Method for producing psicose by sodium alginate-based immobilized whole cells

Abstract

The invention relates to the technical field of rare sugar preparation, and discloses a method for producing psicose from sodium alginate-based immobilized whole cells, which comprises the following steps: (1) culturing recombinant bacteria containing psicose isomerase, centrifuging, discarding supernatant, and collecting cells; (2) mixing the cells with a sodium alginate aqueous solution, adding the obtained mixed solution into a calcium chloride solution to carry out an immobilization reaction, and filtering to obtain sodium alginate-based immobilized whole-cell globules; and (3) adding the sodium alginate-based immobilized whole-cell globules into a fructose aqueous solution of a metal-free buffer salt system, and reacting to obtain the psicose. Whole cells immobilized by sodium alginate are applied to fructose / high fructose corn syrup diluted by water, and metal ions, acid, alkali or a buffer agent and the like are not added in the reaction process, so that the pressure of downstream purification production is reduced, the production process is simplified, the use of acid, alkali and salt is reduced, and the production cost and the environmental pressure are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of rare sugar preparation, and in particular to a method for producing psicose by using sodium alginate-based immobilized whole cells. Background Art

[0002] D-Allulose or D-psicose is a rare six-carbon ketose belonging to the ketose class and a diastereomer of fructose. Its sweetness is similar to that of sucrose, but its calories are much lower than sucrose. Allulose has been recognized as a safe food additive (GRAS) by the U.S. Food and Drug Administration (FDA) and can be used as an ingredient in food or food additives. It is an ideal natural sweetener. Its content in nature is very rare, but it can be converted from fructose by enzyme catalysis. Allulose has many physiological functions that are beneficial to the human body, such as blood sugar regulation: Allulose has a low absorption rate in the human body and can compete with glucose and fructose for transport proteins on the cell membrane surface, thereby reducing the absorption of these sugars, helping to enhance insulin tolerance and inhibiting postprandial blood sugar increase; reducing fat accumulation: It also helps to reduce the accumulation of intra-abdominal fat; preventing diabetes: These characteristics of allulose make it potentially useful in preventing diabetes; allulose also has anti-inflammatory effects, etc.

[0003] The production of allulose mainly relies on bio-enzyme catalysis technology, especially the application of D-psicose 3-epimerase (DPEase). Through this process, high-purity fructose can be converted into allulose. The traditional production process includes enzyme cloning and expression, separation and purification, acid-base adjustment of the reaction solution, fructose isomerization, separation and purification of allulose, and concentration. The reaction process is relatively complicated, and the use of acids, bases, salts and metal ions in the process will also increase environmental pollution pressure.

[0004] The invention patent with publication number CN 116355888 A discloses a D-psicose-3-epimerase immobilized enzyme preparation and a fixation method thereof, wherein a sodium alginate solution containing cobalt chloride is used as a carrier for immobilized cells, a calcium chloride solution is used as a cross-linking agent, and D-psicose-3-epimerase immobilized cells are obtained by embedding-cross-linking method; and then the D-psicose-3-epimerase immobilized cells are adsorbed by resin to obtain a D-psicose-3-epimerase immobilized enzyme preparation. Although this method can improve the conversion rate and enzyme activity recovery rate of the immobilized enzyme preparation, it inevitably uses metal ions, which will not only increase the downstream purification process and environmental pollution pressure, but also greatly increase the cost. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for producing allulose by using sodium alginate-based immobilized whole cells. By using sodium alginate to immobilize cells and optimizing the process, it is helpful to reduce the downstream purification process and promote the green, low-carbon and sustainable development of the rare sugar industry. In addition, the immobilized cells have significant advantages in mechanical strength, preservation state and other properties, and have a higher application prospect.

[0006] The purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for producing psicose by using sodium alginate-based immobilized whole cells, comprising the following steps: (1) culturing a recombinant bacterium containing psicose isomerase, discarding the supernatant by centrifugation, and collecting the cells; wherein the psicose isomerase is derived from Staphylococcus aureus, and its amino acid sequence is shown in SEQ ID NO.1; (2) mixing the cells with a sodium alginate aqueous solution, adding the resulting mixed solution into a calcium chloride solution for immobilization reaction, and filtering to obtain sodium alginate-based immobilized whole-cell pellets; (3) The sodium alginate-based immobilized whole-cell pellets are added to a fructose aqueous solution to obtain psicose after reaction.

[0007] Preferably, the DPEase is derived from Staphylococcus aureus and is referred to as Sa-DPEase (DPEase derived from Staphylococcus aureus), and its amino acid sequence is shown in SEQ ID NO.1.

[0008] Preferably, the recombinant bacteria containing psicose isomerase is Escherichia coli containing a pET-28a plasmid, and the pET-28a plasmid contains a gene encoding psicose isomerase. The gene sequence encoding psicose isomerase is shown in SEQ ID NO.2.

[0009] Preferably, the culturing step comprises: inoculating the recombinant bacteria containing psicose isomerase into an LB medium containing kanamycin, and culturing overnight at 20-37° C. and 150-1200 rpm; then transferring the bacterial solution cultured overnight to an LB medium containing α-lactose at an inoculation rate of 1-20%, culturing at 36-38° C. and 180-240 rpm for 2-3 hours, and then culturing at 16-37° C. and 160-200 rpm for 16-20 hours.

[0010] Kanamycin was used to maintain the resistance of the engineered bacteria, and lactose was used to induce the expression of psicose isomerase.

[0011] More preferably, the culturing step comprises: inoculating the recombinant bacteria containing psicose isomerase into an LB medium containing kanamycin, and culturing overnight at 34-37° C. and 1000-1200 rpm; then transferring the bacterial solution cultured overnight to an LB medium containing α-lactose at an inoculum rate of 10-15%, culturing at 36-38° C. and 180-240 rpm for 2-3 hours, and then culturing at 16-20° C. and 160-200 rpm for 16-20 hours.

[0012] Preferably, the concentration of kanamycin in the LB medium containing kanamycin is 40-60 μg / mL; the mass concentration of α-lactose in the LB medium containing α-lactose is 0.8-1.2%.

[0013] Preferably, the centrifugal speed is 2000-5000 rpm and the time is 5-40 min.

[0014] Preferably, the mass concentration of the sodium alginate aqueous solution is 2-4%; the mixing ratio of the cells (wet weight of the bacteria) to the sodium alginate aqueous solution is 20-100 g:1L.

[0015] Preferably, the mass concentration of the calcium chloride solution is 1-4%.

[0016] Preferably, the immobilization reaction time is 2 to 6 hours.

[0017] Preferably, an immobilization reaction is carried out, filtered and then dried to obtain sodium alginate-based immobilized whole-cell pellets; the drying temperature is 30 to 70° C. and the drying time is 30 to 90 minutes.

[0018] More preferably, the drying temperature is 60° C. and the drying time is 30 to 60 minutes.

[0019] The drying time of sodium alginate-based immobilized whole-cell pellets is too long, which is beneficial to improving the mechanical strength of the immobilized whole cells and reducing the requirements for the storage environment, but it will lead to a large loss in conversion rate.

[0020] Preferably, the concentration of fructose in the fructose aqueous solution is 200-700 g / L; and the ratio of the amount of the sodium alginate-based immobilized whole-cell pellet to the fructose aqueous solution is 1-10 g:10 mL.

[0021] Preferably, the fructose aqueous solution is prepared by diluting fructose or fructose-glucose syrup with water.

[0022] Preferably, the reaction is carried out under a rotation condition at 50-70° C. for 1-3 hours.

[0023] Compared with the prior art, the present invention has the following beneficial effects: the present invention applies the immobilized whole cells (including dried immobilized whole cells) to the fructose / fructose-glucose syrup diluted with water, and the dried immobilized whole cells have a partial loss in conversion rate relative to the undried sodium alginate whole cells, but their volume, mechanical strength, storage state and other properties are significantly improved, and have a higher application prospect. At the same time, the medium in the reaction process is pure water, and no metal ions, acids, bases or buffers are added, which reduces the pressure of downstream purification production, simplifies the production process, reduces the use of acids, bases and salts, and reduces production costs and environmental pressures. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the conversion of fructose to psicose by psicose isomerase.

[0025] Figure 2 This is the SDS-PAGE detection diagram of Sa-DPEase enzyme in the supernatant in Example 3.

[0026] Figure 3 This is a graph showing the relative content of allulose after adding different metal whole cells for catalysis in Example 4.

[0027] Figure 4 It is the HPLC chromatogram after the isomerization reaction of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described below with specific embodiments, but the protection scope of the present invention is not limited thereto: In a specific embodiment of the present invention, trypsin was purchased from Haibo Biotechnology Co., Ltd., α-lactose was purchased from Shanghai McLean Biochemical Technology Co., Ltd., yeast extract was purchased from Thermo Fisher Scientific, sodium chloride was purchased from Guangdong Guanghua Technology Co., Ltd., sodium alginate with a viscosity coefficient of 300-500 was donated by Qingdao Mingyue Seaweed Group Co., Ltd., anhydrous calcium chloride was purchased from Xilong Science, fructose was purchased from Aladdin Technology Co., Ltd., kanamycin was purchased from Shanghai Biotech, SDS-PAGE was purchased from Changzhou Boyi Biotechnology Co., Ltd., and Tris and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] The amino acid sequence of allulose isomerase Sa-DPEase (DPEase enzyme from Staphylococcus aureus) was obtained from the NCBI database or literature reports, and Suzhou Jinweizhi Co., Ltd. was commissioned to optimize and chemically synthesize the amino acid sequence according to the sequence shown in SEQ ID NO.1, and the gene synthesis shown in SEQ ID NO.2 was also commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd.

[0030] SEQ ID NO.1 "Amino acid sequence of Sa-DPEase": MNIGCHGLVWTGNFDAEGIRLAVHKTREAGFDLIEFPLMDPFTFDVSVAKDALEEYDLAVS ASLGLSEATDVTSADPAIAAAGEALLVKAVDVLADLGGEHFCGVIYSAMKKYMEPVTPEGL ASSKAAIARVADHAAARGVSVSLEVVNRYETNVLNTARQALAYIAEVDRPNLGIHIDTYHM NIEESDMFTPVLDAAPALRYVHIGESHRGYLGTGTVDFDTFFKALGRIGYGGPIVFESFSSAV VAPDLSRMLGIWRNLWNDNDELGAHANAYIRDKLVAVDSIHHHHHH SEQ ID NO.2 "Gene sequence of Sa-DPEase": atgaacattggctgccatggcctggtgtggaccggcaactttgatgcggaaggcattcgcctggcggtgcataaaacccgcgaagcgggcttt gacctgattgaatttccgctgatggatccgtttacctttgatgtgagcgtggcgaaagatgcgctggaagaatatgatctggcggtgagcgcgag cctgggcctgagcgaagcgaccgatgtgacgagcgcggatccggcgattgcggccgcgggtgaagcgctgctggtgaaagcggtggatgt gctggcggatctgggcggcgaacatttttgcggcgtgatttatagcgcgatgaaaaaatatatggaaccggtgaccccggaaggcctggcgag cagcaaagcggcgattgcgcgcgtggccgatcatgcggccgcgcgtggcgtgagcgtgagcctggaagtggtgaaccgctatgaaaccaac gtgctgaacaccgcgcgccaagcgctggcgtatattgcggaagtggatcgcccgaacctgggcattcatattgatacctatcacatgaacattga agaaagcgatatgtttaccccggtgctggatgcggcgccggcgctgcgctatgtgcatattggcgaaagccatcgcggctatctgggcaccgg caccgtggattttgataccttttttaaagcgctgggccgcattggctatggcggcccgattgtgtttgaaagctttagcagcgcggtggtggcgcc ggatctgagccgcatgctgggcatttggcgcaacctgtggaacgataacgatgaactgggcgcgcatgcgaacgcgtatattcgcgataaact ggtggcggtggatagcattcgcctgactcatcatcatcatcattaa Example 1 (strain culture) (1) Preparation of recombinant bacteria: Suzhou Jinweizhi Company was commissioned to optimize the sequence and chemically synthesize the psicose isomerase (Sa-DPEase) gene, and then connected to the vector pET-28a(+) using the NdeI and XhoI restriction sites to obtain the recombinant plasmid pET-28a(+)-DPE. The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the transformation product was spread on LB solid culture medium (containing 100 mg·mL-1 kanamycin sulfate), and inverted cultured in a 37°C constant temperature incubator for 12 to 16 hours to obtain recombinant bacteria containing psicose isomerase.

[0031] (2) Inoculation: Use a sterilized toothpick to pick up recombinant E. coli BL21 (DE3) from a -80°C cryotube and inoculate into another 96-well plate containing 500 μL LB (containing 50 μg / mL kanamycin) and culture overnight at 37°C and 1000 rpm.

[0032] (3) Transfer induction: Then, the bacterial solution cultured overnight was inoculated into a 96-well plate containing 1.1 mL of LB medium containing 1% α-lactose at a 10% inoculum volume (100 μL), and cultured at 37°C and 220 rpm for 2 to 3 h; the temperature was changed to 18°C ​​and 180 rpm for 16 to 20 h; (4) Harvesting the cells: Centrifuge the induced bacterial solution at 4500 rpm for 10 min at room temperature, discard the supernatant, and collect the cells.

[0033] Example 2 (Protein soluble expression and activity verification) (1) Reaction: Add 1 mL of 100 g / L fructose aqueous solution to the cells collected in Example 1 and mix thoroughly, then place in a shaker at 60°C and 400 rpm for 1 h. Take 200 μL of the bacterial solution to detect OD600, and heat the remaining whole cell reaction solution in a 100°C water bath for 10 min, and centrifuge at 4000 rpm for 10 min.

[0034] (2) Data collection: The supernatant was taken and filtered through a 0.22 μm filter membrane for HPLC detection.

[0035] (3) Protein solubility verification: After the cultured bacterial solution was used for ultrasonic cell disruption, the cell disruption solution was centrifuged at 8000×g for 20 min at 4°C, and the supernatant was taken for SDS-PAGE (4-12%) detection.

[0036] like Figure 2 As shown, the arrow indicates the location of the Sa-DPEase enzyme protein in the soluble part of the supernatant solution.

[0037] Example 3 (Effect of adding different metal whole cell catalysis on the content of psicose) Control group (control group): 1 mL of 100 g / L fructose aqueous solution was added to the cells collected in Example 1 without adding metal ions. After sufficient mixing, the cells were placed in a shaker at 60°C and 400 rpm for reaction for 1 h, and then heated in a 100°C water bath for 10 min. The resulting reaction solution was centrifuged at 4000 rpm for 10 min, the supernatant was taken, passed through a 0.22 μm filter membrane, and then detected by HPLC.

[0038] Add different metals: add 1 mL of 100 g / L fructose aqueous solution to the cells collected in Example 1, add 5 mM Mg 2+ 、Ni 2+ 、Co 2+ and Mn 4+ After fully mixing, place it in a shaker at 60℃ and 400rpm for reaction for 1h, then transfer it to a 100℃ water bath for heating for 10min. The resulting reaction solution is centrifuged at 4000rpm for 10min, the supernatant is taken, passed through a 0.22μm filter membrane, and then detected by HPLC.

[0039] like Figure 3As shown in the results, the addition of metal ions has no obvious advantage over the control group without metal addition, indicating that the DPEase enzyme Sa-DPEase (DPEase enzyme from Staphylococcus aureus) screened in the present invention can catalyze the reaction without the addition of metal ions, and then the subsequent catalytic reaction without metal addition, coupled with the immobilization effect of sodium alginate, can also obtain a higher conversion rate of DPEase.

[0040] Example 4 (Preparation of Sodium Alginate-Based Immobilized Whole Cell Pellets (Undried)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0041] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the resulting mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle at a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was performed for 4 hours. Afterwards, the reaction solution was filtered and washed twice with water to obtain a sodium alginate-based immobilized whole cell pellet (not dried), which was stored in water at 4°C.

[0042] Example 5 (Preparation of sodium alginate-based immobilized whole-cell pellets (dried for 20 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0043] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle with a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 20 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 20 minutes), which was stored at 4° C.

[0044] Example 6 (Preparation of sodium alginate-based immobilized whole-cell pellets (dried for 30 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0045] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle with a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 30 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 30 minutes), which was stored at 4° C.

[0046] Example 7 (Preparation of sodium alginate-based immobilized whole-cell pellets (dried for 40 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0047] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle at a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 40 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 40 minutes), which was stored at 4° C.

[0048] Example 8 (Preparation of sodium alginate-based immobilized whole-cell pellets (dried for 50 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0049] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) through a 25MM / 22G four-tube needle using a Watson-Marlow 5300u peristaltic pump at a flow rate of 7.5 mL / min, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 50 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 50 minutes), which was stored at 4° C.

[0050] Example 9 (Preparation of sodium alginate-based immobilized whole-cell pellets (dried for 60 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0051] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle with a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 60 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 60 minutes), which was stored at 4° C.

[0052] Example 10 (Preparation of Sodium Alginate-Based Immobilized Whole Cell Pellets (Drying for 70 min)) Sodium alginate (viscosity coefficient 300-500) was dissolved in 80°C water at a mass fraction of 2%, and stirred continuously until it was completely dissolved into a uniform and transparent solution, and then returned to room temperature.

[0053] The cells collected in Example 1 were mixed with a sodium alginate aqueous solution at a ratio of 60 g:1 L (wet weight of the cells: volume of the sodium alginate aqueous solution), stirred until mixed into a uniform solution, and the mixed solution was squeezed into a stirred calcium chloride solution (mass concentration of 2%) using a 25MM / 22G four-tube needle at a flow rate of 7.5 mL / min using a Watson-Marlow 5300u peristaltic pump, and the immobilization reaction was carried out for 4 hours. Afterwards, the reaction solution was filtered, washed twice with water, and dried in an oven at 60° C. for 60 minutes to obtain a sodium alginate-based immobilized whole cell pellet (dried for 70 minutes), which was stored at 4° C.

[0054] Example 11 (Reuse of immobilized cells) 5 g of sodium alginate-based immobilized whole-cell pellets (Example 5) were weighed and added to 10 mL of diluted fructose syrup aqueous solution (content 500 g / L). After rotating at 60° C. for 1 h, the reaction solution was filtered, HPLC detection was performed, and the fructose conversion rate was calculated.

[0055] The alginate-based immobilized whole cell pellets were taken out after the reaction, washed with water, and excess water was removed with filter paper, and then 10 mL of new fructose syrup aqueous solution (content 500 g / L) was added, and the process was repeated for 8 batches. Each batch of samples was tested by HPLC, and the fructose conversion rate was calculated.

[0056] Example 12 (Reuse of immobilized cells) 5 g of sodium alginate-based immobilized whole-cell pellets (Example 9) were weighed and added to 10 mL of diluted fructose syrup aqueous solution (content of 500 g / L). After rotating at 60° C. for 1 h, the reaction solution was filtered, HPLC detection was performed, and the fructose conversion rate was calculated.

[0057] The alginate-based immobilized whole cell pellets after the reaction were taken out, washed with water, and excess water was removed with filter paper, and then 10 mL of new fructose syrup aqueous solution (content 500 g / L) was added, and the process was repeated for 8 batches. Each batch of samples was tested by HPLC, and the fructose conversion rate was calculated.

[0058] HPLC detection: High performance liquid chromatograph: Waters Alliance HPLC e2695 chromatography system Chromatographic column: Waters Sugar-Pak Column, 9 μm, 6.5 × 300 mm Column temperature: 90°C Detector: Waters 2414 differential refractometer Detection temperature: 50℃ Mobile phase: 50mg / L calcium disodium EDTA Mobile phase speed: 0.5mL / min Injection volume: 10 μL Table 1 like Figure 1 FIG. 1 is a schematic diagram showing the conversion of fructose to psicose by psicose isomerase. In the present invention, the filtrate of the reaction solution of fructose / high fructose syrup and sodium alginate-based immobilized whole cell beads is subjected to HPLC detection. Part of the HPLC detection chromatogram is shown in FIG. Figure 4 As shown, the fructose and allulose standards were tested by HPLC according to the above parameters, and the retention times were around 12.235min and 18.460min, respectively. Figure 4 The results show that the present invention can successfully react to obtain psicose. The peak area of ​​the target peak is obtained by HPLC detection of the chromatogram, and the content and conversion rate of psicose in the reaction product are calculated based on the standard curve.

[0059] As shown in Table 1, the dried sodium alginate-based immobilized whole-cell pellets have some loss in conversion rate compared to the undried sodium alginate-based immobilized whole-cell pellets, but the dried sodium alginate-based immobilized whole-cells have a smaller volume, higher mechanical strength, and longer storage time. Since the conversion rate during the reaction of batch 1 is relatively low, the activity of the allulose isomerase is better after heating. Therefore, the conversion rate of batch 2 is taken as the actual conversion rate of batch 1, and the difference between the conversion rate of subsequent batches and the conversion rate of batch 2 is calculated, indicating that the dried sodium alginate-based immobilized whole-cells can obtain higher reaction stability, and can still be stable at a higher conversion rate during multiple repeated reactions, and the difference fluctuation is small.

[0060] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for producing psicose by using sodium alginate-based immobilized whole cells, characterized in that: The steps include: (1) Cultivating a recombinant bacterium containing psicose isomerase, discarding the supernatant by centrifugation, and collecting the cells; wherein the psicose isomerase is derived from Staphylococcus aureus, and its amino acid sequence is shown in SEQ ID NO.1; (2) mixing the cells with a sodium alginate aqueous solution, adding the resulting mixed solution into a calcium chloride solution for immobilization reaction, and filtering to obtain sodium alginate-based immobilized whole-cell pellets; (3) The sodium alginate-based immobilized whole-cell pellets are added to a fructose aqueous solution to obtain allulose after reaction.

2. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1, characterized in that: The recombinant bacteria containing psicose isomerase is Escherichia coli containing a pET-28a plasmid, and the pET-28a plasmid contains a gene encoding psicose isomerase.

3. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 2, characterized in that: The gene sequence encoding psicose isomerase is shown in SEQ ID NO.

2.

4. The method for producing psicose by using sodium alginate-based immobilized whole cells according to any one of claims 1 to 3, characterized in that: The culturing step includes: inoculating the recombinant bacteria containing psicose isomerase into an LB medium containing kanamycin, and culturing overnight at 20-37° C. and 150-1200 rpm; then transferring the bacterial solution cultured overnight to an LB medium containing α-lactose at an inoculum amount of 1-20%, culturing at 36-38° C. and 180-240 rpm for 2-3 hours, and then culturing at 16-37° C. and 160-200 rpm for 16-20 hours.

5. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 4, characterized in that: The concentration of kanamycin in the LB culture medium containing kanamycin is 40-60 μg / mL; the mass concentration of α-lactose in the LB culture medium containing α-lactose is 0.8-1.2%.

6. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1, characterized in that: The centrifugal speed is 2000-5000 rpm and the time is 5-40 min.

7. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1, characterized in that: The mass concentration of the sodium alginate aqueous solution is 2-4%; the mixing ratio of the cells to the sodium alginate aqueous solution is 20-100 g:1L.

8. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1, 6 or 7, characterized in that: The mass concentration of the calcium chloride solution is 1-4%.

9. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1, characterized in that: The immobilization reaction time is 2 to 6 hours.

10. The method for producing psicose by using sodium alginate-based immobilized whole cells according to claim 1 or 9, characterized in that: The immobilization reaction is carried out, and the mixture is filtered and dried to obtain sodium alginate-based immobilized whole-cell pellets; the drying temperature is 30-70° C. and the drying time is 30-90 min.

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