CN105950601A - Cell immobilization method for tolerating toxic products and process for producing 1, 5-pentanediamine by immobilized cells - Google Patents
Cell immobilization method for tolerating toxic products and process for producing 1, 5-pentanediamine by immobilized cells Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 69
- 210000001822 immobilized cell Anatomy 0.000 title claims abstract description 38
- 231100000331 toxic Toxicity 0.000 title claims abstract description 8
- 230000002588 toxic effect Effects 0.000 title claims abstract description 8
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 title abstract description 52
- 230000008569 process Effects 0.000 title abstract description 14
- 238000007444 cell Immobilization Methods 0.000 title abstract 2
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 24
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 24
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000000661 sodium alginate Substances 0.000 claims abstract description 19
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 19
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 19
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 19
- 238000007710 freezing Methods 0.000 claims description 18
- 230000008014 freezing Effects 0.000 claims description 18
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 11
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 11
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 230000001120 cytoprotective effect Effects 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 230000003100 immobilizing effect Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 239000003431 cross linking reagent Substances 0.000 claims description 7
- 229960003237 betaine Drugs 0.000 claims description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 230000002572 peristaltic effect Effects 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 2
- 239000002585 base Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 235000009508 confectionery Nutrition 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 21
- 238000004519 manufacturing process Methods 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 11
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000203 mixture Substances 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000001569 carbon dioxide Substances 0.000 abstract description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 238000010924 continuous production Methods 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract 1
- 230000010307 cell transformation Effects 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 230000003472 neutralizing effect Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 6
- 229910001628 calcium chloride Inorganic materials 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000004327 boric acid Substances 0.000 description 4
- 230000002308 calcification Effects 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229960003646 lysine Drugs 0.000 description 3
- 235000018977 lysine Nutrition 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- KJOMYNHMBRNCNY-UHFFFAOYSA-N pentane-1,1-diamine Chemical compound CCCCC(N)N KJOMYNHMBRNCNY-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 238000004026 adhesive bonding Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- CXMXRPHRNRROMY-UHFFFAOYSA-N sebacic acid Chemical compound OC(=O)CCCCCCCCC(O)=O CXMXRPHRNRROMY-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 1
- 229920000305 Nylon 6,10 Polymers 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- -1 cadaverine Chemical compound 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 230000005078 fruit development Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
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Abstract
The invention relates to the technical field of microorganisms, in particular to a cell immobilization method for tolerating toxic products and a continuous production process for producing 1, 5-pentanediamine by immobilized cell transformation. The proportion of polyvinyl alcohol and sodium alginate and a corresponding gelling method are researched and optimized, and the prepared immobilized cell has the advantages of tolerance to damage of high-concentration 1, 5-pentanediamine to somatic cells, better expansion performance, tolerance to influence of release of a large amount of carbon dioxide gas on the structure in the conversion process, high mechanical strength, long service life and the like; the activity is kept above 98% after the composition is used for 6 times in batches, and the activity is kept above 70% after the composition is continuously used for 12 hours. The continuous production process provided by the invention requires less equipment, is convenient to operate, and has the advantages of easy production scale amplification and high automation degree. No neutralizing agent is added in the production process; the product is clear and easy to separate and purify.
Description
Technical field
The present invention relates to microbial technology field, particularly to tolerating the method for immobilizing cell of toxic products and fixing
Change cell and produce 1,5-pentanediamine technique.
Background technology
1,5-pentanediamine (abbreviation pentanediamine), i.e. cadaverine, is that having of being widely present in organism is bioactive nitrogenous
Alkali, the product generated when there is decarboxylic reaction under decarboxylation for lysine during protein corruption.In agricultural production, 1,
5-pentanediamine can regulate plant senescence process as plant growth regulator, stimulates plant growing, promotes fruit development；This
Outward, 1,5-pentanediamine can also improve the cold resistance of plant；Pharmaceutically, 1,5-pentanediamine is clinically for treating iron poisoning
The precursor of medicine DFO；And the application of the current most worthy of 1,5-pentanediamine be as nylon monomer and succinic acid, oneself two
The biological nylon such as polymerization synthesis PA5.4, PA5.6, the PA5.10 such as acid, decanedioic acid.
In recent years, deeply developing and applying due to biological nylon, the demand of 1,5-pentanediamine is continuously increased.Current 1,
The acquisition methods of 5-pentanediamine mainly has extraction method, chemical method, fermentation method and whole-cell catalysis.Extraction method limited source, mistake
Journey is complicated, it is impossible to industrialized production；Chemical method is with nitrile as raw material, and yield is high, is suitable for large-scale industrial production, but it reacted
Journey is violent, pollutes relatively big, does not meets the requirement of Green Development；Fermentation method raw material sources are extensive and renewable, low cost, pollute also
Less, but its regulation process is more complicated, and yield is relatively low.Whole-cell catalysis refers to utilize complete biological cell as urging
Agent carries out chemical conversion, and its essence is to utilize intracellular enzyme to be catalyzed, be between fermentation method and extract enzyme catalysis method it
Between a kind of biocatalysis technology.Comparing fermentation method, it is multiple that whole-cell catalytic overcomes fermentative Production cycle length, metabolite
Miscellaneous, substrate conversion efficiency is low, product separation and Extraction difficulty and the shortcoming such as energy consumption is high.Compare pure enzymic catalytic reaction, each enzyme in full cell
System maintains original state and ad-hoc location, and enzyme stability is more preferable, and the half-life is longer, and adaptability is higher, is more easy to realize energy and auxiliary
The in-situ regeneration of enzyme；Intracellular complete multi-enzyme system can realize the cascade reaction of enzyme, and catalytic efficiency is high.
But due to the product 1 of high concentration, 5-pentanediamine has significantly damage to somatic cells, causes thalline with entering of converting
Go and gradually crush.On the one hand, broken somatic cells 1,5-pentanediamine production efficiency is remarkably decreased, and seriously constrains 1,5-penta
The yield of diamidogen；On the other hand, somatic cells cannot be reused, thus need to repeat to cultivate to obtain substantial amounts of somatic cells,
Thus cause the increase of production cost.Additionally, the thalline vigor of every batch also can be variant, cause the 1 of every batch output, 5-penta
Diamine contents difference, thus affect later separation purification process.Thus, develop a kind of method that can maintain thalline vigor for a long time
Have great importance.
Immobilized cell technology is a kind of method of conventional protection cell, has certain work keeping cell viability
With.Additionally, immobilized cell can reduce production cost with Reusability.In method for immobilizing cell, investment is because of it
Easy and simple to handle, be prone to amplify and the reason such as versatility is most widely used.The kind of carrier being applicable to investment is more, such as alginic acid
Sodium, polyvinyl alcohol, carrageenan, agarose etc., be wherein most widely used is polyvinyl alcohol and sodium alginate, owing to individually making
The easy adhesion of gel ball prepared with polyvinyl alcohol, thus generally the two is used in mixed way by a certain percentage.But the proportioning of the two
Relatively big, particularly similar on the activity of immobilized cell and the mechanical strength impact of immobilized spherule with becoming gluing method accordingly
1B is converted into 1, during the decarboxylic reaction of 5-pentanediamine, and the substantial amounts of carbon dioxide release machine to immobilized spherule
Tool intensity effect is the most notable, and existing method cannot meet the demand of industrialized production.
Summary of the invention
Present invention aim to overcome that in 1,5-pentanediamine production process due to the cell of high concentration product 1,5-pentanediamine
Cellular lysate and production capacity that toxicity causes decline.
Present invention firstly provides the process for fixation of a kind of cell, the method is possible not only to be effectively protected cell with resistance to
Toxic products by high concentration, it is also possible to discharge the gas impact on immobilization spheroid mechanical strength in tolerance conversion process.
The technical solution used in the present invention is:
The present invention provides a kind of method for immobilizing cell tolerating toxic products, comprises the steps:
(1) after cold water addition polyvinyl alcohol post-heating dissolves to 98 DEG C, adding sodium alginate, autoclave heating is dissolved, and stirs
Mix and be cooled to 40-50 DEG C, add the bacteria suspension containing cytoprotective, stir；
(2) with syringe or peristaltic pump, step (1) described mixed liquor is added drop-wise in cross-linking agent solution, forms diameter 0.3-
The bead of 0.5cm, after room temperature stands 6-12h ,-45～-80 DEG C of freezing 8-16h；After freezing terminates, room temperature is slowly melted, with life
Reason saline washs 2 times, and Refrigerator store is used for converting.
Preferably, described cytoprotective is glycerol, glycine betaine, dimethyl sulfoxide (DMSO), polyvinylpyrrolidone
(PVP), any one in mannitol；Preferably PVP or glycine betaine.
Preferably, count in mass ratio, sodium alginate: polyvinyl alcohol: somatic cells (weight in wet base): cytoprotective be 0.5～
6.5:2～10:1～5:1～10
Preferably, described thalline is 3% (by mass percentage).
Preferably, described cross-linking agent is (by mass percentage) 0.5～the calcium chloride solution of 6.5%.
Present invention also offers a kind of immobilized cell obtained by the claims 1-4 either method.
Present invention provides a kind of described immobilized cell and produce 1 continuously, the technique of 5-pentanediamine, its feature exists
In, comprise the steps:
(1) being loaded in column reactor by described immobilized cell, the blade diameter length ratio of tytosis is 1:5～1:10.
(2) with 4～10g(1B)/g(wet cell weight)1B solution is pumped into the fixation cell of step (1) by the flow velocity of/h
In born of the same parents' column type reactor.Wherein, column type reactor can be organized serial or parallel connection more and is applied in combination.
The beneficial effects are mainly as follows:
(1) process for fixation provided by the present invention has that the cost of material is low, simple to operate, the feature such as favorable reproducibility.
(2) immobilized cell prepared has following remarkable advantage: 1. can tolerate high concentration 1,5-pentanediamine thin to thalline
The damage of born of the same parents.2. there is preferable expansion character, can tolerate the release of the great amount of carbon dioxide gas shadow to structure in conversion process
Ring.3. mechanical strength is high, can tolerate fermentation tank continuous stirring 12h at 500 rpm and does not crushes, can pressure 0.2～0.4MPa.④
Service life is long.After using 6 times, vigor is maintained at more than 98% in batches, and after using 12h continuously, vigor is maintained at more than 70%.
(3) continuous production technology equipment needed thereby provided by the present invention is few, it is simple to operation, and production scale is amplified easily,
Automaticity is high.Production process is without any nertralizer；Product clarify, isolated and purified easily.
Therefore the present invention can also be widely used in course of reaction producing the life destroying thalline vigor because of toxic product
Thing converts and produces, it is possible to be applied in other courses of reaction the preparation of the immobilized cell having gas to produce.
Accompanying drawing explanation
Fig. 1 is that polyvinyl alcohol (PVA) becomes the gluing method impact on thalline vigor.
Fig. 2 is the relation of biomass and immobilized bacterium vitality of subject.
Fig. 3 is the checking of response phase method (CCD design) result of the test.
Fig. 4 is that cytoprotective adds test.
Fig. 5 is that immobilized cell reuses test.
Fig. 6 is that immobilized cell column type reactor produces 1,5-pentanediamine continuously.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate the present invention and need not
In limiting the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or press
According to the condition proposed by manufacturer.Unless otherwise defined, all specialties used in literary composition are ripe with this area with scientific words
Practice the same meaning that personnel are familiar with.Additionally, any method similar or impartial to described content and material all can be applicable to
In the inventive method.Preferable implementation described in literary composition only presents a demonstration with material and is used.
Embodiment 1
The PVA of use variable concentrations and the mixture of 2% sodium alginate, as fixing material, use boric acid cross-linking method respectively
Immobilized cell is prepared with freezing method.Concrete operation method is: after cold water addition polyvinyl alcohol post-heating dissolves to 98 DEG C, add
Sodium alginate, autoclave heating is dissolved, and stirs and is cooled to 40-50 DEG C, adds bacteria suspension extremely final concentration of 10g(wet bacterium weight)/ L, fully
Stir.It is added drop-wise in cross-linking agent solution obtain the little of diameter 0.3～0.5cm by above-mentioned mixed liquor with syringe or peristaltic pump
Ball.Boric acid cross-linking method cross-linking agent uses the saturated boric acid solution containing 3% (mass percent) calcium chloride；Freezing method cross-linking agent is
The calcium chloride solution of 3% (mass percent).The bead prepared for boric acid cross-linking method, after room temperature stands 8h, uses normal saline
Wash 2 times, Refrigerator store, be used for converting.The bead that freezing method is prepared, after first room temperature stands 8h ,-80 DEG C of freezing 8h.Cold
After freezing bundle, room temperature is slowly melted, and with brine 2 times, Refrigerator store, is used for converting.
Above-mentioned immobilized cell is used to carry out conversion test with L-lysine hydrochloride for substrate.Transformation system includes: bacterium
Somatic cell 0.1g (wet bacterium weight), 1B amount 150g/l.Conversion condition is 37 DEG C, 200rpm, detects 1,5-after converting 1 hour
Pentanediamine content.Matched group uses the free cell of equal in quality to replace immobilized cell, and remaining condition is identical.Immobilized cell
Relative activity obtain with the 1,5-pentanediamine Production rate of the 1,5-pentanediamine yield specific ionization cell of immobilized cell.
Result is as shown in Figure 1.It is respectively adopted immobilized cell relative activity relatively borate method prepared by-80 DEG C of freezing methods and has aobvious
The raising write, wherein, cell relative viability fixing for 2% sodium alginate-2%PVA reaches 91%.Illustrate that boric acid is to affect thalline
The main cause of vigor.Additionally, immobilized cell relative activity and PVA content are negative correlation trend.
Additionally, Experimental Comparison freezing method plastic and room temperature plastic (the most only use 3%CaCl2Solution plastic, room temperature stands
Difference 8h), as it is shown in figure 1, room temperature plastic relative activity is held in about 60%, without significant difference between different PVA content,
But relatively freezing method have dropped 10%-30%.
Being loaded by immobilized cell after 7.5L fermentation tank stirs 12h at 500 rpm, result shows, three kinds of process for fixation
Prepared immobilized spherule all retains completely.
Embodiment 2
Freezing method in embodiment 1 is used to prepare immobilized cell, wherein sodium alginate mass fraction 2.0%, polyvinyl alcohol
Mass fraction 2.0%, biomass and the mass volume ratio (g/100mL) 1～5 of gel solution, measure the relative of immobilized spherule
Vigor, in conversion condition outside the degerming scale of construction, remaining condition is with embodiment 1.Result as in figure 2 it is shown, in conversional solution 1,5-pentanediamine
Concentration increase with carrying the increase of bacterium amount, but unit mass thalline 1,5-pentanediamine productivity carry bacterium amount more than 3% after do not have
It is obviously improved, and when the load bacterium amount of 3%, the unit mass thalline 1 of immobilized cell, 5-pentanediamine productivity 4.41 ±
0.09g/g(wet bacterium weight)/ h, and compares (4.89 ± 0.46g/g(wet bacterium weight)/ h) compare without significant difference, the load bacterium amount of 3% is described
Saturated immobilized spherule.
Embodiment 3
Freezing method in embodiment 1 is used to prepare immobilized cell, by the center combination design in response phase method analytic process
(CCD) test is to fixation support sodium alginate (SA), the mass fraction of polyvinyl alcohol (PVA), forming agent calcium chloride solution
(CaCl2) mass fraction, the factor such as calcification time and cooling time is optimized.Each factor level is as shown in table 1, totally 32 groups
Test, conversion reaction conditions is with embodiment 1.Result of the test is as shown in table 2.The modeled analysis of result of the test obtains regression equation:
Log10 (loudness vigor)=+ 1.38-1.81*SA+2.21E-003*PVA-0.26*CaCl2During+0.039* calcification
M-0.028* cooling time+0.074*SA*PVA+0.32*SA*CaCl2+ 0.053*SA* cooling time+0.27*SA2-0.014*
PVA2-0.036*CaCl2 2-1.64E-003* calcification the time2-1.94E-003* cooling time2-0.01*SA2*PVA-0.046*
SA2*CaCl2-7.73E-003*SA2* cooling time
Model reaches pole significant level (F=14.44, p < 0.0001), model coefficient R2=0.939, Adj R2=
0.874, illustrate that model can be used for the simulation of actual value.According to the fixing condition that model prediction is optimal it is: sodium alginate
3.62%, polyvinyl alcohol 4.71%, calcium chloride solution 4.21%, calcification 11.84 hours ,-80 freezings 15.96 hours.According to mould
Intending carrying out conversion test after predicted condition carries out cell fixation, conversion reaction conditions is with embodiment 1.Result as shown in Figure 3, carefully
Born of the same parents' relative activity after immobilization is maintained at more than 97%.
Table 1
Table 2
Embodiment 4
Freezing method in embodiment 1 is used to prepare immobilized cell, wherein sodium alginate mass fraction 2.0%, polyvinyl alcohol
Mass fraction 2.0%, biomass 3% (wet bacterium weight).Add final concentration of 10% glycerol respectively, 10% glycine betaine, 7%DMSO,
5%PVP, the cytoprotective such as 1% mannitol.It is matched group without protective agent group.Transformation system includes: somatic cells 0.1g
(wet bacterium weight), 1B amount 50g/l.Conversion condition is 37 DEG C, 200rpm, converts 2 hours, adds 5M hydrochloric acid therebetween and maintains pH
Stable.1B content in periodic detection conversional solution in conversion process, when lysine is consumed to 10g/l, adds substrate extremely
50g/l。
Shown in result such as Fig. 4 (a), the immobilized cell thalline vigor after converting 2h continuously that with the addition of cytoprotective is equal
It is maintained at more than 90%, wherein glycine betaine (10%) and the most pronounced effects of PVP (5%)；Additionally, as shown in Fig. 4 (b), 1,5-
The concentration of pentanediamine is obviously improved with the carrying out converted, and wherein uses the immobilized cell adding glycine betaine and PVP all to convert
To 1,5-pentanediamine 101 ± 2g/L, it is significantly higher than matched group 77 ± 2g/L.
Embodiment 5
Freezing method in embodiment 1 is used to prepare immobilized cell, wherein sodium alginate mass fraction 4%, polyvinyl alcohol matter
Amount mark 5%, 5%PVP, biomass 3% (wet bacterium weight).Transformation system includes: somatic cells 0.1g (wet bacterium weight), 1B
Amount 50g/l.Conversion condition is 37 DEG C, 200rpm, adds 5M hydrochloric acid therebetween and maintains pH stable.In conversion process, periodic detection converts
1B content in liquid, when lysine is consumed to 10g/l, adds substrate to 50g/l.Immobilization is separated after converting 2 hours
Cell, adds fresh 50g/l 1B solution and converts, reuse Cell of Anmrobe 6 times.Result of the test is such as
Shown in Fig. 5, immobilized cell after converting 6 times in batches, and unit thalline 1,5-pentanediamine productivity is the poorest between batch
Different (p=0.471, α=0.05), relative activity is maintained at more than 98%.
Embodiment 6
Freezing method in embodiment 1 is used to prepare immobilized cell, wherein sodium alginate mass fraction 4%, polyvinyl alcohol matter
Amount mark 5%, 5%PVP, biomass 3% (wet bacterium weight).Above-mentioned immobilized cell is loaded in column reactor, tytosis
Blade diameter length ratio be 1:6.300g/L 1B solution is pumped into solid with the flow velocity of 4g (1B)/g (wet cell weight)/h
Surely change in cell column reactor, after solution fills up column type reactor, flow velocity is gradually increased to that 10g (1B)/(cell is wet for g
Weight)/h.Every 1h sampling detection 1,5-pentanediamine content.Result as shown in Figure 6, thalline than conversion rate front four little time interior
Being gradually increasing, this is owing to immobilized spherule progressively expands with the carrying out converted, thus mass-transfer efficiency steps up, after converting 4h
Reaching maximum, now the conversion rate of unit thalline reaches 6.19 ± 0.01g 1,5-pentanediamine/g(wet bacterium weight)/ h, then thalline
Vigor has a declining tendency, but still keeps more than the 70% of the highest vigor after 12h, and in conversional solution 1, the content of 5-pentanediamine can be tieed up
Hold at more than 100g/L.Result of the test shows, immobilized cell prepared by the method utilizing the present invention to provide has and connects for a long time
Continuous conversion production 1, the ability of 5-pentanediamine, and owing to this technique is prone to amplify, by increasing immobilized cell amount and material liquid
The concentration of 1B can obtain 1,5-pentanediamine conversional solution and the conversion ratio of higher 1B of higher concentration.
The scope of the present invention is not limited by the specific embodiments described, and it is each that described embodiment is only used as illustrating the present invention
The single example of individual aspect, also includes method and the component of functional equivalent in the scope of the invention.It practice, except as herein described
Outside content, those skilled in the art can easily grasp the multiple improvement to the present invention with reference to described above and accompanying drawing.Institute
State within improvement also falls into the scope of the appended claims.
Claims (7)
1. the method for immobilizing cell tolerating toxic products, it is characterised in that comprise the steps:
(1) after cold water addition polyvinyl alcohol post-heating dissolves to 98 DEG C, adding sodium alginate, autoclave heating is dissolved, and stirs cold
But to 40-50 DEG C, add the bacteria suspension containing cytoprotective, stir;
(2) with syringe or peristaltic pump, step (1) described mixed liquor is added drop-wise in cross-linking agent solution, forms diameter 0.3-0.5
The bead of cm, after room temperature stands 6-12 h ,-45 ~-80 DEG C of freezing 8-16 h;After freezing terminates, room temperature is slowly melted, with life
Reason saline washs 2 times, and Refrigerator store is used for converting.
Method for immobilizing cell the most according to claim 1, it is characterised in that described cytoprotective is glycerol, sweet
Dish alkali, dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP), any one in mannitol；Preferably PVP or glycine betaine.
Method for immobilizing cell the most according to claim 1, it is characterised in that sodium alginate: polyvinyl alcohol: somatic cells
(weight in wet base): cytoprotective is 0.5 ~ 6.5:2 ~ 10:1 ~ 5:1 ~ 10.
Method for immobilizing cell the most according to claim 1, it is characterised in that described thalline is that 3%(is by percent mass
Than).
Method for immobilizing cell the most according to claim 1, it is characterised in that described cross-linking agent is (by percent mass
Than) calcium chloride solution of 0.5 ~ 6.5%.
6. the immobilized cell that the claims 1-5 either method obtains.
7. the immobilized cell described in a claim 6 produces 1 continuously, the technique of 5-pentanediamine, it is characterised in that include as
Lower step:
(1) being loaded in column reactor by described immobilized cell, the blade diameter length ratio of tytosis is 1:5 ~ 1:10.
(2) with 4 ~ 10 g(1B)/g(wet cell weight)1B solution is pumped into the immobilized cell post of step (1) by the flow velocity of/h
In reactor.
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