WO2023279565A1 - 一种用于塔格糖生产的固定化细胞的制备方法及其应用 - Google Patents

一种用于塔格糖生产的固定化细胞的制备方法及其应用 Download PDF

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WO2023279565A1
WO2023279565A1 PCT/CN2021/123610 CN2021123610W WO2023279565A1 WO 2023279565 A1 WO2023279565 A1 WO 2023279565A1 CN 2021123610 W CN2021123610 W CN 2021123610W WO 2023279565 A1 WO2023279565 A1 WO 2023279565A1
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tagatose
phosphate
thermostable
glucose
immobilized
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马延和
石婷
韩平平
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中国科学院天津工业生物技术研究所
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Priority to EP21949052.1A priority patent/EP4368711A1/en
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Definitions

  • the present disclosure relates to the technical field of bioengineering, in particular to the field of production and preparation of tagatose.
  • Tagatose is a rare monosaccharide that occurs naturally and is the ketose form of galactose, the epimer of fructose.
  • the sweetness properties of tagatose are similar to that of sucrose, providing very fresh and pure sweetness, but with only one-third of the calories of sucrose, it is called a low-calorie sweetener.
  • tagatose has important physiological functional properties such as low calorie, low glycemic index, anti-caries, anti-oxidation, prebiotics, improvement of intestinal function, immune regulation, and drug precursors, and can be widely used in food, beverages, medicine, Health care and other fields have huge economic value (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76: 1-8).
  • the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences used cheap corn starch, cellulose, maltodextrin, sucrose, etc. as raw materials, and used whole-cell catalysis to prepare tagatose (CN107988286A).
  • This process reduces the need for multi-enzyme purification
  • the process steps reduce production cost and environmental pollution, and improve the yield of tagatose.
  • the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences further improved the safety production of food preparations and provided a method for producing high-concentration tagatose by catalyzing high-concentration starch with whole cells of Bacillus subtilis (CN112342179B).
  • the permeabilized Bacillus subtilis is immobilized to obtain immobilized whole cells, which are then used for the production of tagatose, thereby realizing the recycling of the whole cells and reducing the production cost.
  • this method still has some shortcomings in the preparation of immobilized cells: first, this method needs to go through multiple cumbersome steps such as bacterial cell collection, bacterial resuspension, and bacterial cell permeabilization treatment before immobilization.
  • This method is based on the granulation and immobilization of permeable cells, and the cell permeabilization treatment is likely to cause the leakage of heterologous proteins expressed in the cells, which in turn leads to the loss of heterologous proteins and enzyme immobilization during the immobilization process. decrease in chemical efficiency.
  • this method only obtains immobilized enzyme granules by simple extrusion granulation, and the obtained immobilized enzyme granules are not uniform.
  • the purpose of this disclosure is to provide a method for immobilized cells to produce tagatose, simplify the production steps of enzymes in the production process of tagatose, simplify the separation and purification of products and enzymes in the production process of tagatose, and realize the cycle of multiple enzymes use, reduce the production cost of tagatose, and realize the industrialized production of tagatose.
  • a method for preparing immobilized cells for tagatose production characterized in that it comprises the steps of:
  • Escherichia coli expressing ⁇ -glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase were obtained by fermentation or Bacillus subtilis fermented liquid, the above-mentioned fermented liquid is mixed to obtain a fermented mixed liquid;
  • the filter cake is obtained after vacuum filtration, and the filter cake is extruded and granulated into long strips by a rotary granulator, and then cut into short and uniform particles by a spherical shot blasting machine;
  • the obtained granules were boil dried to obtain immobilized cells for tagatose production.
  • a method for preparing immobilized cells for tagatose production characterized in that it comprises the steps of:
  • Escherichia coli expressing ⁇ -glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase were obtained by fermentation or Bacillus subtilis fermented liquid, mixing the above-mentioned fermented liquid to obtain a fermented mixed liquid;
  • the filter cake is obtained after vacuum filtration, and the filter cake is extruded into long strips with a rotary granulator, and then the long strips of immobilized cells are cut into long and short uniform particles by a spherical shot blasting machine;
  • the obtained particles are boiled and dried to obtain immobilized cells for tagatose production, wherein the temperature of the air inlet of the boiled drying is controlled at 60-90°C.
  • Fermentation can use any exogenous protein-labeled medium, including but not limited to LB medium, SR medium, TB medium, etc.
  • the ⁇ -glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, 6-phosphate tagatose epimerase and 6-phosphate tagatose phosphatase are resistant to Thermo ⁇ -glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphoisomerase, thermostable tagatose 6-phosphate isomerase and thermostable tagatose 6-phosphate phosphatase.
  • the heat-resistant ⁇ -glucan phosphorylase refers to the temperature above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or An enzyme that phosphorylates starch into glucose-1-phosphate (G1P) above 80°C.
  • the heat-resistant ⁇ -glucan phosphorylase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus upirothermophilum, Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.; or the amino acid sequence of the thermostable ⁇ -glucan phosphorylase derived from the thermostable ⁇ -glucan
  • the sugar phosphorylases have at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%
  • thermostable glucose phosphomutase refers to the enzyme that has the function of phosphomutase above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or above 80°C.
  • thermostable glucose phosphomutase has at least 70% with the thermostable glucose phosphomutase derived from the thermophilic microorganism
  • thermostable glucose phosphoisomerase refers to a enzyme that has the ability to be used at temperatures above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or above 80°C.
  • thermostable glucose phosphate to fructose-6-phosphate (F6P) functional enzyme.
  • G6P to fructose-6-phosphate
  • F6P fructose-6-phosphate
  • thermostable 6-phosphate tagatose epimerase is activated at temperatures above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or An enzyme capable of isomerizing fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) above 80°C.
  • F6P fructose-6-phosphate
  • T6P tagatose-6-phosphate
  • thermostable tagatose 6-phosphate epimerase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus moth Caldicellulosiruptor kronotskyensis, Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.;
  • the thermostable tagatose 6-phosphate epimerase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity. More preferably, the tagatose
  • the 6-phosphate tagatose phosphatase refers to above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, or above 80°C
  • the above enzymes have the function of removing phosphate groups from tagatose-6-phosphate (T6P) to produce tagatose (Tagatose).
  • T6P tagatose-6-phosphate
  • Tagatose tagatose-6-phosphate
  • T6P tagatose-6-phosphate
  • thermostable ⁇ -glucan phosphorylase thermostable glucose phosphomutase, thermostable glucose phosphoisomerase, thermostable 6-phosphate tagatose epimerase and thermostable 6 -
  • the ratio of wet cells of phosphotagatose phosphatase is (0.1-10):(0.1-10):(0.1-10):(0.1-10) for mixing, and after mixing
  • the OD600 of the bacterial suspension is between 10-150.
  • the inorganic soil includes but not limited to montmorillonite, diatomite, kaolin and bentonite, etc.
  • the inorganic soil is diatomite.
  • the flocculant includes but is not limited to polyethyleneimine, chitosan, polydimethyldiallylammonium chloride (PDADMAC), polyacrylamide, etc., preferably, the flocculant is polyethylene imine and PDADMAC, preferably, the polyethyleneimine has a molecular weight of 600-70000.
  • the crosslinking agent includes but not limited to glutaraldehyde, trihydroxymethylphosphine, N,N-methylenebisacrylamide, epichlorohydrin, etc., preferably, the crosslinking agent is glutaraldehyde .
  • the method further includes the step of sieving the obtained immobilized cells to obtain immobilized cells with uniform morphology.
  • the present disclosure therefore also provides a method for producing tagatose by immobilized cells, characterized in that starch or starch derivatives are converted into tagatose by using the above-mentioned immobilized cells.
  • a step of filtering and recovering the immobilized cells is also included.
  • the biotransformation reaction system comprises starch or starch derivatives 50-300g/L, a buffer solution with a pH value of 5.0-8.0, 10-50mM inorganic phosphate, 3-7mM divalent magnesium ions and immobilized cells.
  • the buffer can be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, etc.
  • the inorganic phosphate can be sodium phosphate or potassium phosphate.
  • the present disclosure has the following beneficial effects: the method for producing tagatose by immobilized cells, compared with the multi-enzyme catalyzed reaction, not only simplifies the production and preparation process of the enzyme, but also overcomes the multi-enzyme and product Difficulty in separation, which is beneficial to the separation and purification of the product tagatose.
  • the immobilized cells and the reaction solution can be separated by simple filtration, which simplifies the separation of the enzyme and the product compared with the whole-cell catalytic reaction, realizes the repeated use of the enzyme, and is conducive to improving the utilization rate of cells and reducing the rate of tagatose. production cost.
  • the cells are reused, avoiding environmental pollution caused by multiple fermentations and simplifying the operation steps.
  • the more important point is that in the present disclosure, after the fermented liquid expressing the enzyme is prepared and mixed, the fermented mixed liquid is directly used for granulation, and the steps of collecting fermented liquid cells, bacterial resuspension and cell permeabilization treatment are omitted ( The step of recovering the bacteria from the fermentation broth is omitted, the immobilization process is simplified and the operability of the process is improved; the cell permeabilization treatment step is omitted).
  • the fermentation mixture is used for direct immobilization.
  • the cell membrane or cell wall of the cells is almost not damaged, and the expressed enzyme after immobilization treatment will hardly leak, so a higher enzyme immobilization efficiency can be obtained.
  • immobilized cells after immobilization first use a rotary extrusion granulator to prepare strips with controllable thickness, and then use a spherical shot blasting machine to shorten the prepared strips into particles with uniform length, After boiling and drying at high temperature, the granules are processed (to achieve the purpose of cell permeabilization treatment), and the immobilized enzyme granules with uniform particle size are obtained after sieving, which can be more effectively used in the production of tagatose.
  • the granulation process adopted in the present disclosure is not only more conducive to the subsequent permeabilization treatment and the uniformity of the granules, but also can simplify the previous bacterial cell collection steps (as shown in FIG. 1 , the specific process of immobilized cells in the present disclosure).
  • the effect obtained by the present disclosure is very significant.
  • the immobilized Bacillus subtilis of the present disclosure continuously catalyzes the reaction, the initial product yield can reach up to 75%, and after 65 batches of continuous catalysis, the product yield can still maintain 61%.
  • the immobilized Escherichia coli of the present disclosure continuously catalyzes the reaction, the initial product yield can reach up to 74%, and after 65 batches of continuous catalysis, the product yield can still maintain 60%.
  • Fig. 1 is a schematic flow chart of the specific process of immobilizing cells in the present disclosure.
  • Figure 2 is the effect of immobilized Bacillus subtilis in producing tagatose in Example 3.
  • Fig. 3 is the effect of immobilized Escherichia coli producing tagatose in Example 11.
  • Fig. 4 is the effect of producing tagatose by Bacillus subtilis in Comparative Example 1.
  • Figure 5 is the effect of producing tagatose by Escherichia coli in Comparative Example 2.
  • thermostable ⁇ -glucan phosphorylase gene express thermostable glucose phosphate mutase gene, express thermostable glucose phosphate isomerase gene, express thermostable 6-phosphate tagatose epimerase gene, Bacillus subtilis recombinant engineered bacteria expressing the heat-resistant 6-phosphate tagatose phosphatase gene (the starting bacterium is SCK6, see CN112342179B), and inoculated in LB medium respectively, and cultured overnight at 37°C with shaking.
  • the culture was transferred to LB medium with a 1% inoculum size, and cultured overnight at 37°C with shaking to obtain Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase, and Bacillus subtilis expressing thermostable glucose phosphotransmutation Fermentation broth of Bacillus subtilis expressing thermostable glucose phosphate isomerase, fermentation broth of Bacillus subtilis expressing thermostable 6-phosphate tagatose epimerase, fermentation broth of Bacillus subtilis expressing thermostable 6-phosphate Bacillus subtilis fermentation broth of tagatose phosphatase.
  • Embodiment 2 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • Embodiment 3 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • Embodiment 4 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • 1% w/v polydimethyldiallylammonium chloride PDADMAC aqueous solution was added to flocculate at room temperature.
  • 0.05% v/v glutaraldehyde aqueous solution was added to crosslink for 3 h at room temperature.
  • the filter cake is obtained after vacuum filtration, and the filter cake is extruded and granulated into a long strip with a particle size of 1.0mm by a rotary granulator, and then the long strip is cut off by a spherical shot blasting machine to obtain particles of uniform length; the obtained immobilized cells The particles were boiled and dried at 90°C to obtain immobilized cells.
  • Embodiment 5 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation liquid expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • Embodiment 6 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • Embodiment 7 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • Embodiment 8 Production of tagatose by immobilized Bacillus subtilis
  • the Bacillus subtilis fermentation broth expressing thermostable ⁇ -glucan phosphorylase prepared in Example 1 and the Bacillus subtilis expressing thermostable glucose phosphomutase were fermented Bacillus subtilis fermentation broth expressing thermostable glucose phosphate isomerase, Bacillus subtilis fermentation broth expressing thermostable 6-phosphate tagatose epimerase, expressing thermostable 6-phosphate tagatose phosphatase
  • thermostable ⁇ -glucan phosphorylase gene express thermostable glucose phosphate mutase gene, express thermostable glucose phosphate isomerase gene, express thermostable 6-phosphate tagatose epimerase Gene, Escherichia coli recombinant engineered bacteria expressing the heat-resistant 6-phosphate tagatose phosphatase gene (BL21(DE3) was selected as the starting bacteria, see CN107988286B), and inoculated in LB medium respectively, and cultured overnight at 37°C with shaking. The culture was transferred to LB medium with 1% inoculum, induced with IPTG at 18°C, and cultured with shaking overnight to obtain E.
  • thermostable ⁇ -glucan phosphorylase and thermostable glucose Escherichia coli fermentation broth expressing phosphomutase
  • Escherichia coli fermentation broth expressing thermostable glucose phosphate isomerase
  • Escherichia coli fermentation broth expressing thermostable 6-phosphate tagatose epimerase
  • thermostable 6-phosphate E. coli fermentation broth of tagatose phosphatase.
  • Example 10 Production of tagatose by immobilized Escherichia coli
  • Example 11 Production of tagatose by immobilized Escherichia coli
  • Example 12 Production of tagatose by immobilized Escherichia coli
  • 1% w/v polydimethyldiallylammonium chloride PDADMAC aqueous solution was added to flocculate at room temperature.
  • 0.5% v/v glutaraldehyde aqueous solution was added to crosslink for 3 h at room temperature.
  • the filter cake is obtained, and the filter cake is extruded and granulated into a long strip with a particle size of 1.0mm by a rotary granulator, and then cut into long and uniform particles by a spherical shot blasting machine; the obtained immobilized cell particles are subjected to 90°C Immobilized cells were obtained after boiling and drying.
  • Example 13 Production of tagatose by immobilized Escherichia coli
  • Example 14 Production of tagatose by immobilized Escherichia coli
  • Example 15 Production of tagatose by immobilized Escherichia coli
  • Example 16 Production of tagatose by immobilized Escherichia coli
  • thermostable ⁇ -glucan phosphorylase whole cells expressing thermostable glucose phosphomutase
  • thermostable ⁇ -glucan phosphorylase whole cells expressing thermostable glucose phosphomutase
  • Whole cells expressing thermostable glucose phosphate isomerase, whole cells expressing thermostable 6-phosphate tagatose epimerase, and whole cells expressing thermostable 6-phosphate tagatose phosphatase were added to the above cells respectively 50mM sodium phosphate buffer (pH 7.5), resuspend the bacteria to OD 600 200. Heat-treat the resuspended bacteria at 75°C for 90 min.
  • thermostable ⁇ -glucan phosphorylase gene express thermostable glucose phosphate mutase gene, express thermostable glucose phosphate isomerase gene, express thermostable 6-phosphate tagatose epimerase gene, Bacillus subtilis recombinant engineered bacteria expressing the heat-resistant 6-phosphate tagatose phosphatase gene, and inoculated in LB medium respectively, and cultured overnight at 37°C with shaking.
  • thermostable ⁇ -glucan phosphorylase expressing Whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable glucose phosphoisomerase, whole cells expressing thermostable tagatose 6-phosphate epimerase, expressing thermostable tagatose 6-phosphate Whole cells of phosphatases.
  • Add 50mM sodium phosphate buffer (pH 7.5) to the above cells respectively, and resuspend the cells to OD600 200. Heat-treat the resuspended bacteria at 75°C for 90 min.
  • the filter cake was obtained after vacuum filtration, and the filter cake was extruded and granulated into particles with a particle size of 0.4 mm by a rotary granulator; the obtained immobilized cell particles were dried at 30° C. to obtain immobilized cells.
  • thermostable ⁇ -glucan phosphorylase gene express thermostable glucose phosphate mutase gene, express thermostable glucose phosphate isomerase gene, express thermostable 6-phosphate tagatose epimerase Gene, Escherichia coli recombinant engineered bacteria expressing the heat-resistant 6-phosphate tagatose phosphatase gene, were inoculated in LB medium, and cultured overnight at 37°C with shaking.
  • thermostable ⁇ -glucan phosphorylase whole cells expressing thermostable ⁇ -glucan phosphorylase.
  • cells whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable glucose phosphoisomerase, whole cells expressing thermostable 6-phosphate tagatose epimerase, whole cells expressing thermostable 6-phosphate Whole cells of tagatose phosphatase.
  • the filter cake was obtained after vacuum filtration, and the filter cake was extruded and granulated into particles with a particle size of 1.0 mm by a rotary granulator; the obtained immobilized cell particles were dried at 30° C. to obtain immobilized cells.

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Abstract

提供了一种生产塔格糖的固定化细胞的制备方法及其生产塔格糖的方法。所述固定化细胞的制备方法包括：将表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的大肠杆菌或枯草芽孢杆菌发酵液进行混合得发酵混合液，加入无机土后搅拌均匀，再加入絮凝剂絮凝菌体，随后加入交联剂交联，经真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成长条状，然后经过球形抛丸机截断成长短均匀的颗粒，经沸腾干燥后得到用于塔格糖生产的固定化细胞。本发明简化了塔格糖生产中所需酶的分离纯化步骤，提高酶回收利用率，实现了酶的循环使用。

Description

一种用于塔格糖生产的固定化细胞的制备方法及其应用 技术领域
本公开涉及生物工程技术领域，尤其涉及塔格糖的生产制备领域。
背景技术
塔格糖是天然存在的一种稀有单糖，是半乳糖的酮糖形式，果糖的差向异构体。塔格糖的甜味特性与蔗糖相似，提供非常新鲜和纯净的甜度，但热量只有蔗糖的三分之一，被称为低热量甜味剂。研究表明塔格糖具有低热量、低升糖指数、抗龋齿、抗氧化、益生元、改善肠道功能、免疫调节、药物前体等重要生理功能特性，可以广泛应用于食品、饮料、医药、保健等领域，具有巨大的经济价值(Oh D-K:Tagatose:properties,applications,and biotechnological processes.App.Microbiol.Biotechnol.2007,76:1-8)。
中国科学院天津工业生物技术研究所以廉价玉米淀粉、麦芽糊精、蔗糖等为原料，成功建立了塔格糖体外多酶合成新路线，从根本上改变了现有塔格糖的生产工艺(CN106399427A)。该多酶合成新路径不仅打破了化学法合成塔格糖能耗高，产物复杂，纯化困难，副反应多，化学污染大的限制；而且突破了生物法合成塔格糖的原料昂贵受限，转化率低，分离工艺复杂的缺陷。在此基础上，中国科学院天津工业生物技术研究所以廉价玉米淀粉、纤维素、麦芽糊精、蔗糖等为原料，利用全细胞催化制备塔格糖的方法(CN107988286A)，该工艺减少了多酶纯化工艺步骤，降低了生产成本低和环境污染，提高了塔格糖产率。中国科学院天津工业生物技术研究所进一步针对食品制剂的安全生产问题进行改进，提供了一种利用枯草芽孢杆菌全细胞催化高浓度淀粉制备生产高浓度塔格糖的方法(CN112342179B)。该方法将透性化处理的枯草芽孢杆菌进行固定化，获得固定化全细胞，然后用于塔格糖的生产，实现了全细胞的循环使用，降低生产成本。但是经过研究分析表明该方法在制备固定化细胞时仍然存在一些不足：一是该方法在进行固定化前需要经过菌体收集，菌体重悬以及菌体透性化处理等多个繁琐步骤。二是该方法在透性细胞的基础上进行制粒固定化，而细胞透性化处理易造成胞内表达的异源蛋白的泄漏，进而在固定化进程中导致异源蛋白的损失以及酶固定化效率的降低。三是该方法只是通过简单挤压制粒的方式得到固定化酶颗粒，获得的固定化酶颗粒并非均一的。
因此，亟需在此基础上开发一种可简便获得均匀的固定化细胞的方法，既能简化酶的生产工序；又能避免或降低透性细胞在固定化过程中造成的酶的泄漏和损失，提高酶的固定化效率，实现酶的循环利用，再进一步降低塔格糖的生产成本，实现塔格糖的工业化生产。
发明内容
针对现有的固定化细胞制备塔格糖的方法所存在的问题如酶的生产步骤繁琐、透性化处理造成酶的损失及固定化效率降低、制备得到的固定化酶颗粒度不均匀等问题，本公开的目的在于提供一种固定化细胞生产塔格糖的方法，简化塔格糖生产过程中酶的生产步骤，简便塔格糖生产过程中产品和酶的分离纯化，实现多酶的循环使用，降低塔格糖的生产成本，实现塔格糖的工业化生产。
为解决上述技术问题，本公开采用以下技术方案：
一种用于塔格糖生产的固定化细胞的制备方法，其特征在于，包括下述步骤：
通过发酵分别获得表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的大肠杆菌或枯草芽孢杆菌发酵液，将上述发酵液进行混合得发酵混合液；
向所述发酵混合液中加入无机土，搅拌均匀；
再向所述发酵混合液中加入絮凝剂絮凝菌体，随后加入交联剂交联；
真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成长条状，然后经过球形抛丸机截断成长短均匀的颗粒；
获得的颗粒经沸腾干燥后得到用于塔格糖生产的固定化细胞。
在一个具体的实施方式中，本公开采用以下技术方案：
一种用于塔格糖生产的固定化细胞的制备方法，其特征在于，包括下述步骤：
通过发酵分别获得表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的大肠杆菌或枯草芽孢杆菌发酵液，将上述发酵液混合得到发酵混合液；
向所述发酵混合液中加入1-10％w/v无机土，搅拌均匀；
再向所述发酵混合液中加入0.1-2％w/v絮凝剂絮凝菌体，随后加入0.05-3％v/v交联剂交联1-4h；
真空过滤后得到滤饼，滤饼用旋转造粒机挤压成长条状，然后采用球形抛丸机将长条状固定化细胞截断成长短均匀的颗粒；
获得的颗粒经沸腾干燥后得到用于塔格糖生产的固定化细胞，其中所述沸腾干燥的进风口温度控制在60-90℃。
对于所述发酵液的制备使用本领域已知的方法进行。发酵可使用任何外源蛋白标的培养基，包括但不限于LB培养基、SR培养基、TB培养基等。
优选地，所述的α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷 酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶分别是耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热塔格糖6-磷酸异构酶和耐热塔格糖6-磷酸磷酸酶。
具体地，所述耐热α-葡聚糖磷酸化酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将淀粉磷酸化为葡萄糖-1-磷酸(G1P)功能的酶。进一步优选地，所述耐热α-葡聚糖磷酸化酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热α-葡聚糖磷酸化酶的氨基酸序列与来源于所述嗜热微生物的耐热α-葡聚糖磷酸化酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热α-葡聚糖磷酸化酶来源于Thermococcus kodakarensis。
具体地，耐热葡萄糖磷酸变位酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将葡萄糖-1-磷酸(G1P)变位为葡萄糖-6-磷酸(G6P)功能的酶。进一步优选地，所述耐热葡萄糖磷酸变位酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热葡萄糖磷酸变位酶的氨基酸序列与来源于所述嗜热微生物的耐热葡萄糖磷酸变位酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热葡萄糖磷酸变位酶来源于Thermococcus kodakarensis。
具体地，耐热葡萄糖磷酸异构酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将葡萄糖-6-磷酸(G6P)变位为果糖-6-磷酸(F6P)功能的酶。进一步优选地，所述耐热葡萄糖磷酸异构酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor  kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热葡萄糖磷酸异构酶的氨基酸序列与来源于所述嗜热微生物的耐热葡萄糖磷酸异构酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热葡萄糖磷酸异构酶来源于Thermus thermophilus。
具体地，耐热6-磷酸塔格糖差向异构酶在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有果糖-6-磷酸(F6P)异构为塔格糖-6-磷酸(T6P)功能的酶。进一步优选地，所述耐热6-磷酸塔格糖差向异构酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热6-磷酸塔格糖差向异构酶的氨基酸序列与来源于所述嗜热微生物的耐热6-磷酸塔格糖差向异构酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐6-磷酸塔格糖差向异构酶来源于Thermoanaerobacter indiensis。
具体地，所述6-磷酸塔格糖磷酸酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有塔格糖-6-磷酸(T6P)脱掉磷酸基团为产物塔格糖(Tagatose)功能的酶。进一步优选地，所述6-磷酸塔格糖磷酸酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述6-磷酸塔格糖磷酸酶的氨基酸序列与来源于所述嗜热微生物的6-磷酸塔格糖磷酸酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述6-磷酸塔格糖磷酸酶来源于Archaeoglobus fulgidus。
进一步优选地，分别表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的湿菌体的按比例为(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)进行混合，且混合后的菌悬液OD600在10-150之间。
进一步，所述无机土包括但并不限于蒙脱土、硅藻土、高岭土和膨润土等，优选地，所述无机土为硅藻土。
进一步，所述絮凝剂包括但并不限于聚乙烯亚胺、壳聚糖、聚二甲基二烯丙基氯化铵(PDADMAC)、聚丙烯酰胺等，优选地，所述絮凝剂为聚乙烯亚胺和PDADMAC，优选地，所述聚乙烯亚胺的分子量为600-70000。
进一步，所述交联剂包括但不限于戊二醛、三羟甲基磷、N,N-亚甲基双丙烯酰胺、环氧氯丙烷等，优选地，所述交联剂为戊二醛。
所述方法进一步包括将获得的固定化细胞经过筛分得到形态均匀的固定化细胞的步骤。
本公开因此还提供一种固定化细胞生产塔格糖的方法，其特征在于：利用上述固定化细胞将淀粉或淀粉衍生物转化为塔格糖。
进一步地，反应结束后还包括过滤回收固定化细胞的步骤。
在具体实施方式中，其中生物转化反应体系中包含淀粉或淀粉衍生物50-300g/L，pH值为5.0-8.0的缓冲液，10-50mM无机磷酸根，3-7mM二价镁离子和固定化细胞。
进一步，所述缓冲液可为HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液、醋酸盐缓冲液等。所述无机磷酸根可为磷酸钠或磷酸钾。
与现有技术相比，本公开具有如下有益效果：所述固定化细胞生产塔格糖的方法，与多酶催化反应相比，不仅简化了酶的生产制备过程，而且克服了多酶与产品的分离困难，有利于产物塔格糖的分离纯化。所述固定化细胞与反应液可通过简单过滤进行分离，与全细胞催化反应相比，更加简化了酶与产品的分离，实现酶的重复使用，有利于提高细胞的利用率，降低塔格糖的生产成本。同时，细胞重复使用，避免了多次发酵产生的环境污染及简化操作步骤。其中，比较重要之处在于，本公开在制备得到表达酶的发酵液混合后，直接将发酵混合液用于制粒，省略发酵液菌体收集，菌体重悬及细胞透性化处理的步骤(省去发酵液菌体回收步骤，简便了固定化过程，提高了过程的可操作性；省去细胞透性化处理步骤)。而采用发酵混合液直接进行固定化，此时细胞的细胞膜或细胞壁几乎没有破损，经过固定化处理后表达的酶几乎不会泄漏，因此能够获得较高的酶固定化效率。而对于本公开在固定化后获得固定化细胞，首先采用旋转挤压制粒机制备出粗细可控的长条，然后用球形抛丸机将制备得到的长条截短成长度均匀的颗粒，再经过沸腾干燥高温干燥处理颗粒(以达到细胞透性化处理的目的)，筛分后得到粒径均匀的固定化酶颗粒，如此能更有效的用于塔格糖的生产。其中本公开采用的制粒流程不仅更有利于后面的透性化处理和颗粒的均匀度，而且更能简化前面的菌体收集步骤(如图1所示本公开固定化细胞的具体流程)。经实验表明，本公开获得的效果十分显著，本公开 的固定化枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持61％。本公开的固定大肠杆菌连续催化反应时，初始产物得率最高可达74％，连续催化65批次后，产物得率仍可维持60％。
附图说明
图1为本公开固定化细胞的具体流程示意图。
图2为实施例3固定化枯草芽孢杆菌生产塔格糖效果。
图3为实施例11固定化大肠杆菌生产塔格糖效果。
图4为对比例1枯草芽孢杆菌生产塔格糖效果。
图5为对比例2大肠杆菌生产塔格糖效果。
具体实施方式
术语定义
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
除非另有定义，本文所用的所有技术和科学术语与本公开所属领域普通技术人员通常理解的意义相同。虽然可利用与本文所述相似或等价的任何方法和材料来实施或检验本公开，但优选本文所述的方法和材料。
为更进一步阐述本公开所采取的技术手段及其效果，以下通过具体实施例来进一步说明本公开的技术方案。但是应理解所述实施例仅是范例性的，不对本公开的范围构成任何限制。本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本公开的保护范围。
实施例1：枯草芽孢杆菌全细胞的制备
分别挑取表达耐热α-葡聚糖磷酸化酶基因、表达耐热葡萄糖磷酸变位酶基因、表达耐热葡萄糖磷酸异构酶基因、表达耐热6-磷酸塔格糖差向异构酶基因、表达耐热6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌重组工程菌(出发菌选用SCK6，见CN112342179B)，并分别接种于LB培养基中，37℃振荡过夜培养。将培养物以1％的接种量转接于LB培养基中，37℃震荡培养过夜，分别获得表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液。
实施例2：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入1％w/v蒙脱土，搅拌均匀。随后，加入0.5％w/v分子量为10000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入2％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经60℃沸腾干燥后得到固定化细胞。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲(pH 7.0)以及固定化枯草芽孢杆菌，使OD600＝20，在70℃水浴摇床反应。反应过程中用高效液相色谱分析塔格糖含量。反应结束后通过简单过滤收集固定化枯草芽孢杆菌，经缓冲液洗涤后进行下一批次反应。实验结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持58％。
实施例3：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.1％w/v分子量为70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入1％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成3.0mm粒径的的长条状，然后 经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经75℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持61％。
实施例4：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入10％w/v硅藻土，搅拌均匀。随后，加入1％w/v聚二甲基二烯丙基氯化铵PDADMAC水溶液在室温条件下絮凝。然后，加入0.05％v/v戊二醛水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经90℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果如图1所示，结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持59％。
实施例5：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:5:5将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.5％w/v分子量为600的聚乙烯亚胺在室温条件下絮凝。然后，加入0.3％v/v戊二醛水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成0.4mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经80℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持56％。
实施例6：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷 酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入6％w/v硅藻土，搅拌均匀。随后，加入1％w/v分子量为600的聚乙烯亚胺在室温条件下絮凝。然后，加入1％v/v三羟甲基磷水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成0.4mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经80℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持59％。
实施例7：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入3％w/v硅藻土，搅拌均匀。随后，加入0.5％w/v聚丙烯酰胺在室温条件下絮凝。然后，加入2.0％v/v N,N-亚甲基双丙烯酰胺水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经90℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持57％。
实施例8：固定化枯草芽孢杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例1中制备的表达耐热α-葡聚糖磷酸化酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸变位酶的枯草芽孢杆菌发酵液、表达耐热葡萄糖磷酸异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的枯草芽孢杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的枯草芽孢杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入1％w/v硅藻土，搅拌均匀。随后，加入0.1％w/v分子量70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入0.5％v/v环氧氯丙烷水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经75℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定枯草芽孢杆菌连续催化 反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持58％。
实施例9：大肠杆菌全细胞的制备
分别挑取表达耐热α-葡聚糖磷酸化酶基因、表达耐热葡萄糖磷酸变位酶基因、表达耐热葡萄糖磷酸异构酶基因、表达耐热6-磷酸塔格糖差向异构酶基因、表达耐热6-磷酸塔格糖磷酸酶基因的大肠杆菌重组工程菌(出发菌选用BL21(DE3)，见CN107988286B)，并分别接种于LB培养基中，37℃振荡过夜培养。将培养物以1％的接种量转接于LB培养基中，18℃用IPTG诱导，震荡培养过夜，分别获得表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液。
实施例10：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入1％w/v蒙脱土，搅拌均匀。随后，加入0.5％w/v分子量为10000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入2％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机截断成长短均匀的颗粒；获得的固定化细胞颗粒经60℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达74％，连续催化65批次后，产物得率仍可维持55％。
实施例11：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.1％w/v分子量为70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入1％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成3.0mm粒径的长条状，然后经过球形抛丸机截断成长短均匀的颗粒；获得的固定化细胞颗粒经75℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果如图2所示，结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达74％，连续催化65批次后，产物得率仍可 维持60％。
实施例12：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入10％w/v硅藻土，搅拌均匀。随后，加入1％w/v聚二甲基二烯丙基氯化铵PDADMAC水溶液在室温条件下絮凝。然后，加入0.5％v/v戊二醛水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机截断成长短均匀的颗粒；获得的固定化细胞颗粒经90℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达74％，连续催化65批次后，产物得率仍可维持57％。
实施例13：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:5:5将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入4％w/v硅藻土，搅拌均匀。随后，加入1％w/v分子量为600的聚乙烯亚胺在室温条件下絮凝。然后，加入0.3％v/v戊二醛水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成0.4mm粒径的长条状，然后经过球形抛丸机截断成长短均匀的颗粒；获得的固定化细胞颗粒经80℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达74％，连续催化65批次后，产物得率仍可维持58％。
实施例14：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.8％w/v分子量为10000的聚乙烯亚胺在室温条件下絮凝。然后，加入0.7％v/v三羟甲基磷水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经80℃沸腾干燥后得到固定 化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持59％。
实施例15：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100左右，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.3％w/v聚丙烯酰胺在室温条件下絮凝。然后，加入1.0％v/v N,N-亚甲基双丙烯酰胺水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经90℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持57％。
实施例16：固定化大肠杆菌生产塔格糖
按照OD600比例1:1:1:1:1将实施例9中制备的表达耐热α-葡聚糖磷酸化酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸变位酶的大肠杆菌发酵液、表达耐热葡萄糖磷酸异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖差向异构酶的大肠杆菌发酵液、表达耐热6-磷酸塔格糖磷酸酶的大肠杆菌发酵液进行混合，使得OD 600＝100，向菌悬液中加入2％w/v硅藻土，搅拌均匀。随后，加入0.2％w/v分子量70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入1.0％v/v环氧氯丙烷水溶液在室温条件下交联3h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的长条状，然后经过球形抛丸机将长条截断后得到长短均匀的颗粒；获得的固定化细胞颗粒经75℃沸腾干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定大肠杆菌连续催化反应时，初始产物得率最高可达75％，连续催化65批次后，产物得率仍可维持55％。
对比例1：枯草芽孢杆菌生产塔格糖
将实施例1中制备得到的发酵液于5500rpm离心10min，弃去上清，分别得到表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞，分别向上述细胞中加入50mM磷酸钠缓冲液(pH 7.5)，重悬菌体至OD 600＝200。将重悬的菌体75℃热处理90min。用pH 7.0磷酸钠缓冲液按照比例 1:1:1:1:1将上述全细胞进行混合，使得OD600＝200。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲(pH 7.0)以及上述混合的枯草芽孢杆菌OD 600＝20，在70℃水浴摇床反应。反应过程中用高效液相色谱分析塔格糖含量。反应结束后通过离心获得沉淀菌体，经缓冲液洗涤后进行下一批次反应。实验结果如图3所示，结果表明，固定枯草芽孢杆菌连续催化反应时，初始产物得率最高可达73％，但连续催化2批次后，产物得率仅剩余20％。
对比例2：大肠杆菌生产塔格糖
将实施例9中制备得到的发酵液于5500rpm离心10min，弃去上清，分别获得表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞，分别向上述细胞中加入50mM磷酸钠缓冲液(pH 7.5)，重悬菌体至OD 600＝200。将重悬的菌体75℃热处理90min。用pH 7.0磷酸钠缓冲液按照比例1:1:1:1:1将上述全细胞进行混合，使得OD600＝200。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲(pH 7.0)以及上述混合的大肠杆菌OD 600＝20，在70℃水浴摇床反应。反应过程中用高效液相色谱分析塔格糖含量。反应结束后通过离心获得沉淀菌体，经缓冲液洗涤后进行下一批次反应。实验结果如图4所示，结果表明，大肠杆菌连续催化反应时，初始产物得率最高可达73％，但连续催化2批次后，产物得率仅剩余15％。
对比例3:固定化透性枯草芽孢杆菌生产塔格糖
分别挑取表达耐热α-葡聚糖磷酸化酶基因、表达耐热葡萄糖磷酸变位酶基因、表达耐热葡萄糖磷酸异构酶基因、表达耐热6-磷酸塔格糖差向异构酶基因、表达耐热6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌重组工程菌，并分别接种于LB培养基中，37℃振荡过夜培养。将培养物以1％的接种量转接于LB培养基中，37℃震荡培养过夜，5500rpm离心10min，弃去上清，分别获得表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞。分别向上述细胞中加入50mM磷酸钠缓冲液(pH 7.5)，重悬菌体至OD 600＝200。将重悬的菌体75℃热处理90min。
用pH 7.0磷酸钠缓冲液按照比例1:1:1:1:1将上述透性全细胞进行混合，使得OD 600＝100，向菌悬液中加入5％w/v硅藻土，搅拌均匀。随后，加入0.5％w/v分子量为70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入0.5％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成0.4mm粒径的颗粒； 获得的固定化细胞颗粒经30℃干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定化透性枯草芽孢杆菌连续催化反应时，初始产物得率最高可达73％，连续催化65批次后，产物得率为43％。
对比例4：固定化透性大肠杆菌生产塔格糖
分别挑取表达耐热α-葡聚糖磷酸化酶基因、表达耐热葡萄糖磷酸变位酶基因、表达耐热葡萄糖磷酸异构酶基因、表达耐热6-磷酸塔格糖差向异构酶基因、表达耐热6-磷酸塔格糖磷酸酶基因的大肠杆菌重组工程菌，并分别接种于LB培养基中，37℃振荡过夜培养。将培养物以1％的接种量转接于LB培养基中，18℃IPTG诱导，震荡培养过夜，5500rpm离心10min，弃去上清，分别获得表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞。分别向上述细胞中加入50mM磷酸钠缓冲液(pH 7.5)，重悬菌体至OD 600＝200。将重悬的菌体75℃热处理90min。
用pH 7.0磷酸钠缓冲液按照比例1:1:1:1:1将上述透性全细胞进行混合，使得OD600＝100，向菌悬液中加入1％w/v硅藻土，搅拌均匀。随后，加入0.5％w/v分子量为70000的聚乙烯亚胺水溶液在室温条件下絮凝。然后，加入1％v/v戊二醛水溶液在室温条件下交联2h。真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成1.0mm粒径的颗粒；获得的固定化细胞颗粒经30℃干燥后得到固定化细胞。
按实施例2的方法进行塔格糖的生产。实验结果表明，固定化透性大肠杆菌连续催化反应时，初始产物得率最高可达72％，连续催化65批次后，产物得率为40％。

Claims (14)

  1. 一种用于塔格糖生产的固定化细胞的制备方法，其特征在于，包括下述步骤：
    通过发酵分别获得表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的大肠杆菌或枯草芽孢杆菌发酵液，将上述发酵液进行混合得发酵混合液；
    向所述发酵混合液中加入无机土，搅拌均匀；
    再向所述发酵混合液中加入絮凝剂絮凝菌体，随后加入交联剂交联；
    真空过滤后得到滤饼，滤饼用旋转造粒机挤压制粒成长条状，然后经过球形抛丸机截断成长短均匀的颗粒；
    获得的颗粒经沸腾干燥后得到用于塔格糖生产的固定化细胞。
  2. 一种用于塔格糖生产的固定化细胞的制备方法，其特征在于，包括下述步骤：
    通过发酵分别获得表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的大肠杆菌或枯草芽孢杆菌发酵液，将上述发酵液混合得到发酵混合液；
    向所述发酵混合液中加入1-10％w/v无机土，搅拌均匀；
    再向所述发酵混合液中加入0.1-2％w/v絮凝剂絮凝菌体，随后加入0.05-3％v/v交联剂交联1-4h；
    真空过滤后得到滤饼，滤饼用旋转造粒机挤压成长条状，然后采用球形抛丸机将长条状固定化细胞截断成长短均匀的颗粒；
    获得的颗粒经沸腾干燥后得到用于塔格糖生产的固定化细胞，其中所述沸腾干燥的进风口温度控制在60-90℃。
  3. 如权利要求1或2所述的方法，其特征在于，所述的α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶分别是耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热塔格糖6-磷酸异构酶和耐热塔格糖6-磷酸磷酸酶。
  4. 如权利要求3所述的方法，其特征在于，所述耐热是指在40℃以上具有酶的活性。
  5. 如权利要求4所述的方法，其特征在于，分别表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热塔格糖6-磷酸异构酶和耐热塔格糖6-磷酸磷酸酶的湿菌体的按比例为(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)进行混合，且混合后的菌悬液OD600在10-150之间。
  6. 如权利要求1或2所述的方法，其特征在于，所述无机土选自蒙脱土、硅藻土、高 岭土或膨润土。
  7. 如权利要求1或2所述的方法，其特征在于，所述絮凝剂选自聚乙烯亚胺、壳聚糖、聚二甲基二烯丙基氯化铵、聚丙烯酰胺。
  8. 如权利要求7所述的方法，其特征在于，所述絮凝剂为聚乙烯亚胺或PDADMAC，且所述聚乙烯亚胺的分子量为600-70000。
  9. 如权利要求1或2所述的方法，其特征在于，所述交联剂选自戊二醛、三羟甲基磷、N,N-亚甲基双丙烯酰胺或环氧氯丙烷。
  10. 如权利要求1或2所述的方法，其特征在于，进一步包括将获得的固定化细胞经过筛分得到形态均匀的固定化细胞的步骤。
  11. 一种固定化细胞生产塔格糖的方法，其特征在于：利用如权利要求1至10任一项所述方法得到的固定化细胞将淀粉或淀粉衍生物转化为塔格糖。
  12. 如权利要求11所述的方法，其特征在于，进一步地，反应结束后还包括过滤回收固定化细胞的步骤。
  13. 如权利要求11所述的方法，其特征在于，其中生物转化反应体系中包含淀粉或淀粉衍生物50-300g/L，pH值为5.0-8.0的缓冲液，10-50mM无机磷酸根，3-7mM二价镁离子和固定化细胞。
  14. 如权利要求11所述的方法，其特征在于，所述缓冲液为HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液或醋酸盐缓冲液；所述无机磷酸根可为磷酸钠或磷酸钾。
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