CN116676166A - Method for continuously preparing D-psicose by using bacillus licheniformis - Google Patents
Method for continuously preparing D-psicose by using bacillus licheniformis Download PDFInfo
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- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 title claims abstract description 41
- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 25
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 49
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 239000003054 catalyst Substances 0.000 claims abstract description 39
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims abstract description 31
- 238000001471 micro-filtration Methods 0.000 claims abstract description 29
- 229930091371 Fructose Natural products 0.000 claims abstract description 19
- 239000005715 Fructose Substances 0.000 claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000005086 pumping Methods 0.000 claims abstract description 6
- 238000010924 continuous production Methods 0.000 claims abstract description 5
- 238000007670 refining Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000006555 catalytic reaction Methods 0.000 claims description 8
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 5
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- PCTMTFRHKVHKIS-BMFZQQSSSA-N (1s,3r,4e,6e,8e,10e,12e,14e,16e,18s,19r,20r,21s,25r,27r,30r,31r,33s,35r,37s,38r)-3-[(2r,3s,4s,5s,6r)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-19,25,27,30,31,33,35,37-octahydroxy-18,20,21-trimethyl-23-oxo-22,39-dioxabicyclo[33.3.1]nonatriaconta-4,6,8,10 Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2.O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 PCTMTFRHKVHKIS-BMFZQQSSSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
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- 108030002106 D-psicose 3-epimerases Proteins 0.000 description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
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- 241000186226 Corynebacterium glutamicum Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
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- 239000005720 sucrose Substances 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
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- 238000011084 recovery Methods 0.000 description 2
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- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N D-fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
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- 210000003608 fece Anatomy 0.000 description 1
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- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
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- 239000012510 hollow fiber Substances 0.000 description 1
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- 239000007924 injection Substances 0.000 description 1
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- 238000005580 one pot reaction Methods 0.000 description 1
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- 150000008163 sugars Chemical class 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
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- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Sustainable Development (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for continuously preparing D-psicose by using bacillus licheniformis, and belongs to the technical field of biology. The method comprises the following steps: fermenting the recombinant bacillus licheniformis, and recovering cells to serve as a catalyst; preparing D-fructose solution in a preparation tank; mixing the fructose solution with a cell catalyst, and pumping into a microfiltration device; allowing clear liquid obtained by microfiltration to enter a simulated moving bed connected in series with a microfiltration device to separate a substrate and a product, and refluxing mixed liquid containing a cell catalyst to a reaction tank for continuous reaction; the D-psicose obtained by simulated moving bed separation is further roughly mentioned for refining, and D-fructose and mixed liquor are refluxed to a batching tank for continuous reaction. The bacillus licheniformis cell catalyst adopted by the invention can be stably and continuously reused at high temperature. The micro-filtration device is connected with the simulated moving bed in series, so that the balance of the reversible reaction is continuously pushed to the direction of product synthesis, the reaction efficiency is high, and the method is suitable for industrial-scale continuous production.
Description
Technical Field
The invention relates to a method for continuously preparing D-psicose by using bacillus licheniformis, belonging to the technical field of biology.
Background
D-psicose has a molecular weight of 180.16, a melting point of 96 ℃, is easily dissolved in water, has a sweetness of about 70% of sucrose, and is structurally different from that of the C-3 position of D-fructose. Unlike conventional sweeteners, 70% of the D-psicose is directly discharged through urine or feces after being ingested into a human body, and the energy generated is only 0.3% of the same amount of sucrose, so that no digestion burden is caused, and no health threat is caused to the human body.
Early D-psicose was obtained by chemical synthesis. The bioconversion method has the advantages of mild reaction conditions, few byproducts, simple purification steps, environmental protection and the like, and has become the main direction of the synthesis of the D-psicose. At present, D-Psicose bioconversion mainly utilizes hosts such as escherichia coli, corynebacterium glutamicum or bacillus subtilis to express D-Psicose 3-epimerase, then utilizes cell disruption to obtain pure enzyme, and uses D-fructose as a substrate to convert and synthesize D-Psicose. In this production mode, the pure enzyme as a catalyst can be used only once, and inconvenience is brought to separation of the product.
At present, the report of synthesizing D-psicose by using whole cell transformation is relatively few, and the host cells mainly used are escherichia coli, corynebacterium glutamicum, bacillus subtilis and the like. Whole cell catalysis omits the step of cell disruption after fermentation, and the catalyst can be conveniently separated from the product by centrifugation or filtration. However, cells of E.coli, corynebacterium glutamicum or Bacillus subtilis are easily lysed at a catalytic temperature of 60-70 ℃; even if the cells are recovered, phage are extremely susceptible to infection during the process. Therefore, recycling of whole cell catalysts is difficult.
On the other hand, the reaction of D-psicose 3-epimerase catalyzed D-fructose to D-psicose is a reversible reaction, and about half of fructose is not converted when the reaction reaches equilibrium. Based on the structural similarity of the two sugars, the extraction and purification of the product after the reaction is a technical difficulty in production.
The simulated moving bed chromatographic separation technology is a modern separation technology proposed in the 60 th century, adopts a multi-column countercurrent operation mode, overcomes the defects of high solvent consumption and low production efficiency of single-column intermittent preparation chromatography, and is one of chromatographic technologies most suitable for continuous large-scale industrial production. Patent CN202111164114.6 (a method for separating psicose from mixed syrup using a simulated moving bed) discloses a method for separating psicose from mixed syrup using a simulated moving bed. However, the method needs to obtain the mixed syrup containing D-psicose through catalysis, and after the reversible reaction reaches the balance, the mixed syrup is separated and extracted by using a simulated moving bed, and the production process is not continuous.
Disclosure of Invention
The first object of the present invention is to provide a method for culturing recombinant Bacillus licheniformis cells which can perform multi-batch, high-temperature reaction and long-time microfiltration treatment.
The invention provides a device for continuously producing D-psicose, which consists of a material mixing tank, a reaction tank, a microfiltration device, a simulated moving bed, a catalytic supplementing device agent and other components, wherein the material mixing tank is used for preparing fructose solution with a certain concentration, the reaction tank is used for mixing and reacting the fructose solution with a cell catalyst, the microfiltration device is used for intercepting and recovering the cell catalyst in reaction liquid, the simulated moving bed is used for separating substrate fructose and product psicose in the reaction liquid, and the components are connected through stainless steel pipelines.
The simulated moving bed comprises 6 chromatographic columns and a plurality of valves, wherein molecular sieve resin is filled in each chromatographic column, the chromatographic columns are connected end to end through a high-pressure gas pipe, a circulating pump is arranged behind the 6 th chromatographic column, and a separation component containing substrate D-fructose is refluxed to a batching tank for continuous catalysis; introducing hot water into each chromatographic column through a jacket for circulation and heat preservation; each chromatographic column was controlled by 4 solenoid valves for 4 feed and discharge ports, respectively, which were pure water inlet (a) for elution, inlet (B) for inflow of clear liquid obtained by microfiltration, outlet (C) for outflow of D-fructose rich substrate component and outlet (D) for outflow of product D-psicose rich component.
A second object of the present invention is to provide a continuous preparation method of D-psicose coupling a cell catalytic reaction and a product chromatography separation, comprising the steps of:
(1) Fermenting the recombinant bacillus licheniformis, and recovering the recombinant bacillus licheniformis cells as a catalyst;
(2) Preparing D-fructose solution in a preparation tank by using buffer solution; can be prepared with a Co content of 5mM 2+ And Mn of 2+ 50% (w/w) fructose solution was prepared in 50mM HEPES buffer (pH 8.5);
(3) Mixing the D-fructose solution with a certain amount of cell catalyst, pumping into a reaction tank at a certain flow rate, and pumping into a microfiltration device from the reaction tank; the clear liquid obtained by microfiltration enters a simulated moving bed connected in series behind a microfiltration device to separate a substrate D-fructose and a product D-psicose, and the mixed liquid containing the cell catalyst trapped by the microfiltration flows back to a reaction tank to carry out continuous reaction;
after the D-fructose solution is mixed with the cell catalyst, the mass concentration of the D-fructose is 16.7-30%, and the cell density is OD 600 ＝5；
The control conditions of the simulated moving bed can be AB flow ratio of 2:1-5:4, CD flow ratio of 2:1-1:2, and outlet flow velocity V of D-psicose solution p =0.6-1.5L/h, the back mixing flow is 2-3L/h, the solenoid valve switching time is 269-762 s; the solenoid valve switching time refers to the time interval from the opening of the AB valve to the switching to the opening of the CD valve.
(4) The D-psicose obtained by simulated moving bed separation is further roughly mentioned for refining, and D-fructose is refluxed together with the mixed liquor to a batching tank for continuous reaction. The crude reference to refining includes drying and crystallization.
The microfiltration device is used for intercepting and recovering the cell catalyst in the reaction liquid so as to separate the cell catalyst from the sugar liquid, and can be selected from plate type, tubular type (inner pressure tube type and outer pressure tube type), coiled type, hollow fiber type and other types.
Preferably, the inlet A has a water inflow of 1.42L/h, the inlet B has a clear liquid feed of 0.57L/h, the AB flow ratio is 5:2, the outlet C has a D-fructose outlet flow of 1.33L/h, the outlet D has a D-psicose outlet flow of 0.67L/h, the back mixing flow is 2L/h, the solenoid valve switching time is 739s, and the operating temperature is 60 ℃.
[ advantageous effects ]
Through the technical scheme, the continuous recycling of the cell catalyst is realized, and the use cost of the catalyst is reduced (100 kg of psicose can be prepared by the cell catalyst obtained by 1L of fermentation liquor); and the concentration of the product D-psicose in the reaction system is continuously reduced to be less than 30% of the reaction equilibrium concentration by coupling a continuous separation system based on a simulated moving bed, so that the concentration of a substrate D-fructose is improved, the equilibrium of the reversible reaction is pushed to the direction of product synthesis, and the catalytic efficiency is remarkably improved.
Bacillus licheniformis is a widely used production host for food enzyme preparations and important nutritional chemicals, the product of which is FDA certified as "generally regarded as safe" (GRAS) safety grade. On the other hand, the strain is a typical heat-resistant microorganism, can grow at 50℃and the cultured cells are excellent in stability at 60-70℃with few problems of phage infection. By using the recombinant bacillus licheniformis as a cell catalyst, the stability is obviously improved compared with a cell catalyst machine based on model microorganisms (such as escherichia coli and bacillus subtilis), and the continuous production of the D-psicose can be realized.
Drawings
FIG. 1 example 1 cell growth during the preparation of recombinant Bacillus licheniformis cells using a fermenter.
FIG. 2 example 1 shows the trend of the change in enzyme activity in terms of conversion rate in the preparation of recombinant Bacillus licheniformis cells using a fermenter, wherein the left column is one pot data and the right column is two pot data in two columns at the same time point.
FIG. 3 shows an apparatus for continuous production of D-psicose.
Detailed Description
The recombinant Bacillus licheniformis BLA1 used in the examples below was used to express the gene encoding D-psicose-3-epimerase using Bacillus licheniformis CICICIM B1341 as a host and pHY300-PLK as an expression vector. The bacillus licheniformis CICIM B1341 is a bacterial strain stored in a China university industrial microorganism resource platform. In particularThe recombinant bacillus licheniformis BLA1 can be constructed with reference to example 1 described in CN116064619 a: taking the a1 gene fragment expression cassette fused with the promoter and the terminator obtained through codon optimization as a template, amplifying the gene to obtain a gene for encoding D-psicose-3-epimerase; PCR conditions: denaturation at 94℃for 2min, denaturation at 98℃for 30s, annealing at 50℃for 30s, and extension at 68℃for 1min. pHY300-PLK vector (sources are same as Li, Y.; jin, K.; zhang, L.; ding, Z.; gu, Z.; shi, G.development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose OPERON. Journal of Agricultural and Food Chemistry2018,66, 9456-9464.) and the a1 gene fragment obtained by PCR amplification were digested at 37℃and ligated with T4 ligase at 16℃and the resulting ligation product was transformed into E.coli DH5a, screened with ampicillin-resistant solid seed medium plates, transformants were picked for plasmid extraction, restriction and gene sequencing, and the sequenced correct plasmid was named pHY300-P lan -a1; extraction of recombinant plasmid pHY300-P from E.coli lan A1, according to Li, y; jin, k; zhang, l.; ding, z.; gu, z.; bacillus licheniformis was introduced by the method of Shi, G.development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose OPERon. Journal of Agricultural and Food Chemistry2018,66,9456-9464 to obtain recombinant Bacillus licheniformis BLA1.
The medium used consisted of (g/L): corn flour 70, cottonseed protein 30, corn steep liquor dry powder 5, tryptone 10, yeast powder 10 and K 2 HPO 4 ·3H 2 O 9.12、KH 2 PO 4 1.36、(NH 4 ) 2 HPO 4 10。
The whole cell catalyst activity detection method comprises the following steps: mu.L of 100g/L D-fructose solution (prepared with 50mM HEPES buffer pH7.5, containing Co at a final concentration of 5mM each) was used as a substrate 2+ And Mn of 2+ ) 200. Mu.L of recombinant Bacillus licheniformis cells after the resuspension was added to the cell OD in the reaction solution 600 =5, forming a catalytic reaction system, at 60 ℃, ph7.5And (3) accurately reacting for 10min, boiling to inactivate enzyme, and refrigerating at 4 ℃. The concentration of the substrate and the product is detected by HPLC, and the enzyme activity is characterized by the conversion rate, and the calculation formula is as follows: conversion = product concentration (g/L)/(substrate concentration (g/L) +product concentration (g/L)).
The method for measuring the product comprises the following steps: the catalytic reaction system was centrifuged at 13000rpm for 20min, the supernatant was diluted 2-fold with absolute ethanol, and after standing at 4℃for 2h, it was filtered with a 0.22. Mu.M water membrane, and the presence or absence of the production of D-psicose product was detected by HPLC. HPLC detection conditions: using chromatographic columns Dikma CarboPac Ca 2+ The mobile phase was pure water at 6 μm 300x 8.0mm, the detector was a differential detector, the flow rate was 1ml/min, the column temperature was 40℃and the differential detector cell temperature was 40 ℃.
Example 1
(1) Preparation of recombinant Bacillus licheniformis cells Using fermentors
The initial media are shown in table 1 below:
TABLE 1 initial Medium
The preparation method of the feed medium comprises the following steps:
a:250g sucrose was diluted with water to 600ml and placed in a blue-cap bottle for sterilization.
B: corn steep liquor dry powder 20g and tryptone 80g are diluted with water to volume 800ml, and placed in a blue cap bottle for sterilization.
C:2L empty feeding bottle, and sterilizing after binding. After sterilization, A, B was poured into an empty feed bottle in an ultra clean bench as feed medium.
2 fermentation tanks of 5L are adopted, namely a first tank and a second tank, and the initial culture medium liquid loading amounts in the 2 fermentation tanks are all 2L. Before inoculation, aeration rate was maintained at 0.8vvm, rotation speed was 600rpm, pH was controlled at 6.8, temperature was maintained at 37℃and antibiotics were added to correct for 100%. After inoculation, the ventilation was unchanged. And (3) feeding: tank one DO is maintained at 30%, and dissolved oxygen is associated with feeding; the second tank was inoculated for 8 hours and fed at a constant rate of 30 ml/h. Hair brushIn the fermentation process, intermittent sampling is performed to detect the cell concentration and the activity of the whole cell catalyst. The whole cell catalyst activity detection method comprises the following steps: mixing the cells with fructose solution prepared from HEPES buffer solution with pH7.5 until the OD of the cells is 2 and the final concentration of fructose is 100g/L, and adding CoCl with final concentration of 5mmol/L 2 The reaction mixture was added and the reaction mixture obtained was thoroughly mixed and reacted at 65℃for 10 minutes, followed by heating in a boiling water bath for 5 minutes to stop the reaction. The activity of the whole-cell catalyst for producing 1. Mu. Mol of D-psicose per unit time (1 min) under the above conditions was defined as 1U.
As shown in figures 1 and 2, the cell concentration of the recombinant bacillus licheniformis can reach OD respectively under two different fermentation control conditions adopted by a first tank and a second tank 600 162 and OD 600 148, the enzyme activities per unit cell (per OD) reached a maximum at 24h and 72h, respectively, of 113U/mL and 116U/mL, respectively. The cells cultured under both conditions can be used as a high-efficiency whole-cell catalyst for producing D-psicose.
(2) Setting up continuous reaction device
As shown in fig. 3 below, the continuous reaction apparatus includes: the device comprises a dosing tank, a reaction tank, a microfiltration device, a simulated moving bed, a catalyst supplementing device and other components, wherein the dosing tank is used for preparing fructose solution with a certain concentration, the reaction tank is used for mixing and reacting the fructose solution and a cell catalyst, the microfiltration device is used for intercepting and recovering the cell catalyst in the reaction solution, the simulated moving bed is used for separating substrate fructose and product psicose in the reaction solution, and the components are connected through stainless steel pipelines.
The simulated moving bed adopts a 6-column multi-valve automatic control sequential simulated moving bed, and comprises 6 chromatographic columns and a plurality of valves, wherein molecular sieve resin is filled in each chromatographic column, the chromatographic columns are connected end to end through a high-pressure gas pipe, a circulating pump is arranged behind the 6 th chromatographic column, and a separation component containing D-fructose is refluxed to a batching tank for continuous catalysis. The columns were kept warm by jacket-fed hot water circulation, and each column was controlled by 1 set of a total of 4 solenoid valves, with a total of 4 inlet and outlet ports for ABCD, the 4 inlet and outlet ports being respectively a pure water inlet (a) for elution, an inlet (B) for inflow of the clear liquid obtained by microfiltration, an outlet (C) for outflow of the D-fructose rich substrate component, and an outlet (D) for outflow of the product-rich D-psicose component.
(3) With 5mM Co 2+ And Mn of 2+ 50% (w/w) fructose solution was prepared in 50mM HEPES buffer (pH 8.5), and the fructose solution was mixed with the cell catalyst at 60℃to a mass concentration S=30% of D-fructose and a cell density of OD 600 =5, pumping the microfiltration device at a certain flow rate; finishing the reaction in the continuous operation process of the materials, and finishing the reaction when the reaction liquid enters the micro-filtration device;
(4) The clear liquid obtained by microfiltration enters a simulated moving bed connected in series with a microfiltration device to separate substrates and products, the sample injection temperature is 60 ℃, the subsequent drying and crystallization of the product D-psicose are carried out, the fructose solution of the substrates which is not fully reacted is returned to a batching tank for continuous reaction, and the cell catalyst trapped by the microfiltration is returned to a reaction tank.
The automatic control program controls the opening and closing of each 1 group of solenoid valves, and the switching time of each 1 group of solenoid valves is changed to match with the forward pushing speed of the clear liquid in the chromatographic column under the action of the eluent; and the flow rate of the inlets A and B, the flow rate of the outlets C and D and the back mixing flow (the flow rate of fructose solution which is obtained by separating clear liquid through a simulated moving bed and flows back to a preparation tank) are regulated, so that the components C and D are always at the optimal point of separation and purification, and the pressure of the whole system is controlled to be about 0.3 MPa.
The purity of D-psicose obtained by simulated moving bed separation was checked by HPLC.
TABLE 2 optimization results of continuous Process conditions for preparing psicose
25 combinations of process conditions were selected to investigate the effect on the continuous preparation of D-psicose. The results show that under the conditions of 1.42L/h of water inflow rate and 0.57L/h of clear liquid inflow rate (AB flow ratio is 5:2), 1.33L/h of D-fructose outlet flow rate, 0.67L/h of D-psicose outlet flow rate and 2L/h of back mixing flow rate, the switching time of the electromagnetic valve is 739s, the operating temperature is 60 ℃, the D-psicose realizes efficient conversion and separation, the product concentration in the D-psicose outlet effluent reaches 98.34%, the recovery rate reaches 82.48%, and meanwhile, the cell catalyst and the D-fructose are recycled. The process technology is simple and convenient to operate, no chemical reagent is used in the separation process, no pollution is caused, the environment is protected, the operation cost is low, the purity of the obtained product is high, the recovery rate is high, and the industrial production can be carried out after the amplification.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The device for continuously producing the D-psicose is characterized by comprising a batching tank, a reaction tank, a microfiltration device, a simulated moving bed and a catalyst supplementing device, wherein the batching tank is used for preparing a fructose solution, the reaction tank is used for mixing and reacting the fructose solution and a cell catalyst, the microfiltration device is used for intercepting and recovering the cell catalyst in a reaction solution, and the simulated moving bed is used for separating substrate fructose and product psicose in the reaction solution;
the simulated moving bed comprises a plurality of chromatographic columns and a plurality of valves, wherein molecular sieve resin is filled in each chromatographic column, the chromatographic columns are connected end to end through a high-pressure gas pipe, a circulating pump is arranged behind the last 1 chromatographic column, and a separation component containing substrate D-fructose is refluxed to a batching tank for continuous catalysis; each chromatographic column is respectively controlled by 4 electromagnetic valves to control 4 feeding and discharging ports of ABCD, wherein the 4 feeding and discharging ports are respectively a pure water inlet A for elution, an inlet B for flowing in clear liquid obtained by microfiltration, an outlet C for flowing out D-fructose substrate-rich components and an outlet D for flowing out product D-psicose-rich components.
2. The apparatus for continuous production of D-psicose as claimed in claim 1, wherein the number of the chromatographic columns is 6.
3. A method for continuously preparing D-psicose using the apparatus of claim 1 or 2, characterized by comprising the steps of:
(1) Fermenting the recombinant bacillus licheniformis, and recovering the recombinant bacillus licheniformis cells as a catalyst;
(2) Preparing D-fructose solution in a preparation tank by using buffer solution;
(3) Mixing the D-fructose solution with a cell catalyst, pumping into a reaction tank, and pumping into a microfiltration device from the reaction tank; the clear liquid obtained by microfiltration enters a simulated moving bed connected in series behind a microfiltration device to separate a substrate D-fructose and a product D-psicose, and the mixed liquid containing the cell catalyst trapped by the microfiltration flows back to a reaction tank to carry out continuous reaction;
(4) The D-psicose obtained by simulated moving bed separation is further roughly mentioned for refining, and D-fructose is refluxed together with the mixed liquor to a batching tank for continuous reaction.
4. A process according to claim 3, wherein the catalyst is selected from the group consisting of 5mM Co 2+ And Mn of 2+ The buffer of 50mM HEPES pH 8.5 was prepared as a fructose solution of 50% (w/w).
5. A method according to claim 3, wherein the microfiltration device is of the plate, tube, roll or hollow fibre type.
6. The method according to claim 3, wherein after the D-fructose solution is mixed with the cell catalyst, the mass concentration of D-fructose is 30%, and the cell density is OD 600 ＝5。
7. A method according to claim 3, wherein the flow ratio of the AB inlet of the chromatographic column of the simulated moving bed is 2:1-5:4, the flow ratio of the cd outlet is 2:1-1:2, the solenoid valve switching time is 269-762 s; the solenoid valve switching time refers to the time interval from the opening of the AB valve to the switching to the opening of the CD valve.
8. A process according to claim 3, wherein the back-mixing rate of D-fructose with the mixture is 2-3L/h.
9. The method according to claim 7, wherein the inlet water flow rate of inlet A is 1.42L/h, the clear liquid feed rate of inlet B is 0.57L/h, the D-fructose outlet flow rate of outlet C is 1.33L/h, the D-psicose outlet flow rate of outlet D is 0.67L/h, the back mixing flow rate is 2L/h, the solenoid valve switching time is 739s, and the operating temperature is 60 ℃.
10. A method according to claim 3, wherein the coarse-mentioned refining comprises drying and crystallization.
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| WO2024067086A1 (en) * | 2022-09-26 | 2024-04-04 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for conversion and synthesis of d-allulose |
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