously, the use of a simulated moving bed enables the recycling of the substrate D-xylose and the intermediate products D-xylulose and L-ribulose, which helps reduce production costs and thus promotes the industrial production of L-arabinose.

Description

Technical Field

[0001] This invention belongs to the field of functional small molecule sugar separation and preparation, specifically relating to a method for obtaining L-arabinose. Background Technology

[0002] L-Arabinose, also known as L-arabinose or L-pentose, has the molecular formula C5H10H2O. 10 O5 is a pentose aldose. L-arabinose itself is a low-energy natural sugar, a zero-calorie natural sweetener approved by the U.S. Food and Drug Administration (FDA) with 50% of the sweetness of sucrose. Studies have shown that L-arabinose is also a natural sucrase inhibitor, binding to sucrase in the small intestine and inhibiting its activity through non-competitive action, preventing sucrose hydrolysis and thus inhibiting sucrose absorption. Simultaneously, L-arabinose has intestinal regulatory functions, acting as a prebiotic that promotes the growth of beneficial bacteria. As a functional sugar ingredient, L-arabinose is widely used in various health foods and beverages, with a promising market prospect. Furthermore, L-arabinose is also used as a starting material for synthetic drugs and a biochemical product widely used in molecular biology experiments and industrial fermentation.

[0003] L-arabinose exists in the hemicellulose of higher plants in the forms of arabinogalactan, arabinoxylan, and arabinogalactan. L-arabinose can be obtained by alkaline extraction of hemicellulose from corn fiber, gum arabic, and beet pulp, followed by acid hydrolysis (Chinese Patent CN105061521A); alternatively, it can be obtained simultaneously by acid hydrolysis of corn cobs to produce D-xylose (Chinese Patents CN107383116A, CN111004824A). Traditional extraction methods involve acid-alkali hydrolysis of plant fibers followed by purification processes such as filtration, ultrafiltration, ion exchange, concentration, and crystallization. While this method is relatively mature, the purification steps are complex, and the production process uses large amounts of acid and alkali, potentially causing environmental pollution.

[0004] Chinese patent CN113122528A discloses a method for preparing L-arabinose using D-xylose as a substrate through a three-enzyme catalysis process, passing through intermediates D-xylulose and L-ribulose. This method features a simple production process and introduces an innovative three-enzyme catalytic system, opening up a new technological pathway in the field of carbohydrate conversion. However, regarding the separation and preparation of L-arabinose, only a conceptual approach using a simulated moving bed (SMB) to separate L-arabinose from the unused substrate D-xylose and the intermediates L-ribulose and D-xylulose is proposed; the specific separation process is not studied. Therefore, it is necessary to investigate the separation and purification of L-arabinose in this reaction system. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] Addressing the shortcomings of existing technologies in the complex and environmentally polluting separation and purification of L-arabinose, the inventors researched the separation process for L-arabinose prepared using the three-enzyme catalytic system described in Chinese patent CN113122528A. Previous studies indicated that the one-pot method using CN113122528A yielded a product containing the final product L-arabinose, the substrate xylose, and the intermediate products D-xylulose and L-ribulose. Conventional separation methods cannot effectively separate L-arabinose from these components. Therefore, this invention provides a novel method for preparing and separating L-arabinose.

[0007] Solution for solving the problem

[0008] [1]. A method for obtaining L-arabinose, wherein the method comprises the following steps:

[0009] S1. Using D-xylose isomerase as a catalyst, D-xylose is converted into D-xylulose to obtain a mixture containing D-xylose and D-xylulose.

[0010] S2. Using a simulated moving bed, the mixture containing D-xylose and D-xylulose obtained in step S1 is separated to obtain an adsorbent solution BD1 containing D-xylose and an adsorbent solution AD1 containing D-xylulose; the adsorbent solution BD1 containing D-xylose is returned to step S1 for catalytic reaction.

[0011] S3. Using D-xylulose 4-epimerase and L-arabinose isomerase as catalysts, the D-xylulose in the adsorbent solution AD1 containing D-xylulose obtained in step S2 is converted into L-arabinose, to obtain a mixture containing D-xylulose, L-ribulose and L-arabinose.

[0012] S4. Using a simulated moving bed, separate the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 to obtain an adsorbent solution BD2 containing L-arabinose and an adsorbent solution AD2 containing D-xylulose and L-ribulose; return the adsorbent solution AD2 containing D-xylulose and L-ribulose to step S3 for catalytic reaction.

[0013] [2]. According to the method described in [1], wherein the enzyme-catalyzed conversion reaction of step S1 and / or step S3 is carried out under aerobic, microaerobic or anaerobic conditions, preferably under anaerobic conditions;

[0014] The enzyme-catalyzed conversion reaction in step S1 and / or step S3 is carried out at 30°C-90°C, preferably at 40°C-80°C, and more preferably at 50°C-70°C.

[0015] The enzyme-catalyzed conversion reaction system in step S1 and / or step S3 contains metal ions; optionally, the metal ions are one or more divalent metal ions; preferably, the metal ions are Cu. 2+ Fe 2+ Zn 2+ Ca 2+ Mg 2+ Co 2 + Ni 2+ Mn 2+ One, two, three or more of them.

[0016] [3]. According to the method described in [1] or [2], wherein the packing material of the simulated moving bed in steps S2 and S4 is a sodium-type cation exchange resin;

[0017] Optionally, the sodium-type ion exchange resin is selected from Biorad Chelex. TM One, two, three, or more of the following: 100 sodium-type ion exchange resin, Amberlite IR120 sodium-type ion exchange resin, Dowex 50-X8 sodium-type ion exchange resin, 001×7 sodium-type ion exchange resin, Lewatit S-100 sodium-type ion exchange resin, and Diaion SK-1B sodium-type ion exchange resin.

[0018] [4]. The method according to any one of [1] to [3], wherein in steps S2 and S4, chromatographic separation is performed using a simulated moving bed, and the temperature of the chromatographic separation is controlled at 50-80℃; preferably 55-70℃.

[0019] [5]. The method according to any one of [1] to [4], wherein in steps S2 and S4, chromatographic separation is performed using a simulated moving bed and pure water is used as the eluent for chromatographic separation.

[0020] [6]. The method according to any one of [1] to [5], wherein the method for obtaining L-arabinose further includes:

[0021] Step A1, a step prior to step S2, to concentrate the mixture containing D-xylose and D-xylulose obtained in step S1; and / or

[0022] Step B1 is a step of concentrating the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 before step S4.

[0023] [7]. According to the method of [6], wherein the method for obtaining L-arabinose further includes:

[0024] Step A2, a desalting step of the mixture containing D-xylose and D-xylulose obtained in step S1 prior to step A1; and / or

[0025] Step B2 is a desalting step performed on the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 prior to step B1.

[0026] [8]. According to the method of [7], wherein the method for obtaining L-arabinose further comprises:

[0027] Step A3, a step of decolorizing the mixture containing D-xylose and D-xylulose obtained in step S1 prior to step A2; and / or

[0028] Step B3 is a step of decolorizing the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 prior to step B2.

[0029] [9]. According to the method of [8], wherein the method for obtaining L-arabinose further includes:

[0030] Step A4, prior to step A3, involves removing bacterial cells and / or proteins from the mixture containing D-xylose and D-xylulose obtained in step S1; and / or

[0031] Step B4: Prior to step B3, the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 is subjected to a step of removing bacterial cells and / or proteins.

[0032]

[10] . The method according to any one of [1] to [9], wherein the method for obtaining L-arabinose further includes the step of concentrating and crystallizing the absorbent BD2 containing L-arabinose in step S4.

[0033] The effects of the invention

[0034] Based on the specific separation of substrates, products, and intermediates in the L-arabinose reaction solution, and considering material balance and equipment investment in each unit process, the inventors of this invention have developed a stepwise enzyme-catalyzed coupling process for L-arabinose production based on simulated moving bed separation. This process has the advantages of simple separation and purification, environmental friendliness, and the purity of L-arabinose crystals obtained through crystallization is ≥99%. At the same time, the simulated moving bed enables the recycling of substrate D-xylose and intermediates D-xylulose and L-ribulose, which helps to reduce production costs and thus promote the industrial production of L-arabinose. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the L-arabinose separation and preparation process. Wherein, XI: D-xylose isomerase (EC 5.3.1.5); Xu4E: D-xylulose 4-epimerase; AI: L-arabinose isomerase (EC 5.3.1.4); SMB: simulated moving bed.

[0036] Figure 2 The diagrams show the sequential operation of a moving bed, with (a) representing the dual-inlet / dual-outlet stage, (b) the circulation stage, and (c) the inlet / outlet (BD) stage. AD represents the adsorbate after separation in the simulated moving bed, and BD represents the residual adsorbate after separation in the simulated moving bed.

[0037] Figure 3 This is an HPLC chromatogram showing the content of D-xylose and D-xylulose in the reaction solution at equilibrium of the D-xylose to D-xylulose conversion reaction.

[0038] Figure 4 This is an HPLC chromatogram of the detection of D-xylitol in adsorbent AD1 after sequential simulated moving bed separation.

[0039] Figure 5 This is an HPLC chromatogram of D-xylose detection in the residual liquid BD1 after sequential simulated moving bed separation. Detailed Implementation

[0040] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0041] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0042] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0043] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0045] To enable those skilled in the art to understand the features and effects of this invention, the terms and expressions used in the specification and claims are generally described and defined below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding this invention, and in case of conflict, the definitions in this specification shall prevail.

[0046] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0047] In this invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0048] In this invention, unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0049] The terms “preferred” and “ideal” as used herein are not intended to limit the scope of the claimed invention or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to emphasize alternative or additional features that may or may not be used in particular embodiments of the invention.

[0050] In this invention, "optional" and "optionally" mean that the event or situation described below may or may not occur, and the description includes both cases in which the event or situation occurs and cases in which the event or situation does not occur.

[0051] In this invention, the range of values ​​represented by "value A to value B" refers to the range that includes the endpoint values ​​A and B.

[0052] Unless otherwise specified, the term "comprising" as used in this invention can be either open-ended or closed-ended. For example, "comprising" may mean that it may also include other components not listed, or it may include only the listed components.

[0053] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0054] In this invention, when "room temperature" or "room temperature" is used, the temperature can be 23±2℃.

[0055] As used herein, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0056] As used in this invention, although other organic or inorganic catalysts may be used, the term "converting" refers to a chemical transformation from one molecule to another catalyzed primarily by one or more polypeptides (enzymes); it may also refer to the ratio (in %) between the molar amount of the desired product and the molar amount of the limited substrate.

[0057] In this invention, the "adsorption liquid" refers to the liquid containing the components selectively adsorbed by the adsorbent during the adsorption or separation process.

[0058] In this invention, the "residual liquid" refers to the liquid that flows out directly without being adsorbed by the adsorbent during the adsorption or separation process.

[0059] The technical solution of the present invention will be described in detail below:

[0060] Based on the enzymatic method for producing L-arabinose disclosed in the prior patent CN113122528A, the product produced by the one-pot method in this patent includes the final product L-arabinose, the substrate xylose, the intermediate product D-xylulose and L-ribulose, and it is necessary to separate L-arabinose from other intermediate products and substrates. However, the inventors discovered through research that when separating the products of the one-pot reaction, on various ion exchange resins, either the final product L-arabinose cannot be baseline separated from the intermediate products D-xylulose and L-ribulose, as well as the substrate xylose, or the final product L-arabinose overlaps with the intermediate products D-xylulose and / or L-ribulose, making it difficult to separate the final product L-arabinose in one step. Therefore, this invention effectively combines enzyme reaction with substance separation, utilizing the high efficiency of enzyme reaction while also effectively separating the substrate, intermediate products, and final product. Furthermore, the substrate and intermediate products separated during the reaction can be added back to the enzyme reaction system to continue the reaction, achieving efficient utilization of the substrate and intermediate products.

[0061] <Methods for preparing and isolating L-arabinose>

[0062] This invention provides a method for obtaining L-arabinose, as illustrated in the schematic diagram below. Figure 1 As shown.

[0063] In some specific implementations, the method includes the following steps:

[0064] S1: Using D-xylose isomerase as a catalyst, D-xylose is converted into D-xylulose to obtain a mixture containing D-xylose and D-xylulose;

[0065] S2: The mixture containing D-xylose and D-xylulose obtained in step S1 is separated using a simulated moving bed to obtain an adsorbent solution BD1 containing D-xylose and an adsorbent solution AD1 containing D-xylulose. The adsorbent solution BD1 containing D-xylose is returned to step S1 for catalytic reaction.

[0066] S3: Using D-xylulose 4-epimerase and L-arabinose isomerase as catalysts, the D-xylulose in the D-xylulose-rich adsorbent solution AD1 obtained in step S2 is converted into L-arabinose, resulting in a mixture containing D-xylulose, L-ribulose and L-arabinose.

[0067] S4: Using a simulated moving bed, the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 is separated to obtain an adsorbent BD2 containing L-arabinose and an adsorbent AD2 containing the mixture of D-xylulose and L-ribulose. The adsorbent AD2 containing the mixture of D-xylulose and L-ribulose is returned to step S3 for catalytic reaction.

[0068] In some embodiments of the present invention, separation is performed using a simulated moving bed in steps S2 and S4, wherein the packing material of the simulated moving bed is a sodium-type cation exchange resin. The sodium-type cation exchange resin can be Biorad Chelex. TM One or more of the following: 100 sodium-type ion exchange resin, Amberlite IR120 sodium-type ion exchange resin, Dowex 50-X8 sodium-type ion exchange resin, 001×7 sodium-type ion exchange resin, Lewatit S-100 sodium-type ion exchange resin, Diaion SK-1B sodium-type ion exchange resin.

[0069] In some embodiments of the present invention, in steps S2 and S4, chromatographic separation is performed using a simulated moving bed, and the temperature of the chromatographic separation, i.e., the column temperature of the simulated moving bed column, is controlled at 50-80°C, preferably 55-70°C. Further, pure water is used as the eluent for chromatographic separation.

[0070] In some embodiments of the present invention, in steps S2 and S4, the feed sugar concentration of the simulated moving bed is not less than 30% (mass fraction percentage), preferably not less than 40% (mass fraction percentage), and more preferably not less than 45% (mass fraction percentage).

[0071] In some optional embodiments of the present invention, the mixture containing D-xylose and D-xylulose obtained in step S1, and the mixture containing D-xylulose, L-ribulose, and L-arabinose obtained in step S3, can be concentrated. The method of concentration is not particularly limited in the present invention and can be obtained by conventional methods in the art, such as evaporation concentration, ultrafiltration concentration, etc., to remove water from the solution containing the mixture so that the feed reaches the corresponding sugar concentration. For example, depending on the specific catalytic reaction, the method for separating and preparing L-arabinose further includes a concentration step, specifically a step of concentrating the mixture containing D-xylose and D-xylulose obtained in step S1 before step S2 and / or the mixture containing D-xylulose, L-ribulose, and L-arabinose obtained in step S3 before step S4.

[0072] In some optional embodiments of the present invention, depending on the specific catalytic reaction, the method for separating and preparing L-arabinose further includes a desalting step before the concentration step, specifically a desalting step of the mixture containing D-xylose and D-xylulose obtained in step S1 and / or the mixture containing D-xylulose, L-ribulose, and L-arabinose obtained in step S3. The desalting can be performed using cation and anion exchange resins, electrodialysis, etc. In some specific embodiments, the desalting is performed using a continuous ion exchange desalting system. The continuous ion exchange desalting system includes separation units comprising a gel-type strong cation exchange resin and a macroporous anion exchange resin, wherein the separation units are connected in series. After desalting, the conductivity of the sugar mixture is not higher than 150 μS / cm, not higher than 130 μS / cm, not higher than 110 μS / cm, not higher than 90 μS / cm, or not higher than 70 μS / cm.

[0073] In some optional embodiments of the present invention, depending on the specific catalytic reaction, the method for separating and preparing L-arabinose further includes a decolorization step, specifically a step of decolorizing the mixture containing D-xylose and D-xylulose obtained in step S1 and / or the mixture containing D-xylulose, L-ribulose, and L-arabinose obtained in step S3 before the desalting step. The decolorization can be performed using a decolorizing agent, such as powdered activated carbon, granular activated carbon, activated carbon fiber, macroporous anion exchange resin, etc. More preferably, the decolorization is performed using activated carbon. In the activated carbon decolorization step, the amount of activated carbon added is 0.1%-5% (mass fraction) of the sugar content, preferably 0.5%-1%; the activated carbon decolorization time is 15-60 min, preferably 30 min; and the decolorization temperature is 10-70°C, preferably room temperature.

[0074] In some embodiments of the present invention, depending on the specific catalytic reaction, the method for obtaining L-arabinose further includes removing bacterial cells and / or proteins from the mixture containing D-xylose and D-xylulose obtained in step S1 and / or the mixture containing D-xylulose, L-ribulose, and L-arabinose obtained in step S3 before the decolorization step. For example, bacterial cells and proteins in the reaction solution can be removed by microfiltration and / or ultrafiltration, or immobilized enzymes or immobilized cells in the reaction solution can be removed by filtration and / or centrifugation. The microfiltration can use a hollow fiber membrane, preferably a hollow fiber membrane with a pore size of 50-250 nm; or a ceramic membrane, preferably a ceramic membrane with a pore size of 20-200 nm. The ultrafiltration can use an ultrafiltration membrane with a molecular weight cutoff of 1-10 kD, preferably an ultrafiltration membrane with a molecular weight cutoff of 3-6 kD.

[0075] In some embodiments of the present invention, the method for separating and preparing L-arabinose further includes a concentration and crystallization step of the L-arabinose-rich blot solution BD2 from step S4, thereby obtaining L-arabinose crystals. The purity of the L-arabinose crystals can reach 99%.

[0076] <enzyme>

[0077] In this invention, there are no specific limitations on D-xylose isomerase, xylulose 4-epimerase, and xylulose 4-epimerase. The D-xylose isomerase has the function of converting D-xylose to D-xylulose; the xylulose 4-epimerase has the function of converting D-xylulose to L-ribulose; and the L-arabinose isomerase has the function of converting L-ribulose to L-arabinose. All proteins with corresponding functions from microorganisms, plants, animals, etc., are also included. That is, newly discovered enzymes are included as long as they have the corresponding function, and all can be applied to this invention.

[0078] In some alternative embodiments, the enzymes used in this invention are wild-type enzymes or enzymes that have undergone certain genetic modifications that enhance certain properties of the enzyme catalyst, such as activity, thermal stability, etc. Enzymes with desired properties derived from wild-type enzymes through genetic engineering are referred to as "mutants," "mutant derivatives," or "mutants derived from the corresponding wild-type enzyme." In this invention, the enzyme catalysts used to implement this invention include these performance-enhanced "mutants."

[0079] In some exemplary embodiments, the D-xylose isomerase used can be selected from commercially available immobilized enzymes, such as Novozymes immobilized glucose isomerase. IT can also be selected from D-xylose isomerases derived from microorganisms, such as the D-xylose isomerase derived from Thermus thermophiles disclosed in Chinese patent CN113122528A.

[0080] For the D-xylinosyl 4-epimerase used, one can choose from wild-type D-xylinosyl 4-epimerase from Thermotoga maritima disclosed in Chinese patent CN113122528A, or D-xylinosyl 4-epimerase mutants with excellent performance, such as D-xylinosyl 4-epimerase mutants M4 to M87 disclosed in CN113122528A.

[0081] The L-arabinose isomerase used can be selected from the L-arabinose isomerase derived from *Geobacillus stearothermophilus* disclosed in Chinese patent CN113122528A.

[0082] <Forms of Enzyme Use>

[0083] In this invention, the D-xylose isomerase and / or D-xylulose 4-epimerase and / or L-arabinose isomerase can be used directly as pure enzymes (i.e., free enzymes), or the pure enzymes can be immobilized (i.e., immobilized enzymes) to maintain stability and recyclability; alternatively, whole-cell microorganisms or cultures of whole-cell microorganisms (i.e., fermentation products) containing the D-xylose isomerase and / or D-xylulose 4-epimerase and / or L-arabinose isomerase can be used directly, or the whole cells can be immobilized (i.e., immobilized cells) to maintain stability and recyclability. Immobilization methods are known in the art; for example, immobilization carriers such as activated carbon, silica gel, quartz sand, sodium alginate, and ion exchange resins can be used to immobilize crude enzyme solutions, pure enzymes, or whole cells.

[0084] <Enzyme-catalyzed reaction conditions>

[0085] In some embodiments of the present invention, the enzymatic conversion reaction of step S1 and / or step S3 can be carried out under aerobic, microaerobic, or anaerobic conditions. In other embodiments of the present invention, the enzymatic conversion reaction of step S1 and / or step S3 is carried out under anaerobic conditions (i.e., in the absence of detectable oxygen).

[0086] In some embodiments of the present invention, the enzymatic conversion reaction in step S1 and / or step S3 is carried out at 30°C-90°C. In some specific embodiments of the present invention, the enzymatic conversion reaction in step S1 and / or step S3 is carried out at 40°C-80°C. In some more specific embodiments of the present invention, the enzymatic conversion reaction in step S1 and / or step S3 is carried out at 50°C-70°C.

[0087] In some embodiments of the present invention, the enzyme-catalyzed conversion reaction system of step S1 and / or step S3 may contain metal ions. The metal ions may be one or more metal ions. For example, the metal ions may be selected from Cu. 2+ Fe 2+ Zn 2+ Ca 2+ Mg 2+ Co 2+ Ni 2+ or Mn 2+ .

[0088] Example

[0089] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of the invention) are given for illustrative purposes only, as various changes and modifications made within the spirit and scope of the invention will become apparent to those skilled in the art upon reading this detailed description.

[0090] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. Unless otherwise stated, the raw materials and reagents used in the following embodiments are commercially available products, or can be prepared by known methods.

[0091] Enzyme preparation

[0092] D-xylose isomerases are commercially available immobilized enzymes, such as Novozymes immobilized glucose isomerase. IT. The D-xylinose 4-epimerase is the wild-type D-xylinose 4-epimerase and / or its mutants from *Thermotoga maritima*, as disclosed in Chinese Patent CN113122528A, such as the D-xylinose 4-epimerase mutant M87 disclosed in CN113122528A. The L-arabinose isomerase is the L-arabinose isomerase from *Geobacillus stearothermophilus*, as disclosed in Chinese Patent CN113122528A. The above enzymes are incorporated herein by reference.

[0093] The above enzymes can be purified by heat treatment (50-80℃, 10-60 min). After heat treatment and centrifugation, the supernatant of the cell lysate is the enzyme used in the enzyme-catalyzed reaction that produces D-xylulose and L-arabinose.

[0094] Intermediate and product detection methods

[0095] The concentrations of D-xylose and D-xylulose were determined using a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a differential refractive index detector. The chromatographic column was a Bio-Rad Aminex HPX-87H sugar analysis column, the column temperature was 60℃, the mobile phase was 5 mM sulfuric acid, the flow rate was 0.6 mL / min, and the injection volume was 20 μL.

[0096] The concentrations of D-xylulose, L-ribulose, and L-arabinose were determined using a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a differential refractive index detector. The chromatographic column was a Shodex Sugar KS-801 sodium ion-exchange column, the column temperature was 70°C, the mobile phase was deionized water, the flow rate was 0.5 mL / min, and the injection volume was 10 μL.

[0097] Simulated mobile bed

[0098] The simulated moving bed separation used in the substance separation steps all employs a sequential simulated moving bed. The sequential simulated moving bed chromatographic separation system is equipped with n chromatographic columns, which are connected in series, forming a closed system, and are sequentially designated as column 1 to column n, where n ≥ 6. The volume of each column is preferably 400–800 mL. Each column is equipped with an inlet, a water inlet, a circulation outlet, and an outlet. The columns are preferably insulated with jackets to maintain a constant system temperature. The sequential simulated moving bed chromatographic separation system is controlled by a computer system. The operating program of the sequential simulated moving bed chromatographic separation system includes several separation cycles; each separation cycle includes a sequentially performed dual-inlet / dual-outlet stage, a circulation stage, and a water-inlet / BD-outlet stage. The operational diagrams of the dual-inlet / dual-outlet stage, circulation stage, and water-inlet / BD-outlet stage are shown below. Figure 2 As shown.

[0099] In this embodiment, the sequential simulated moving bed chromatography system is equipped with six chromatographic columns connected in series, each with a volume of 700 mL, and the packing material is Biorad Chelex. TM Sodium-type ion exchange resin.

[0100] In this embodiment, the process of separating a mixture containing D-xylose and D-xylulose using a simulated moving bed is as follows: Figure 2 As shown:

[0101] In the first separation cycle, the dual-inlet dual-outlet stage involves water entering through the inlet of column 1, a mixture of D-xylose and D-xylulose entering through the feed inlet of column 4, an adsorbent solution AD1 rich in D-xylulose being collected at the outlet of column 1, and a residual adsorbent solution BD1 rich in D-xylose being collected at the outlet of column 5. During the circulation stage, no material enters or exits, and the material circulates within the column. In the water-inlet-BD-outlet stage, water enters through the inlet of column 2, and a residual adsorbent solution BD1 rich in D-xylose is collected at the outlet of column 6. After the first separation cycle is completed, the inlet for the mixture of D-xylose and D-xylulose, the inlet for water, and the outlet for receiving D-xylulose or D-xylulose are sequentially moved to the next column from column 1 to column n. The double-in, double-out, circulation, and water-in-BD-out stages are repeated to complete the next separation cycle until separation is finished. For example, when there are 6 columns, in the second separation cycle, water is introduced into the inlet of column 2 during the double-in, double-out stage, the mixture of D-xylose and D-xylulose is introduced into the inlet of column 5, the adsorbate AD1 rich in D-xylulose is received at the outlet of column 2, and the residual adsorbate BD1 rich in D-xylose is received at the outlet of column 6. During the circulation stage, no material enters or exits, and the material circulates within the column. During the water-in-BD-out stage, water is introduced into the inlet of column 3, and the residual adsorbate BD1 rich in D-xylose is received at the outlet of column 1. The third cycle follows the same pattern until separation is finished. In this invention, completing the above-mentioned double-inlet double-outlet stage, circulation stage, and water inlet BD outlet stage once is recorded as a small cycle. When all n chromatographic columns in the simulated moving bed chromatography separation system have completed one small cycle, it is recorded as one large cycle. After one large cycle is completed, the same procedure is followed for the next large cycle until the material processing is completed.

[0102] In this embodiment, the process of separating a mixture containing D-xylulose, L-ribulose, and L-arabinose using a simulated moving bed is as follows:

[0103] In the first separation cycle, the dual-inlet dual-outlet stage involves water entering through the inlet of column 1, a mixture of D-xylulose, L-ribulose, and L-arabinose entering through the feed inlet of column 4, an adsorbent solution AD2 rich in the mixture of D-xylulose and L-ribulose being collected at the outlet of column 1, and a residual adsorbent solution BD2 rich in L-arabinose being collected at the outlet of column 5. During the circulation stage, no material enters or exits, and the material circulates within the column. In the water-inlet-BD-outlet stage, water enters through the inlet of column 2, and a residual adsorbent solution BD2 rich in L-arabinose is collected at the outlet of column 6. After the first separation cycle is completed, the inlet for the mixture of D-xylulose, L-ribulose, and L-arabinose, the inlet for water, and the outlet for receiving the mixture of D-xylulose and L-ribulose or L-arabinose are sequentially moved to the next column from column 1 to column n. This process of double-inlet / double-outlet, circulation, and water-inlet / BD-outlet stages is repeated to complete the next separation cycle until separation is finished. For example, when there are 6 columns, in the second separation cycle, water is introduced into the inlet of column 2 during the double-inlet / double-outlet stage, the mixture of D-xylulose, L-ribulose, and L-arabinose is introduced into the inlet of column 5, and the mixture rich in D-xylulose and L-ribulose is received at the outlet of column 2. The adsorbent AD2 of the compound is collected at the outlet of column 6, and the residual liquid BD2 rich in L-arabinose is received. During the circulation phase, the material does not enter or exit, and the material circulates within the chromatographic column. During the water-in-BD-out phase, water is introduced at the inlet of column 3, and the residual liquid BD2 rich in L-arabinose is received at the outlet of column 1. The third cycle is repeated in this manner until the separation is completed. In this invention, the completion of the above-mentioned double-in-double-out phase, circulation phase, and water-in-BD-out phase is recorded as a small cycle. When all n chromatographic columns in the simulated moving bed chromatography separation system have completed one small cycle, it is recorded as a large cycle. After the completion of a large cycle, the same procedure is followed for the next large cycle until the material processing is completed.

[0104] Example 1

[0105] Enzymatic preparation of D-xylulose: In a 50 L reaction system, the reaction mixture contained 2 M D-xylose substrate and 5 mM Mg 2+ 50 g / L immobilized D-xylose isomerase and 50 mM HEPES buffer (pH 7.5) were used for anaerobic reaction at 70 °C. After the reaction reached equilibrium, the contents of D-xylose and D-xylulose in the reaction solution were detected by HPLC. The HPLC results are shown below. Figure 3 As shown in the figure. The results showed that when the reaction reached equilibrium, the D-xylose content was 1.3 ± 0.13 M and the D-xylulose content was 0.7 ± 0.07 M.

[0106] Decolorization and desalting of the reaction solution: After the reaction, the immobilized enzyme was removed by centrifugation, yielding a supernatant of a mixture of D-xylose and D-xylulose. Activated carbon (1% by weight of total sugar) was added to the supernatant, and the solution was decolorized for 30 min at room temperature. The activated carbon was then filtered using a plate and frame filter press to obtain a filtrate of the mixture of D-xylose and D-xylulose. The filtrate was then desalted sequentially through a continuous ion exchange system containing a gel-type strong cation exchange resin (001X7, hydrogen form) and a macroporous weakly basic anion exchange resin (D380, hydroxide form) at a flow rate of 30 mL / min. The conductivity at the outlet of the anion exchange unit after passing through the continuous ion exchange system was 40 μS / cm. Resin regeneration was performed simultaneously with desalting: 5% hydrochloric acid was used for regeneration of the cation exchange resin at a feed rate of 50 mL / min; 5% sodium hydroxide was used for regeneration of the anion exchange resin at a feed rate of 50 mL / min.

[0107] Separation of the mixture: The desalted mixture of D-xylose and D-xylulose was concentrated to a sugar concentration of approximately 50% (mass fraction percentage), and then chromatographic separation was performed using a sequential simulated moving bed packed with sodium-type ion exchange resin. Pure water was used as the eluent, and the temperature was controlled at 65°C during the separation process. The chromatographic separation control parameters are shown below.

[0108] step Time s Feed flow rate mL / min Inlet flow rate (mL / min) Circulation flow rate (mL / min) Double in, double out 543 37.4 40 - cycle 1166 - - 40 Water inlet and BD outlet 111 - 40 -

[0109] After sequential simulated moving bed separation, the purity of D-xylulose in adsorption solution AD1 was 99%, and the purity of D-xylulose in adsorption residue BD1 was also 99%. The HPLC detection results are as follows: Figure 4 and Figure 5 As shown.

[0110] The D-xylose in the adsorbent BD1 can be returned to the enzyme-catalyzed D-xylulose preparation step for the D-xylulose preparation reaction, while the D-xylulose in the adsorbent AD1 is used as a substrate for the preparation of L-arabinose.

[0111] Example 2

[0112] Enzymatic preparation of L-arabinose: In a 100 L reaction system, the reaction mixture contained 0.5 M D-xylitol substrate and 0.2 mM Co 2+ 1mM Mn 2+ The reaction mixture was prepared using 45 U / mL D-xylinose 4-epimerase mutant M87, 40 U / mL L-arabinose isomerase, and 100 mM Tris buffer (pH 8.5). An anaerobic reaction was carried out at 50 °C. After equilibrium was reached, the L-arabinose content in the reaction solution was determined by HPLC. The results showed that the L-arabinose content was 0.23 M at equilibrium.

[0113] Decolorization and desalting of the reaction solution: After the reaction, the reaction solution was filtered through a hollow fiber column (hollow fiber membrane with a pore size of 220 nm). The resulting filtrate was then ultrafiltered through a 10 kDa ultrafiltration membrane. After passing through the hollow fiber column and ultrafiltration, almost all proteins in the reaction solution were removed, and most pigments were also removed. The mixture containing D-xylulose, L-ribulose, and L-arabinose after passing through the hollow fiber column and ultrafiltration was then passed sequentially at a flow rate of 40 mL / min through a continuous ion exchange system containing a gel-type strong cation exchange resin (001X7) and a macroporous weakly basic anion exchange resin (D380). The conductivity at the outlet of the anion exchange unit after passing through the continuous ion exchange system was 66 μS / cm. Resin regeneration was performed simultaneously with desalting: 5% hydrochloric acid was used for regeneration of the cation exchange resin at a feed rate of 50 mL / min; 5% sodium hydroxide was used for regeneration of the anion exchange resin at a feed rate of 50 mL / min.

[0114] Separation of the mixture: The desalted mixture of D-xylulose, L-ribulose, and L-arabinose was concentrated to a sugar concentration of approximately 45% (mass fraction percentage), and then chromatographic separation was performed using a sequential simulated moving bed packed with sodium-type ion exchange resin. Pure water was used as the eluent, and the temperature was controlled at 55°C during the separation process. The chromatographic separation control parameters are shown below.

[0115] step Time s Feed flow rate mL / min Inlet flow rate (mL / min) Circulation flow rate (mL / min) Double in, double out 300 25 35 - cycle 1560 - - 35 Water inlet and BD outlet 95 - 35 -

[0116] After sequential simulated moving bed separation, the purity of L-arabinose in the residual liquid BD2 was 97%.

[0117] The mixture of D-xylulose and L-ribulose in the adsorbent AD2 was used as a substrate in the enzyme-catalyzed preparation of L-arabinose.

[0118] Example 3

[0119] The L-arabinose solution obtained after chromatographic separation was evaporated and concentrated to a sugar concentration of approximately 63% under a vacuum of 0.08 MPa and a feed temperature of 60 °C. After stabilizing at 65 °C for 30 min, a programmed cooling process was initiated, with a cooling rate of 4 °C / h. When the temperature dropped to 57 °C, seed crystals containing 0.3% (mass fraction) of total sugar were added, and the temperature was further reduced to 25 °C at a cooling rate of 4 °C / h. The crystals were collected by centrifugation, and the obtained L-arabinose crystals were dried in a vacuum drying oven at 40 °C for 8 h. The purity of the L-arabinose crystals was determined to be 99.9% by HPLC.

Claims

1. A method for obtaining L-arabinose, wherein, The method includes the following steps: S1. Using D-xylose isomerase as a catalyst, D-xylose is converted into D-xylulose to obtain a mixture containing D-xylose and D-xylulose. S2. Using a simulated moving bed, the mixture containing D-xylose and D-xylulose obtained in step S1 is separated to obtain an adsorbent solution BD1 containing D-xylose and an adsorbent solution AD1 containing D-xylulose; the adsorbent solution BD1 containing D-xylose is returned to step S1 for catalytic reaction. S3. Using D-xylulose 4-epimerase and L-arabinose isomerase as catalysts, the D-xylulose in the adsorbent solution AD1 containing D-xylulose obtained in step S2 is converted into L-arabinose, to obtain a mixture containing D-xylulose, L-ribulose and L-arabinose. S4. Using a simulated moving bed, separate the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 to obtain an adsorbent solution BD2 containing L-arabinose and an adsorbent solution AD2 containing D-xylulose and L-ribulose; return the adsorbent solution AD2 containing D-xylulose and L-ribulose to step S3 for catalytic reaction.

2. The method according to claim 1, wherein, The enzyme-catalyzed conversion reaction in step S1 and / or step S3 is carried out under aerobic, microaerobic or anaerobic conditions, preferably under anaerobic conditions. The enzyme-catalyzed conversion reaction in step S1 and / or step S3 is carried out at 30°C-90°C, preferably at 40°C-80°C, and more preferably at 50°C-70°C. The enzyme-catalyzed conversion reaction system in step S1 and / or step S3 contains metal ions; optionally, the metal ions are one or more divalent metal ions; preferably, the metal ions are Cu. 2+ Fe 2+ Zn 2+ Ca 2+ Mg 2+ Co 2+ Ni 2 + Mn 2+ One, two, three or more of them.

3. The method according to claim 1 or 2, wherein, The packing material for the simulated moving bed in steps S2 and S4 is sodium-type cation exchange resin. Optionally, the sodium-type ion exchange resin is selected from Biorad Chelex. TM One, two, three, or more of the following: 100 sodium-type ion exchange resin, Amberlite IR120 sodium-type ion exchange resin, Dowex 50-X8 sodium-type ion exchange resin, 001×7 sodium-type ion exchange resin, Lewatit S-100 sodium-type ion exchange resin, and Diaion SK-1B sodium-type ion exchange resin.

4. The method according to any one of claims 1 to 3, wherein, In steps S2 and S4, chromatographic separation is performed using a simulated moving bed, and the temperature of the chromatographic separation is controlled at 50-80℃; preferably 55-70℃.

5. The method according to any one of claims 1 to 4, wherein, In steps S2 and S4, chromatographic separation is performed using a simulated moving bed, with pure water as the eluent.

6. The method according to any one of claims 1 to 5, wherein, The method for obtaining L-arabinose further includes: Step A1, a step prior to step S2, to concentrate the mixture containing D-xylose and D-xylulose obtained in step S1; and / or Step B1 is a step of concentrating the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 before step S4.

7. The method according to claim 6, wherein, The method for obtaining L-arabinose further includes: Step A2, a desalting step of the mixture containing D-xylose and D-xylulose obtained in step S1 prior to step A1; and / or Step B2 is a desalting step performed on the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 prior to step B1.

8. The method according to claim 7, wherein, The method for obtaining L-arabinose further includes: Step A3, a step of decolorizing the mixture containing D-xylose and D-xylulose obtained in step S1 prior to step A2; and / or Step B3 is a step of decolorizing the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 prior to step B2.

9. The method according to claim 8, wherein, The method for obtaining L-arabinose further includes: Step A4, prior to step A3, involves removing bacterial cells and / or proteins from the mixture containing D-xylose and D-xylulose obtained in step S1; and / or Step B4: Prior to step B3, the mixture containing D-xylulose, L-ribulose and L-arabinose obtained in step S3 is subjected to a step of removing bacterial cells and / or proteins.

10. The method according to any one of claims 1 to 9, wherein, The method for obtaining L-arabinose further includes the steps of concentrating and crystallizing the absorbent solution BD2 containing L-arabinose from step S4.

Images