Method for producing tagatose using biomimetic silicon mineralization microcapsules immobilized with multiple enzymes

Abstract

Provided is a biomimetic silicon mineralized microcapsule-immobilized multienzyme, a manufacturing method thereof, and a method for producing tagatose using the same, the manufacturing method comprising the steps of: (1) premixing glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase solutions and adding them to a calcium chloride solution, then injecting sodium carbonate solution and stirring to obtain calcium carbonate microspheres containing multienzymes; (2) mixing the calcium carbonate microspheres with a polyethyleneimine solution and separating to obtain polyethyleneimine-calcium carbonate microspheres; (3) mixing the polyethyleneimine-calcium carbonate microspheres with a silicate solution and separating to obtain biomimetic silicon mineralized-calcium carbonate microspheres; and (4) mixing and reacting the biomimetic silicon mineralized-calcium carbonate microspheres with ethylenediaminetetraacetic acid to remove calcium carbonate, and separating to obtain biomimetic silicon mineralized microcapsule-immobilized multienzyme.

Description

【Technical Field】 【0001】 The present invention relates to the field of production of tagatose, and specifically to a method for producing tagatose by biomimetic silicon mineralized microcapsule-immobilized multi-enzymes. 【Background Art】 【0002】 Tagatose is a rare, naturally occurring monosaccharide, the ketose form of galactose and an epimer of fructose. While its sweetness is similar to sucrose, it contains only one-third the calories and is therefore considered a low-calorie sweetener. Tagatose provides a very fresh and pure sweetness, and its flavor profile is similar to fructose. Studies have shown that tagatose possesses significant physiological functional properties, including low calories, a low glycemic index, anti-carcinogenicity, antioxidant properties, prebiotic properties, improved gut function, immunomodulation, and drug precursor properties, and has been shown to have enormous economic value, with widespread applications in food, beverages, pharmaceuticals, and health (Oh DK:Tagatose:properties, applications, and biotechnological processes.App.Microbiol.Biotechnol.2007,76:1-8). In 2001, the U.S. Food and Drug Administration (FDA) confirmed the safety of tagatose and approved it as a GRAS (Generally Regarded As Safe) product. The FDA approved tagatose as a tooth-friendly ingredient in December 2002 and as a food additive in October 2003, allowing its use as a sweetener in the food and beverage and pharmaceutical sectors. In 2001, the Joint FEA (Joint Expert Committee on Food Additives) of the United Nations Food and Agriculture Organization / World Health Organization (JECFA) recommended tagatose as a new low-calorie sweetener that could be used as a food additive. At its 63rd meeting in 2004, it was announced that there was no need to limit the acceptable daily intake (ADI) of tagatose, and that an "unspecified" ADI would be assigned to the safest list of food ingredients that JECFA could arrange. South Korea, the Commonwealth of Australia (Australia and New Zealand), and the European Union approved the launch of tagatose in their respective regions in 2003, 2004, and 2005, respectively, and in December 2005, tagatose was officially approved in the European Union as a new food ingredient with no restrictions on use. China also approved tagatose as a new food ingredient in May 2014. Currently, tagatose is approved in more than 30 countries worldwide, as well as by the WHO / FAO and the Codex Alimentarius Commission, and there are no restrictions on its acceptable daily intake or uses. 【0003】 Currently, the mainstream method for producing tagatose involves steps such as galactose isomerization, desalting, decolorization, separation, concentration, and crystallization to produce pure tagatose. However, this method also has drawbacks. It cannot completely convert galactose into tagatose, and the final product is a mixture of galactose and tagatose. This makes the tagatose separation process complex, resulting in a low conversion rate and high separation costs. Furthermore, the price of raw material galactose is not cheap, ultimately leading to high production costs for tagatose. (Rhimi M,Aghajari N,Juy M,Chouayekh H,Maguin E,Haser R,Bejar S:Rational design of Bacillus stearothermophilus US100l-arabinose isomerase:Potential applications for d-tagatose production.Biochim.2009,91:650-653.Oh HJ,Kim HJ,Oh DK:Increase in d-tagatose production rate by site-directed mutagenesis of l-arabinose isomerase from Geobacillus thermodenitrificans.Biotechnol.Lett.2006,28:145-149.Bosshart A,Hee CS,Bechtold M,Schirmer T,Panke S:Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.ChemBioChem (2015, 16:592-601.) CJ Corporation of South Korea and the Institute of Biotechnology of the Chinese Academy of Sciences in Tianjin have both developed new pathways for in vitro multi-enzyme tagatose synthesis (WO2018004310A1, CN109790524A, CN107988286A, CN109666620A, CN106399427A).The novel in vitro multi-enzyme pathway for tagatose synthesis uses starch, maltodextrin, and sucrose as raw materials and synthesizes tagatose through a multi-step enzymatic catalytic reaction, fundamentally changing the existing production process for tagatose isomerization. However, this new synthesis pathway involves multiple enzyme molecules, requiring cumbersome extraction and purification before use in the catalytic reaction, thus increasing the production cost of the enzymes. Furthermore, since biological enzymes are water-soluble molecules, it is difficult to recover and reuse the water-soluble enzyme molecules after the catalytic reaction is complete, leading to enzyme waste. Due to these factors, the production cost of the new pathway for tagatose synthesis is high, making it urgent to develop a method for producing tagatose using immobilized multi-enzymes that allows for repeated use of the enzymes and reduces production costs. 【0004】 Microcapsules are widely studied as immobilized enzyme carriers. The hollow core of a microcapsule can not only embed a large number of enzyme molecules, but also provide the embedded enzyme molecules with good physical and chemical fine structure, thereby increasing their stability. Because the capsule wall of a microcapsule is semipermeable, substrate / product transfer can be achieved, contributing to the progress of enzymatic reactions. Some researchers use protamine as an inducer to in-situ produce biomimetic silicon mineralization microcapsules, and then embed these biomimetic silicon mineralization microcapsules in calcium alginate spheres for enzyme immobilization. (Jiang Y, Sun Q, Zhang L, et al. Capsules-in-bead scaffold: a rational architecture for spatially separated multienzyme cascade system [J]. Journal of Materials Chemistry, 2009, 19(47):9068.) Other researchers use polyethyleneimine as an inducer to induce silicon precursors and produce silicon oxide particles, and then load these silicon oxide particles into the capsule walls of microcapsules. (Shi J, Jiang Z. An efficient and recyclable enzyme catalytic system constructed through the synergy between biomimetic mineralization and polyamine-salt aggregate assembly [J]. Journal of Materials Chemistry B, 2014, 2(28).) Protamine can induce silicon precursors in situ to produce silicon mineralization microcapsules, but the high cost of protamine makes it unsuitable for industrial-scale production. Polyethyleneimine is an inexpensive industrial reagent, and using polyethyleneimine as a inducer to induce silicon precursors in situ and produce silicon mineralization microcapsules for enzyme immobilization significantly reduces the production cost of immobilized enzymes, contributing to the industrial production of immobilized enzymes. [Overview of the project] 【0005】 The object of the present invention is to provide a method for producing tagatose using biomimetic silicon mineralization microcapsules immobilized with multiple enzymes. This method uses biomimetic silicon mineralization microcapsules as immobilized enzyme carriers, immobilizes multiple enzymes catalytically converted by the tagatose enzymatic method, obtains immobilized multiple enzymes, and uses these immobilized multiple enzymes to catalytically produce tagatose, thereby enabling the recovery and reuse of enzymes. Since the immobilized multiple enzymes can be recycled multiple times, the amount of enzymes that need to be used in the production of tagatose is significantly reduced, thereby lowering production costs. 【0006】 This invention co-immobilizes five key enzyme molecules involved in the production of tagatose—glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase—using biomimetic silicon mineralized microcapsules. The hollow core of the microcapsule can not only embed a large number of enzyme molecules but also provide the embedded enzyme molecules with good physical and chemical fine structure, thereby enhancing the stability of the enzyme molecules. Since the capsule wall of the microcapsule is semipermeable, substrate / product transfer can be achieved, contributing to the progress of the enzymatic reaction. 【0007】 Therefore, the present invention provides biomimetic silicon mineralized microcapsule-immobilized polyenzymes for producing tagatose. Step (1) involves pre-mixing solutions of five enzymes involved in tagatose production: glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase, adding the enzyme solutions to a calcium chloride solution, then injecting a sodium carbonate solution into the solution and stirring to separate the solid and liquid, collecting calcium carbonate microspheres containing multiple enzymes as a solid product. Step (2) involves mixing the aforementioned calcium carbonate microspheres containing multiple enzymes with a polyethyleneimine solution, performing solid-liquid separation, and collecting the solid product, polyethyleneimine-calcium carbonate microspheres. Step (3) involves mixing the polyethyleneimine-calcium carbonate microspheres with a silicate solution, performing solid-liquid separation, and collecting the biomimetic silicon mineralization-calcium carbonate microspheres, which are the solid product. The product is manufactured by a method comprising the step (4) of mixing biomimetic silicon mineralization-calcium carbonate microspheres with ethylenediaminetetraacetic acid to remove calcium carbonate, performing solid-liquid separation, and collecting the solid product, biomimetic silicon mineralization microcapsule-immobilized polyenzymes. 【0008】 Preferably, the mass ratio of the amounts used of the glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase is 1-2:1-2:1-2:2-4:2-4. 【0009】 In a specific embodiment, the concentrations of each enzyme solution in step (1) are as follows: 0.05-0.15 mg / ml for glucan phosphorylase, 0.05-0.15 mg / ml for glucose phosphomutase, 0.05-0.15 mg / ml for glucose phosphate isomerase, 0.1-0.3 mg / ml for tagatose 6-phosphate 4-epimerase, and 0.1-0.3 mg / ml for tagatose 6-phosphate phosphatase. 【0010】 In a preferred embodiment, the concentration of polyethyleneimine is 0.1 to 1.0 g / L, and the molecular weight of polyethyleneimine is 600 to 70000. 【0011】 Preferably, the concentration of the silicate in step (3) is 2 to 10 g / L. More preferably, the silicate in step (3) is sodium silicate, and its concentration is 8 g / L. 【0012】 In specific embodiments, steps (2) and (3) are repeated 1 to 3 times. Preferably, steps (2) and (3) are repeated twice (i.e., steps (2) and (3) are performed twice). This allows for the achievement of better results. 【0013】 In a specific embodiment, the ratio of the mass of polyethyleneimine to the mass of the calcium carbonate microspheres containing multiple enzymes, as described above, is 20 to 50:1. 【0014】 In a specific embodiment, the ratio of the mass of the silicate to the mass of the calcium carbonate microspheres containing multiple enzymes, as described above, is 20 to 50:1. 【0015】 In a preferred embodiment, each enzyme solution from step (1) is added to a calcium chloride solution with a concentration of 0.2 to 0.4 M, and equimolar and equivolute sodium carbonate solutions are injected into the calcium chloride solution at a rotation speed of 600 to 1500 r / min, reacting for 20 to 30 s, then centrifuged at a rotation speed of 3000 r / min to remove the supernatant, and then washed with deionized water until the supernatant is free of sodium ions and chloride ions to obtain calcium carbonate microspheres containing enzymes. 【0016】 In a preferred embodiment, the concentration of the ethylenediaminetetraacetic acid solution in step (4) is adjusted to 0.03-0.05 M, its pH to 5.0-6.0, the ethylenediaminetetraacetic acid solution and the obtained microspheres are uniformly mixed in a mass ratio of 20-50:1, shaken for 10-20 minutes, centrifuged at 3000 r / min to remove the supernatant, washed with ethylenediaminetetraacetic acid 3-4 times, and washed with deionized water until the supernatant no longer contains ethylenediaminetetraacetic acid to obtain biomimetic silicon mineralized microcapsule-immobilized multi-enzymes. 【0017】 As a preferred solution of the present invention, the biomimetic silicon mineralized microcapsule-immobilized polyenzymes are produced by a method comprising the following steps. 【0018】 (1) Five major enzyme molecules involved in the production of tagatose—glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase—are pre-mixed in specific enzyme usage amounts, with the usage amounts being 0.05-0.15 mg / ml for glucan phosphorylase, 0.05-0.15 mg / ml for glucose phosphomutase, 0.05-0.15 mg / ml for glucose phosphate isomerase, 0.1-0.3 mg / ml for tagatose 6-phosphate 4-epimerase, and 0.1-0.3 mg / ml for tagatose 6-phosphate phosphatase. The enzyme solution is added to a calcium chloride solution with a concentration of 0.2 to 0.4 M, and equimolar and equivolute sodium carbonate solution is injected into the calcium chloride solution at a rotation speed of 600 to 1500 r / min. The reaction is allowed to proceed for 20 to 30 s, and the mixture is centrifuged at a rotation speed of 3000 r / min. The supernatant is removed, and the mixture is washed with deionized water until the supernatant is free of sodium and chloride ions to obtain calcium carbonate microspheres containing the enzyme. 【0019】 (2) Prepare a silicate solution with a concentration of 2 to 10 g / L, and prepare a polyethyleneimine solution with a molecular weight of 600 to 70000, adjusting its concentration to 0.1 to 1.0 g / L. Mix the polyethyleneimine solution and calcium carbonate microspheres containing the enzyme uniformly for 10 to 20 minutes at a mass ratio of 20 to 50:1, centrifuge at a rotation speed of 3000 r / min, remove the supernatant, and wash with deionized water until the supernatant no longer contains polyethyleneimine. Add the silicate solution to the microspheres at a mass ratio of 20 to 50:1 and mix uniformly for 10 to 20 minutes, centrifuge at a rotation speed of 3000 r / min, remove the supernatant, and wash with deionized water until the supernatant no longer contains silicate ions. 【0020】 (3) Prepare an ethylenediaminetetraacetic acid solution with a concentration of 0.03 to 0.05 M, adjust its pH to 5.0 to 6.0, uniformly mix the ethylenediaminetetraacetic acid solution and the obtained microspheres in a mass ratio of 20 to 50:1, shake for 10 to 20 minutes, centrifuge at 3000 r / min to remove the supernatant, wash with ethylenediaminetetraacetic acid 3 to 4 times, and wash with deionized water until the supernatant no longer contains ethylenediaminetetraacetic acid to obtain biomimetic silicon mineralized microcapsule-immobilized multi-enzymes. 【0021】 The multi-enzyme for producing tagatose according to the present invention includes glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase. When the five types of enzymes are co-immobilized in the biomimetic silicon mineralization microcapsule, the catalytic efficiency of the enzymes themselves is ensured, and the enzymes can be reused, thereby significantly reducing production costs. In the present invention, the enzyme proportions are preferably such that the amount of glucan phosphorylase used is 0.05 to 0.15 mg / ml, the amount of glucose phosphomutase used is 0.05 to 0.15 mg / ml, the amount of glucose phosphate isomerase used is 0.05 to 0.15 mg / ml, the amount of tagatose 6-phosphate 4-epimerase used is 0.1 to 0.3 mg / ml, and the amount of tagatose 6-phosphate phosphatase used is 0.1 to 0.3 mg / ml. This ensures that when actually producing tagatose, the immobilized multi-enzyme can achieve good catalytic efficiency and multiple recovery uses with appropriate dosages. 【0022】 The present invention further claims a method for producing tagatose using the immobilized multi-enzyme, the method of producing tagatose by enzyme-catalyzed conversion using starch or a derivative thereof as a raw material and the immobilized multi-enzyme. 【0023】 As a preferred solution of the present invention, specifically, the method is to carry out an enzyme-catalyzed conversion reaction at 40-70°C on a reaction solution containing 50-150 g / L of starch or starch derivative, 80-120 mM of HEPES buffer with a pH value of 6.0-7.0, 10-50 mM of inorganic phosphate, 3-7 mM of divalent magnesium ions, 0.3-0.7 mM of zinc ions or manganese ions, 3-7 U / ml of a debranching enzyme, and 1-5 mg / ml of an immobilized multi-enzyme. 【0024】 After the reaction is completed, solid-liquid separation is carried out to collect the immobilized multi-enzyme, which can be recycled for the production of tagatose. The immobilized multi-enzyme is recycled 1-10 times. 【0025】 Compared with the prior art, the biomimetic silicon mineralized microcapsule-immobilized multi-enzyme of the present invention uses the biomimetic silicon mineralized microcapsule as an immobilized enzyme carrier, with a simple process and mild conditions. In particular, by producing tagatose with the immobilized multi-enzyme, the recycling of the enzyme can be realized. By recycling the enzyme multiple times, the amount of enzyme used for multiple productions of tagatose can be significantly reduced, thereby reducing the production cost. 【Brief Description of the Drawings】 【0026】 [Figure 1] Figure 1 is a conversion curve of tagatose produced by an immobilized multi-enzyme with 1 layer of biomimetic silicon mineralization according to Example 1. [Figure 2] Figure 2 is a conversion curve of tagatose produced by an immobilized multi-enzyme with 2 layers of silicon mineralization according to Example 2. [Figure 3] Figure 3 is a conversion curve of tagatose produced by an immobilized multi-enzyme with 3 layers of silicon mineralization according to Example 3. [Figure 4] Figure 4 is a conversion curve of tagatose produced by an immobilized multi-enzyme with 2 layers of silicon mineralization and a molecular weight of 600 of polyethyleneimine according to Example 6. [Figure 5]Figure 5 is a histogram showing the recycling of immobilized multi-enzymes for producing tagatose in Example 6 in Example 8. [Modes for carrying out the invention] 【0027】 The present invention discloses a method for producing tagatose using immobilized polyenzymes, which can be realized by those skilled in the art by appropriately modifying process parameters with reference to the contents of this specification. In particular, all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method of the present invention is illustrated by preferred embodiments, and it will be apparent to those skilled in the art that the technology of the present invention can be realized and applied by modifying or appropriately changing and combining the methods and applications described herein without departing from the content, spirit and scope of the present invention. 【0028】 Example 1 The biomimetic silicon mineralized microcapsule-immobilized multi-enzymes of this example were prepared by the following method. 【0029】 (1) Five major enzyme molecules involved in the production of tagatose—glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase—were pre-mixed in specific enzyme usage amounts, with glucan phosphorylase being 0.1 mg / ml, glucose phosphomutase being 0.1 mg / ml, glucose phosphate isomerase being 0.1 mg / ml, tagatose 6-phosphate 4-epimerase being 0.2 mg / ml, and tagatose 6-phosphate phosphatase being 0.2 mg / ml. The enzyme solution described above was added to a 0.33 M calcium chloride solution, and equimolar and equivolume sodium carbonate solution was injected into the calcium chloride solution at a rotation speed of 700 r / min. The reaction was allowed to proceed for 20-30 seconds, and the mixture was centrifuged at a rotation speed of 3000 r / min. After removing the supernatant, the mixture was washed with deionized water until the supernatant was free of sodium and chloride ions to obtain calcium carbonate microspheres containing the enzyme. 【0030】 (2) Prepare a sodium silicate solution with a concentration of 8.5 g / L, and prepare a polyethyleneimine solution with a molecular weight of 1800, and adjust its concentration to 0.5 g / L. Mix the polyethyleneimine solution and calcium carbonate microspheres containing the enzyme uniformly in a mass ratio of 30:1 for 10-20 minutes, centrifuge at 3000 r / min, remove the supernatant, and wash with deionized water until the supernatant no longer contains polyethyleneimine. 【0031】 (3) A sodium silicate solution was added to the above microspheres in a mass ratio of 30:1 and mixed uniformly for 10 to 20 minutes. The mixture was then centrifuged at a rotational speed of 3000 r / min, the supernatant was removed, and the supernatant was washed with deionized water until no silicate ions were present. The resulting capsule wall had one layer. 【0032】 (4) Prepare a 0.05 M ethylenediaminetetraacetic acid solution, adjust its pH to 5.8, and uniformly mix the ethylenediaminetetraacetic acid solution with the microspheres obtained above in a mass ratio of 30:1. Shake for 10-20 minutes, centrifuge at 3000 r / min to remove the supernatant, wash with ethylenediaminetetraacetic acid 3-4 times, and wash with deionized water until the supernatant no longer contains ethylenediaminetetraacetic acid to obtain biomimetic silicon mineralized microcapsule-immobilized multi-enzymes. 【0033】 Example 2 Comparing the biomimetic silicon mineralized microcapsule-immobilized multi-enzyme of this example with that of Example 1, the only difference is that the number of layers in the capsule wall of the biomimetic silicon mineralized microcapsule changes from one to two. Specifically, steps (2) and (3) of Example 1 are repeated twice. In other words, after obtaining calcium carbonate microspheres containing enzymes with one capsule wall layer in step (2) of Example 1, polyethyleneimine solution and the obtained calcium carbonate microspheres containing enzymes with one capsule wall layer are uniformly mixed for 10-20 minutes at a mass ratio of 30:1, centrifuged at 3000 r / min, the supernatant is removed, and the supernatant is washed with deionized water until polyethyleneimine is no longer present. Then, sodium silicate solution is added to the microspheres at a mass ratio of 30:1 and mixed uniformly for 10-20 minutes, centrifuged at 3000 r / min, the supernatant is removed, and the supernatant is washed with deionized water until silicate ions are no longer present, resulting in capsules with two layers. 【0034】 Example 3 Comparing the biomimetic silicon mineralized microcapsule-immobilized multi-enzyme of this example with that of Example 1, the only difference is that the number of layers in the capsule wall of the biomimetic silicon mineralized microcapsule changes from one to three. Specifically, steps (2) and (3) of Example 1 were repeated three times, meaning that steps (2) and (3) in Example 2 were repeated to obtain calcium carbonate microspheres containing enzymes with two layers in the capsule wall. Finally, polyethyleneimine solution and calcium carbonate microspheres containing enzymes were uniformly mixed for 10-20 minutes at a mass ratio of 30:1, centrifuged at 3000 r / min, the supernatant was removed, and the supernatant was washed with deionized water until polyethyleneimine was no longer present. Sodium silicate solution was then added to the microspheres at a mass ratio of 30:1 and mixed uniformly for 10-20 minutes, centrifuged at 3000 r / min, the supernatant was removed, and the supernatant was washed with deionized water until silicate ions were no longer present, resulting in capsules with three layers in the capsule wall. 【0035】 Example 4 This embodiment provides biomimetic silicon mineralized microcapsule-immobilized polyenzymes, and compared to Example 1, the only difference is that the molecular weight of polyethyleneimine changes from 1800 to 600. 【0036】 Example 5 This embodiment provides biomimetic silicon mineralized microcapsule-immobilized polyenzymes, and compared to Example 1, the only difference is that the molecular weight of polyethyleneimine changes from 1800 to 70000. 【0037】 Example 6 This embodiment provides a biomimetic silicon mineralized microcapsule-immobilized polyenzyme, and compared to Example 1, the only differences are that the molecular weight of polyethyleneimine changes from 1800 to 600, and the number of layers in the capsule wall of the biomimetic silicon mineralized microcapsule changes from one layer to two layers (i.e., steps (2) and (3) are repeated twice). 【0038】 Comparative Example This comparative example provides an unimmobilized multi-enzyme mixture for producing tagatose, having the same composition as the multi-enzyme mixture in Example 1. 【0039】 Example 7 Tagatose was produced using the immobilized polyenzymes described in Examples 1 to 6 and the unimmobilized polyenzyme mixture described in the Comparative Example, respectively, by the following method. 【0040】 A mixture of 100 g / L starch, 100 mM HEPES buffer with a pH of 6.5, 20 mM inorganic phosphate, 5 mM divalent magnesium ions, 0.5 mM zinc or manganese ions, 5 U / ml debranching enzyme, and 3 mg / ml of immobilized polyenzyme (or unimmobilized polyenzyme mixture) according to each of the above examples or comparative examples was subjected to an enzyme-catalyzed conversion reaction at 70°C, and the concentration of tagatose was detected by high-performance liquid chromatography. 【0041】 The product was prepared using the immobilized multi-enzyme according to Example 1. After 37 hours of reaction, the reaction approached equilibrium, and as shown in Figure 1, the tagatose production concentration was 40 g / L and the conversion rate was 40%. The biomimetic silicon mineralization microcapsule of Example 1 had one layer in the capsule wall, and the conversion rate was relatively high. However, because the capsule wall had only one layer, there was a possibility that the enzyme embedded in the microcapsule might leak out. Therefore, based on Example 1, the number of layers in the capsule wall could be further increased to further improve the conversion rate. 【0042】 The product was prepared using the immobilized multi-enzyme described in Example 2. After 37 hours of reaction, the reaction approached equilibrium, and as shown in Figure 2, the tagatose production concentration was 60 g / L and the conversion rate was 60%. As a result, it was found that the conversion rate was significantly improved compared to Example 1. 【0043】 The biomimetic silicon mineralization microcapsules of Example 3 were used for production, and after 37 hours of reaction, the reaction approached equilibrium. As shown in Figure 3, the tagatose production concentration was 10 g / L and the conversion rate was 10%. The biomimetic silicon mineralization microcapsules of Example 3 had three layers in the capsule wall, compared to the biomimetic silicon mineralization microcapsules of Example 2, which had two layers. As a result, the capsule wall of Example 3 was thicker, and a serious mass transfer problem existed between the enzyme embedded in the microcapsule and the external substrate. Consequently, the conversion rate of tagatose produced by the immobilized multi-enzyme of Example 3 was lower, and consequently lower than that of the immobilized multi-enzyme with only one capsule wall produced in Example 1. 【0044】 The product was prepared using the immobilized multi-enzyme according to Example 4. After 21 hours of reaction, the reaction approached equilibrium, and the tagatose production concentration was 70 g / L, with a conversion rate of 70%. Compared to Example 1, the molecular weight of polyethyleneimine in Example 4 decreased, and the thickness of the induced capsule wall increased. Although it was only one layer, it was still possible to suppress leakage of the enzyme embedded in the microcapsule, thereby improving the conversion rate of tagatose. 【0045】 The product was prepared using the immobilized multi-enzyme according to Example 5. After 21 hours of reaction, the reaction approached equilibrium, and the tagatose production concentration was 35 g / L, with a conversion rate of 35%. Compared to Example 1, the molecular weight of polyethyleneimine in Example 5 increased, the thickness of the induced capsule wall decreased, and leakage of the enzyme embedded in the microcapsule became more likely, resulting in a relative decrease in the tagatose conversion rate. 【0046】 The sample was prepared using the immobilized multi-enzyme according to Example 6. After 20 hours of reaction, the reaction approached equilibrium, and as shown in Figure 4, the tagatose production concentration was 74 g / L and the conversion rate was 74%. The molecular weight of polyethyleneimine used in Example 4 was lower, and compared to the case where the molecular weight of polyethyleneimine is high, the thickness of the capsule wall increased, but one layer was insufficient. Therefore, compared to Example 4, the number of layers in the capsule wall of the biomimetic silicon mineralization microcapsule in Example 6 was increased to two. This increase in the number of layers in the capsule wall further suppressed leakage of the enzyme embedded in the microcapsule and further improved the conversion rate of tagatose. 【0047】 The product was prepared using the non-immobilized multi-enzyme mixture of the comparative example. After 10 hours of reaction, the reaction approached equilibrium, with a tagatose production concentration of 72 g / L and a conversion rate of 72%. 【0048】 A comparison of the effects of Examples 1 to 6 revealed that the highest concentration of tagatose was achieved when producing tagatose using the immobilized multi-enzyme according to Example 6. A comparison of the number of layers in the capsule wall of the biomimetic silicon mineralization microcapsules revealed that the optimal production effect of tagatose was achieved when the capsule wall had two layers, when there was one layer the embedded enzyme leaked out of the microcapsule and the production concentration of tagatose decreased, and when there were three layers the mass transfer resistance increased and the production concentration of tagatose decreased. 【0049】 Example 8 1. Recycling of immobilized multi-enzymes in Example 6 After producing tagatose using the method described in Example 7, solid-liquid separation was performed to collect immobilized multi-enzymes, which were then recycled for tagatose production. The concentration of tagatose produced in each cycle was detected by high-performance liquid chromatography, and the results were expressed as the relative tagatose production concentration, with the tagatose concentration produced in the first cycle reaction set to 100%. 【0050】 The product was manufactured using the immobilized multi-enzyme according to Example 6, and after 10 recycling cycles, the immobilized multi-enzyme maintained a relative tagatose production concentration of 45%, and the results are shown in Figure 5. 【0051】 2. Recycling of the unimmobilized multi-enzyme mixture of the comparative example. After producing tagatose using the method described in the comparative example, the mixture was ultrafiltered to recover the unimmobilized multi-enzyme mixture, which was then recycled for tagatose production. After two cycles, the unimmobilized multi-enzyme mixture recovered by ultrafiltration maintained a relative tagatose production concentration of 10%. 【0052】 A comparison of the cycle effects revealed that the production of tagatose using immobilized multi-enzymes enables repeated recovery and reuse of the enzymes. Compared to catalytic conversion with pure enzymes, recycling immobilized multi-enzymes multiple times significantly reduces the amount of enzyme needed for multiple production cycles of tagatose, thereby lowering production costs. While pure enzymes have a high conversion rate when not reused, the immobilized enzymes of this invention have a significant advantage over pure enzymes when considering repeated cycle use. 【0053】 However, although the present invention has been described in detail above using a general description, specific embodiments and tests, several modifications or improvements can be made based on the present invention, which will be obvious to those skilled in the art. Accordingly, any such modifications or improvements made without departing from the spirit of the present invention will fall within the scope of the patent protection of the present invention.

Claims

[Claim 1] A method for producing biomimetic silicon mineralized microcapsule-immobilized multi-enzymes for producing tagatose, Step (1) involves pre-mixing solutions of five enzymes involved in tagatose production: glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase, adding the enzyme solutions to a calcium chloride solution, then injecting a sodium carbonate solution into the solution and stirring to separate the solid and liquid components, collecting calcium carbonate microspheres containing multiple enzymes as a solid product. Step (2) involves mixing the calcium carbonate microspheres containing multiple enzymes with the polyethyleneimine solution, performing solid-liquid separation, and collecting the solid product, polyethyleneimine-calcium carbonate microspheres. Step (3) involves mixing the polyethyleneimine-calcium carbonate microspheres with a silicate solution, performing solid-liquid separation, and collecting the biomimetic silicon mineralization-calcium carbonate microspheres, which are the solid product. Step (4) involves mixing the biomimetic silicon mineralization-calcium carbonate microspheres with ethylenediaminetetraacetic acid to remove calcium carbonate, then performing solid-liquid separation to collect the solid product, which is biomimetic silicon mineralization microcapsule-immobilized multi-enzymes. A manufacturing method that includes this. [Claim 2] The manufacturing method according to claim 1, characterized in that the mass ratio of the amounts used of the glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase is 1-2:1-2:1-2:2-4:2-4. [Claim 3] The manufacturing method according to claim 1, characterized in that the concentrations of each enzyme solution in step (1) are 0.05 to 0.15 mg / ml for the amount of glucan phosphorylase used, 0.05 to 0.15 mg / ml for the amount of glucose phosphomutase used, 0.05 to 0.15 mg / ml for the amount of glucose phosphate isomerase used, 0.1 to 0.3 mg / ml for the amount of tagatose 6-phosphate 4-epimerase used, and 0.1 to 0.3 mg / ml for the amount of tagatose 6-phosphate phosphatase used. [Claim 4] The manufacturing method according to claim 1, characterized in that the concentration of polyethyleneimine is 0.1 to 1.0 g / L, and the molecular weight of polyethyleneimine is 600 to 70000. [Claim 5] The manufacturing method according to claim 1, characterized in that the concentration of the silicate in step (3) is 2 to 10 g / L. [Claim 6] The manufacturing method according to claim 1, characterized in that the silicate in step (3) is sodium silicate and its concentration is 8 g / L. [Claim 7] The manufacturing method according to claim 1, characterized in that the operations of step (2) and step (3) are repeated once, twice, or three times. [Claim 8] The manufacturing method according to any one of claims 1 to 7, characterized in that the ratio of the mass of polyethyleneimine to the mass of the calcium carbonate microspheres containing multiple enzymes described above is 20 to 50:

1. [Claim 9] The manufacturing method according to any one of claims 1 to 7, characterized in that the ratio of the mass of the silicate to the mass of the calcium carbonate microspheres containing multiple enzymes described above is 20 to 50:

1. [Claim 10] The manufacturing method according to claim 1, characterized in that each enzyme solution in step (1) is added to a calcium chloride solution with a concentration of 0.2 to 0.4 M, an equimolar concentration and equivolume of sodium carbonate solution is injected into the calcium chloride solution at a rotation speed of 600 to 1500 r / min, the reaction is allowed to proceed for 20 to 30 s, the mixture is centrifuged at a rotation speed of 3000 r / min, the supernatant is removed, and the supernatant is washed with deionized water until it does not contain sodium ions and chloride ions to obtain calcium carbonate microspheres containing enzymes. [Claim 11] The manufacturing method according to claim 1, characterized in that, in step (4), the concentration of the ethylenediaminetetraacetic acid solution is adjusted to 0.03 to 0.05 M, its pH is adjusted to 5.0 to 6.0, the ethylenediaminetetraacetic acid solution and the obtained microspheres are uniformly mixed in a mass ratio of 20 to 50:1, the mixture is shaken for 10 to 20 min, the supernatant is removed by centrifugation at a rotation speed of 3000 r / min, the mixture is washed with ethylenediaminetetraacetic acid 3 to 4 times, and the supernatant is washed with deionized water until ethylenediaminetetraacetic acid is no longer present in the supernatant, thereby obtaining biomimetic silicon mineralized microcapsule-immobilized polyenzymes. [Claim 12] A method for producing tagatose using an immobilized multi-enzyme produced by the production method described in any one of claims 1 to 11, characterized in that tagatose is produced by an enzyme-catalyzed conversion method using the immobilized multi-enzyme as a raw material, with starch or a derivative thereof as the raw material. [Claim 13] The method described above is characterized by carrying out an enzyme-catalyzed conversion reaction at 40 to 70°C with a reaction solution comprising 50 to 150 g / L of starch or starch derivative, 80 to 120 mM of HEPES buffer with a pH of 6.0 to 7.0, 10 to 50 mM of inorganic phosphate, 3 to 7 mM of divalent magnesium ions, 0.3 to 0.7 mM of zinc ions or manganese ions, 3 to 7 U / ml of debranching enzyme, and 1 to 5 mg / ml of immobilized polyenzyme, and collecting tagatose after the reaction is completed.

Images