Strains, cell immobilized particles and catalytic processes for the production of d-psicose-3-epimerase

Abstract

The application belongs to the technical field of bioengineering and enzyme engineering, and discloses a D-allosucrose-3-epimerase-producing strain, a cell immobilized particle and a catalysis method, which is classified and named as halobacillus sp. Bacillus halotolerans ), which is preserved in the China General Microbiological Culture Collection Center on December 26, 2022, and has a preservation number of CGMCC No. 26285. The cell is loaded on a support carrier through a cross-linking agent, and the cross-linking and drying process is slow drying. The obtained cell immobilized particle has the advantages of large mechanical strength and water insolubility.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering and enzyme engineering, and particularly relates to a D-psicose-3-epimerase-producing strain, a cell immobilized particle and a catalysis method. BACKGROUND

[0002] The statements herein are provided only to enhance understanding of the present application and are not necessarily intended to constitute the prior art.

[0003] D-psicose (D-allulose) is a C-3 epimer of D-fructose and a rare natural ketohexose in nature. Compared with fructose, D-psicose has the functions of reducing blood sugar and preventing obesity. The melting point of D-psicose is 109℃, and it is extremely soluble in water. The sugar degree of D-psicose is 70% of sucrose, and the heat is 0.3% of sucrose, so it is called non-calorie sugar. At present, it has been approved in many countries to be used as a food additive or food raw material.

[0004] D-psicose is difficult to obtain in nature, which restricts its application in actual production. At present, the preparation methods of D-psicose mainly include chemical synthesis and biological transformation, and the biological transformation method is mainly used. The main research focuses on the modification of D-psicose-3-epimerase in nature and the expression of heterologous hosts.

[0005] D-psicose-3-epimerase has a wide biological source. In the biological transformation process of D-psicose, the biological enzyme cannot be reused, and the process cost is high. Compared with the traditional immobilized enzyme technology, the cell immobilization technology is more simple and efficient, and can greatly compress the fermentation and subsequent separation cost. In the prior art, a calcium alginate embedding method for producing D-psicose-3-epimerase engineering strain is used to catalyze fructose, and the preparation process is complex, the particle hardness is poor, and the enzyme or cell is easy to leak, which is difficult to be used for industrial scale production. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide a D-psicose-3-epimerase-producing strain, a cell immobilized particle and a catalysis method.

[0007] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0008] In a first aspect, the present application provides a D-psicose-3-epimerase-producing strain SYNY-019, which is classified and named as halotolerant bacillus (Bacillus halodurans) SYNY-019. Bacillus halotolerans), and was preserved in China General Microbiological Culture Collection Center on December 26, 2022, with a preservation number of CGMCC No. 26285.

[0009] In a second aspect, the present application provides a cell immobilized particle, comprising a strain, a support carrier and a cross-linking agent, the strain is loaded on the support carrier by the cross-linking agent, and the strain is the strain producing D-allulose-3-epimerase.

[0010] In some embodiments, the particle size of the cell immobilized particle is 425-850 μm, and it can pass through a 20-40 mesh sieve.

[0011] In some embodiments, the support carrier has a loose porous structure and is neutral or weakly alkaline after being dispersed in an aqueous phase.

[0012] Preferably, the support carrier is at least one of diatomite, activated carbon or kaolin.

[0013] Further preferably, the support carrier is a mixture of diatomite, activated carbon and kaolin, and the mass ratio of diatomite, activated carbon and kaolin is 2-4:1-2:3-5.

[0014] The inventors have found through experiments that when the support carrier is a single substance, the mechanical strength of the single support carrier is not good, which is manifested as uneven mechanical strength, and the loaded cell particles are easily broken into powder. When three kinds of support carriers are mixed as a composite carrier according to a certain mass ratio, the mechanical strength of the cell immobilized particle can be effectively increased.

[0015] In some embodiments, the cross-linking agent is at least one of polyvinyl alcohol, chitosan, polyvinyl alcohol, glutaraldehyde, polyacrylamide, polyethyleneimine, N,N'-methylene bisacrylamide or polydimethyl diallyl ammonium chloride.

[0016] Preferably, the cross-linking agent is a mixture of chitosan, polyethyleneimine and glutaraldehyde, and the mass ratio of chitosan, polyethyleneimine and glutaraldehyde is 1-5:1-10:1-3. Cross-linking is generated by various characteristic chemical reactions of amino or amine groups with aldehyde groups, and after sufficient dehydration, the cell immobilized particle as a whole is not easily dissolved in water.

[0017] In some embodiments, the preparation method of the cell immobilized particle is as follows:

[0018] The fermentation broth of the strain producing D-allulose-3-epimerase is mixed with the support carrier, mixed uniformly, and then the cross-linking agent is added to the fermentation broth, mixed thoroughly to obtain a viscous solid-liquid mixture;

[0019] The solid-liquid mixture is separated, the solid is granulated, and the granulated product is dried at 40-60 DEG C to obtain the immobilized cell granules.

[0020] Preferably, the fermentation broth of the D-alloketone-3-epimerase-producing strain is mixed with diatomite, kaolin and activated carbon respectively.

[0021] Then, chitosan, polyethylene imine and glutaraldehyde are sequentially added to the mixed system.

[0022] The chitosan provides amino groups, and the polyethylene amide provides amine groups, and the two substances do not react when mixed, and after the addition of glutaraldehyde, the aldehyde groups of the glutaraldehyde crosslink with the amine groups and the amino groups. By adjusting the ratio of the three crosslinking agents, the strength and water resistance of the immobilized cell granules can be improved.

[0023] Preferably, the mass / volume ratio of the support carrier to the fermentation broth is 1%-15%, g / mL.

[0024] Preferably, the mass / volume ratio of the crosslinking agent to the fermentation broth is 5%-20%, g / mL.

[0025] In a third aspect, the application provides a catalytic method, comprising the following steps: adding the immobilized cell granules to a fructose solution, heating in a water bath, and catalyzing the production of D-allulose from fructose.

[0026] In some embodiments, the temperature of the water bath heating is 50-60 DEG C.

[0027] The application achieves the following beneficial effects through one or more embodiments:

[0028] The support carrier of the application is a mixed carrier of diatomite, activated carbon and kaolin, which can effectively improve the mechanical strength of the immobilized cell granules.

[0029] Chitosan, polyethylene imine and glutaraldehyde are used as a composite crosslinking agent, and crosslinking is generated by various characteristic chemical reactions of amino or amine groups and aldehyde groups. In combination with a slow dehydration process, the immobilized cell granules are not easily dissolved in water. BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings accompanying the specification of the application form a part of the application and serve to provide further understanding of the application. The exemplary embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation of the application.

[0031] Figure 1 The figure shows the influence of pH value on the fructose conversion rate during whole-cell catalysis in the embodiments of the application;

[0032] Figure 2For the embodiment of the present application, the temperature has an influence on the trend chart of the conversion rate of fructose when the whole cell catalyzes;

[0033] Figure 3 For the embodiment of the present application, the conversion rate of fructose catalyzed by the immobilized cell particles at different temperatures is compared in the chart;

[0034] Figure 4 For the embodiment of the present application, the catalytic effect chart of the immobilized cell particles suitable for industrial production is shown in the chart;

[0035] Figure 5 For the embodiment of the present application, the liquid separation chromatogram of the catalysis of the immobilized cell particles is shown in the chart;

[0036] Figure 6 For the embodiment of the present application, the flow chart of the immobilization of the cell is shown in the chart. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0038] Definitions:

[0039] The mass volume fraction is the ratio of the mass of a substance to the volume of a liquid when the substance is added to the liquid, such as adding 10 g of diatomite to 1 L of fermentation liquid, the mass volume fraction of diatomite is 1%.

[0040] The present application will be further described below in conjunction with examples.

[0041] Example 1

[0042] Strain screening and fermentation:

[0043] Enrichment medium: 0.5% glucose, 0.5% peptone, 0.25% yeast extract, 0.25% sodium chloride, 0.25% ammonium sulfate, 0.05% MgSO4·7H2O, 0.001% FeSO4·7H2O and 0.001% MnSO4·4H2O (pH 7.0).

[0044] Purification medium: 0.5% glucose, 0.5% peptone, 0.25% sodium chloride, 0.25% yeast extract (pH 7.0).

[0045] Screening medium: 0.5% glucose, 0.5% peptone, 0.25% sodium chloride, 1% fructose (pH 7.0).

[0046] Seed medium: 1.0% glucose, 0.5% yeast extract, 1.0% peptone (pH 7.0).

[0047] Fermentation medium: 1.0% glucose, 0.3% ammonium sulfate, 1.0% yeast extract, 1.0% peptone, 0.1% KH2PO4, 0.3% K2HPO4, 0.05% MgSO4·7H2O, 0.001% FeSO4·7H2O and 0.001% MnSO4·4H2O (pH 7.0).

[0048] Fifteen soil samples (300g each) were collected from farmland around Binzhou Port. A suitable amount of each sample was added to 100mL of enrichment medium and incubated at 30℃ and 220r / min on a shaker for 24 hours. The enriched bacterial culture was then centrifuged, and the supernatant was serially diluted with water to a concentration equal to 10 times the original supernatant concentration. -3 Take an appropriate amount and spread it on a purification medium plate to ensure that a single clone can be selected from the medium plate. Incubate at 30°C for 2 days. Select a variety of single colonies with typical characteristics and incubate them in liquid screening medium. At the same time, repeat the isolation and purification in the purification medium. Repeat this process 3 times to finally obtain a series of strains. After incubating in the purification medium at 30°C for 2 days, store them in a refrigerator at 4°C.

[0049] The selected strains were inoculated into fermentation medium and cultured in liquid with shaking at 30℃ and a shaking speed of 220 r / min for 3 days. The culture medium was centrifuged at 12000g for 5 min, and the bacterial cells and supernatant were collected separately.

[0050] The bacterial cells were suspended in an equal volume of phosphate buffer (pH 7.0), and fructose was added to both the bacterial cells and the supernatant. The reaction was carried out at 50°C, and the relative content of D-allulose produced was determined by HPLC. Stranded culture was performed on strains with high enzyme activity, ultimately yielding a D-allulose-producing strain, SYNY-019, with a high conversion rate.

[0051] The selected D-allulose-producing strain was inoculated into seed culture medium and cultured at 30℃ for 16 h. Then, it was transferred to fermentation medium at an inoculation rate of 5% and cultured at 30℃ for 2 days. The fermentation broth was then collected.

[0052] Bacterial cell immobilization process, such as Figure 6 As shown, the main steps include adding a support carrier to the fermentation broth, mixing well, adding a cross-linking agent, mixing well again, and drying to obtain immobilized bacterial particles. Specifically, the steps are as follows:

[0053] Step 1: Take fresh Bacillus halotoleransThe CGMCC No.26285 fermentation liquor is sequentially added with 2% diatomite, low-temperature stirring for 10 minutes, 3% kaolin, low-temperature stirring for 30 minutes, 1% activated carbon, and low-temperature stirring for 1 hour, so as to ensure the sufficient mixing of the fermentation liquor and the three supporting carriers.

[0054] Step 2, 2% chitosan is sequentially added to the mixed fermentation liquor in step 1 according to the mass volume fraction, normal-temperature stirring for 2 hours, 2% polyethyleneimine is added, normal-temperature stirring for 1 hour, 1% glutaraldehyde is added, normal-temperature stirring for 1 hour, and a high-viscosity solid-liquid mixture formed by the sufficient mixing of the fermentation liquor, the supporting carrier and the crosslinking agent is obtained.

[0055] Step 3, the solid-liquid mixture in step 2 is separated by pressure filtration, and a filter cake with low water content is obtained. The precipitate or filter cake is made into granules by a granulator, and the granules are slowly dried at 50 DEG C. In the dehydration process, the granules are further crosslinked and finally form the immobilized cell granules, which are filtered through a 40-mesh sieve.

[0056] The immobilized cell granules as catalysts for catalyzing fructose:

[0057] In a water bath chromatographic column with an inner diameter of 18*300mm, 40g of the immobilized cell granules are added, a 60% fructose solution is used as a substrate, and the conversion reaction of fructose is carried out under water bath conditions at a flow rate of 6 times the mass volume of the granules per hour (240mL / h). The conversion rate of the discharged fructose and the overall operation time are determined.

[0058] The pH value of the fixed reaction system is 7.0, and the fructose conversion rate trends of the immobilized cell granules under different temperatures are compared. Figure 3 It can be seen that the conversion rate of fructose is the highest when the reaction temperature is 60 DEG C.

[0059] After the immobilization of the cells, the conversion rate can be maintained above 28% at a pH value of 7.0 and a temperature of 60 DEG C, and the stable operation can be maintained for at least 540 hours, as shown in the following table. Figure 4

[0060] It can be seen from the above table that the immobilized cell granules prepared in the present application have a long service life, which indicates that no enzyme or cell leakage occurs in the fermentation process. Figure 3 Figure 4 When the hardness of the immobilized cell enzyme granules is low, the chromatographic column will be easily blocked in the continuous production process, the column pressure will be increased, and the continuous reaction time will be short. The immobilized cell enzyme in the present application can be continuously used for a long time, which indicates that the prepared immobilized cell enzyme granules have good mechanical strength and are not easily dissolved in water.

[0061]

[0062] ​​​Figure 5 Liquid chromatogram of the reaction solution for preparing D-psicose from fructose in Example 1.

[0063] Example 2

[0064] Using the fermentation broth obtained in Example 1, the cell immobilization process mainly includes adding a support carrier to the fermentation broth, mixing, then adding a crosslinking agent, mixing, and drying to obtain cell immobilized particles. The specific steps include the following:

[0065] Step 1: Take fresh fermentation broth, and add 4% diatomite, 5% kaolin, and 2% activated carbon in the order of mass fraction, and stir at low temperature for 10 min, 10 min, and 30 min, respectively, to ensure the full mixing of the fermentation broth and the three support carriers.

[0066] Step 2: To the fully mixed fermentation broth in Step 1, add 5% chitosan, 4% polyethyleneimine, and 2% glutaraldehyde in the order of mass fraction, and stir at room temperature for 2 h, 10 min, and 30 min, respectively, to obtain a high-viscosity solid-liquid mixture fully mixed by the fermentation broth, support carrier, and crosslinking agent.

[0067] Step 3: Separate the solid-liquid mixture in Step 2 by pressure filtration to obtain a filter cake with low water content. Use a granulator to make the precipitate or filter cake into granules, and slowly dry the granules at 50°C. Further crosslinking occurs during the dehydration process, and finally the cell immobilized particles are formed. Sieve the particles through a 40-mesh sieve.

[0068] Catalysis of the cell immobilized particles as a catalyst on fructose:

[0069] Add 40 g of cell immobilized particles to an inner diameter of 18*300 mm water bath chromatographic column, use a 60% fructose solution as the substrate, and perform the fructose conversion reaction at a water bath temperature of 55°C and a flow rate of 6 times the particle mass volume per hour (240 mL / h). The conversion rate of the discharged fructose is 27%, and the continuous operation time is more than 540 h.

[0070] Example 3

[0071] Using the fermentation broth obtained in Example 1, the cell immobilization process mainly includes adding a support carrier to the fermentation broth, mixing, then adding a crosslinking agent, mixing, and drying to obtain cell immobilized particles. The specific steps include the following:

[0072] Step 1, take fresh fermentation broth, add 3% diatomite by mass fraction, low temperature stirring for 10 min. Add 3% kaolin, low temperature stirring for 30 min. Add 1% activated carbon, low temperature stirring for 1 h, to ensure the fermentation broth and the three kinds of support carrier are fully mixed.

[0073] Step 2, to the fermentation broth mixed in step 1, add 1% chitosan by mass fraction, normal temperature stirring for 2 h, add 3% polyethyleneimine, normal temperature stirring for 1 h, add 2% glutaraldehyde, normal temperature stirring for 1 h, to obtain a high viscosity solid-liquid mixture of fermentation broth, support carrier and crosslinking agent.

[0074] Step 3, separate the solid-liquid mixture in step 2 by pressure filtration to obtain a filter cake with low water content. The precipitate or filter cake is made into granules by a granulator, and the granules are slowly dried at 50°C. In the dehydration process, the granules are further crosslinked and finally form a cell immobilized granule, which is passed through a 40 mesh sieve.

[0075] The cell immobilized granule as a catalyst for catalyzing fructose:

[0076] In a water bath chromatography column with an inner diameter of 18*300 mm, 40 g of cell immobilized granules were added, and a 60% fructose solution was used as the substrate. The water bath temperature was 55°C, and the reaction was carried out at a flow rate of 6 times the granule mass volume per hour (240 mL / h). The conversion rate of the discharged fructose was 28%, and the continuous operation time was at least 540 h.

[0077] Comparative Example 1

[0078] Whole cell catalysis:

[0079] Take an appropriate amount of fresh fermentation broth and centrifuge the cell to a wet weight of 100 g / L. Mix 0.5 mL of the cell with 50% fructose solution and react at 50°C for 3 h to determine the fructose conversion rate under different buffer conditions. Test the fructose conversion rate at different temperatures in a pH 7.0 phosphate buffer. Under incomplete reaction conditions, the relative enzyme activity is reflected by the conversion rate, and the optimal fructose conversion conditions are determined. From the whole cell catalysis results Figure 1 and Figure 2 It can be seen that the optimal conditions for the conversion of fructose to allulose are pH 6-8 and temperature 60-75°C.

[0080] Comparative Example 2

[0081] The difference between Example 1 and Comparative Example 2 is that kaolin is replaced by diatomite in equal amount, and the others are the same as Example 1.

[0082] The cell immobilized granule catalyzes the preparation of D-psicose from fructose, and the conversion rate can be maintained at 28% at pH 7.0 and 60 DEG C, and the stable operation can be maintained for about 240 hours.

[0083] Comparative Example 3

[0084] The difference from Example 1 is that the activated carbon is replaced by kaolin in equal amount, and the other conditions are the same as those in Example 1.

[0085] The cell immobilized granule catalyzes the preparation of D-psicose from fructose, and the conversion rate can be maintained at 27% at pH 7.0 and 60 DEG C, and the stable operation can be maintained for about 400 hours.

[0086] Comparative Example 4

[0087] The difference from Example 1 is that the activated carbon and diatomite are replaced by kaolin in equal amount, and the other conditions are the same as those in Example 1.

[0088] The cell immobilized granule catalyzes the preparation of D-psicose from fructose, and the conversion rate can be maintained at 22% at pH 7.0 and 60 DEG C, and the stable operation can be maintained for about 180 hours.

[0089] Comparative Example 5

[0090] The difference from Example 1 is that the chitosan is replaced by polyethylene imine in equal amount, and the other conditions are the same as those in Example 1.

[0091] The cell immobilized granule catalyzes the preparation of D-psicose from fructose, and the conversion rate can be maintained at 28% at pH 7.0 and 60 DEG C, and the stable operation can be maintained for about 440 hours.

[0092] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cell immobilized particle, characterized by: The application relates to a D-allulose-3-epimerase producing strain, a support carrier and a crosslinking agent, wherein the strain is loaded on the support carrier through the crosslinking agent, the strain is classified as a salt-tolerant Bacillus ( Bacillus halotolerans ) SYNY-019, which is preserved in the China General Microbiological Culture Collection Center on December 26, 2022, and has a preservation number of CGMCC No.26285.​ The support carrier is a mixture of diatomite, activated carbon and kaolin, and the mass ratio of diatomite, activated carbon and kaolin is 2-4:1-2:3-5; The cross-linking agent is a mixture of chitosan, polyethyleneimine and glutaraldehyde, and the mass ratio of chitosan, polyethyleneimine and glutaraldehyde is 1-5:1-10:1-3.

2. The bacterial immobilized pellet according to claim 1, characterized in that: The preparation method of the bacterial immobilized particles is as follows: The fermentation broth of the strain producing D-allulose-3-epimerase is mixed with the support carrier, mixed uniformly, then the cross-linking agent is added into the fermentation broth, and mixed thoroughly to obtain a viscous solid-liquid mixture; The solid-liquid mixture is separated, the solid is granulated, and after drying at 40-60℃, the bacterial immobilized particles are obtained.

3. The bacterial immobilized pellet according to claim 2, characterized in that: The mass / volume ratio of the support carrier to the fermentation broth is 1%-15%, g / ml; The mass / volume ratio of the cross-linking agent to the fermentation broth is 5%-20%, g / ml.

4. A catalytic process characterized by: The method comprises the following steps: adding the bacterial immobilized particles of any one of claims 1-3 into a fructose solution, heating in a water bath, and catalyzing the production of D-allulose from fructose.

5. The catalytic process of claim 4, wherein: The temperature of the water bath heating is 50-60℃.

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