Strain for producing D-psicose-3-epimerase, thallus immobilized particles and catalytic method

Abstract

The invention belongs to the technical field of bioengineering and enzyme engineering, and discloses a strain for producing D-psicose-3-epimerase, thallus immobilized particles and a catalytic method, the strain is classified and named as Bacillus halotolans, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 26, 2022, the preservation number is CGMCC No.26285, and the preservation number is CGMCC No.26285. The invention further discloses a preparation method of the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase. The thalli are loaded on the supporting carrier through the cross-linking agent, slow drying is achieved in the cross-linking drying process, and the obtained thalli immobilized particles have the advantages of being large in mechanical strength and insoluble in water.

Description

Technical Field

[0001] The present invention belongs to the technical fields of bioengineering and enzyme engineering, and particularly relates to a strain producing D-allulose-3-epimerase, an immobilized cell particle, and a catalytic method. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] D-allulose (D-psicose / D-allulose) is an epimer of D-fructose at C-3, a relatively rare natural hexulose in nature. Compared with fructose, it has the functions of reducing blood sugar and preventing obesity. The melting point of D-allulose is 109 °C, it is extremely soluble in water, the sugar degree is 70% of sucrose, and the calorie is 0.3% of sucrose, so it is called calorie-free sugar. It has been approved in many countries for use as a food additive or food raw material.

[0004] The content of D-allulose in nature is very small and it is difficult to obtain, which restricts its application in actual production. At present, the preparation methods of D-allulose mainly include chemical synthesis method and biological conversion method. In fact, the biological conversion method is mainly used, mainly focusing on the modification of natural D-allulose-3-epimerase and heterologous host expression.

[0005] The biological sources of D-allulose-3-epimerase are extensive. In the biological conversion process of D-allulose, the biological enzyme cannot be reused and the process cost is relatively high. Compared with the traditional immobilized enzyme technology, the cell immobilization technology is more simple and efficient, and can greatly reduce the fermentation and subsequent separation costs. In the prior art, an immobilization method using calcium alginate to entrap an engineering strain producing allulose 3-epimerase is used for catalyzing fructose. The preparation process is complex, the particle hardness is poor, and it is easy to cause leakage of enzymes or cells, so it is difficult to be used in industrial scale production. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a strain producing D-allulose-3-epimerase, an immobilized cell particle, and a catalytic method.

[0007] To achieve the above purpose, the present invention is realized through the following technical solutions: In the first aspect, the present invention provides a strain SYNY-019 producing D-allulose-3-epimerase, which is classified and named as Bacillus halotolerans ( Bacillus halotolerans ), deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, with the deposit number CGMCC No. 26285.

[0008] In a second aspect, the present invention provides an immobilized cell particle, comprising a strain, a support carrier, and a crosslinking agent. The strain is loaded on the support carrier through the crosslinking agent, and the strain is the strain producing D-allulose-3-epimerase.

[0009] In some embodiments, the immobilized cell particle has a particle size of 425-850 μm and passes through a 20-40 mesh sieve.

[0010] In some embodiments, the support carrier has a loose porous structure and is neutral or weakly basic after being dispersed in an aqueous phase.

[0011] Preferably, the support carrier is at least one of diatomite, activated carbon, or kaolin.

[0012] More 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.

[0013] The inventors found through experiments that when the support carrier is a single substance, the mechanical strength of the single support carrier is not good, manifested as non-uniform mechanical strength, and the loaded cell particles are easily broken into powder. When the three support carriers are mixed as a composite carrier in a certain mass ratio, the mechanical strength of the immobilized cell particle can be effectively increased.

[0014] In some embodiments, the crosslinking agent is selected from at least one of polyvinyl alcohol, chitosan, polyvinyl alcohol, glutaraldehyde, polyacrylamide, polyethyleneimine, N,N'-methylenebisacrylamide, or polydimethyldiallylammonium chloride.

[0015] Preferably, the crosslinking 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. Crosslinking is achieved through various characteristic chemical reactions between amino or amine groups and aldehyde groups. After sufficient dehydration, the whole immobilized cell particle is not easily soluble in water.

[0016] In some embodiments, the preparation method of the immobilized cell particle is as follows: Mix the fermentation broth of the strain producing D-allulose-3-epimerase with the support carrier, and then add a crosslinking agent to the fermentation broth and mix well to obtain a viscous solid-liquid mixture; Separate the solid and liquid of the viscous solid-liquid mixture, granulate the solid, and dry it at 40-60 °C to obtain the immobilized cell particle.

[0017] Preferably, the fermentation broth of the strain producing D-allulose-3-epimerase is fully mixed with diatomite, kaolin, and activated carbon respectively; Then, chitosan, polyethyleneimine, and glutaraldehyde are sequentially added to the mixed system.

[0018] Chitosan provides amino groups, and polyethyleneamide provides amine groups. These two substances do not react when mixed. Finally, after adding glutaraldehyde, the aldehyde groups of glutaraldehyde react with amine groups and amino groups to form crosslinks. By adjusting the ratio of the three crosslinking agents, the strength and water resistance of the immobilized cell particles can be improved preferably.

[0019] Preferably, the mass-volume ratio of the support carrier to the fermentation broth is 1% - 15%, g / mL.

[0020] Preferably, the mass-volume ratio of the crosslinking agent to the fermentation broth is 5% - 20%, g / mL.

[0021] In a third aspect, the present invention provides a catalytic method, which includes the following steps: adding the immobilized cell particles to a fructose solution, heating in a water bath, and catalyzing fructose to produce D - allulose.

[0022] In some embodiments, the temperature of the water bath heating is 50 - 60 °C.

[0023] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows: The support carrier of the present invention is a mixed carrier of diatomite, activated carbon, and kaolin, which can effectively improve the mechanical strength of the immobilized cell particles.

[0024] Using chitosan, polyethyleneimine, and glutaraldehyde as a composite crosslinking agent, relying on various characteristic chemical reactions between amino or amine groups and aldehyde groups to form crosslinks, and cooperating with a slow dehydration process, the immobilized cell particles are not easily dissolved in water. Description of the Drawings

[0025] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0026] Figure 1 It is a trend graph of the influence of pH value on the fructose conversion rate during whole-cell catalysis in the embodiment of the present invention; Figure 2 It is a trend graph of the influence of temperature on the fructose conversion rate during whole-cell catalysis in the embodiment of the present invention; Figure 3 It is a comparison graph of the conversion rates of fructose catalyzed by immobilized cell particles at different temperatures in the embodiment of the present invention; Figure 4 It is an effect graph of the catalysis by immobilized cell particles suitable for industrial production in the embodiment of the present invention; Figure 5In the embodiments of the present invention, the chromatogram of the liquid phase separation catalyzed by the immobilized cell particles; Figure 6 In the embodiments of the present invention, the flowchart of the immobilization of the cells. Detailed implementation manners

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0028] Definition: 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. For example, when 10 g of diatomaceous earth is added to 1 L of fermentation broth, the mass - volume fraction of diatomaceous earth is 1%.

[0029] The present invention will be further described below in conjunction with embodiments.

[0030] Embodiment 1 Strain screening and fermentation: Enrichment medium: 0.5% glucose, 0.5% peptone, 0.25% yeast extract, 0.25% sodium chloride, 0.25% ammonium sulfate, 0.05% MgSO 4 ·7H 2 O, 0.001% FeSO 4 ·7H 2 O and 0.001% MnSO 4 ·4H 2 O (pH 7.0).

[0031] Purification medium: 0.5% glucose, 0.5% peptone, 0.25% sodium chloride, 0.25% yeast extract (pH 7.0).

[0032] Screening medium: 0.5% glucose, 0.5% peptone, 0.25% sodium chloride, 1% fructose (pH 7.0).

[0033] Seed medium: 1.0% glucose, 0.5% yeast extract, 1.0% peptone (pH 7.0).

[0034] Fermentation medium: 1.0% glucose, 0.3% ammonium sulfate, 1.0% yeast extract, 1.0% peptone, 0.1% KH 2 PO 4 ,0.3% K 2 HPO 4 ,0.05% MgSO 4 ·7H 2 O, 0.001% FeSO4 7H 2 O and 0.001% MnSO 4 ·4H 2 O (pH 7.0).

[0035] Fifteen soil samples were collected from farmland soil around Binzhou Port, each weighing 300 g. An appropriate amount of soil sample was added to 100 mL of enrichment medium and cultured in a shaking incubator at 30°C and 220 r / min for 24 h. The enriched bacterial solution was then centrifuged and the supernatant was diluted with water to a concentration of 10% of the original supernatant. -3 , and spread an appropriate amount on the purification culture medium plate to ensure that pure single clones can be selected from the culture medium plate, culture in a 30℃ incubator for 2 days, pick out a variety of single colonies with typical characteristics and culture them in liquid screening culture medium, and repeat the separation and purification in the purification culture medium for 3 times to finally obtain a series of strains. After culturing in the purification culture medium at 30℃ for 2 days, store them in a refrigerator at 4℃.

[0036] The selected strains were inoculated into the fermentation medium for liquid shaking culture at a temperature of 30°C, a shaking speed of 220 r / min, and a culture time of 3 days. The culture solution was centrifuged at 12000g for 5 minutes, and the bacterial cells and supernatant were collected respectively.

[0037] The bacterial cells were suspended in an equal volume of phosphate buffer with a pH value of 7.0, and fructose was added to the supernatant to react at 50°C. The relative content of the generated D-psicose was determined by HPLC. Strains with high enzyme activity were selected for streaking and subculture, and finally a D-psicose production strain SYNY-019 with a high conversion rate was obtained.

[0038] The screened D-psicose-producing strain was inoculated into a seed culture medium, cultured at 30°C for 16 h, and then transferred to a fermentation medium at a 5% inoculation rate, cultured at a constant temperature of 30°C for 2 days, and the fermentation broth was collected.

[0039] Bacteria immobilization process, such as Figure 6 As shown, it mainly includes adding a support carrier to the fermentation liquid, mixing it, adding a cross-linking agent thereto, mixing it, and drying it to obtain bacterial cell immobilization particles. Specifically, it includes the following steps: Step 1: Take fresh Bacillus halotolerans CGMCC No.26285 fermentation liquid, add 2% diatomaceous earth in order by mass volume fraction, stir at low temperature for 10 minutes, add 3% kaolin, stir at low temperature for 30 minutes. Add 1% activated carbon, stir at low temperature for 1 hour to ensure that the fermentation liquid and the three support carriers are fully mixed.

[0040] Step 2: Add 2% chitosan to the well-mixed fermentation broth in Step 1, stir at room temperature for 2 h, add 2% polyethyleneimine, stir at room temperature for 1 h, add 1% glutaraldehyde, and stir at room temperature for 1 h to obtain a highly viscous solid-liquid mixture in which the fermentation broth, support carrier, and crosslinking agent are well mixed.

[0041] Step 3: Separate the solid and liquid in the highly viscous solid-liquid mixture in Step 2 by pressure filtration to obtain a filter cake with a low water content. Make the precipitate or filter cake into granules through a granulator, slowly dry the granules at 50 °C, further crosslink fully during the dehydration process, and finally form immobilized cell particles. Pass through a 40-mesh sieve to obtain the product.

[0042] Catalysis of fructose by the immobilized cell particles as a catalyst: Add 40 g of the immobilized cell particles to a water bath chromatography column with an inner diameter of 18 * 300 mm. Use a 60% fructose solution as the substrate. Under water bath conditions, carry out the conversion reaction of fructose at a flow rate of 6 times the particle mass volume per hour (240 mL / h), and measure the conversion rate of the outlet fructose and the overall operation time.

[0043] Fix the pH value of the reaction system at 7.0, and compare the trend of the fructose conversion rate when the immobilized cell particles continuously catalyze the production of D-allulose at different temperatures. It can be seen from Figure 3 that when the reaction temperature is 60 °C, the conversion rate of fructose is the highest.

[0044] After the cells are immobilized, at a pH value of 7.0 and a temperature of 60 °C, the conversion rate can be maintained above 28%, and stable operation can be carried out for at least 540 h, as Figure 4 shown.

[0045] Through Figure 3 and Figure 4 it can be seen that the immobilized cell particles prepared by the present invention have a long service life, indicating that no leakage of enzymes or cells occurs during the fermentation process.

[0046] When the hardness of the immobilized enzyme particles is small, during continuous production, it will easily cause blockage of the chromatography column, increase the column pressure, and the continuous reaction time is short. The immobilized enzyme in the present invention can have a long continuous time, indicating that the prepared immobilized enzyme particles have good mechanical strength and are not easily soluble in water.

[0047] Figure 5 It is the liquid chromatogram of the reaction solution for preparing D-allulose from fructose in Example 1.

[0048] Example 2 Using the fermentation broth obtained in Example 1, the process of immobilizing the bacteria mainly includes adding a supporting carrier to the fermentation broth, mixing evenly, then adding a crosslinking agent, mixing evenly, and drying to obtain the immobilized bacteria particles. The specific steps are as follows: Step 1: Take fresh fermentation broth, and successively add 4% diatomaceous earth according to the mass-volume fraction, stir at low temperature for 10 min. Add 5% kaolin, stir at low temperature for 10 min. Add 2% activated carbon, stir at low temperature for 30 min to ensure sufficient mixing of the fermentation broth with the three supporting carriers.

[0049] Step 2: To the fermentation broth that is sufficiently mixed in Step 1, successively add 5% chitosan according to the mass-volume fraction, stir at room temperature for 2 h, add 4% polyethyleneimine, stir at room temperature for 10 min, add 2% glutaraldehyde, stir at room temperature for 30 min to obtain a highly viscous solid-liquid mixture in which the fermentation broth, the supporting carrier, and the crosslinking agent are fully mixed.

[0050] Step 3: Separate the solid and liquid in the highly viscous solid-liquid mixture in Step 2 by pressure filtration to obtain a filter cake with a lower water content. Make the precipitate or filter cake into particles through a granulator, slowly dry the particles at 50 °C, further fully crosslink during the dehydration process and finally form the immobilized bacteria particles, and pass through a 40-mesh sieve.

[0051] Catalysis of fructose by the immobilized bacteria particles as a catalyst: Add 40 g of the immobilized bacteria particles to a water bath chromatography column with an inner diameter of 18 * 300 mm. Using a 60% fructose solution as the substrate, under the condition of a water bath temperature of 55 °C, carry out the conversion reaction of fructose at a flow rate of 6 times the particle mass volume per hour (240 mL / h), and measure that the conversion rate of the outlet fructose is 27%, and the sustainable operation time is more than 540 h.

[0052] Example 3 Using the fermentation broth obtained in Example 1, the process of immobilizing the bacteria mainly includes adding a supporting carrier to the fermentation broth, mixing evenly, then adding a crosslinking agent, mixing evenly, and drying to obtain the immobilized bacteria particles. The specific steps are as follows: Step 1: Take fresh fermentation broth, and successively add 3% diatomaceous earth according to the mass-volume fraction, stir at low temperature for 10 min. Add 3% kaolin, stir at low temperature for 30 min. Add 1% activated carbon, stir at low temperature for 1 h to ensure sufficient mixing of the fermentation broth with the three supporting carriers.

[0053] Step 2: To the fermentation broth that is sufficiently mixed in Step 1, successively add 1% chitosan according to the mass-volume fraction, stir at room temperature for 2 h, add 3% polyethyleneimine, stir at room temperature for 1 h, add 2% glutaraldehyde, stir at room temperature for 1 h to obtain a highly viscous solid-liquid mixture in which the fermentation broth, the supporting carrier, and the crosslinking agent are fully mixed.

[0054] Step 3: Separate the solid and liquid in the high-viscosity solid-liquid mixture in Step 2 by pressure filtration to obtain a filter cake with a lower water content. Make the precipitate or filter cake into granules through a granulator, slowly dry the granules at 50°C, further fully crosslink during the dehydration process, and finally form immobilized cell particles. Pass through a 40-mesh sieve to obtain the product.

[0055] Catalysis of fructose by immobilized cell particles: Add 40 g of immobilized cell particles into a water bath chromatography column with an inner diameter of 18 * 300 mm. Use a 60% fructose solution as the substrate. Under the condition of a water bath temperature of 55°C, carry out the conversion reaction of fructose at a volumetric flow rate of 6 times the particle mass per hour (240 mL / h). Measure the conversion rate of the outlet fructose to be 28%, and the sustainable operation time is at least 540 h.

[0056] Comparative Example 1 Whole-cell catalysis: Take an appropriate amount of fresh fermentation broth, centrifuge and concentrate the cells until the wet cell weight is 100 g / L. Take 0.5 mL of cells and mix with a 50% fructose solution. React at 50°C for 3 h to measure the fructose conversion rate under different buffer solutions; measure the fructose conversion rate at different temperatures in a pH 7.0 phosphate buffer solution. Reflect the relative enzyme activity through the conversion rate under incomplete reaction conditions to determine the optimal fructose conversion conditions. From the results of whole-cell catalysis 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.

[0057] Comparative Example 2 The difference from Example 1 is that kaolin is replaced with diatomite in equal amount, and the others are the same as in Example 1.

[0058] When using immobilized cell particles to catalyze the preparation of D-allulose from fructose, at a pH value of 7.0 and a temperature of 60°C, the conversion rate can be maintained at 28%, and it can operate stably for about 240 h.

[0059] Comparative Example 3 The difference from Example 1 is that activated carbon is replaced with kaolin in equal amount, and the others are the same as in Example 1.

[0060] When using immobilized cell particles to catalyze the preparation of D-allulose from fructose, at a pH value of 7.0 and a temperature of 60°C, the conversion rate can be maintained at 27%, and it can operate stably for about 400 h.

[0061] Comparative Example 4 The difference from Example 1 is that activated carbon and diatomite are replaced with kaolin in equal amount, and the others are the same as in Example 1.

[0062] When the immobilized cell particles catalyze the preparation of D-allulose from fructose, at a pH value of 7.0 and a temperature of 60 °C, the conversion rate can be maintained at 22%, and stable operation can be maintained for about 180 h.

[0063] Comparative Example 5 The difference from Example 1 is that chitosan is replaced with polyethyleneimine in equal amounts, and the others are the same as in Example 1.

[0064] When the immobilized cell particles catalyze the preparation of D-allulose from fructose, at a pH value of 7.0 and a temperature of 60 °C, the conversion rate can be maintained at 28%, and stable operation can be maintained for about 440 h.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A strain producing D-psicose-3-epimerase, characterized in that: The classification name is Bacillus halodurans ( Bacillus halotolerans ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is December 26, 2022, and the deposit number is CGMCC No.26285.

2. A bacterial cell immobilization particle, characterized in that: The method comprises a strain, a support carrier and a cross-linking agent, wherein the strain is loaded on the support carrier through the cross-linking agent, and the strain is the strain producing D-psicose-3-epimerase according to claim 1.

3. The bacterial cell immobilization particles according to claim 2, characterized in that: The support carrier is at least one of diatomaceous earth, activated carbon or kaolin.

4. The bacterial cell immobilization particle according to claim 3, characterized in that: The support carrier is a mixture of diatomaceous earth, activated carbon and kaolin, and the mass ratio of diatomaceous earth, activated carbon and kaolin is 2-4:1-2:3-5.

5. The bacterial cell immobilization particles according to claim 2, characterized in that: The cross-linking agent is selected from at least one of polyvinyl alcohol, chitosan, polyvinyl alcohol, glutaraldehyde, polyacrylamide, polyethyleneimine, N,N'-methylenebisacrylamide or polydimethyldiallylammonium chloride.

6. The bacterial cell immobilization particle according to claim 5, characterized in that: 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.

7. The bacterial cell immobilization particle according to any one of claims 2 to 6, characterized in that: The preparation method of the bacterial immobilized particles is as follows: The fermentation broth of the strain producing D-psicose-3-epimerase is mixed with the support carrier, and then a cross-linking agent is added to the fermentation broth, and the mixture is fully mixed to obtain a viscous solid-liquid mixture; The viscous solid-liquid mixture is separated into solid and liquid, the solid is granulated, and dried at 40-60° C. to obtain the product.

8. The bacterial cell immobilization particles according to claim 7, characterized in that: The mass volume ratio of the support carrier to the fermentation liquid is 1%-15%, g / ml; The mass volume ratio of the cross-linking agent to the fermentation broth is 5%-20%, g / ml.

9. A catalytic method, characterized in that: The method comprises the following steps: adding the bacterial cell immobilized particles according to any one of claims 2 to 6 into a fructose solution, heating the solution in a water bath, and catalyzing the fructose to produce D-psicose.

10. The catalytic method according to claim 9, characterized in that: The water bath is heated at 50-60°C.

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