Pulullan production method

Abstract

The invention provides a brand-new method for producing and separating pullulan by a conversion method, which mainly takes aureobasidium pullulans EP1001 as a starting strain, carries out conversion culture on saccharides which contain molecular markers and take glucose as a basic unit, and carries out specific directional separation on the pullulan with the molecular markers in a conversion solution by a conversion product under the action of magnetic force, thereby obtaining the pullulan with the molecular markers. Magnetic beads and a pullulan polysaccharide compound are dissociated from the separated product through enzymatic hydrolysate, molecular markers are further decomposed, and high-purity pullulan polysaccharide is finally obtained after organic solvent precipitation. The separation and extraction process does not need the steps of flocculant pretreatment, activated carbon adsorption decoloration, oxidation decoloration, protease decomposition deproteinization, ion exchange desalination and the like, the experimental process is simple, the product yield and purity are high, and the quality is excellent. The yield of the finally obtained pullulan is as high as 94.58 g / L, the purity is 99.80%, and the whiteness value is 99.62.

Description

Technical Field

[0001] The invention belongs to the field of microbial pharmacy, and specifically relates to a method for producing pullulan by a budding Aureobasidium pullulans biotransformation method and a method for separating and purifying pullulan. Background Art

[0002] Pullulan is a new type of polymer biomaterial with excellent film-forming, fiber-forming, gas barrier, bonding, easy processing, non-toxicity and other properties. It has been widely used in food, medicine, light industry, chemical industry and petroleum. Studies have shown that pullulan is first combined with glucose by α-1, 4 glycosidic bonds to form maltotriose, and then the two ends are combined with α-1, 6 glycosidic bonds to combine maltotriose, and the ratio of α-(1-4) bond to α-(1-6) bond is 2:1. Therefore, carbon source is a significant factor in the process of pullulan production by budding Aureobasidium pullulans. The carbon sources that can be used in the synthesis of pullulan include glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin and agricultural waste, etc. They can be used by cells as nutrients and can also participate in the construction of pullulan molecules. Sucrose has been proven to be the most ideal carbon source for pullulan fermentation production.

[0003] The disclosed methods for extracting pullulan include precipitation method and direct drying method. The most mature methods in the precipitation method are alcohol precipitation method and salt precipitation method. Commonly used alcohol precipitants include methanol, ethanol, isopropanol, etc.; Commonly used salt precipitants mainly include quaternary ammonium salts, calcium chloride, calcium hydroxide, and aluminum chloride, etc.; The advantage of the salt precipitation method is that the amount of alcohol used is 2-3 times less than that of the alcohol precipitation method, but the product quality is slightly worse. The direct drying method is to evaporate the water from the fermentation liquid and directly dry it into a solid product. Freeze drying in the laboratory is the best way to achieve this goal. The products obtained by industrial methods such as drum baking, blast drying, and spray drying are all crude pullulan products, which contain a large number of impurities such as bacteria, inorganic salts, and organic residues. The product has poor water solubility, dark color, and poor rheology, and its application range is limited.

[0004] The extraction steps of pullulan mainly include pretreatment, decolorization, deproteinization and desalination. The main decolorization methods include activated carbon adsorption, ion exchange, oxidative decolorization and metal complex method. Although activated carbon decolorization is more thorough, the adsorption of polysaccharides by activated carbon will cause the loss of polysaccharides; the ion exchange method uses weak alkaline resin to adsorb pigments. This method is effective for free negative ion pigments, but not for pigments bound to sugar; the oxidative decolorization method mainly uses oxidants to oxidize and remove pigments under appropriate conditions. The main deproteinization methods include Sevage method, trichloroacetic acid method, tannic acid method, trifluorotrichloroethane method and protease method. The decolorized and deproteinized polysaccharide solution can be separated by ethanol graded precipitation, and membrane technology can be used to remove salts and small molecules.

[0005] At present, most of the production methods of pullulan are still based on budding Aureobasidium as the starting strain, with organic carbon sources such as glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin, etc. as the skeleton, and pullulan is produced by microbial fermentation. The fermentation broth undergoes a series of flocculant pretreatment, activated carbon adsorption decolorization, oxidation decolorization, protease decomposition deproteinization, ion exchange desalination, acetone or ethanol precipitation and other steps. The whole production process is very complicated. Usually, in order to improve the purity of the product, the precipitated pullulan is subjected to multiple dissolution and precipitation processes, which consumes a large amount of organic solvents, has a long processing cycle and a high production cost. If hot air or drum drying is used, if the temperature is not well controlled, it is easy to cause the product color to turn yellow, affecting the product quality. Summary of the invention

[0006] In order to overcome the above problems, one of the purposes of the present invention is to provide a method for producing pullulan by transformation of Aureobasidium pullulans, which is produced by transforming molecularly labeled sugars with glucose as the basic unit as a carbon source. The Aureobasidium pullulans strain was deposited in the China General Microbiological Culture Collection Center (CGMCC for short) on April 19, 2022; the deposit number is CGMCC No.40158, and the classification name is Aureobasidium pullulans, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Another object of the present invention is to provide a conversion liquid composition for producing pullulan by the budding Aureobasidium pullulans transformation method. Nuclease inhibitors and / or protease inhibitors are added to the conversion liquid, mainly to inhibit the activity of ribonucleases and / or deoxyribonucleases and / or proteases produced during the transformation of budding Aureobasidium pullulans, to prevent ribonucleases and / or deoxyribonucleases and / or proteases from decomposing the molecular markers linked to the substrate (molecularly labeled sugars with glucose as the basic unit) and / or the product (pululan), thereby affecting the separation and extraction of the pullulan product.

[0008] Another object of the present invention is to provide a method for separating and extracting pullulan, wherein the molecular markers coated on the surface of magnetic beads are combined with the pullulan with molecular markers in the product to form a complex, and under the action of an external magnetic force, the pullulan in the conversion liquid is specifically and directionally separated, and the molecular markers of the separated product are dissociated by an enzymatic solution to finally obtain high-purity pullulan.

[0009] Another object of the present invention is to provide a composition of an enzymatic hydrolyzate for decomposing molecular markers, wherein nuclease and / or protease are added to the enzymatic hydrolyzate, the main purpose of which is to dissociate the magnetic beads and pullulan complex and further decompose the molecular markers linked to the pullulan molecules, so as to prepare for the subsequent precipitation of high-quality pullulan with an organic solvent.

[0010] The pullulan polysaccharide extraction method provided by the present invention is that the nucleic acid and / or protein and / or antibody and / or other identifiable markers coated on the surface of the magnetic beads are combined with the nucleic acid and / or protein and / or antibody and / or other identifiable pullulan polysaccharides in the product to form a complex, and under the action of magnetic force, the pullulan polysaccharides with molecular markers in the conversion liquid are specifically and directional separated, and the separated product is dissociated from the magnetic beads and the pullulan polysaccharide complex by an enzymatic solution, and the molecular markers are further decomposed, and after precipitation with an organic solvent, a high-purity pullulan polysaccharide is finally obtained; the entire separation and extraction process does not require flocculant pretreatment, activated carbon adsorption decolorization, oxidation decolorization, protease decomposition deproteinization, ion exchange desalting, and other steps, the experimental process is simple, the product yield and purity are high, and the quality is excellent. The product is adsorbed, desorbed, decomposed by the enzymatic solution, and ethanol precipitated, and the final pullulan polysaccharide has a yield of up to 94.58g / L, a purity of 99.80%, and a whiteness value of 99.62 (Table 1).

[0011] The invention provides a method for producing pullulan, which mainly comprises the following steps.

[0012] The steps of producing pullulan by the Aureobasidium pullulans transformation method.

[0013] Seed plate culture: The EP1001 strain, which is deposited in the China General Microbiological Culture Collection Center and has a deposit number of CGMCC No. 40158, is inoculated into the PDA plate culture medium by streaking with an inoculation needle for activation culture; the specific steps are: take out the budding Acidis pulvinus strain EP1001 from the minus 80 refrigerator, naturally dissolve it on ice, transfer it to the PDA plate culture medium on a sterile clean bench with an inoculation needle, and culture it in a constant temperature incubator at 30°C in the dark for 72 hours.

[0014] Seed liquid culture: The budding Aureobasidium strain EP1001 activated in the above step was transferred to a liquid seed culture medium and cultured for 24 hours. The specific steps are: a single colony of the culture activated and cultured on the PDA plate medium for 72 hours was inoculated into a 500ml Erlenmeyer flask containing 100ml of liquid seed culture medium, the culture temperature was 30°C, the shaker speed was 220rpm, and the shaking culture was performed for 24 hours to obtain a liquid culture of the budding Aureobasidium strain.

[0015] Transformation culture: 5.0L bioreactor, liquid volume 0.5L-2.0L, inoculation size 5%-25%, stirring speed controlled at 200rpm-600rpm, ventilation volume 1.0:1.0-2.0 (V / V), tank pressure 0.01Mpa-0.02Mpa, temperature 27℃-28℃, pH value 2.8-3.0, at the beginning of fermentation, the transformation liquid is fed at a flow rate of 1.0g / L / h-3.0g / L / h (based on sugars with molecular labeled glucose as the basic unit), and the transformation culture is 60 hr -72hr.

[0016] Furthermore, the conversion solution is composed of 0.0 g / L-100 g / L molecularly labeled sugars with glucose as the basic unit, 100 U / L-2500 U / L nuclease inhibitors, and 1 μmol / L-1.0 mmol / L protease inhibitors.

[0017] Furthermore, the concentration of the molecularly labeled sugar with glucose as the basic unit in the conversion solution is 0.0g / L-100g / L.

[0018] Furthermore, when the sugar containing the molecular marker and glucose as the basic unit is added into the bioreactor container at one time, the concentration thereof in the conversion liquid is 100 g / L.

[0019] Furthermore, when a molecularly labeled sugar with glucose as the basic unit is added to a bioreactor by continuous flow addition, its initial concentration in the conversion solution is 0.0 g / L, the final concentration is 100 g / L, and the flow rate is 1.0 g / L / h-3.0 g / L / h (based on the sugar with molecularly labeled glucose as the basic unit).

[0020] Furthermore, when the rate of addition of molecularly labeled glucose-based sugars during the conversion process is 1.0 g / L•h-3.0 g / L•h, the final volume of the conversion solution is 2.5 L-3.5 L, and the conversion period is 60 hr-72 hr.

[0021] Furthermore, 100 U / L-2500 U / L nuclease inhibitor and / or 1 μmol / L -1.0 mmol / L protease inhibitor in the transformation liquid is mainly used to inhibit the activity of ribonucleases and / or deoxyribonucleases and / or proteases produced during the transformation of Aureobasidium pullulans, to prevent ribonucleases and / or deoxyribonucleases and / or proteases from decomposing the molecular markers linked to the substrate (molecularly labeled sugars with glucose as the basic unit) and / or the product (pululan), thereby affecting the final separation and extraction of pullulan.

[0022] Furthermore, the molecules used to label carbohydrates include nucleic acids, proteins, antibodies, polypeptides, etc., which can be labeled individually or in the form of a mixture of two or more.

[0023] Furthermore, nucleic acids used to label sugars include DNA, RNA, cDNA, RLFP, VNTR, SSR, RAPD, AFLP, oligonucleotides, etc., which can be labeled individually or in the form of a mixture of two or more.

[0024] Furthermore, proteins used to label carbohydrates include 6xHIS, Flag, GST, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, SUMO, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, etc., which can be labeled individually or in combination of two or more.

[0025] Furthermore, the antibody molecule used to label the carbohydrate is prepared by using the protein molecule in the above step as an immunogen.

[0026] Furthermore, the molecular marker is located at the OH group of C2 and / or C3 and / or C6 of the basic structural unit of glucose. - and / or H + Location.

[0027] Furthermore, sugars with glucose as the basic unit, including glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin, honey and the like, which can be converted by Aureobasidium pullulans, can be used alone or in combination of two or more.

[0028] Furthermore, the Aureobasidium pullulans strain used for transformation was deposited in the China General Microbiological Culture Collection Center (CGMCC for short) on April 19, 2022; the deposit number is CGMCC No.40158, and the classification name is Aureobasidium pullulans, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] Separation and extraction steps of pullulan.

[0030] S1 bacterial liquid separation: Take 10 ml of transformation liquid, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new sample tube for later use.

[0031] S2 Magnetic Bead Activation: Magnetic bead solution coated with nucleic acid, protein or antibody at a concentration of 1.0mg / mL-10.0mg / mL, fully mix the magnetic bead solution to make the magnetic beads in the tube completely and evenly suspended. Adsorption is through the interaction of the molecular markers coated on the surface of the magnetic beads with the molecular markers in the pullulan product to form a complex, and the complex in the conversion solution is separated by the magnetic device and the magnetic bead gravitational adsorption. The diameter of the magnetic beads is 50nm-1000nm.

[0032] S3 Preparation of complex: Take 1.0-5.0 times the volume of the supernatant and add the magnetic bead solution to the separated transformation liquid supernatant, blow or vortex for 5S-15S, and let it stand at room temperature for 5 min-30min.

[0033] S4 adsorption of complex: Place the sample tube containing the complex on the magnetic device for about 1min-10min or until the magnetic beads are completely adsorbed by the magnetic device, keep the sample tube still on the magnetic rack, use a pipette to aspirate and discard the supernatant, and completely remove all the liquid in the tube. Avoid touching the magnetic bead cluster during the operation. The central magnetic induction intensity of the magnetic device should be ≥50 mT.

[0034] S5 separation complex: remove the sample tube from the magnetic device, place it at room temperature for 5 min-30 min, add 100μL-1000μL distilled water to the tube for elution, and gently blow 5-10 times with a pipette to resuspend and mix the magnetic beads, and place it at room temperature for 5 min-30 min. Care should be taken to avoid bubbles during the operation.

[0035] S6 complex to remove molecular markers: The complex solution separated and collected in the above step is used to decompose the nucleic acids and / or proteins and / or antibodies coated on the surface of the magnetic beads with an enzymatic solution, wherein the concentration of the nuclease used is 50 U / L-2000 U / L, the concentration of the protease used is 10μmol / L-2.5mmol / L, and the enzymatic hydrolysis is carried out at 37℃ for 1.0hr-5.0hr.

[0036] Obtaining S7 pullulan: centrifuge the delabeled complex solution obtained in the above step at 4°C, 12000rpm for 15 minutes, and the precipitate is the dissociated magnetic beads; transfer the supernatant to a new sample tube, add 1.5-5.0 times the volume of the supernatant anhydrous ethanol, mix by inverting 30 times, centrifuge at 12000rpm in a high-speed centrifuge for 20 minutes, discard the supernatant, dry the precipitate at 120°C for 24 hours, and weigh it to obtain crude pullulan P1.

[0037] S7 Determination of pullulan purity: Weigh 50.0 mg of the above-mentioned dried crude pullulan P1 to prepare a 50.0 mg / L pullulan solution. The total sugar was determined by the sulfuric acid-anthrone method, with reference to the literature (Zhang Shuihua. Food Analysis. Beijing: China Light Industry Press, 2004). The amount of pullulan P2 (based on total sugar) was calculated according to the standard curve, and the pullulan purity % = P2 / P1*100.

[0038] Determination of S8 pullulan whiteness: using a blue light whiteness meter, the determination was carried out in accordance with GBT22427.6-2008.

[0039] In the present invention, the term "pullulan" refers to a polysaccharide composed of maltotriose units connected, glucose units are connected with α-1, 4 bonds to produce maltotriose, and maltotriose units are connected with α-1, 6 bonds to produce pullulan, also known as pullulan, pullulan, and pullulan.

[0040] In the present invention, the term "Aureobasidium pullulans" is a yeast-like fungus widely distributed in nature.

[0041] In the present invention, the term "conversion" refers to a biochemical reaction that uses certain enzymes or enzyme systems in the microbial metabolism process to convert a compound into a product containing a special functional group. Specifically, microbial conversion is the structural modification of a complex substrate by microbial cells to convert it into another organic compound with a similar structure.

[0042] In the present invention, the term "sugar with glucose as the basic unit" refers to various disaccharides, oligosaccharides, polysaccharides, etc. composed of a single glucose unit. Any sugar containing a single glucose structure is a sugar with glucose as the basic unit.

[0043] In the present invention, the term "RFLP" refers to Restriction Fragment Length Polymorphism; the term "VNTR" refers to Variable Number of Tandem Repeats, which is also called minisatellite DNA; the term "RAPD" refers to Random Amplified Polymorphism DNA; the term "AFLP" refers to Amplified Fragment Length Polymorphism. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The raw materials used in the present invention, unless otherwise specified, are all conventional commercial products, the methods used in the present invention, unless otherwise specified, are all conventional methods in the art, and the quality of each substance used in the present invention is the quality of conventional use. Unless otherwise specified, they are purchased from commercial channels or have been disclosed. Example 1

[0050] For seed culture steps, see Section

[0013] for seed plate culture and Section

[0014] for seed liquid culture.

[0051] 500 ml of the activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion solution for pullulan production. The concentration of glucose in the conversion solution was 100 g / L, and the total reaction volume was 2.5 L.

[0052] Transformation culture control process: In a 5.0L bioreactor, the stirring speed was controlled at 600rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01Mpa, the temperature was 28°C, and the pH value was controlled at 2.8 using 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution, respectively. The transformation culture was completed after 72 hours.

[0053] Pullulan polysaccharide separation and extraction: 10 ml of transformation culture medium was taken, centrifuged at 12000 rpm for 15 minutes, the supernatant was transferred to a new sample tube, 3.0 times the volume of the supernatant was added with anhydrous ethanol, mixed by inversion 30 times, centrifuged at 12000 rpm for 20 minutes in a high-speed centrifuge, the supernatant was discarded, the precipitate was dried at 120°C for 24 hours and weighed, that is, the yield of pullulan polysaccharide was 83.6 g / L, and the purity was only 85.12%. (Table 1).

[0054] The whiteness of pullulan was measured using a blue light whiteness meter in accordance with GBT22427.6-2008. The whiteness value of the product was 65.5 (Table 1). Example 2

[0055] Seed culture steps are shown in Example 1.

[0056] 500 ml of the activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion solution for pullulan production. The concentration of glucose (the C2 hydroxyl group was replaced by 9 thymines (5'-TTTTTTTTT-3')) in the conversion solution was 100 g / L, the concentration of nuclease inhibitors was 100 U / L, the concentration of protease inhibitors was 1 μmol / L, and the total reaction volume was 2.5 L.

[0057] Transformation culture control process: same as Example 1.

[0058] Pullulan was isolated and extracted in two ways. The control group extracted pullulan in the way of Example 1, with a pullulan yield of 85.56 g / L, a purity of 80.29%, and a whiteness value of 61.56 (Table 1). The experimental group was carried out in the following way.

[0059] For S1 bacterial culture separation, take 10 ml of transformation culture medium, centrifuge at 12000 rpm for 15 minutes, and transfer the supernatant to a new sample tube for later use.

[0060] S2 magnetic beads were activated. The surface of the magnetic beads was coated with a magnetic bead solution of 9 adenines (5'-AAAAAAAAA3') at a concentration of 5.0 mg / mL. The magnetic bead solution was fully mixed and shaken to make the magnetic beads in the tube completely and evenly suspended.

[0061] S3 prepares the complex. Take a magnetic bead solution twice the volume of the supernatant separated in S1 and add it to the supernatant. Pipette or vortex for 15 seconds and leave it at room temperature for 30 minutes to bind.

[0062] S4 adsorbs the complex, places the sample tube on the magnetic rack for 10 minutes or until the magnetic beads are completely adsorbed by the magnetic rack device, keeps the sample tube stationary on the magnetic rack, uses a pipette to absorb the supernatant and discard it, and completely removes the liquid in the tube.

[0063] S5 separation complex, remove the sample tube from the magnetic stand and place it at room temperature for 30 min; add 1000 μL of distilled water to the tube for elution, and gently blow 5-10 times with a pipette to resuspend and mix the magnetic beads, and place it at room temperature for 30 min. Care should be taken to avoid generating bubbles during the operation.

[0064] The S6 complex was delabeled. The complex separated and collected in the above step was treated with 2000 U / L nuclease to decompose the adenine (5'-AAAAAAAAA3') coated on the surface of the magnetic beads and the thymine (5'-TTTTTTTTT-3') labeled on the pullulan product. The reaction was carried out at 37°C for 1.0 hr.

[0065] To obtain S7 pullulan, the delabeled complex solution obtained in the above step was centrifuged at 4°C, 12000rpm for 15 minutes. The precipitate was the magnetic beads without molecular markers. The supernatant was transferred to a new centrifuge tube, and 5.0 times the volume of the supernatant was added with anhydrous ethanol. After inversion 30 times to mix, the tube was centrifuged at 12000rpm in a high-speed centrifuge for 20 minutes. The supernatant was discarded, and the precipitate was dried at 120°C for 24 hours and weighed. The yield of pullulan was 84.93 g / L, and the purity was 97.95% (Table 1).

[0066] S8 The whiteness of pullulan was measured using a blue light whiteness meter in accordance with GBT22427.6-2008, and the whiteness value was 99.26 (Table 1). Example 3

[0067] Seed culture steps are shown in Example 1.

[0068] 500 ml of the activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion solution for pullulan production. The concentration of glucose (the C3 hydroxyl group was replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide) in the conversion solution was 100 g / L, the concentration of nuclease inhibitor was 2500 U / L, the concentration of protease inhibitor was 1 mmol / L, and the total reaction volume was 2.5 L.

[0069] Transformation culture control process: see Example 1.

[0070] Separation and extraction of Rulan polysaccharide.

[0071] S1 bacterial liquid separation, see S1 in Example 2.

[0072] Activate S2 magnetic beads. Coat the surface of the magnetic beads with the antibody magnetic bead solution of Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide. The concentration is 1.0 mg / mL. Fully mix and shake the magnetic bead solution to make the magnetic beads in the tube completely and evenly suspended.

[0073] S3 prepares the complex, see Example 2.

[0074] S4 adsorption complex, see Example 2.

[0075] S5 separation complex, remove the sample tube from the magnetic stand and place it at room temperature for 15 min; add 1000 μL PBS buffer (pH 7.0) to the tube for elution, and gently blow 5-10 times with a pipette to resuspend and mix the magnetic beads, and place it at room temperature for 30 min. Care should be taken to avoid generating bubbles during the operation.

[0076] The S6 complex was delabeled and 1000ul of the complex separated and collected in the above step was used to decompose the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide labeled on the pullulan product with 1.0mmol / L protease to separate the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide and the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu antibody. The reaction was carried out at 37°C for 4.0 hours.

[0077] The preparation of S7 pullulan can be seen in S7 of Example 2. The yield of pullulan is 87.98 g / L and the purity is 98.89% (Table 1).

[0078] S8 Determination of pullulan whiteness, see Example 2, the whiteness value is 99.35 (Table 1). Example 4

[0079] Seed culture steps are shown in Example 1.

[0080] 500 ml of the activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion solution for pullulan production. The concentration of glucose (the C3 hydroxyl group was replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide, and the C6 hydroxyl group was replaced by 9 thymines (5'-TTTTTTTTT-3')) in the conversion solution was 100 g / L, the concentration of nuclease inhibitor was 100 U / L, the concentration of protease inhibitor was 1 μmol / L, and the total reaction volume was 2.5 L.

[0081] Transformation culture control process: see Example 2.

[0082] Separation and extraction of Rulan polysaccharide.

[0083] S1 bacterial liquid separation, see S1 in Example 2.

[0084] S2 magnetic beads were activated. The surface of the magnetic beads was coated with antibodies to the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide and a magnetic bead solution with 9 adenines (5'-AAAAAAAAA3') at a concentration of 2.0 mg / mL. The magnetic bead solution was fully mixed and shaken to make the magnetic beads in the tube completely and evenly suspended.

[0085] S3 prepares the complex, see Example 2.

[0086] S4 adsorption complex, see Example 2.

[0087] S5 separation complex, see S5 in Example 3.

[0088] The S6 complex was delabeled. 1000ul of the complex separated and collected in the above step was used to decompose the nine thymines labeled on the pullulan product (5'-TTTTTTTTT-3' and Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide) with 50 U / L nuclease and 10 μmol / L protease, respectively, and the reaction was carried out at 37°C for 3.0 hr.

[0089] The preparation of S7 pullulan is shown in Example 2, i.e., the yield of pullulan is 88.36 g / L and the purity is 99.56% (Table 1).

[0090] S8 Determination of pullulan whiteness, see Example 2, the whiteness value is 99.59 (Table 1). Example 5

[0091] Seed culture steps are shown in Example 1.

[0092] 2000 ml of the activated liquid seed culture was centrifuged at 12000 rpm for 15 minutes to separate the supernatant and the bacteria; the bacteria were transferred to a 5.0 L bioreactor containing 500 ml of transformation solution. The concentration of glucose in the transformation solution (the hydroxyl group at C3 was replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and the hydroxyl group at C6 was replaced by 9 thymines (5'-TTTTTTTTT-3')) was 100 g / L, the concentration of nuclease inhibitor was 500 U / L, the concentration of protease inhibitor was 0.5 mmol / L, the initial reaction volume was 500 ml, 2.0 L of the transformation solution with a concentration of 100 g / L was added at the beginning of the transformation, the flow rate was 2.0 g / L / h, and the transformation culture was completed after 72 hours.

[0093] The transformation culture control process is the same as that in Example 2.

[0094] The separation and extraction of Rulan polysaccharide and the determination of whiteness are the same as in Example 4.

[0095] The final pullulan yield was 94.58 g / L, the purity was 99.80%, and the whiteness value was 99.62 (Table 1).

[0096] Table 1 The test results of pullulan yield, purity and whiteness value in Examples 1 to 5.

[0097] Example Pullulan crude yield (g / L) Pullulan purity (%) Pullulan net yield (g / L) Pullulan Whiteness Consumption of carbon source (g / L) Example 1 83.6 85.12 71.16 65.5 100.0 Example 2 Control group 85.56 80.29 68.69 61.56 100.0 Example 2 Experimental Group 84.93 97.95 83.19 99.26 100.0 Example 3 87.98 98.89 86.99 99.35 100.0 Example 4 88.36 99.56 87.97 99.59 100.0 Example 5 94.58 99.80 94.39 99.62 100.0

[0098] The advantages of the present invention are that a new transformation method for producing pullulan and separating pullulan from a transformation liquid is provided. The method mainly uses EP1001, which is deposited in the China General Microbiological Culture Collection Center and has a deposit number of CGMCC No. 40158, as a starting strain, and transforms and cultivates sugars containing molecular markers and glucose as a basic unit. Under the action of magnetic force, the transformation product specifically and directionally separates the pullulan molecules with molecular markers in the transformation liquid. The separated product is dissociated from the magnetic beads and the pullulan complex by an enzymatic solution, and the molecular markers are further decomposed. After precipitation with an organic solvent, high-purity pullulan is finally obtained. The entire separation and extraction process does not require flocculant pretreatment, activated carbon adsorption decolorization, oxidation decolorization, protease deproteinization, ion exchange desalination, and the like. The experimental process is simple, the product yield and purity are high, and the quality is excellent. The product was subjected to adsorption, desorption, enzymatic decomposition and ethanol precipitation, and the final pullulan yield was as high as 94.58 g / L, the purity was 99.80%, and the whiteness value was 99.62.

Claims

1. A method for producing pullulan, characterized in that: The following steps are involved: S1, Aureobasidium pullulans strain, deposited in China General Microbiological Culture Collection Center (CGMCC) on April 19, 2022; the deposit number is CGMCC No.40158, the classification name is Aureobasidium pullulans, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; S2, transformation culture medium, including 0.0g / L-100g / L molecularly labeled sugars with glucose as the basic unit, 100 U / L-2500 U / L nuclease inhibitor, 1μmol / L-1.0mmol / L protease inhibitor; S3, transformation culture conditions are: 5.0L bioreactor, liquid volume 0.5L-2.0L, inoculation volume 5.0%-25.0%, stirring speed 200rpm-600rpm, ventilation volume 1.0:1.0-2.0 (V / V), tank pressure 0.01Mpa-0.02Mpa, temperature 27℃-28℃, pH value 2.8-3.0, at the beginning of transformation, the transformation liquid is fed at a flow rate of 1.0g / L / h-3.0g / L / h (based on molecular labeled glucose as the basic unit of sugars), and the transformation culture is 60 hr -72hr; S4. The product obtained from the above transformation is subjected to the steps of adsorption, desorption, enzymatic decomposition and organic solvent precipitation to separate and purify pullulan.

2. The method for producing pullulan according to claim 1, characterized in that: The molecular markers used to label carbohydrates include nucleic acids, proteins, antibodies, polypeptides, etc., which can be labeled alone or in combination of two or more.

3. The method for producing pullulan according to claim 2, characterized in that: The nucleic acids used to label sugars include DNA, RNA, cDNA, RLFP, VNTR, SSR, RAPD, AFLP, oligonucleotides, etc., and can be labeled alone or in combination of two or more.

4. The method for producing pullulan according to claim 2, characterized in that: The proteins used to label carbohydrates include 6xHIS, Flag, GST, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, SUMO, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, etc., which can be labeled individually or in combination of two or more.

5. The method for producing pullulan according to claim 2, characterized in that: The antibody for labeling carbohydrates is prepared by using the protein of claim 2 as an immunogen.

6. The method for producing pullulan according to claim 1, characterized in that: The molecular marker is located at the OH group of C2 and / or C3 and / or C6 of the basic structural unit of glucose. - and / or H + Location.

7. The method for producing pullulan according to claim 1, characterized in that: The sugars with glucose as the basic unit include glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin, honey and the like which can be converted by Aureobasidium pullulans and can be used alone or in combination of two or more.

8. The method for producing pullulan according to claim 1, characterized in that: The adsorption is carried out by the interaction of the molecular markers coated on the surface of the magnetic beads with the molecular markers carried in the pullulan product to form a complex, and the complex in the conversion solution is separated by a magnetic device, the diameter of the magnetic beads is 50nm-1000nm, and the central magnetic induction intensity of the magnetic device is ≥50 mT.

9. The method for producing pullulan according to claim 1, characterized in that: The desorption is achieved by eliminating the interaction force between the magnetic device and the magnetic beads.

10. The method for producing pullulan according to claim 1, characterized in that: The enzymatic hydrolysis solution includes 50 U / L-2000 U / L nuclease and 10 μmol / L-2.5 mmol / L protease, and the enzymatic hydrolysis is carried out at 30° C.-37° C. for 1.0 hr-5.0 hr.