CN103865970A - Process and device for continuously producing glycyrrhetinic acid monoglucuronide by using immobilized cells - Google Patents
Process and device for continuously producing glycyrrhetinic acid monoglucuronide by using immobilized cells Download PDFInfo
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- CN103865970A CN103865970A CN201410088328.3A CN201410088328A CN103865970A CN 103865970 A CN103865970 A CN 103865970A CN 201410088328 A CN201410088328 A CN 201410088328A CN 103865970 A CN103865970 A CN 103865970A
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Abstract
The invention discloses a process and device for continuously producing glycyrrhetinic acid monoglucuronide by converting glycyrrhizic acid by using immobilized cells of Li-3(Penicillium purpurogenum Li-3) CGMCC NO.5446 and belongs to the field of biochemical engineering. According to the process, penicillium purpurogenum Li-3 is immobilized by using diatomite and calcium alginate, and the glycyrrhetinic acid monoglucuronide is continuously produced by converting the glycyrrhizic acid in a packed bed reactor with an isolation screen by using the immobilized cells. The packed bed reactor with the isolation screen is manufactured by adding an isolation screen into a common packed bed reactor so as to load beads of the immobilized cells via different layers of a packed bed so that damage caused by the self-weight of the beads is reduced; by adopting the process and the device, the activity of the cells can be kept for a long time, the service life of the beads of the immobilized cells is prolonged, the product purity is improved, the production cost is greatly reduced, and large-scale continuous production of glycyrrhetinic acid monoglucuronide is possible.
Description
Technical field
The invention belongs to biological chemical field, relate to a kind of technique and equipment therefor thereof that utilizes penicillium purpurogenum Li-3CGMCC NO.5446 Cell of Anmrobe Potenlini to produce continuously GAMG.
Background technology
Single glucuronic acid glycyrrhetinic acid (Glycyrrhetinic acid monoglucuronide, GAMG) be Potenlini (Glycyrrhizin, GL) derivative of a glucal acidic group is removed in modification, it is a kind of high sugariness natural function sweeting agent low in calories, and sugariness is more than 1000 times of sucrose.The stable in properties of GAMG, has that high temperature resistant, acidproof and high pressure resistant and sweet sense significantly lags behind, the advantage such as emulsifying property and whipability preferably.Single glucuronic acid glycyrrhetinic acid, possessing outside the anti-inflammatory of Potenlini, antiviral, antineoplastic curative effect, because its polarity is moderate, has good solubleness and the ability of transmembrane transport in vivo, has better bioavailability compared with Potenlini.And the Lethal Dose 50 of GAMG is 5000mg/kg, much larger than the 805mg/kg of Potenlini, without aberration inducing effect.
The preparation method of single glucuronic acid glycyrrhetinic acid has chemical method and biological enzyme, chemical method is mainly by be hydrolyzed the glycosidic link in Potenlini molecular structure under acidic conditions, but acid is not strong to two glycosidic link selectivity in Potenlini molecular structure, often causes a large amount of generations of by product glycyrrhetinic acid.Chemical method is time-consuming, effort, and product is difficult to obtain, and can produce a large amount of sewage, causes environmental pollution, is the friendly type technology of a kind of non-ambient.By contrast, biological enzyme have that speed of reaction is high, substrate specificity is strong, reaction conditions temperature and the advantage such as environmentally friendly.Biological process transforms the enzyme that the synthetic GAMG catalyzer used of Potenlini is mainly microorganism cells or extracts by microorganism cells at present, but the problem often existing is short as enzyme or the somatic cells life-span of catalyzer, impurity in products is many, cause later separation purification step complicated operation, cost is high.
Immobilized cell is exactly the cell that is limited to move freely, and cell is subject to the constraint of the factor such as physical chemistry or is limited in certain space boundary, but intracellular enzyme still retain catalytic activity and have can be by the vigor repeatedly or continuously using.It does not need immobilized cell smudge cells and directly can utilize intracellular enzyme, enzyme is more stable in cell, immobilized cell density is large, adsorptive capacity is high, speed of response is accelerated, improve throughput, thereby reduced production cost, and somatic cells is easily separated in separation and purification process, immobilized cell can be reused.Packed bed reactor (packed bed reactor, PBR) is in immobilized cell technology, to use the most general, most widely used a kind of reactor.The granules of catalyst packing density that packed bed reactor has unit reactor volume is high, simple in structure, and fabrication cost is low, simple to operate, and speed of response is fast, is easy to expand the scale of production, and can realize the advantage that serialization is produced.
Penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) the beta-glucuronidase enzyme of its expression of CGMCC NO.5446 has strict substrate specificity, and the glucal acidic group of hydrolysis Potenlini non-reducing end generates GAMG.Utilizing biological process to transform production GAMG continuously can realize by penicillium purpurogenum Li-3CGMCCNO.5446 immobilized cell.
Summary of the invention
The object of the invention is to overcome the defect of prior art, improve the production efficiency of GAMG, reduce its production cost.
For achieving the above object, the present invention utilizes sodium alginate to embed and diatomite adsorption co-immobilization penicillium purpurogenum Li-3CGMCC NO.5446, optimize after fixing condition, with a fixed column porosity dress post, immobilized cell taking dress after post is as catalyzer, by certain density Potenlini substrate solution, with the certain flow rate packed column of flowing through, under best reaction conditions, transform and generate GAMG; For reacting undesirable in traditional packed bed reactor, in packed bed, the more crackly feature of immobilization particle of bottom, improves traditional packed bed reactor.In modified version packed bed reactor, add isolation screen device particle layering has been spread out, isolation sieve (as shown in Figure 2) comprises that one of two portions are chassis, is made up of grid; Another part is supporter.Every layer of isolation sieve can take out from reactor one end.This reactor has following advantage with respect to traditional pillar packed bed reactor: one, and above having avoided, particle is to the extruding of particle below, and every layer of particle has point of suppon, thereby slowed down the distortion of particle in reaction process.Its two, immobilized cell particle can more effectively contact with reaction solution, thereby is more conducive to the transmission of material in the catalysis of enzyme and reaction process.Its three, be convenient to clean and change particle, if the granule strength in different layers change inconsistent, can replace separately, thus reduce reaction cost.
The technical solution used in the present invention is as follows:
(1) seed liquor cultivation and fermentor cultivation are prepared penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) CGMCC NO.5446 cell;
(2) immobilization penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) preparation of CGMCC NO.5446 cell: cell suspending liquid is mixed with the diatomite of 1-6%, add sodium alginate soln, splash in calcium chloride, make gel beads;
(3) immobilized cell is filled in packed bed reactor;
(4) produce continuously GAMG: the Potenlini solution that is 0.5-3.0g/L with sodium acetate-acetate buffer configuration concentration, and add tween-80, with the flow velocity of the 0.3-1.0mL/min whole packed bed reactor of flowing through, carry out the continuous production of GAMG.
The beneficial effect that the present invention has:
(1) immobilized cell long service life, taking immobilized cell packed bed as catalyzer, can reach 15-30 days by the synthetic GAMG of continuous catalysis, be the 3-6 of free cell (5 days) doubly, this has not only reduced raw material consumption, and saved energy consumption, greatly reduce production costs.
(2) product purity is high, utilizes immobilized cell packed bed to catalyze and synthesize GAMG, can avoid on the one hand free cell in catalytic process because of broken, dead produced albumen and other amino acid impurity; On the other hand, can avoid cell directly to mix with reaction solution, bring difficulty to subsequent products separation and purification.
(3) producing can serialization, packed bed reactor of the present invention, reaction solution flows out from the inflow top, bottom of post, the GAMG that has catalysis to generate in reaction solution out, whole process simplification reactions steps, greatly improved production efficiency.
Brief description of the drawings
Accompanying drawing 1, immobilization penicillium purpurogenum Li-3CGMCC NO.5446 cell are produced the packed bed reactor schematic diagram of GAMG continuously.
Accompanying drawing 2, isolation screen device schematic diagram.
Accompanying drawing 3, immobilized cell dress post process schematic diagram.
Accompanying drawing 4, immobilized cell successive reaction schematic flow sheet.
Wherein, (a) packed bed reactor; (b) substrate solution storage tank; (c) peristaltic pump; (d) product-collecting tank
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.
Embodiment 1
The making of packed bed reactor
The packed bed reactor providing in the present embodiment is synthetic glass post, and its internal diameter is 50mm, is highly 500mm, and reactor volume is 981mL, and isolation screen bottom tray diameter is 48mm, and pillar stiffener height is 50mm.
Embodiment 2
(1) preparation of penicillium purpurogenum Li-3CGMCC NO.5446 cell
By the penicillium purpurogenum Li-3 of slant culture, be linked in seed culture medium, 32 DEG C, 170r/min cultivates 72h, then proceeds to secondary seed medium with 1.5% inoculum size, and 32 DEG C, 170r/min cultivates 24h, obtains secondary seed solution.By cultured secondary seed nutrient solution according to 10%(v/v) inoculum size, be seeded in the 1000mL triangular flask that 300mL fermention medium is housed and carry out fermentation culture, 30 DEG C of leavening temperatures, rotating speed is 170r/min.By the penicillium purpurogenum Li-3 obtaining, centrifugal 10min under 12000rpm, uses distilled water wash thalline 3 times afterwards.
Seed liquor substratum consists of and in every premium on currency, contains 5.0g glucose, 3.0g NaNO
3, 1.0g K
2hPO
4, 0.5gMgSO
47H
2o, 0.5g KCl, 0.01g FeSO
47H
2o; Fermentation tank culture medium consists of and in every premium on currency, contains 4.5g Potenlini, 3.0g NaNO
3, 1.0g K
2hPO
4, 0.5g MgSO
47H
2o, 0.5g KCl, 0.01g FeSO
47H
2o.
(2) preparation of immobilization penicillium purpurogenum Li-3CGMCC NO.5446
The thalline that fermentation is obtained, adds distilled water to prepare 20% bacteria suspension, adds 4% diatomite, after stirring and with 2.5% sodium alginate soln of 121 DEG C of sterilizing 20min of two volumes, mixes.Draw sodium alginate bacteria suspension with syringe, splash into the 2%CaCl on the magnetic stirring apparatus that is placed in low cruise
2being fixed in solution, forms immobilization gel beads of uniform size, diameter maximum difference≤0.5mm, and immobilization 12h, makes the gel beads that diameter is 3mm, and with being stored in 0.9% physiological saline in 4 DEG C of refrigerators.
(3) immobilized cell dress post
Get 440mL immobilized cell, pack in the packed bed reactor of making in embodiment 1, as shown in Figure 3, packed bed reactor porosity is 0.54 to dress post process.
(4) utilize the immobilized cell of dress post to transform continuously production GAMG
The concentration that configures Potenlini with sodium acetate-acetate buffer of pH5.4 is 0.72g/L, add the tween-80 of volume fraction 0.12%, be under 35 DEG C of conditions in water-bath catalyzed reaction temperature, substrate solution flows out (as shown in Figure 4) from inflow top, packed bed bottom with the flow velocity of 0.34mL/min.Under this reaction conditions, can produce continuously GAMG, its output is 0.193g/L, and yield is 34%, and space-time yield is 336 μ mol/ (Ld); Immobilized cell can use 30 days continuously.
Embodiment 3
(1) preparation of penicillium purpurogenum Li-3CGMCC NO.5446 cell is with described in embodiment 2.
(2) preparation of immobilization penicillium purpurogenum Li-3CGMCC NO.5446
The thalline that fermentation is obtained, adds distilled water to prepare 20% bacteria suspension, adds 1% diatomite, after stirring and with 2.5% sodium alginate soln of 121 DEG C of sterilizing 20min of two volumes, mixes.Draw sodium alginate bacteria suspension with syringe, splash into the 2%CaCl on the magnetic stirring apparatus that is placed in low cruise
2being fixed in solution, forms immobilization gel beads of uniform size, diameter maximum difference≤0.5mm, and immobilization 12h, makes the gel beads that diameter is 3mm, and with being stored in 0.9% physiological saline in 4 DEG C of refrigerators.
(3) immobilized cell dress post is with described in embodiment 2.
(4) utilize the immobilized cell of dress post to transform continuously production GAMG
The concentration that configures Potenlini with sodium acetate-acetate buffer of pH5.4 is 1.35g/L, add the tween-80 of volume fraction 0.12%, be under 35 DEG C of conditions in water-bath catalyzed reaction temperature, substrate solution flows out (as shown in Figure 4) from inflow top, packed bed bottom with the flow velocity of 1.0mL/min.Under this reaction conditions, can produce continuously GAMG, its output is 0.18g/L, and yield is 16.9%, and space-time yield is 908 μ mol/ (Ld); Immobilized cell can use 20 days continuously.
Embodiment 4
(1) preparation of penicillium purpurogenum Li-3CGMCC NO.5446 cell is with described in embodiment 2.
(2) preparation of immobilization penicillium purpurogenum Li-3CGMCC NO.5446
The thalline that fermentation is obtained, adds distilled water to prepare 20% bacteria suspension, adds 6% diatomite, after stirring and with 2.5% sodium alginate soln of 121 DEG C of sterilizing 20min of two volumes, mixes.Draw sodium alginate bacteria suspension with syringe, splash into the 2%CaCl on the magnetic stirring apparatus that is placed in low cruise
2being fixed in solution, forms immobilization gel beads of uniform size, diameter maximum difference≤0.5mm, and immobilization 12h, makes the gel beads that diameter is 3mm, and with being stored in 0.9% physiological saline in 4 DEG C of refrigerators.
(3) immobilized cell dress post is with described in embodiment 2.
(4) utilize the immobilized cell of dress post to transform continuously production GAMG
The concentration that configures Potenlini with sodium acetate-acetate buffer of pH5.4 is 3.0g/L, add the tween-80 of volume fraction 0.12%, be under 35 DEG C of conditions in water-bath catalyzed reaction temperature, substrate solution flows out (as shown in Figure 4) from inflow top, packed bed bottom with the flow velocity of 0.66mL/min.Under this reaction conditions, can produce continuously GAMG, its output is 0.2g/L, and yield is 8.5%, and space-time yield is 666 μ mol/ (Ld); Immobilized cell can use 15 days continuously.
Claims (2)
1. immobilized cell is produced technique and the device of GAMG continuously, it is characterized in that: with immobilization penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) CGMCC NO.5446 cell produces GAMG continuously at tool isolation sieve packed bed reactor transfer glycyrrhizic acid, and production process comprises following process steps:
(1) seed liquor cultivation and fermentor cultivation are prepared penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) CGMCC NO.5446 cell;
(2) immobilization penicillium purpurogenum Li-3(Penicillium purpurogenum Li-3) preparation of CGMCC NO.5446 cell: cell suspending liquid is mixed with the diatomite of 1-6%, add sodium alginate soln, splash in calcium chloride, make gel beads;
(3) immobilized cell is filled in packed bed reactor;
(4) produce continuously GAMG: the Potenlini solution that is 0.5-3.0g/L with sodium acetate-acetate buffer configuration concentration, and add tween-80, with the flow velocity of the 0.3-1.0mL/min whole packed bed reactor of flowing through, carry out the continuous production of GAMG.
2. packed bed reactor as claimed in claim 1, comprise reactor shell, packed bed, water distribution system, aerating system and isolation sieve, it is characterized in that: in conventional packed bed reactor, add isolation screen device, isolation sieve is independent of packed bed reactor, it is made up of two portions, a part is chassis, is made up of grid, and another part is supporter.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104087646A (en) * | 2014-07-21 | 2014-10-08 | 江苏天晟药业有限公司 | Method for preparing glycyrrhetinic acid |
| CN104342430A (en) * | 2014-09-30 | 2015-02-11 | 嘉兴学院 | Ionic liquid-loaded hollow liquid core microencapsulation cell and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201313899Y (en) * | 2008-10-27 | 2009-09-23 | 宋秋兰 | Novel circulating packed bed reactor |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201313899Y (en) * | 2008-10-27 | 2009-09-23 | 宋秋兰 | Novel circulating packed bed reactor |
Non-Patent Citations (2)
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| 陈金燕等: "含离子液体体系中固定化细胞转化甘草酸生成单葡萄糖醛酸基甘草次酸", 《生物加工工程》, vol. 9, no. 5, 30 September 2011 (2011-09-30), pages 17 - 21 * |
| 韩振为等: "海藻酸钠－硅藻土包埋石油脱硫菌Rhodococcus sp. H-412", 《化学工业与工程》, vol. 23, no. 6, 19 February 2013 (2013-02-19) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087646A (en) * | 2014-07-21 | 2014-10-08 | 江苏天晟药业有限公司 | Method for preparing glycyrrhetinic acid |
| CN104342430A (en) * | 2014-09-30 | 2015-02-11 | 嘉兴学院 | Ionic liquid-loaded hollow liquid core microencapsulation cell and application thereof |
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Application publication date: 20140618 |