CN109880859A - Method for producing pentanediamine by immobilized lysine decarboxylase - Google Patents
Method for producing pentanediamine by immobilized lysine decarboxylase Download PDFInfo
- Publication number
- CN109880859A CN109880859A CN201910256808.9A CN201910256808A CN109880859A CN 109880859 A CN109880859 A CN 109880859A CN 201910256808 A CN201910256808 A CN 201910256808A CN 109880859 A CN109880859 A CN 109880859A
- Authority
- CN
- China
- Prior art keywords
- lysine decarboxylase
- pentanediamine
- immobilised
- gene
- lysine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010048581 Lysine decarboxylase Proteins 0.000 title claims abstract description 59
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 33
- KJOMYNHMBRNCNY-UHFFFAOYSA-N pentane-1,1-diamine Chemical compound CCCCC(N)N KJOMYNHMBRNCNY-UHFFFAOYSA-N 0.000 title claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 85
- 102000004190 Enzymes Human genes 0.000 claims abstract description 84
- 238000000034 method Methods 0.000 claims abstract description 39
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 229920002101 Chitin Polymers 0.000 claims abstract description 23
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 21
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000004472 Lysine Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- 238000010521 absorption reaction Methods 0.000 claims description 14
- 239000013612 plasmid Substances 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- HZUKSQHMCTUZJL-UHFFFAOYSA-N P(=O)(O)(O)OCC=1C(=C(C(=NC1)C)O)C=O.P(=O)(O)(O)OC=1C(=NC=C(C1C=O)CO)C Chemical compound P(=O)(O)(O)OCC=1C(=C(C(=NC1)C)O)C=O.P(=O)(O)(O)OC=1C(=NC=C(C1C=O)CO)C HZUKSQHMCTUZJL-UHFFFAOYSA-N 0.000 claims description 9
- 239000005515 coenzyme Substances 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 229960003646 lysine Drugs 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 238000010353 genetic engineering Methods 0.000 claims description 5
- 229960005337 lysine hydrochloride Drugs 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 238000004064 recycling Methods 0.000 claims description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 3
- 239000013599 cloning vector Substances 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 235000005979 Citrus limon Nutrition 0.000 claims 2
- 244000248349 Citrus limon Species 0.000 claims 1
- 244000131522 Citrus pyriformis Species 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000006114 decarboxylation reaction Methods 0.000 abstract description 4
- 235000019766 L-Lysine Nutrition 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000001742 protein purification Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 description 12
- 239000004677 Nylon Substances 0.000 description 9
- 229920001778 nylon Polymers 0.000 description 9
- 239000004952 Polyamide Substances 0.000 description 7
- 108010091086 Recombinases Proteins 0.000 description 7
- 102000018120 Recombinases Human genes 0.000 description 7
- 229920002647 polyamide Polymers 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 6
- 235000018977 lysine Nutrition 0.000 description 5
- 229920002521 macromolecule Polymers 0.000 description 5
- 230000001376 precipitating effect Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000036983 biotransformation Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108090000489 Carboxy-Lyases Proteins 0.000 description 3
- 229920002302 Nylon 6,6 Polymers 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 125000003700 epoxy group Chemical group 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 3
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 3
- 229960001327 pyridoxal phosphate Drugs 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 2
- 229920002292 Nylon 6 Polymers 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N Pyridoxal Chemical compound CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- CNPURSDMOWDNOQ-UHFFFAOYSA-N 4-methoxy-7h-pyrrolo[2,3-d]pyrimidin-2-amine Chemical compound COC1=NC(N)=NC2=C1C=CN2 CNPURSDMOWDNOQ-UHFFFAOYSA-N 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 1
- 241000522978 Hafniaceae bacterium Species 0.000 description 1
- 241001640034 Heteropterys Species 0.000 description 1
- 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 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 229960003581 pyridoxal Drugs 0.000 description 1
- 235000008164 pyridoxal Nutrition 0.000 description 1
- 239000011674 pyridoxal Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing pentanediamine by immobilized lysine decarboxylase. The method comprises binding chitinDomain of union: (ChBD) Gene ligation to lysine decarboxylase Gene: (CadA) Constructing a fusion protein at the N end, fermenting the fusion protein to obtain a crude lysine decarboxylase liquid, adding chitin into the crude lysine decarboxylase liquid to obtain immobilized lysine decarboxylase, and finally participating the immobilized lysine decarboxylase in the decarboxylation reaction of L-lysine to prepare the 1, 5-pentanediamine. The invention skillfully utilizes the specific affinity action of substrate chitin and fusion protein to adsorb lysine decarboxylase, the immobilized carrier chitin has low cost, high immobilization efficiency and better activity stability of enzyme, and the specific affinity adsorption can omit the step of protein purification. The invention provides a method for repeatedly producing 1, 5-pentanediamine, which can reduce the production cost and simplify the separation steps of downstream products and enzymes, and has remarkable economic benefit.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of method of immobilised lysine decarboxylase production pentanediamine.
Background technique
Polyamide (PA), also known as nylon are that one kind can be widely applied to the neck such as weaving, automobile, electronic apparatus, medical material
The important polymer in domain.Currently, every annual consumption of global polyamide (nylon, PA) product is about 30,000,000 tons, it is contemplated that arrive
The year two thousand twenty turnover is up to 43,700,000,000 dollars.Wherein, the polyamide material being most widely used is mainly polycaprolactam (PA6)
And polyhexamethylene adipamide (PA66), the consumption figure of the two account for ninety percent or so of polyamide aggregate consumption.The polymerization institute of PA66
One of monomer needed, hexamethylene diamine are a kind of important compounds from fossil fuel, depend on the conjunction with petroleum resources unduly
It certainly will lead to a series of problems, such as environmental pollution, rise of the oil price, climate change etc. at mode.With fossil resource it is exhausted with
And the reinforcement of people's environmental consciousness, start more to be paid attention to using biomass as the environmentally friendly material of raw material, so that finding
A kind of renewable resource substitution Petroleum Production nylon becomes extremely urgent, take 1,5- pentanediamine as the biological poly of monomer with this
Amide 5X series has obtained great concern and development.
1,5- pentanediamine is that one kind is widely present in nature, the polyamines with multiple biological activities, can be by protein
It converts to obtain through lysine decarboxylase when corrupt.As a kind of important synthetic intermediate, it is widely used in various new
The synthesis of material and polymer.Such as the nylon 5.4, nylon 5.6, nylon 5.12 etc. of synthesis are participated in pentanediamine, it is based on penta 2
The nylon material of amine has shown excellent material property, wherein many important material properties have been over conventional nylon material
Material.Therefore, compared with the conventional nylon material derived from petroleum, petroleum resources can be effectively relieved in the production of biological poly amide
Consumption and environmental problem, have strategic development prospect.
The method of production pentanediamine is roughly divided into engineering bacteria direct fermentation and produces pentanediamine and biotransformation method at present.The first
It is by the transformation of the metabolic pathway to microorganism (such as Escherichia coli, Corynebacterium glutamicum), one-step fermentation obtains 1,5- penta 2
Amine.Biotransformation method is that substrate L-lysine is acted on by lysine decarboxylase, in the addition of coenzyme phosphopyridoxal pyridoxal phosphate (PLP)
Under, a step decarboxylation obtains product 1,5- pentanediamine.Biotransformation method is high with the substrate transformation rate, catalytic cycle is short, production process
The advantage of easy easy-regulating, is used widely.
But due to enzymatic during, the not recyclable of enzyme, recycling rate of waterused be low, production cost promotion constrains 1,
The industrialized production of 5- pentanediamine.
Document " preparing 1,5- pentanediamine with immobilization L-lysine decarboxylase cell " (Jiang Lili etc., fine chemistry industry,
2007,24(11), 1080-1084) in report using the fixed cell containing lysine decarboxylase of calcium alginate of 3wt%, this is solid
Surely it is poor to change the fixed cell stability of method, enzyme activity is just remarkably decreased when 2 approving and forwardingization, enzyme activity reduction when 4 approving and forwardingization
To the 38% of about the 1st batch enzyme activity.
Application number 201610726632.5 discloses a kind of bad using the macromolecule carrier preparation immobilization of diazo modification
Propylhomoserin decarboxylase, and the method for preparing 1,5- pentanediamine with it.In the immobilised enzymes preparation process of the process for fixation, it is related to more
The chemical reagent of step reaction and discharge can cause environmental pollution.
Application number 201610726608.1 discloses preparation and the lysine of the epoxy group modified macromolecule carrier of one kind
Decarboxylase react with epoxy group the preparation method for the immobilised enzymes being fixed on macromolecule carrier by covalent bond, Yi Jiyong
The method of immobilised enzymes production 1,5- pentanediamine.The macromolecule carrier of this method prepares and the immobilization of lysine decarboxylase
In the process, the epoxy group modified and covalent bond of design, process is cumbersome, needs the participation of a variety of organic reagents, in whole process
The discharge that will cause biggish chemical pollutant is unfavorable for large-scale industrialization process.
Comprehensive literature and patent report can show that the lysine decarboxylase of the immobilization prepared at present mostly uses macromolecule
Gel carrier, the step in actual mechanical process is cumbersome, big and low there is also the repetitive rate situation of reaction exhaust emission.
Therefore, this field needs a kind of more cheap fixation support, easier, more stable lysine decarboxylase immobilised enzymes
Preparation method.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of immobilised lysine decarboxylase productions penta 2
The method of amine.This method construction of fusion protein bacterial strain pET-29a(+ first)-Chbd-Cada, in the crude enzyme liquid obtained by fermentation, add
Enter reproducible bio-based materials chitin low in cost as fixation support, carries out the affine latter step of absorption and be prepared admittedly
Surely change enzyme.The preparation method of gained immobilised enzymes is easy, enzyme activity loss is small, stability is good, the substrate transformation rate is high, can also simplify production
The separating step of 1,5- pentanediamine and enzyme in product reduces production cost, is the direct catalytic production 1 of enzyme process, and 5- pentanediamine, which is realized, to be produced
Industryization has certain impetus.
To solve prior art problem, the technical scheme adopted by the invention is as follows:
A kind of method of immobilised lysine decarboxylase production pentanediamine, comprising the following steps:
Step 1, the building of fusion protein plasmid
Extract lysine decarboxylase geneCadAWith chitin binding domain geneChBDGene, to connect method for transformation for gained base
Because segment is sequentially connected in empty plasmid pET-29a (+)ChBDGene andCadAGene, whereinChBDGene is connected toCadA
The N-terminal of gene completes plasmid pET-29a(+)-Chbd-CadaAfter building, it is transferred to cloning vector Trans1-T1, by LB plate
It after primary dcreening operation, chooses and to be sequenced after carrying out bacterium colony PCR verifying, and after correct strain culturing, upgrading grain will be sequenced, be transformed into expression and carry
Body BL21(DE3) to get the genetic engineering bacterium containing lysine decarboxylase fusion protein；
Step 2, the preparation of lysine decarboxylase crude enzyme liquid
The genetic engineering bacterium containing lysine decarboxylase fusion protein is cultivated, seed liquor is connect with volume fraction 1%-5% inoculum concentration
Enter in LB culture medium, cultivates at 37 DEG C to strain density OD600When for 0.4-0.6, fermentation is collected after inducer induction 12-14h is added
Liquid, then 6000-8000g centrifugation is carried out in refrigerated centrifuge, bacterial strain is collected, smudge cells after being washed with distilled water 2-5 times, low temperature
Supernatant is collected after centrifugation to get lysine decarboxylase crude enzyme liquid in centrifuge 6000-8000g；
Step 3, the affine absorption of lysine decarboxylase and chitin
It is added the chitin that mass fraction is 1-4% into lysine decarboxylase crude enzyme liquid, 4-37 DEG C, stir under 100-400rpm,
PH to 5.6-8.0 is adjusted again, and affine absorption 10-120min is to get immobilised enzymes；
Step 4, the production of 1,5- pentanediamine
Immobilised enzymes is separated from crude enzyme liquid, addition substrate lysine hydrochloride and coenzyme phosphopyridoxal pyridoxal phosphate, at 42 DEG C,
The mixing speed of 200-400rpm obtains pentanediamine conversion fluid after carrying out conversion reaction 2-5 hours, and recycles the fixation in conversion fluid
Change enzyme, it is spare；The substrate lysine hydrochloric acid salinity is 100g/L-200g/L, and coenzyme phosphopyridoxal pyridoxal phosphate concentration is 0.05-
0.2mM。
It is the high-pressure homogeneous processed chitin powder of chitin as improved.
It is the chitin binding domain gene as improvedChBDFrom bacterial strainChitinolyticbacter meiyuanensi SYBC-H1；The lysine decarboxylase geneCadAFrom Escherichia coli (Escherichia coli)
MG1655。
It is that the method for separating immobilised enzymes in step 4 from crude enzyme liquid or conversion fluid is centrifugation or filters as improved.
It is that the concentration of substrate lysine hydrochloride is 100g/L-200g/L, coenzyme phosphorus in the step 4 as improved
Sour pyridoxal concentration is 0.1mM.
It is the immobilised enzymes distilled water recycled in step 4 or the punching of citrate-phosphate disodium hydrogen buffer as improved
It washes.
It is further improved to be, it is characterised in that: the concentration of the citrate-phosphate disodium hydrogen buffer is 100mM,
PH is 6.2-6.8.
The utility model has the advantages that
(1) the lysine decarboxylase fusion protein that the genetic engineering bacterium constructed in the present invention is expressed, can the affine suction of specificity
It is attached on chitin, realizes the immobilization of zymoprotein, immobilised lysine decarboxylase is prepared.Prepared lysine decarboxylase
It may be directly applied to the enzymatic production of 1,5- pentanediamine.
(2) immobilised enzymes of the present invention has many advantages, such as that excellent adsorption, immobilization efficiency are high, enzyme activity loss is small, and repeatedly returns
Receiving to use can all keep high enzyme living.Solve the not recyclable of resolvase, stability difference and the full cell fixation side of tradition
The separation of the low disadvantage of the repetitive rate of method, product and enzyme is easy, simplifies separating technology, reduces production cost.
(3) immobilization material used in immobilised enzymes of the present invention is reproducible biological material chitin, low in cost,
The cost of traditional immobilization material is substantially reduced, and suction type is simple, the preparation process of being simplified to fix enzyme.
(4) immobilised lysine decarboxylation substrate for enzymatic activity generates high, the simplified downstream product point of productivity of 1,5- pentanediamine
Separating process reduces production cost, has certain impetus to the industrialization of biotransformation method production 1,5- pentanediamine.
Detailed description of the invention
Fig. 1 shows fusion protein pET-29a(+)-Chbd-CadaSDS-PAGE analysis:
M- albumen Marker,
1- fusion protein supernatant,
2- fusion protein precipitating,
Supernatant after the absorption of 3- fusion protein,
After 4- ankyrin repeats reaction 9 times, SDS elutes albumen；
Fig. 2 is the active situation of lysine decarboxylase and immobilised enzymes under different temperatures；
Fig. 3 is the temperature stability of lysine decarboxylase and immobilised enzymes；
Fig. 4 is the active situation of lysine decarboxylase and immobilised enzymes under different pH；
Fig. 5 is the pH stability of lysine decarboxylase and immobilised enzymes；
Fig. 6 is immobilized enzyme catalysis response situation；
Fig. 7 is the recycling situation of immobilised enzymes in embodiment 7；
Fig. 8 be embodiment 6 produce 1,5- pentanediamine liquid phase analysis figure.
Specific embodiment
The following examples can make those skilled in the art that the present invention be more fully understood, but not limit this in any way
Invention.
The building of 1 fusion protein plasmid of embodiment
It is extracted using bacterial genomes extracts kit (TianGen) and is purchased from the vast spirit life in Wuhan from Escherichia coli MG1655(
Object Science and Technology Ltd.) and gift from the Kui of Southern Yangtze University, Southern Yangtze University HaoChitinolyticbacter meiyuanensis SYBC-H1(ATCC BAA-2140T) genome, then lysine decarboxylation is obtained by PCR amplification respectively
Enzyme gene CadA and chitin binding domain gene C hBD.
Use following primer
1:5 '-CGGGATCCTTCTTTCAATACCTTAACGG -3 '
2:5 '-CATGCCATGGATGAACGTTATTGCAATATTG -3 '
WithE.coliThe total DNA of MG1655 be template carry out PCR amplification, PCR after reaction, in Ncol I and Bam HI enzyme
Digestion obtains the target fragment that size is 2.1kb at enzyme siteCadA, pass through plastic recovery kit after the verifying of nucleic acid glue
(TianGen) target gene is recycled.
Use following primer
3:5 '-CCCAAGCTTCGGCGTCGGAGTAGCAACAACC-3 '
4:5 '-CGGGATCCACGCCGGTTGCTGCCACC-3 '
WithChitinolyticbacter meiyuanensi The total DNA of SYBC-H1 is that template carries out PCR amplification, PCR reaction
After, digestion obtains the target fragment that size is 0.4kb at Bam HI and Hind III digestion siteChBD, nucleic acid glue tests
Target gene is recycled by plastic recovery kit (TianGen) after card.
Plasmid pET29a(+) is subjected to double digestion (restriction enzyme site is Hind III and Ncol I), by gained genetic fragmentCadAWithChBDIt connect to obtain recombinant plasmid pET-29a(+ with the good plasmid of digestion with T4 DNA ligase)-Chbd-Cada。
Recombinant plasmid resistance is kanamycins, promoter T7, terminator rrnB.By recombinant plasmid by recombinant plasmid
PET-29a(+)-Chbd-Cada(10 μ l) is mixed with Escherichia coli Trans1-T1 competent cell (20 μ L) and is placed on ice
25-30 min, then heat shock 42-45s, the LB culture medium of 800 μ L is added in super-clean bench, is placed in 37 on shaking table after the completion of heat shock
DEG C culture 1 h.4000 g of centrifuge is centrifuged 4min after the completion of culture, sucks supernatant (700 μ l) after the completion of centrifugation, will be remaining
100 μ L are coated (plate LB, resistance are kanamycins, and concentration is 0.5 mM) and are placed in 30-37 DEG C of incubator and train
Support 10-18 h.Bacterium colony PCR verifying is carried out to the bacterium colony on gained plate, is positive bacterium to cementing fruit is run, sequencing is sent to complete just
Sieve.Correct bacterium is sequenced in picking, is inoculated in LB culture medium, after cultivating 12-14h, upgrading grain.By plasmid (10 μ l) and large intestine bar
After bacterium BL21 (DE3) competent cell (20 μ l) in kind completes conversion, the bacterium of lysine decarboxylase fusion protein is obtained
Strain.
The formula of LB culture medium used is as follows: 10g/L peptone, 5g/L yeast powder, 5g/L sodium chloride.
The preparation of 2 lysine decarboxylase crude enzyme liquid of embodiment
The seed liquor for the lysine decarboxylase production bacterium that preparation building obtains, culture medium is LB culture medium, at 37 DEG C, 200rpm
6-7h is cultivated, seed liquor is inoculated in the fermentation medium equipped with 500mlLB with volume fraction 2%, at 37 DEG C, 200rpm
Culture is 0.6 to OD, adds inducer IPTG with 1 ‰ additive amount, and end fermentation after 12h is induced to receive at 30 DEG C, 200rpm
Collect fermentation liquid.
8000g is carried out to fermentation liquid, thallus is collected after centrifugation, after being washed with distilled water twice, is resuspended in 25ml citric acid-
In disodium hydrogen phosphate (100mM pH6.8), 8000g centrifugation is carried out after ultrasonication, collecting supernatant is that lysine decarboxylase is thick
Enzyme solution, 4 DEG C of storages are spare.
The affine absorption of 3 lysine decarboxylase of embodiment and chitin
Gained lysine decarboxylase crude enzyme liquid in 10ml embodiment 2 is taken, the high-pressure homogeneous processed chitin powder of 0.1g is added
Carry out the affine absorption of lysine decarboxylase.6000g centrifugation removes supernatant and collects precipitating after the completion of absorption, and precipitating is for I
Needed for the chitin powder (immobilised enzymes) for being adsorbed with lysine decarboxylase.After being washed with distilled water twice, washing is collected
Immobilised lysine decarboxylase afterwards, preservation is spare at 4 DEG C, and absorption environmental parameter is 20 DEG C of temperature, speed of agitator 200rpm,
pH6.8。
4 fusion protein pET-29a(+ of embodiment)-Chbd-CadaSDS-PAGE analysis
Gained immobilised enzymes in crude enzyme liquid and embodiment 3 obtained in Example 2 respectively, PAGE gel electricity is carried out to it
Its protein expression is analyzed in swimming.200 μ l crude enzyme liquids are taken, after 12000g is centrifuged, it is spare to collect supernatant；It leaves away after supernatant, precipitating is used
Distilled water is mended to 200 μ l systems, and concussion is spare after being resuspended；Take the supernatant in 200 μ l embodiments 3 after immobilised enzymes absorption spare；
Immobilised enzymes after taking 200 μ l enzyme amount to adsorb, is denaturalized it with the SDS solution of 1% concentration of 200 μ l, Denaturing 99
DEG C, 10min.It is spare that supernatant is collected by centrifugation in denaturation end 12000g.Take ready crude enzyme liquid supernatant (i.e. fusion protein supernatant),
Crude enzyme liquid precipitates (i.e. fusion protein precipitating), immobilised enzymes absorption supernatant (i.e. supernatant after fusion protein absorption), immobilised enzymes SDS
Albumen is eluted, after completing thermal denaturation with 4X protein SDS loading buffer(TaKARa), carries out PAGE gel
Electrophoretic analysis, concrete analysis result are as shown in Figure 1.
The comparison of the zymologic property of 5 immobilised enzymes of embodiment and resolvase
The crude enzyme liquid and immobilised enzymes for taking equivalent enzyme activity respectively are added in the reflection system of a 2ml, and the substrate L- of system relies
Propylhomoserin concentration is 100g/L, and coenzyme phosphopyridoxal pyridoxal phosphate concentration is 0.01mM.In 25 DEG C to 60 DEG C of temperature range, it is surveyed respectively
Optimum temperature and temperature stability, system pH are 6.8, are stirred as 200rpm；According to same system, in pH4.0-8.0
In range, the optimal pH and pH stability of crude enzyme liquid and immobilised enzymes are surveyed respectively, and in system, reaction temperature is 42 DEG C, is stirred
It mixes as 200rpm.The optimal reactive temperature of resolvase and immobilised enzymes is respectively 42 DEG C and 47 DEG C, optimal pH be respectively 6.2 with
6.8.In comparison, immobilised enzymes is able to maintain higher enzyme activity in 47 DEG C to 60 DEG C of interval range, at 47 DEG C, resolvase
Enzyme activity rapid drawdown and immobilised enzymes are able to maintain the enzyme activity more than 85%, and at 57 DEG C, immobilised enzymes can also remain above 75% enzyme activity.?
When pH range is 6.8-8.0, immobilised enzymes is able to maintain better stability, when pH is 8.0, can remain above 83% enzyme activity
Property.
Embodiment 61, the production of 5- pentanediamine
Conversion reaction carries out in 50ml centrifuge tube, in the lysine decarboxylase immobilised enzymes being prepared in embodiment 3, adds
Enter lysine hydrochloric acid salting liquid and coenzyme phosphopyridoxal pyridoxal phosphate (PLP), with citrate-phosphate dihydro sodium solution (100mM, pH6.8)
It mends to the reaction system of 10ml, wherein the final concentration of the lysine hydrochloride and phosphopyridoxal pyridoxal phosphate (PLP) of reaction system is respectively
100g/L and 0.01mM converts 3h at 42 DEG C under the conditions of 200rpm, obtain pentanediamine conversion fluid, and molar yield reaches
98.3%。
1,5- pentanediamine detection method is HPLC, and testing conditions are 1260 Infinity System of Agilgent；Show difference
Detector；Chromatographic column: YMC Carotenoid chromatographic column, 250 × 4.6mml.D.S-5 μm of (product article No.: CT99S05-
2546WT)；Flow phase constituent :+5 mL trifluoroacetic acid of 50 mL acetonitrile adds water constant volume to 1 L；Flow velocity: 0.8 mL/min；Sample introduction
Amount: 10 μ L, column temperature: 35 DEG C.It is specific as shown in Figure 8.
The recycling of 7 immobilised enzymes of embodiment
Under 6000g revolving speed, chitin obtained in embodiment 5, after being washed twice repeatedly with distilled water, 6000g is collected by centrifugation
It is collected by centrifugation to obtain immobilised enzymes progress conversion reaction next time, the immobilised enzymes of this method preparation is gone back after repeating conversion 9 times
It can keep 75% or more initial enzyme activity.
Compared with immobilised enzymes used in the present invention, when using resolvase carry out decarboxylic reaction when, resolvase only into
Once catalysis reaction can not simultaneously be reused row, and immobilised enzymes is with stable compared to the better thermal stability of resolvase and pH
Property.This method provides one kind, and a step purifying-absorption immobilization strategy has adsorption efficiency height, proteopexy securely, specifically
Property adsorbs strong advantage.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Sequence table
<110>Nanjing University of Technology
<120>a kind of method of immobilised lysine decarboxylase production pentanediamine
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cgggatcctt ctttcaatac cttaacgg 28
<210> 2
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
catgccatgg atgaacgtta ttgcaatatt g 31
Claims (7)
1. a kind of method of immobilised lysine decarboxylase production pentanediamine, which comprises the following steps:
Step 1, the building of fusion protein plasmid
Extract lysine decarboxylase geneCadAWith chitin binding domain geneChBDGene, to connect method for transformation for gained base
Because segment is sequentially connected in empty plasmid pET-29a (+)ChBDGene andCadAGene, whereinChBDGene is connected toCadA
The N-terminal of gene completes plasmid pET-29a(+)-Chbd-CadaAfter building, it is transferred to cloning vector Trans1-T1, by LB plate
It after primary dcreening operation, chooses and to be sequenced after carrying out bacterium colony PCR verifying, and after correct strain culturing, upgrading grain will be sequenced, be transformed into expression and carry
Body BL21(DE3) to get the genetic engineering bacterium containing lysine decarboxylase fusion protein；
Step 2, the preparation of lysine decarboxylase crude enzyme liquid
The genetic engineering bacterium containing lysine decarboxylase fusion protein is cultivated, seed liquor is connect with volume fraction 1%-5% inoculum concentration
Enter in LB culture medium, cultivates at 37 DEG C to strain density OD600When for 0.4-0.6, fermentation is collected after inducer induction 12-14h is added
Liquid, then 6000-8000g centrifugation is carried out in refrigerated centrifuge, bacterial strain is collected, smudge cells after being washed with distilled water 2-5 times, low temperature
Supernatant is collected after centrifugation to get lysine decarboxylase crude enzyme liquid in centrifuge 6000-8000g；
Step 3, the affine absorption of lysine decarboxylase and chitin
It is added the chitin that mass fraction is 1-4% into lysine decarboxylase crude enzyme liquid, 4-37 DEG C, stir under 100-400rpm,
PH to 5.6-8.0 is adjusted again, and affine absorption 10-120min is to get immobilised enzymes；
Step 4, the production of 1,5- pentanediamine
Immobilised enzymes is separated from crude enzyme liquid, addition substrate lysine hydrochloride and coenzyme phosphopyridoxal pyridoxal phosphate, at 42 DEG C,
The mixing speed of 200-400rpm obtains pentanediamine conversion fluid after carrying out conversion reaction 2-5 hours, and recycles the fixation in conversion fluid
Change enzyme, it is spare；The substrate lysine hydrochloric acid salinity is 100g/L-200g/L, and coenzyme phosphopyridoxal pyridoxal phosphate concentration is 0.05-
0.2mM。
2. the method for immobilised lysine decarboxylase production pentanediamine according to claim 1, it is characterised in that: described
Chitin is the chitin powder of high-pressure homogeneous processing.
3. the method for immobilised lysine decarboxylase production pentanediamine according to claim 1, it is characterised in that: described
Chitin binding domain geneChBDFrom bacterial strainChitinolyticbacter meiyuanensi SYBC-H1；The lysine
Decarboxylase geneCadAFrom Escherichia coli (Escherichia coli) MG1655.
4. the method for immobilised lysine decarboxylase production pentanediamine according to claim 1, it is characterised in that: step 4
In from crude enzyme liquid or conversion fluid separate immobilised enzymes method be centrifugation or filter.
5. the method for immobilised lysine decarboxylase production pentanediamine according to claim 1, it is characterised in that: the step
The concentration of substrate lysine hydrochloride is 100g/L-200g/L in rapid 4, and coenzyme phosphopyridoxal pyridoxal phosphate concentration is 0.1mM.
6. the method for immobilised lysine decarboxylase production pentanediamine according to claim 1, it is characterised in that: step 4
The immobilised enzymes distilled water or citrate-phosphate disodium hydrogen buffer of middle recycling rinse.
7. the method for immobilised lysine decarboxylase production pentanediamine according to claim 5, it is characterised in that: the lemon
Lemon acid-disodium hydrogen phosphate buffer concentration is 100mM, pH 6.2-6.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910256808.9A CN109880859A (en) | 2019-04-01 | 2019-04-01 | Method for producing pentanediamine by immobilized lysine decarboxylase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910256808.9A CN109880859A (en) | 2019-04-01 | 2019-04-01 | Method for producing pentanediamine by immobilized lysine decarboxylase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109880859A true CN109880859A (en) | 2019-06-14 |
Family
ID=66935489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910256808.9A Pending CN109880859A (en) | 2019-04-01 | 2019-04-01 | Method for producing pentanediamine by immobilized lysine decarboxylase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109880859A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779446A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN111117940A (en) * | 2019-12-04 | 2020-05-08 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
| CN112522246A (en) * | 2020-12-02 | 2021-03-19 | 南京工业大学 | Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application |
| CN113061158A (en) * | 2021-03-30 | 2021-07-02 | 南京工业大学 | Method for purifying and immobilizing protein by using divalent metal ions and histidine tag and application thereof |
| CN113621603A (en) * | 2021-08-18 | 2021-11-09 | 南京工业大学 | Co-immobilization continuous catalysis method for cofactor and cofactor-dependent enzyme |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073480A (en) * | 2014-07-04 | 2014-10-01 | 深圳市海普瑞药业股份有限公司 | High-immobilization-tendency heparinase I coding gene and protein thereof |
| CN105316270A (en) * | 2014-06-27 | 2016-02-10 | 中国科学院微生物研究所 | Engineering bacteria for catalytically producing 1,5-pentanediamine and application thereof |
| CN106222231A (en) * | 2016-07-12 | 2016-12-14 | 南京工业大学 | Method for rapidly producing high-optical-purity D-lysine |
| CN107779445A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN107779446A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN108795916A (en) * | 2018-07-16 | 2018-11-13 | 南京工业大学 | Lysine decarboxylase mutant, coding gene thereof, expression and application thereof |
| CN109266633A (en) * | 2018-09-14 | 2019-01-25 | 华东理工大学 | Carbohydrate binding module and its application in immobilised enzymes preparation and fusion protein purification |
-
2019
- 2019-04-01 CN CN201910256808.9A patent/CN109880859A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316270A (en) * | 2014-06-27 | 2016-02-10 | 中国科学院微生物研究所 | Engineering bacteria for catalytically producing 1,5-pentanediamine and application thereof |
| CN104073480A (en) * | 2014-07-04 | 2014-10-01 | 深圳市海普瑞药业股份有限公司 | High-immobilization-tendency heparinase I coding gene and protein thereof |
| CN106222231A (en) * | 2016-07-12 | 2016-12-14 | 南京工业大学 | Method for rapidly producing high-optical-purity D-lysine |
| CN107779445A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN107779446A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN108795916A (en) * | 2018-07-16 | 2018-11-13 | 南京工业大学 | Lysine decarboxylase mutant, coding gene thereof, expression and application thereof |
| CN109266633A (en) * | 2018-09-14 | 2019-01-25 | 华东理工大学 | Carbohydrate binding module and its application in immobilised enzymes preparation and fusion protein purification |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG A等: "Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-d-glucosamine production", 《BIOTECHNOLOGY FOR BIOFUELS》 * |
| 居乃琥主编: "《酶工程手册》", 25 January 2019 * |
| 林宇菲: "耐热性β-葡萄糖苷酶及其固定化在洋葱皮槲皮素糖苷生物转化中的应用研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779446A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN111117940A (en) * | 2019-12-04 | 2020-05-08 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
| CN111117940B (en) * | 2019-12-04 | 2022-06-28 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
| CN112522246A (en) * | 2020-12-02 | 2021-03-19 | 南京工业大学 | Method for artificially constructing chitin corpuscle multienzyme complex scaford-ChiC-ChiA-sg and application |
| CN113061158A (en) * | 2021-03-30 | 2021-07-02 | 南京工业大学 | Method for purifying and immobilizing protein by using divalent metal ions and histidine tag and application thereof |
| CN113621603A (en) * | 2021-08-18 | 2021-11-09 | 南京工业大学 | Co-immobilization continuous catalysis method for cofactor and cofactor-dependent enzyme |
| CN113621603B (en) * | 2021-08-18 | 2024-01-26 | 南京工业大学 | Co-immobilization continuous catalysis method for cofactor and cofactor dependent enzyme |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109880859A (en) | Method for producing pentanediamine by immobilized lysine decarboxylase | |
| CN109652356A (en) | A kind of lysine decarboxylase bacterial strain and its application in pentanediamine production | |
| CN108060114B (en) | A kind of Escherichia coli of fermenting and producing l-Alanine and its application | |
| CN105624128B (en) | Immobilized monoamine oxidase and application thereof in synthesis of chiral azabicyclo compound | |
| CN115216414B (en) | Pichia pastoris strain with high methanol conversion rate and high protein and preparation method thereof | |
| CN112831488B (en) | Glutamic acid decarboxylase and gamma-aminobutyric acid high-yield strain | |
| CN104178536A (en) | Biological preparation method for R-3-aminopiperidine | |
| CN104878024A (en) | Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method | |
| CN104131048A (en) | Biological preparation method of R-3-aminobutanol | |
| CN101481681A (en) | Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria | |
| CN105801675B (en) | A kind of High-activity chitosanase control gene csn and the method using gene production High-activity chitosanase | |
| CN100392075C (en) | Glutamine synthetase and its dedicated expression engineered bacteria and uses | |
| CN108504617A (en) | A kind of Escherichia coli recombinant strain and its construction method of high-yield L-lysine | |
| CN104513839A (en) | Biocatalysis preparation method of D-tert-leucine | |
| CN103243032B (en) | Candida sake and fermentation process thereof | |
| CN106191089B (en) | A method of accelerating the production of 5- aminovaleric acid bioanalysis | |
| CN104830744A (en) | Method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase | |
| CN107916246A (en) | The method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum | |
| CN110760533B (en) | Gene for coding glutamate decarboxylase, recombinant engineering bacterium and application thereof | |
| CN107400667A (en) | A kind of process for fixation of the cell of glucose isomerase containing recombination high temperature-resistant | |
| CN107723317A (en) | A kind of method that itaconic acid is produced in Escherichia coli | |
| CN113930376A (en) | Engineering bacterium for catalytic production of D-p-hydroxyphenylglycine, high-density culture method and catalytic production method | |
| CN112941003A (en) | Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells | |
| CN106916840A (en) | A kind of nitrilase gene engineering bacteria and its construction method, fermentation culture method and application | |
| CN101892228B (en) | Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190614 |
|
| RJ01 | Rejection of invention patent application after publication |