Method for producing immobilized cells for tagatose production and use thereof

Abstract

The present invention provides a method for producing immobilized cells that produce tagatose, and a method for producing tagatose using the same. The method for producing the immobilized cells includes the steps of: mixing Escherichia coli or Bacillus subtilis fermentation liquids expressing α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase to obtain a fermentation mixture; adding inorganic soil and stirring uniformly; further adding a flocculant to flocculate the cells; then adding a crosslinking agent to crosslink the cells; vacuum filtering to obtain a filter cake; extruding the filter cake into strands using a rotary granulator to granulate them; then cutting the filter cake into pellets of uniform length using a spheronizer; and fluidizing and drying the resulting mixture to obtain immobilized cells for producing tagatose. The present invention simplifies the steps for separating and purifying enzymes required for tagatose production, increases the enzyme recovery and utilization rate, and realizes enzyme recycling.

Description

[Technical Field]

[0001] The present disclosure relates to the technical field of biotechnology, and in particular to the field of tagatose production and manufacturing. [Background technology]

[0002] Tagatose is a naturally occurring, rare monosaccharide, a ketose form of galactose and an epimer of fructose. Tagatose's sweetening properties are similar to those of sucrose, providing a refreshingly pure sweetness. However, it contains only one-third the calories of sucrose, making it a low-calorie sweetener. Research has shown that tagatose possesses important physiological properties, including low calorie content, low glycemic index, anti-caries properties, antioxidant properties, probiotic properties, intestinal function improvement properties, immunomodulatory properties, and drug precursor properties. Its wide application in food, beverages, medicine, and health maintenance has enormous economic value (Oh DK: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76:1-8).

[0003] The Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, has successfully established a new in vitro multienzyme synthesis route for tagatose using inexpensive corn starch, maltodextrin, sucrose, and other raw materials, fundamentally changing existing tagatose production technologies (CN106399427A). This new multienzyme synthesis route not only overcomes the limitations of chemical tagatose synthesis, such as high energy consumption, complex products, difficult purification, numerous side reactions, and severe chemical pollution, but also resolves the shortcomings of biological tagatose synthesis, such as expensive and limited raw materials, low conversion rates, and complex separation processes. Based on this, the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, has developed a whole-cell catalyst tagatose production method (CN107988286A) using inexpensive corn starch, cellulose, maltodextrin, sucrose, and other raw materials. This process reduces the number of steps in the multienzyme purification process, reduces production costs, reduces environmental pollution, and increases tagatose yields. The Tianjin Institute of Industrial Biology, Chinese Academy of Sciences, has further addressed the issue of safe production of food formulations by providing a method for producing high-concentration tagatose using Bacillus subtilis as a whole-cell catalyst with high-concentration starch (CN112342179B). This method involves immobilizing permeabilized Bacillus subtilis to obtain immobilized whole cells, which are then used to produce tagatose, allowing for whole-cell recycling and reducing production costs. However, research and analysis have revealed that this method still has several shortcomings when producing immobilized cells. First, this method requires many tedious steps, such as cell collection, cell resuspension, and cell permeabilization, prior to immobilization. Second, this method relies on permeabilized cells for granulation immobilization, but cell permeabilization is prone to leakage of heterologous proteins expressed within the cells, resulting in loss of heterologous proteins and reduced enzyme immobilization efficiency during the immobilization process. Furthermore, this method merely obtains immobilized enzyme particles by simple extrusion granulation, and the resulting immobilized enzyme particles are not uniform.

[0004] Therefore, there is an urgent need to develop a method for easily obtaining uniform immobilized cells that can simplify the enzyme production process, avoid or reduce leakage or loss of enzymes from permeable cells due to immobilization, increase the enzyme immobilization efficiency, realize enzyme recycling, reduce the production cost of tagatose, and enable industrial production of tagatose. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to provide a method for producing tagatose using immobilized cells, which simplifies the enzyme production steps in tagatose production, simplifies the separation and purification of products and enzymes in tagatose production, enables the recycling of multiple enzymes, reduces the production costs of tagatose, and enables industrial production of tagatose, in response to the problems present in conventional methods for producing tagatose using immobilized cells, such as complicated enzyme production steps, permeabilization treatment causing enzyme loss and reduced immobilization efficiency, and the particle size of the produced immobilized enzyme not being uniform. [Means for solving the problem]

[0006] To solve the above technical problems, the present disclosure adopts the following technical solutions:

[0007] A method for producing immobilized cells for producing tagatose, comprising: Obtaining fermentation liquids of Escherichia coli or Bacillus subtilis that express α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase by fermentation, and mixing the fermentation liquids to obtain a fermentation mixture; Adding inorganic soil to the fermentation mixture and stirring uniformly; adding a flocculant to the fermentation mixture to flocculate the bacterial cells, and then adding a cross-linking agent to cross-link the bacterial cells; Vacuum filtering to obtain a filter cake, extruding the filter cake into strands using a rotary granulator to granulate them, and then cutting the extrudate into pellets with a uniform length using a spheronizer; and a step of fluidizing and drying the pellet to obtain immobilized cells for producing tagatose.

[0008] In one particular embodiment, the present disclosure adopts the following technical solutions:

[0009] A method for producing immobilized cells for producing tagatose, comprising: Obtaining fermentation liquids of Escherichia coli or Bacillus subtilis that express α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase by fermentation, and mixing the fermentation liquids to obtain a fermentation mixture; adding 1 to 10% w / v inorganic soil to the fermentation mixture and stirring uniformly; Further, adding 0.1 to 2% w / v of a flocculant to the fermentation mixture to flocculate the bacterial cells, and then adding 0.05 to 3% v / v of a cross-linking agent to cross-link the bacterial cells for 1 to 4 hours; filtration under vacuum to obtain a filter cake, extruding the filter cake into strands using a rotary granulator, and then cutting the strands of immobilized cells into pellets with a uniform length using a spheronizer; and a step of fluidizing and drying the pellets by controlling the temperature at the blowing port to 60 to 90°C to obtain immobilized cells for producing tagatose.

[0010] The fermentation broth can be produced by a method known in the art. Any medium suitable for the production of foreign proteins may be used for the fermentation, including, but not limited to, LB medium, SR medium, TB medium, etc.

[0011] Preferably, the α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase are thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose phosphate isomerase, thermostable tagatose-6-phosphate isomerase, and thermostable tagatose-6-phosphate phosphatase, respectively.

[0012] Specifically, the thermostable α-glucan phosphorylase refers to an enzyme that has the function of phosphorylating starch to glucose-1-phosphate (G1P) at temperatures of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. More preferably, the thermostable α-glucan phosphorylase is produced from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor cronotskiiensis, or the like. or the amino acid sequence of said thermostable α-glucan phosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical to that of the thermostable α-glucan phosphorylase derived from said thermophilic microorganism.More preferably, the thermostable α-glucan phosphorylase is derived from Thermococcus kodakarensis.

[0013] Specifically, thermostable phosphoglucomutase refers to an enzyme that has the function of converting glucose-1-phosphate (G1P) to glucose-6-phosphate (G6P) at temperatures of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. More preferably, the thermostable phosphoglucomutase is derived from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor cronotskiiensis, or the like. or the amino acid sequence of the thermostable phosphoglucomutase is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical to that of the thermostable phosphoglucomutase derived from the thermophilic microorganism.More preferably, the thermostable phosphoglucomutase is derived from Thermococcus kodakarensis.

[0014] Specifically, thermostable glucose phosphate isomerase refers to an enzyme that has the function of converting glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P) at temperatures of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. More preferably, the thermostable glucose phosphate isomerase is produced from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor kronotskyensis, The thermostable glucose phosphate isomerase is derived from Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, or the like, or the amino acid sequence of the thermostable glucose phosphate isomerase has at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identity with the thermostable glucose phosphate isomerase derived from the thermophilic microorganism.More preferably, the thermostable glucose phosphate isomerase is derived from Thermus thermophilus.

[0015] Specifically, the thermostable tagatose-6-phosphate epimerase refers to an enzyme that has the function of isomerizing fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) at 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. More preferably, the thermostable tagatose-6-phosphate epimerase is produced from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor cronotskiiensis, or the like. or the amino acid sequence of the thermostable tagatose-6-phosphate epimerase derived from a thermophilic microorganism is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical to that of the thermostable tagatose-6-phosphate epimerase derived from a thermophilic microorganism.More preferably, the thermostable tagatose-6-phosphate epimerase is derived from Thermoanaerobacter indiensis.

[0016] Specifically, the tagatose-6-phosphate phosphatase refers to an enzyme that has the function of removing a phosphate group from tagatose-6-phosphate (T6P) to produce tagatose as a product at temperatures of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. More preferably, the tagatose-6-phosphate phosphatase is produced from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor cronotskiiensis, or the like. or the tagatose-6-phosphate phosphatase is derived from a thermophilic microorganism such as Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, or Archaeoglobus profundus, or the amino acid sequence of the tagatose-6-phosphate phosphatase has at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identity with the tagatose-6-phosphate phosphatase derived from the thermophilic microorganism.More preferably, the tagatose-6-phosphate phosphatase is derived from Archaeoglobus fulgidus.

[0017] More preferably, wet bacterial cells expressing thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose phosphate isomerase, thermostable tagatose-6-phosphate epimerase, and thermostable tagatose-6-phosphate phosphatase are mixed in a ratio of (0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10), and the OD600 of the bacterial suspension after mixing is between 10 and 150.

[0018] Furthermore, the mineral soil includes, but is not limited to, montmorillonite, diatomaceous earth, kaolin, bentonite, etc., and preferably, the mineral soil is diatomaceous earth.

[0019] Furthermore, the flocculant includes, but is not limited to, polyethyleneimine, chitosan, polydimethyldiallylammonium chloride (PDADMAC), polyacrylamide, etc., and preferably, the flocculant is polyethyleneimine or PDADMAC, and preferably, the molecular weight of the polyethyleneimine is 600 to 70,000.

[0020] Furthermore, the cross-linking agent includes, but is not limited to, glutaraldehyde, tris(hydroxymethyl)phosphine, N,N-methylenebisacrylamide, epichlorohydrin, etc., and preferably, the cross-linking agent is glutaraldehyde.

[0021] The method further comprises the step of screening the obtained immobilized cells to obtain immobilized cells with a uniform morphology.

[0022] Therefore, the present disclosure also provides a method for producing tagatose using immobilized cells, which comprises converting starch or a starch derivative into tagatose using the immobilized cells.

[0023] After the reaction is completed, the method further includes a step of recovering the immobilized cells by filtration.

[0024] In a specific embodiment, the bioconversion reaction system includes 50-300 g / L of starch or starch derivative, a buffer solution having a pH value of 5.0-8.0, 10-50 mM inorganic phosphate, 3-7 mM divalent magnesium ions, and immobilized cells.

[0025] Furthermore, the buffer may be a HEPES buffer, a phosphate buffer, a Tris buffer, an acetate buffer, etc. The inorganic phosphate may be sodium phosphate or potassium phosphate. [Effects of the Invention]

[0026] Compared with the prior art, the present disclosure has the following beneficial effects. The method for producing tagatose using immobilized cells not only simplifies the enzyme production and manufacturing process compared to multienzyme catalytic reactions, but also overcomes the difficulty of separating the multienzyme from the product, facilitating the separation and purification of the product, tagatose. The immobilized cells and the reaction solution can be separated by simple filtration, which makes enzyme separation from the product easier than whole-cell catalytic reactions and allows for repeated use of the enzyme, thereby improving cell utilization and reducing tagatose production costs. Furthermore, repeated use of cells avoids environmental pollution caused by multiple fermentations and simplifies operational steps. The present disclosure produces and mixes a fermentation broth containing expressed enzymes, and then uses the fermentation mixture directly for granulation. This important step eliminates the steps of collecting the fermentation broth, resuspending the fermentation broth, and permeabilizing the fermentation broth (omitting the fermentation broth ... On the other hand, when immobilization was performed directly using a fermentation mixture, the cell membrane and cell wall of the cells were hardly damaged, and the enzyme expressed after immobilization was less likely to leak, resulting in high enzyme immobilization efficiency. In the present disclosure, to obtain immobilized cells by immobilization, strands of controlled thickness were first produced using a rotary extrusion granulator. The strands were then cut into uniform-length particles using a spheronizer. The particles were then dried at high temperature using a fluidized bed dryer (to achieve the purpose of cell permeabilization), and sieved to obtain immobilized enzyme particles with uniform particle sizes, which can be more effectively used in tagatose production. The granulation process used in the present disclosure is not only advantageous for the subsequent permeabilization treatment and particle uniformity, but also simplifies the previous cell collection step (the specific process of cell immobilization in the present disclosure is shown in Figure 1). Experimental results showed that the effects of the present disclosure were very significant, with continuous catalytic reactions using the immobilized Bacillus subtilis of the present disclosure achieving a maximum initial product yield of 75%, and even after 65 batches of continuous catalytic reactions, the product yield could be maintained at 61%. Continuous catalytic reactions using the immobilized E. coli of the present disclosure achieved a maximum initial product yield of 74%, and even after 65 batches of continuous catalytic reactions, the product yield could be maintained at 60%. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram showing a specific flow of cell immobilization according to the present disclosure. [Figure 2] 1 shows the effect of tagatose production by immobilized Bacillus subtilis in Example 3. [Figure 3] 1 shows the effect of tagatose production by immobilized E. coli in Example 11. [Figure 4] 1 shows the effect of tagatose production by Bacillus subtilis in Comparative Example 1. [Figure 5] 1 shows the effect of tagatose production by Escherichia coli in Comparative Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0028] Definition of Terms The terms "a" or "an," when used in the claims and / or specification, along with the term "comprising," can mean "one," but can also mean "one or more," "at least one," or "one or more."

[0029] When used in the claims and the specification, the words "comprise," "have," "comprise," or "contain" are meant to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0030] Although the disclosed subject matter supports the definition of the term "or" as merely alternatives or "and / or," the term "or" in the claims means "and / or" unless explicitly stated as merely alternatives or mutually exclusive between alternatives.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, the methods and materials described herein are preferred.

[0032] To further illustrate the technical means and their effects employed in the present disclosure, the technical solutions of the present disclosure are further described below using specific examples. However, it should be understood that the above examples are merely illustrative and do not limit the scope of the present disclosure. Unless otherwise specified, all experimental techniques and methods used in the present examples are conventional technical methods. For example, experimental methods for which specific conditions are not described in the following examples generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, all materials and reagents used in the examples are commercially available. Those skilled in the art may modify or substitute the details and forms of the technical solutions of the present disclosure without departing from the spirit and scope of the present disclosure, and it should be understood that all such modifications and substitutions fall within the scope of the present invention.

[0033] Example 1: Preparation of Bacillus subtilis whole cells Recombinant Bacillus subtilis strains (starting strain SCK6, see CN112342179B) expressing the thermostable α-glucan phosphorylase gene, the thermostable phosphoglucomutase gene, the expressed thermostable glucose phosphate isomerase gene, the expressed thermostable tagatose-6-phosphate epimerase gene, and the thermostable tagatose-6-phosphate phosphatase gene were each collected, inoculated into LB medium, and cultured overnight at 37°C with shaking. The culture was transferred to LB medium at an inoculation amount of 1% and cultured overnight with shaking at 37°C to obtain a Bacillus subtilis fermentation liquid expressing thermostable α-glucan phosphorylase, a Bacillus subtilis fermentation liquid expressing thermostable phosphoglucomutase, a Bacillus subtilis fermentation liquid expressing thermostable glucose phosphate isomerase, a Bacillus subtilis fermentation liquid expressing thermostable tagatose-6-phosphate epimerase, and a Bacillus subtilis fermentation liquid expressing thermostable tagatose-6-phosphate phosphatase, respectively.

[0034] Example 2: Production of tagatose by immobilized Bacillus subtilis The fermentation broths of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, Bacillus subtilis expressing the thermostable phosphoglucomutase, Bacillus subtilis expressing the thermostable glucose phosphate isomerase, Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 1% w / v montmorillonite was added to the bacterial suspension and stirred uniformly. Next, a 0.5% w / v aqueous solution of polyethyleneimine with a molecular weight of 10,000 was added and the mixture was allowed to flocculate at room temperature. A 2% v / v aqueous solution of glutaraldehyde was then added and the mixture was allowed to crosslink at room temperature for 2 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 60°C to obtain immobilized cells.

[0035] A 1L reaction system was mixed with 100g / L starch, 50mM sodium phosphate buffer (pH 7.0), and immobilized Bacillus subtilis to a final concentration of 20. The reaction was performed in a shaker at 70°C in a water bath. The tagatose content was analyzed during the reaction by high-performance liquid chromatography. After the reaction was completed, the immobilized Bacillus subtilis was collected by simple filtration, washed with buffer, and then used in the next batch of reaction. Experimental results showed that in continuous catalytic reactions using immobilized Bacillus subtilis, the initial product yield reached a maximum of 75%, and even after 65 batches of continuous catalytic reactions, the product yield remained at 58%.

[0036] Example 3: Production of tagatose by immobilized Bacillus subtilis The fermentation broths of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, Bacillus subtilis expressing the thermostable phosphoglucomutase, Bacillus subtilis expressing the thermostable glucose phosphate isomerase, Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 0.1% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and the mixture was allowed to flocculate at room temperature. A 1% v / v aqueous solution of glutaraldehyde was then added and the mixture was allowed to crosslink at room temperature for 2 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 3.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 75°C to obtain immobilized cells.

[0037] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial yield of the product reached a maximum of 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 61%.

[0038] Example 4: Production of tagatose by immobilized Bacillus subtilis The fermentation broth of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, the fermentation broth of Bacillus subtilis expressing the thermostable phosphoglucomutase, the fermentation broth of Bacillus subtilis expressing the thermostable glucose phosphate isomerase, the fermentation broth of Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to approximately OD600 = 100. 10% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 1% w / v aqueous solution of polydimethyldiallylammonium chloride (PDADMAC) was added and the mixture was allowed to flocculate at room temperature. A 0.05% v / v aqueous solution of glutaraldehyde was then added and the mixture was allowed to crosslink at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 90°C to obtain immobilized cells.

[0039] Tagatose was produced by the method of Example 2. The experimental results shown in Figure 1 show that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial yield of the product reached a maximum of 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 59%.

[0040] Example 5: Production of tagatose by immobilized Bacillus subtilis The fermentation broths of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, Bacillus subtilis expressing the thermostable phosphoglucomutase, Bacillus subtilis expressing the thermostable glucose phosphate isomerase, Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed at an OD600 ratio of 1:1:1:5:5, and the mixture was adjusted to an OD600 of approximately 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.5% w / v polyethyleneimine with a molecular weight of 600 was added and allowed to flocculate at room temperature. Then, 0.3% v / v aqueous glutaraldehyde solution was added and the mixture was crosslinked at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 0.4 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 80°C to obtain immobilized cells.

[0041] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial product yield reached up to 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 56%.

[0042] Example 6: Production of tagatose by immobilized Bacillus subtilis The fermentation broths of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, Bacillus subtilis expressing the thermostable phosphoglucomutase, Bacillus subtilis expressing the thermostable glucose phosphate isomerase, Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to an OD600 of approximately 100. 6% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 1% w / v polyethyleneimine with a molecular weight of 600 was added and allowed to flocculate at room temperature. Then, a 1% v / v aqueous solution of tris(hydroxymethyl)phosphine was added and the mixture was crosslinked at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 0.4 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 80°C to obtain immobilized cells.

[0043] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial product yield reached up to 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 59%.

[0044] Example 7: Production of tagatose by immobilized Bacillus subtilis The fermentation broths of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, Bacillus subtilis expressing the thermostable phosphoglucomutase, Bacillus subtilis expressing the thermostable glucose phosphate isomerase, Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 3% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.5% w / v polyacrylamide was added and the mixture was allowed to flocculate at room temperature. A 2.0% v / v N,N-methylenebisacrylamide aqueous solution was then added and the mixture was allowed to crosslink at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 90°C to obtain immobilized cells.

[0045] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial product yield reached up to 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 57%.

[0046] Example 8: Production of tagatose by immobilized Bacillus subtilis The fermentation broth of Bacillus subtilis expressing the thermostable α-glucan phosphorylase prepared in Example 1, the fermentation broth of Bacillus subtilis expressing the thermostable phosphoglucomutase, the fermentation broth of Bacillus subtilis expressing the thermostable glucose phosphate isomerase, the fermentation broth of Bacillus subtilis expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of Bacillus subtilis expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 1% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 0.1% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and the mixture was allowed to flocculate at room temperature. Then, a 0.5% v / v aqueous solution of epichlorohydrin was added and the mixture was allowed to crosslink at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 75°C to obtain immobilized cells.

[0047] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial product yield reached up to 75%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 58%.

[0048] Example 9: Production of E. coli whole cells Recombinant engineered Escherichia coli strains expressing the thermostable α-glucan phosphorylase gene, thermostable phosphoglucomutase gene, thermostable glucose phosphate isomerase gene, thermostable tagatose-6-phosphate epimerase gene, and thermostable tagatose-6-phosphate phosphatase gene (BL21(DE3) was used as the starting strain; see CN107988286B) were each collected, inoculated into LB medium, and cultured overnight at 37°C with shaking. The culture was transferred to LB medium at an inoculation amount of 1%, induced with IPTG at 18°C, and cultured overnight with shaking to obtain E. coli fermentation liquid expressing thermostable α-glucan phosphorylase, E. coli fermentation liquid expressing thermostable phosphoglucomutase, E. coli fermentation liquid expressing thermostable glucose phosphate isomerase, E. coli fermentation liquid expressing thermostable tagatose-6-phosphate epimerase, and E. coli fermentation liquid expressing thermostable tagatose-6-phosphate phosphatase, respectively.

[0049] Example 10: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to an OD600 of approximately 100. 1% w / v montmorillonite was added to the bacterial suspension and stirred uniformly. Next, a 0.5% w / v aqueous solution of polyethyleneimine with a molecular weight of 10,000 was added and the mixture was allowed to aggregate at room temperature. A 2% v / v aqueous solution of glutaraldehyde was then added and the mixture was allowed to crosslink at room temperature for 2 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The extruded cake was then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 60°C to obtain immobilized cells.

[0050] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached a maximum of 74%, and even after 65 batches of continuous catalytic reaction, the yield of the product remained at 55%.

[0051] Example 11: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 0.1% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and the mixture was allowed to flocculate at room temperature. Then, a 1% v / v aqueous solution of glutaraldehyde was added and the mixture was allowed to crosslink at room temperature for 2 hours. The filter cake was obtained by vacuum filtration, and the filter cake was extruded and granulated in a rotary granulator into strands with a particle diameter of 3.0 mm. The extruded cake was then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 75°C to obtain immobilized cells.

[0052] Tagatose was produced by the method of Example 2. The experimental results shown in Figure 2 show that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached a maximum of 74%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 60%.

[0053] Example 12: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 10% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 1% w / v aqueous solution of polydimethyldiallylammonium chloride (PDADMAC) was added and the mixture was allowed to flocculate at room temperature. Then, a 0.5% v / v aqueous solution of glutaraldehyde was added and the mixture was allowed to crosslink at room temperature for 3 hours. The filter cake was obtained by vacuum filtration, and the filter cake was extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The extruded cake was then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 90°C to obtain immobilized cells.

[0054] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached a maximum of 74%, and even after 65 batches of continuous catalytic reaction, the yield of the product remained at 57%.

[0055] Example 13: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:5:5, and the mixture was adjusted to OD600 = 100. 4% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 1% w / v polyethyleneimine with a molecular weight of 600 was added and allowed to flocculate at room temperature. Then, 0.3% v / v aqueous glutaraldehyde solution was added and the mixture was crosslinked at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 0.4 mm. The extruded cake was then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 80°C to obtain immobilized cells.

[0056] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial product yield reached a maximum of 74%, and even after 65 batches of continuous catalytic reaction, the product yield remained at 58%.

[0057] Example 14: Production of tagatose by immobilized E. coli The E. coli fermentation broth expressing the thermostable α-glucan phosphorylase produced in Example 9, the E. coli fermentation broth expressing the thermostable phosphoglucomutase, the E. coli fermentation broth expressing the thermostable glucose phosphate isomerase, the E. coli fermentation broth expressing the thermostable tagatose-6-phosphate epimerase, and the E. coli fermentation broth expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to an OD600 of approximately 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.8% w / v polyethyleneimine with a molecular weight of 10,000 was added and the mixture was allowed to aggregate at room temperature. Then, 0.7% v / v aqueous tris(hydroxymethyl)phosphine solution was added and the mixture was allowed to crosslink at room temperature for 2 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 80°C to obtain immobilized cells.

[0058] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached up to 75%, and even after 65 batches of continuous catalytic reaction, the yield of the product remained at 59%.

[0059] Example 15: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to an OD600 of approximately 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.3% w / v polyacrylamide was added and the mixture was allowed to aggregate at room temperature. A 1.0% v / v N,N-methylenebisacrylamide aqueous solution was then added and the mixture was allowed to crosslink at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 90°C to obtain immobilized cells.

[0060] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached up to 75%, and even after 65 batches of continuous catalytic reaction, the yield of the product remained at 57%.

[0061] Example 16: Production of tagatose by immobilized E. coli The fermentation broth of E. coli expressing the thermostable α-glucan phosphorylase prepared in Example 9, the fermentation broth of E. coli expressing the thermostable phosphoglucomutase, the fermentation broth of E. coli expressing the thermostable glucose phosphate isomerase, the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate epimerase, and the fermentation broth of E. coli expressing the thermostable tagatose-6-phosphate phosphatase were mixed to an OD600 ratio of 1:1:1:1:1, and the mixture was adjusted to OD600 = 100. 2% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, a 0.2% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and the mixture was allowed to flocculate at room temperature. Then, a 1.0% v / v aqueous solution of epichlorohydrin was added and the mixture was allowed to crosslink at room temperature for 3 hours. The mixture was filtered under vacuum to obtain a filter cake, which was then extruded and granulated in a rotary granulator into strands with a particle diameter of 1.0 mm. The strands were then cut into pellets of uniform length using a spheronizer, and the resulting immobilized cell pellets were fluidized and dried at 75°C to obtain immobilized cells.

[0062] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized E. coli, the initial yield of the product reached up to 75%, and even after 65 batches of continuous catalytic reaction, the yield of the product remained at 55%.

[0063] Comparative Example 1: Production of tagatose by Bacillus subtilis The fermentation broth prepared in Example 1 was centrifuged at 5,500 rpm for 10 minutes, and the supernatant was discarded to obtain whole cells expressing thermostable α-glucan phosphorylase, whole cells expressing thermostable phosphoglucomutase, whole cells expressing thermostable glucose phosphate isomerase, whole cells expressing thermostable tagatose-6-phosphate epimerase, and whole cells expressing thermostable tagatose-6-phosphate phosphatase. Each of the cells was resuspended in 50 mM sodium phosphate buffer (pH 7.5) to an OD of 200. The resuspended cells were heat-treated at 75°C for 90 minutes. The whole cells were mixed with pH 7.0 sodium phosphate buffer at a ratio of 1:1:1:1:1 to obtain an OD of 200.

[0064] A 1L reaction system was mixed with 100 g / L starch, 50 mM sodium phosphate buffer (pH 7.0), and the above-mentioned mixed Bacillus subtilis to a final concentration of 20. The reaction was performed on a shaker in a 70°C water bath. During the reaction, the tagatose content was analyzed by high-performance liquid chromatography. After the reaction, the precipitated cells were collected by centrifugation, washed with buffer, and then used in the next batch of reaction. The experimental results shown in Figure 3 showed that in the case of continuous catalytic reaction using immobilized Bacillus subtilis, the initial product yield reached a maximum of 73%, but after two batches of continuous catalytic reaction, the product yield was only 20%.

[0065] Comparative Example 2: Production of tagatose by E. coli The fermentation broth prepared in Example 9 was centrifuged at 5,500 rpm for 10 minutes, and the supernatant was discarded to obtain whole cells expressing thermostable α-glucan phosphorylase, whole cells expressing thermostable phosphoglucomutase, whole cells expressing thermostable glucose phosphate isomerase, whole cells expressing thermostable tagatose-6-phosphate epimerase, and whole cells expressing thermostable tagatose-6-phosphate phosphatase. Each of the cells was resuspended in 50 mM sodium phosphate buffer (pH 7.5) to an OD of 200. The resuspended cells were heat-treated at 75°C for 90 minutes. The whole cells were mixed with pH 7.0 sodium phosphate buffer at a ratio of 1:1:1:1:1 to obtain an OD of 200.

[0066] A 1-L reaction system was mixed with 100 g / L starch, 50 mM sodium phosphate buffer (pH 7.0), and the above-mentioned mixed E. coli cells to a final concentration of 20. The reaction was then performed on a shaker in a 70°C water bath. During the reaction, the tagatose content was analyzed by high-performance liquid chromatography. After the reaction, the precipitated cells were collected by centrifugation, washed with buffer, and then used in the next batch of reaction. The experimental results shown in Figure 4 indicate that the initial product yield reached a maximum of 73% in the case of continuous catalytic reaction using E. coli, but after two batches of continuous catalytic reaction, the product yield was only 15%.

[0067] Comparative Example 3: Production of tagatose by immobilized permeabilized Bacillus subtilis Recombinant Bacillus subtilis strains expressing the thermostable α-glucan phosphorylase gene, thermostable phosphoglucomutase gene, thermostable glucose phosphate isomerase gene, thermostable tagatose-6-phosphate epimerase gene, and thermostable tagatose-6-phosphate phosphatase gene were collected and inoculated into LB medium and cultured overnight at 37°C with shaking. The cultures were transferred to LB medium at a 1% inoculum size, cultured overnight at 37°C with shaking, centrifuged at 5500 rpm for 10 minutes, and the supernatant was discarded to obtain whole cells expressing thermostable α-glucan phosphorylase, whole cells expressing thermostable phosphoglucomutase, whole cells expressing thermostable glucose phosphate isomerase, whole cells expressing thermostable tagatose-6-phosphate epimerase, and whole cells expressing thermostable tagatose-6-phosphate phosphatase, respectively. 50 mM sodium phosphate buffer (pH 7.5) was added to each of the above cells, and the cells were resuspended to an OD600 of 200. The heavily suspended cells were heat-treated at 75°C for 90 minutes.

[0068] The permeabilized whole cells were mixed with pH 7.0 sodium phosphate buffer at a 1:1:1:1:1 ratio to achieve an OD600 of 100. 5% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.5% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and allowed to flocculate at room temperature. A 0.5% v / v aqueous solution of glutaraldehyde was then added and allowed to crosslink at room temperature for 2 hours. The filter cake was obtained by vacuum filtration. The filter cake was extruded into pellets with a particle size of 0.4 mm using a rotary granulator. The resulting immobilized cell pellet was dried at 30°C to obtain immobilized cells.

[0069] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized permeabilized Bacillus subtilis, the initial product yield reached up to 73%, and even after 65 batches of continuous catalytic reaction, the product yield was only 43%.

[0070] Comparative Example 4: Production of tagatose by immobilized permeabilized E. coli Recombinant E. coli strains expressing the thermostable α-glucan phosphorylase gene, thermostable phosphoglucomutase gene, thermostable glucose phosphate isomerase gene, thermostable tagatose-6-phosphate epimerase gene, and thermostable tagatose-6-phosphate phosphatase gene were collected and inoculated into LB medium and cultured overnight at 37°C with shaking. The cultures were transferred to LB medium at a 1% inoculum size, induced with IPTG at 18°C, cultured overnight with shaking, and centrifuged at 5500 rpm for 10 min. The supernatant was discarded to obtain whole cells expressing thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose phosphate isomerase, thermostable tagatose-6-phosphate epimerase, and thermostable tagatose-6-phosphate phosphatase, respectively. 50 mM sodium phosphate buffer (pH 7.5) was added to each of the above cells, and the cells were resuspended to an OD600 of 200. The heavily suspended cells were heat-treated at 75°C for 90 minutes.

[0071] The permeabilized whole cells were mixed with pH 7.0 sodium phosphate buffer at a 1:1:1:1:1 ratio to achieve an OD600 of 100. 1% w / v diatomaceous earth was added to the bacterial suspension and stirred uniformly. Next, 0.5% w / v aqueous solution of polyethyleneimine with a molecular weight of 70,000 was added and allowed to flocculate at room temperature. A 1% v / v aqueous solution of glutaraldehyde was then added and allowed to crosslink at room temperature for 2 hours. The filter cake was obtained by vacuum filtration. The filter cake was extruded into pellets with a particle size of 1.0 mm using a rotary granulator. The resulting immobilized cell pellet was dried at 30°C to obtain immobilized cells.

[0072] Tagatose was produced by the method of Example 2. Experimental results showed that in the case of continuous catalytic reaction using immobilized permeabilized E. coli, the initial product yield reached a maximum of 72%, and even after 65 batches of continuous catalytic reaction, the product yield was only 40%.

Claims

1. A method for producing immobilized cells for producing tagatose, comprising: By fermentation, an Escherichia coli or Bacillus subtilis fermentation broth is obtained in which α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase are expressed, Mixing the fermentation broths so that the ratio of OD600 of the Escherichia coli or the Bacillus subtilis expressing α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase, respectively, is (0.1-10):(0.1-10):(0.1-10):(0.1-10) to obtain a fermentation mixture; adding 1-10% w / v inorganic soil to the fermentation mixture and stirring uniformly; Further, adding 0.1 to 2% w / v flocculant to the fermentation mixture to flocculate the bacterial cells, and then adding 0.05 to 3% v / v cross-linking agent to cross-link the bacterial cells for 1 to 4 hours; filtration under vacuum to obtain a filter cake, extruding the filter cake into strands using a rotary granulator, and then cutting the strands of immobilized cells into pellets with a uniform length using a spheronizer; and a step of fluidizing and drying the pellets by controlling the temperature at the blowing port to 60 to 90°C to obtain immobilized cells for tagatose production.

2. The α-glucan phosphorylase, phosphoglucomutase, glucose phosphate isomerase, tagatose-6-phosphate epimerase, and tagatose-6-phosphate phosphatase are thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose phosphate isomerase, thermostable tagatose-6-phosphate isomerase, and thermostable tagatose-6-phosphate phosphatase, respectively; 2. The method according to claim 1, wherein the heat resistance means that the enzyme has enzymatic activity even at temperatures of 40°C or higher.

3. The method according to claim 2, wherein the Escherichia coli or the Bacillus subtilis expressing thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose phosphate isomerase, thermostable tagatose 6-phosphate isomerase, and thermostable tagatose 6-phosphate phosphatase, respectively, are mixed in a ratio of (1-10):(1-10):(1-10):(1-10):(1-10), and the OD600 of the bacterial suspension after mixing is between 10 and 150.

4. 2. The method of claim 1, wherein the mineral soil is selected from montmorillonite, diatomaceous earth, kaolin, or bentonite.

5. 2. The method of claim 1, wherein the flocculating agent is selected from polyethyleneimine, chitosan, polydimethyldiallylammonium chloride, and polyacrylamide.

6. 6. The method according to claim 5, wherein the flocculating agent is polyethyleneimine or PDADMAC, and the molecular weight of the polyethyleneimine is 600 to 70,000.

7. 2. The method of claim 1, wherein the cross-linking agent is selected from glutaraldehyde, tris(hydroxymethyl)phosphine, N,N-methylenebisacrylamide, or epichlorohydrin.

8. 2. The method of claim 1, further comprising the step of screening the obtained immobilized cells to obtain immobilized cells with a uniform morphology.

9. A method for producing tagatose using immobilized cells, comprising: A method for producing tagatose using immobilized cells, characterized in that starch or a starch derivative is converted into tagatose using the immobilized cells obtained by the method according to any one of claims 1 to 8.

10. 10. The method according to claim 9, further comprising the step of recovering the immobilized cells by filtration after the reaction is completed.

11. 10. The method of claim 9, wherein the bioconversion reaction system comprises 50-300 g / L of starch or starch derivative, a buffer solution having a pH value of 5.0-8.0, 10-50 mM inorganic phosphate, 3-7 mM divalent magnesium ions, and immobilized cells.

12. 12. The method of claim 11, wherein the buffer is a HEPES buffer, a phosphate buffer, a Tris buffer, or an acetate buffer, and the inorganic phosphate is sodium phosphate or potassium phosphate.

Images