CN117721010A - Multi-enzyme modularized reactor, preparation method and application thereof - Google Patents
Multi-enzyme modularized reactor, preparation method and application thereof Download PDFInfo
- Publication number
- CN117721010A CN117721010A CN202410004796.1A CN202410004796A CN117721010A CN 117721010 A CN117721010 A CN 117721010A CN 202410004796 A CN202410004796 A CN 202410004796A CN 117721010 A CN117721010 A CN 117721010A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- modularized
- reaction
- concentration
- formic acid
- 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
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 100
- 238000006243 chemical reaction Methods 0.000 claims abstract description 73
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 50
- 235000019253 formic acid Nutrition 0.000 claims abstract description 50
- 102000004190 Enzymes Human genes 0.000 claims abstract description 39
- 108090000790 Enzymes Proteins 0.000 claims abstract description 39
- 108010048581 Lysine decarboxylase Proteins 0.000 claims abstract description 34
- 102000003846 Carbonic anhydrases Human genes 0.000 claims abstract description 33
- 108090000209 Carbonic anhydrases Proteins 0.000 claims abstract description 33
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims abstract description 33
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims abstract description 32
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 29
- -1 pentylene diamine Chemical class 0.000 claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 15
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 14
- 150000001412 amines Chemical class 0.000 claims abstract description 13
- 230000003197 catalytic effect Effects 0.000 claims abstract description 13
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims description 34
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 20
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 20
- KJOMYNHMBRNCNY-UHFFFAOYSA-N pentane-1,1-diamine Chemical compound CCCCC(N)N KJOMYNHMBRNCNY-UHFFFAOYSA-N 0.000 claims description 19
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 17
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 14
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000012876 carrier material Substances 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 229910021645 metal ion Inorganic materials 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000008187 granular material Substances 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 5
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 claims description 5
- YSWBFLWKAIRHEI-UHFFFAOYSA-N 4,5-dimethyl-1h-imidazole Chemical compound CC=1N=CNC=1C YSWBFLWKAIRHEI-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical group [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229920002873 Polyethylenimine Polymers 0.000 claims description 3
- 229960003964 deoxycholic acid Drugs 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- OXFSTTJBVAAALW-UHFFFAOYSA-N 1,3-dihydroimidazole-2-thione Chemical compound SC1=NC=CN1 OXFSTTJBVAAALW-UHFFFAOYSA-N 0.000 claims description 2
- KYWMCFOWDYFYLV-UHFFFAOYSA-N 1h-imidazole-2-carboxylic acid Chemical compound OC(=O)C1=NC=CN1 KYWMCFOWDYFYLV-UHFFFAOYSA-N 0.000 claims description 2
- PQAMFDRRWURCFQ-UHFFFAOYSA-N 2-ethyl-1h-imidazole Chemical compound CCC1=NC=CN1 PQAMFDRRWURCFQ-UHFFFAOYSA-N 0.000 claims description 2
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 2
- 229910001429 cobalt ion Inorganic materials 0.000 claims description 2
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 2
- 229910001431 copper ion Inorganic materials 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 239000003607 modifier Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- 125000001176 L-lysyl group Chemical class [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 claims 1
- 238000006114 decarboxylation reaction Methods 0.000 abstract description 27
- 230000008569 process Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052799 carbon Inorganic materials 0.000 abstract description 6
- 150000007524 organic acids Chemical class 0.000 abstract description 4
- 230000001360 synchronised effect Effects 0.000 abstract description 4
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 24
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 23
- 239000004472 Lysine Substances 0.000 description 14
- 229960003646 lysine Drugs 0.000 description 14
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 10
- 235000019766 L-Lysine Nutrition 0.000 description 9
- 238000006555 catalytic reaction Methods 0.000 description 9
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 8
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 8
- 229960001327 pyridoxal phosphate Drugs 0.000 description 8
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 8
- 230000008929 regeneration Effects 0.000 description 7
- 238000011069 regeneration method Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 229910000019 calcium carbonate Inorganic materials 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 235000018977 lysine Nutrition 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 239000011592 zinc chloride Substances 0.000 description 4
- 235000005074 zinc chloride Nutrition 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 3
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 150000008545 L-lysines Chemical class 0.000 description 1
- 101100276041 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) ctpD gene Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940093740 amino acid and derivative Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021398 atomic carbon Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- QVYARBLCAHCSFJ-UHFFFAOYSA-N butane-1,1-diamine Chemical compound CCCC(N)N QVYARBLCAHCSFJ-UHFFFAOYSA-N 0.000 description 1
- 101150008667 cadA gene Proteins 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 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
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000007769 metal material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of multienzyme reactors, in particular relates to a multienzyme modularized reactor and a preparation method thereof, and also relates to application of the multienzyme modularized reactor in synchronous synthesis of organic acid and organic amine. According to the invention, lysine decarboxylase, carbonic anhydrase, formate dehydrogenase and glutamate dehydrogenase are respectively fixed on two different modularized reaction units according to the catalytic sequence, so that the mutual interference between different reactions in the multi-enzyme cascade process is effectively avoided, and the reaction efficiency is improved. When the multi-enzyme modularized reactor is used for synchronously synthesizing the organic acid and the organic amine, the reaction condition is mild, the selectivity is high, the reaction process is green and friendly, the carbon loss in the biological decarboxylation process and the inhibition of micro-environment rapid alkalization on enzyme activity are avoided on the premise of not influencing the biological decarboxylation, and the formic acid yield and the pentylene diamine yield are obviously improved.
Description
Technical Field
The invention belongs to the field of multienzyme reactors, in particular relates to a multienzyme modularized reactor and a preparation method thereof, and also relates to application of the multienzyme modularized reactor in synchronous synthesis of organic acid and organic amine.
Background
The biological production has the characteristics of low carbon circulation, green and clean, and the like, is widely applied to the fields of chemical industry, medicine, energy, food and agriculture, has huge economic potential and research value, generates remarkable economic and social benefits, and promotes the transformation and upgrading of the traditional industry. Biomass is used as a raw material in biological production, and high-value chemicals are synthesized by a microbial or enzyme catalytic conversion technology, so that the method has a wide development prospect.
The biological decarboxylation reaction has obvious technical route advantages, and compared with chemical decarboxylation, the biological decarboxylation reaction has milder reaction conditions and higher selectivity, and the process is green and friendly, and most of amino acids and derivatives thereof, such as pentanediamine, butanediamine, ethanolamine and the like, are obtained through biological decarboxylation.
Lysine decarboxylase (Lysine decarboxylase, cadA) can directly convert L-lysine into 1, 5-pentanediamine by biological decarboxylation, while releasing CO 2 The method comprises the steps of carrying out a first treatment on the surface of the Formate dehydrogenase (Formate dehydrogenase, FDH) CO 2 Reducing into formic acid, and is suitable for coupling amino acid decarboxylation to generate amine compounds. However, biological decarboxylation reactions such as lysine decarboxylase catalyzed decarboxylation of L-lysineFast release of CO during pentylene diamine 2 It is difficult to be completely trapped in the nano-space rapidly, resulting in carbon loss, reduced atom economy, and low formic acid yield. In addition, the catalytic microenvironment is rapidly alkalized after decarboxylation, which is easy to cause the reduction of the activity of formate dehydrogenase and the regeneration capability of NADH, and further causes the reduction of the yield of formic acid. In addition, the solution after decarboxylation reaction is alkaline, a large amount of exogenous acid is needed to maintain the neutral environment of the biocatalysis reaction, alkali is needed to be added for dissociation during product separation, a large amount of waste salt is generated, and the environmental pressure is high.
Disclosure of Invention
Aims at releasing CO during the decarboxylation reaction catalyzed by lysine decarboxylase in the prior art 2 The invention aims to provide a multi-enzyme modularized reactor, a preparation method and application thereof in synchronous synthesis of organic acid and organic amine.
The invention adopts the steps of respectively fixing and orderly arranging enzymes used in different catalytic reaction stages to prepare the multi-enzyme modularized reactor for promoting CO 2 Rapid full capture to avoid biological decarboxylation of CO 2 Overflow, solve the problem that the formic acid yield is low, the catalysis microenvironment that the modularization setting can avoid biological decarboxylation to cause is fast alkalization to the inhibition of NADH regeneration and formic acid synthesis simultaneously, reaction condition is mild, the selectivity is high, the process is green friendly, under the prerequisite that does not influence biological decarboxylation, avoid the carbon loss in the biological decarboxylation process and the inhibition of microenvironment to enzyme activity that is fast alkalization, improved the product yield.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a multienzyme modularized reactor for synchronously synthesizing pentanediamine and formic acid, the multienzyme modularized reactor comprises a first modularized reaction unit and a second modularized reaction unit which are sequentially connected according to a catalytic sequence, wherein lysine decarboxylase and carbonic anhydrase are synchronously fixed in the first modularized reaction unit, and the mass ratio of the lysine decarboxylase to the carbonic anhydrase is (0.1-10): 1; and the formate dehydrogenase and the glutamate dehydrogenase are synchronously fixed in the second modularized reaction unit, and the mass ratio of the formate dehydrogenase to the glutamate dehydrogenase is (0.1-10): 1.
The invention utilizes lysine decarboxylase and carbonic anhydrase to CO-immobilize, so that a large amount of CO generated by biological decarboxylation 2 CO by carbonic anhydrase (Carbonic anhydrase, CA) 2 Conversion to HCO 3 - Thereby reducing CO 2 Can reduce the impact on the carrier material and the negative effect of the enzyme activity. In order to solve the problem of reduced regeneration capacity of NADH, formate dehydrogenase and glutamate dehydrogenase are co-immobilized, and the glutamate dehydrogenase can provide NADH regeneration, so that the synthesis efficiency of formate is comprehensively improved.
According to the invention, lysine decarboxylase, carbonic anhydrase, formate dehydrogenase and glutamate dehydrogenase are respectively fixed on two different modularized reaction units according to the catalytic sequence, so that the mutual interference between different reactions in the multi-enzyme cascade process is effectively avoided, the reaction efficiency is improved, and the biological decarboxylation CO is avoided 2 The method has the advantages that the overflow is carried out, the atomic carbon loss is reduced, the problem of low formic acid yield is solved, and the formic acid yield can reach 67% and the pentylene diamine yield can reach 95% by regulating and controlling the reaction parameters.
Preferably, the mass ratio of lysine decarboxylase to carbonic anhydrase is (0.3-3): 1; the mass ratio of formate dehydrogenase to glutamate dehydrogenase is (0.3-3): 1.
Experiments show that when the mass ratio of lysine decarboxylase to carbonic anhydrase is (0.3-3): 1, the yield of pentanediamine and formic acid is relatively high, especially when the ratio is 1:1. When the mass ratio of formate dehydrogenase to glutamate dehydrogenase is (0.3-3): 1, the yield of the synthesized pentanediamine is relatively high, especially when the weight ratio of the two is 2:1, the yield of formic acid is also relatively high.
In a second aspect, the invention provides a preparation method of the multi-enzyme modularized reactor, which comprises the following steps:
s1: preparation of enzyme granules of lysine decarboxylase and carbonic anhydrase
Mixing lysine decarboxylase and carbonic anhydrase according to the mass ratio, dissolving in water, adding an imidazole compound and a metal ion source, stirring uniformly, adding an organic amine, kong Xiushi agents and a hydrophilic reagent to form an enzyme mixed solution, stirring and loading, centrifuging, washing and drying to obtain enzyme particles containing the lysine decarboxylase and the carbonic anhydrase;
s2: preparation of enzyme granules of formate dehydrogenase and glutamate dehydrogenase
Mixing and dissolving formate dehydrogenase and glutamate dehydrogenase in water according to the mass ratio, adding an imidazole compound and a metal ion source, stirring uniformly, adding organic amine and a pore modifier to form an enzyme mixed solution, stirring and loading, centrifuging, washing and drying to obtain enzyme particles containing formate dehydrogenase and glutamate dehydrogenase;
s3: preparation of a Multi-enzyme Modular reactor
Taking a reaction column with a hollow channel with the inner diameter of 0.1-100 mm as a reaction container of the modularized reaction unit; mixing the enzyme particles prepared in the steps S1 and S2 with a carrier material to prepare immobilized enzyme particles, and filling the immobilized enzyme particles into hollow channels of a reaction column to prepare a first modularized reaction unit and a second modularized reaction unit respectively; the first modular reaction unit and the second modular reaction unit are connected in series according to a catalytic sequence to form the multi-enzyme modular reactor.
Preferably, the imidazole compound comprises at least one of dimethyl imidazole, mercaptoimidazole, ethyl imidazole and carboxyl imidazole; the metal ion source comprises at least one of a cobalt ion source, a copper ion source and a zinc ion source; the organic amine is polyacrylamide or polyethyleneimine, the Kong Xiushi agent is Cetyl Trimethyl Ammonium Bromide (CTAB) or polyvinylpyrrolidone (PVP), and the hydrophilic agent is polyethylene glycol.
Preferably, the support material in step S3 is prepared by the following method:
dispersing deoxycholate sodium and zinc ion source (such as zinc chloride) in water to form mixed solution containing deoxycholate sodium with concentration of 50-200 mM and zinc ion source (such as zinc chloride) with concentration of 20-50 mM, stirring and mixing the mixed solution at 500rpm for 1h, and freeze-drying to obtain the carrier material.
Preferably, the concentration of lysine decarboxylase in the enzyme mixture in the step S1 is 1-3 mg/mL, and the concentration of carbonic anhydrase is 1-3 mg/mL; the concentration of the imidazole compound is 50-200 mM; the concentration of the metal ion source is 20-100 mM; the concentration of the Kong Xiushi agent is 5-30 mg/mL; the concentration of the hydrophilic reagent is 10-50 mg/mL.
Preferably, the concentration of the formate dehydrogenase in the enzyme mixed solution in the step S2 is 1-3 mg/mL, and the concentration of the glutamate dehydrogenase is 1-3 mg/mL; the concentration of the imidazole compound is 50-200 mM; the concentration of the metal ion source is 20-100 mM; the concentration of the organic amine is 10-50 mg/mL; the concentration of Kong Xiushi agent is 25-45 mg/mL.
Preferably, the weight ratio of enzyme particles to carrier material in the immobilized enzyme particles of step S3 is 1-3:1.
In a third aspect, the invention provides a method for synchronously synthesizing pentanediamine and formic acid by using the multi-enzyme modularized reactor, comprising the following steps:
introducing a substrate solution containing 50-200 g/L L-lysine salt solution into the beginning end of the multi-enzyme modularized reactor at a rate of 1-100 mL/min, and performing enzymatic reaction under the reaction conditions of 30-40 ℃ and pH of 4.5-7.5 and reaction pressure of 1-3 atmospheres to synchronously synthesize the pentanediamine and the formic acid.
Preferably, the substrate solution also contains 5-20 mM NADH and 0-1 mM pyridoxal phosphate (PLP), the yield of pentyenediamine is up to 95%, and the yield of formic acid is up to 67%.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, lysine is used as a reaction substrate, immobilized enzyme particles of lysine decarboxylase and carbonic anhydrase and immobilized enzyme particles of formate dehydrogenase and glutamate dehydrogenase are respectively constructed according to catalytic sequences of synthesizing pentanediamine and formic acid sequentially through catalysis of lysine decarboxylase, carbonic anhydrase, formate dehydrogenase and glutamate dehydrogenase, and the two immobilized enzyme particles are respectively constructed into different modularized reaction units, so that a multienzyme module reactor is manufactured through orderly serial distribution, the mutual interference between different reactions in the multienzyme cascade process is effectively avoided, and the reaction efficiency is improved.
The invention adopts two immobilized enzyme particles with different functions to carry outOrderly arranged to prepare a multi-enzyme modularized reactor for promoting CO 2 Rapid full capture to avoid biological decarboxylation of CO 2 Overflow, avoiding the inhibition of NADH regeneration and formic acid synthesis caused by rapid alkalization of catalytic microenvironment due to biological decarboxylation, and solving the problem of low formic acid yield. The reaction condition is mild, the selectivity is high, the process is green and friendly, the carbon loss in the biological decarboxylation process and the inhibition of micro-environment rapid alkalization on the enzyme activity are avoided on the premise of not influencing the biological decarboxylation, and the product yield is improved. The multienzyme modularized reactor combines proper reaction conditions, so that the yield of the pentanediamine is up to 95 percent, and the yield of the formic acid is up to 67 percent.
Drawings
FIG. 1 is a schematic diagram of a multi-enzyme modular reactor;
FIG. 2 is a graph of product yield versus catalytic time;
FIG. 3 is a graph of CO corresponding to different lysine concentrations 2 A fixed rate graph;
FIG. 4 is a graph showing the relative enzyme activity of an immobilized enzyme as a function of the number of recycles;
FIG. 5 is a graph showing the change in the yields of products corresponding to the weight ratios of lysine decarboxylase and carbonic anhydrase;
FIG. 6 is a graph showing the change in the yield of a product corresponding to the weight ratio of formate dehydrogenase to glutamate dehydrogenase.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
In the examples lysine decarboxylase was obtained from BL21 (DE 3)/pCDF-dur-CadA strain expressing E.coli-derived L-lysine decarboxylase, which strain has been published in patent CN 201810195975.2; carbonic anhydrase was purchased from Shanghai Yuan Yes Biotechnology Co., ltd., cat: s10157-50mg; formate dehydrogenase was purchased from Shanghai Seiyaka Biotechnology Co., ltd., cat: s25081-50u; glutamate dehydrogenase is purchased from Shanghai Yuan Yes Biotechnology Co., ltd., cat: s10067-100ku.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1
The schematic diagram of the multi-enzyme modularized reactor is shown in fig. 1, and the multi-enzyme modularized reactor comprises a first modularized reaction unit and a second modularized reaction unit which are sequentially connected in series, wherein the first modularized reaction unit and the second modularized reaction unit are cylinders with hollow channels, the inner diameter of each hollow channel is 1-10 mm, and the cylinders are made of glass, metal or composite materials. The column body of the first modularized reaction unit is filled with immobilized enzyme particles containing lysine decarboxylase and carbonic anhydrase, and the weight ratio of the lysine decarboxylase to the carbonic anhydrase is 1:1; the column body of the second modularized reaction unit is filled with immobilized enzyme particles containing formate dehydrogenase and glutamate dehydrogenase, and the weight ratio of the formate dehydrogenase to the glutamate dehydrogenase is 1:1.
The embodiment provides a preparation method of the multi-enzyme modularized reactor, which comprises the following steps:
1.1 construction of enzyme particle A
The synthesis system is 10mL, 10mg of lysine decarboxylase and 10mg of carbonic anhydrase are respectively taken and dissolved in deionized water, stirring is carried out for 1-2 min at 300rpm, the reaction solution is uniformly mixed, dimethylimidazole with the final concentration of 160mM and cobalt chloride hexahydrate with the final concentration of 40mM are added, the stirring speed is increased to 500rpm and stirred for 10min, polyethylenimine with the final concentration of 15mg/mL, polyvinylpyrrolidone with the final concentration of 35mg/mL and polyethylene glycol with the final concentration of 10mg/mL are added, stirring is carried out for 30min, centrifugation is carried out, part of supernatant is taken to determine the protein concentration, the precipitate is washed for 2-3 times by deionized water, and a vacuum freeze dryer is used for drying, so that enzyme particles A are obtained.
1.2 construction of enzyme granules B
The synthesis system is 10ml, 10mg of formate dehydrogenase and 10mg of glutamate dehydrogenase are respectively taken and dissolved in deionized water, stirring is carried out for 1-2 min at 300rpm, the reaction solution is uniformly mixed, dimethylimidazole with the final concentration of 160mM and zinc chloride with the final concentration of 40mM are added, stirring speed is increased to 500rpm and stirring is carried out for 10min, polyacrylamide with the final concentration of 20mg/ml and polyvinylpyrrolidone with the final concentration of 35mg/ml are added, stirring is carried out for 30min, centrifugation is carried out, part of supernatant is taken to measure protein concentration, the precipitate is washed for 2-3 times by deionized water, and a vacuum freeze dryer is used for drying, thus obtaining enzyme particles B.
1.3 construction of a Multi-enzyme Module reactor
A final concentration of 50mM surfactant (sodium deoxycholate) and 25mM zinc chloride were added to 20ml of deionized water, and the mixture was stirred at 500rpm for 1 hour to synthesize a polymer carrier material, which was freeze-dried using a vacuum freeze-dryer to obtain a polymer carrier material.
Mixing a polymer carrier material with enzyme particles A according to a weight ratio of 1:1 to prepare immobilized enzyme particles A containing lysine decarboxylase and carbonic anhydrase, and loading the immobilized enzyme particles A into a column to obtain a first modularized reaction unit.
And mixing the polymer material with enzyme particles B according to the weight ratio of 1:1 to prepare immobilized enzyme particles B containing formate dehydrogenase and glutamate dehydrogenase, and loading the immobilized enzyme particles B into a column to obtain a second modularized reaction unit.
The first modular reaction unit and the second modular reaction unit are connected in series to form a multi-enzyme modular reactor (shown in figure 1).
Example 2
This example provides a method for catalytic production of pentanediamine and formic acid using the multi-enzyme modular reactor prepared in example 1, comprising the steps of:
the multi-enzyme modular reactor prepared in example 1 was placed in a 37℃water bath, pyridoxal phosphate (PLP) having a final concentration of 0.1mM, NADH having a final concentration of 10mM and L-lysine hydrochloride solution having a final concentration of 50mM were introduced into the multi-enzyme modular reactor from its initial end (the end filled with immobilized enzyme particles A was the initial end) by a pump at a flow rate of 0.2mL/min, the reaction pH was controlled at about 7.0, the reaction pressure was 2 atm, and the end of the multi-enzyme modular reactor was connected to the reaction solution.
Index detection in the reaction process:
(1) Consumption of L-lysine
And selecting a lysine detection mode to detect the consumption of the L-lysine by using an SBA-40E double-channel biosensor.
(2) Synthesis amount of 1, 5-pentanediamine
The concentration of 1, 5-pentanediamine is determined by adopting an Agilent 1290 liquid chromatography system and Agilent TC-C 18 Chromatographic column (4.6X1250 mm). Column temperature 40+ -1deg.C, flow rate 1.0mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the The sample injection amount is 10 μl, the excitation wavelength of the fluorescence detector is 350nm, the emission wavelength is 520nm, and the ultraviolet detection wavelength is 250nm.
(3) NADH content
The NADH content was detected at 340nm using an enzyme-labeled instrument.
(4) Formic acid concentration determination
Agilent 1260 liquid chromatography system and an Aminex HPX-87H column (300X 7.88 mm) were used. Column temperature 65+ -1deg.C, flow rate 0.6mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount is 10 μl; the ultraviolet detector wavelength was 210nm, and the formic acid concentration in the reaction solution was detected.
(5)CO 2 Is fixed rate of (2)
Calcium carbonate is generated by adding a calcium chloride solution into the reaction liquid, and the quality of the calcium carbonate is weighed to detect CO 2 Is fixed rate of CO 2 The conversion was calculated as follows:
the catalysis time is 5-60 min, the production amount of formic acid and pentanediamine is detected by a liquid chromatograph, and the CO is detected by weighing the mass of the calcium carbonate 2 During the catalytic reaction, CO 2 The fixed ratio of (2) was substantially unchanged, and about 45% of the amounts of formic acid and pentamethylene diamine produced were as shown in FIG. 2, and the amounts of formic acid and pentamethylene diamine produced were increased with time, and at the time of catalytic reaction for 60 minutes, the formic acid yield was 67% and the pentamethylene diamine yield was 81.6%.
Example 3
This example differs from example 2 in that the inlet is made by means of a pump from the beginning of the multienzyme modular reactorThe concentrations of L-lysine in the mixture were 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM and 40mM in this order, and the other parts not mentioned were the same as in example 2. Detecting the formation of formic acid and pentanediamine in the catalytic reaction for 20min by a liquid chromatograph, and detecting CO by weighing the mass of calcium carbonate 2 As a result, the fixation ratio of (2) was shown in FIG. 3, CO was increased with the increase of the L-lysine concentration 2 Large amount of released CO is difficult to be captured in time 2 The fixed ratio of (2) becomes low, resulting in a decrease in the yield of formic acid.
The yields of pentamethylenediamine and formic acid corresponding to different substrate concentrations are shown in Table 1, and the concentration of lysine has no influence on the yield of pentamethylenediamine, and as the concentration of lysine increases, CO is captured 2 The amount is increased, the yield of formic acid is correspondingly increased, and when the concentration of the L-lysine in the substrate is 30mM, the yield of formic acid is relatively highest and can reach 53.3 percent.
TABLE 1 yield of Pentanediamine and formic acid corresponding to L-lysine concentration in different substrates
Example 4
A mixed solution of pyridoxal phosphate (PLP) with a final concentration of 0.1mM, NADH with a final concentration of 10mM and L-lysine hydrochloride with a final concentration of 30mM is introduced from the beginning of the multi-enzyme modular reactor prepared in example 1 by a pump, after the reaction is completed for 20min, the enzyme reactor is rinsed by a PBS buffer solution, the rinsed reactor is repeatedly introduced with the above substrate mixed solution, the production amount of formic acid and pentamethylene diamine is detected by a liquid chromatograph through repeated experiments, and CO is detected by weighing the mass of calcium carbonate 2 As a result, the reaction was repeated 1 time, the amount of formic acid produced was 4.8mM, the amount of pentamethylene diamine produced was 15.8mM, and CO was found as shown in FIG. 4 2 The immobilization ratio of (2) was 78%, and after repeated use for 7 times, L-lysine was substantially absent, with the first reaction enzyme activity being 100%Consumption, CO 2 The immobilization ratio of (2) is decreased, the production amount of formic acid and pentylene diamine is not substantially increased, and the immobilized enzyme loses activity.
Example 5
The difference between this example and example 1 is that the weight ratio of lysine decarboxylase to carbonic anhydrase in enzyme granule a is adjusted to be 3:1, 2:1, 1:0, 1:1, 1:2, 1:3 in order, and the content of the synthesized pentanediamine and formic acid is detected during the catalytic reaction for 20min by the method described in example 2, and the result is shown in fig. 5, when the weight ratio of lysine decarboxylase to carbonic anhydrase is 1:1, the yield of pentanediamine is 83.05%, and the yield of formic acid is 53.7%; at the same time, compared with the case (1:0) where carbonic anhydrase is not added, CO 2 Can not be captured in time, and the yield of formic acid is obviously reduced.
Example 6
The difference between this example and example 1 is that the weight ratio of formate dehydrogenase and glutamate dehydrogenase in enzyme pellet B is adjusted in this example as follows: 3:1, 2:1, 1:0, 1:1, 1:2, 1:3, and the content of synthesized pentanediamine and formic acid was measured at 20min of catalytic reaction according to the method described in example 2, and as shown in fig. 6, when the weight ratio of formate dehydrogenase to glutamate dehydrogenase was 2:1, the yield of pentanediamine was 83.05%, and the yield of formic acid was 54.8%, and at the same time, compared with the case where glutamate dehydrogenase was not added, the yield of formic acid was significantly reduced due to slow regeneration rate of NADH.
Example 7
In this example, a multi-enzyme modular reactor prepared by a mass ratio of lysine decarboxylase to carbonic anhydrase of 1:1 and a mass ratio of formate dehydrogenase to glutamate dehydrogenase of 2:1 was placed in a water bath at 37℃and a mixed solution of pyridoxal phosphate (PLP) having a final concentration of 0.1mM, NADH having a final concentration of 10mM and L-lysine hydrochloride solution having a final concentration of 30mM was introduced thereto from the start end (the end filled with immobilized enzyme particles A) of the multi-enzyme modular reactor by a pump at a flow rate of 0.2mL/min, a reaction pH of about 7.0 and a reaction pressure of 2 atm, and the end of the multi-enzyme modular reactor was connected to the reaction solution. The synthesis of pentamethylenediamine and formic acid was examined as described in example 2, with a pentamethylenediamine yield of 95% and a formic acid yield of 53.6%.
In summary, the preparation method of the multi-enzyme module reactor and the application thereof in continuous synchronous synthesis of the invention constructs immobilized particles modified by organic amine in which lysine decarboxylase and carbonic anhydrase are CO-immobilized on a hydrophilic porous structure, immobilized particles modified by cationic polymer in which formate dehydrogenase and glutamate dehydrogenase are CO-immobilized on a porous structure, and simultaneously orderly arranges two immobilized particles with new functions to prepare the module multi-enzyme reactor, thereby promoting CO 2 Rapid full capture to avoid biological decarboxylation of CO 2 The overflow solves the problems of low formic acid yield, avoids the inhibition of rapid alkalization of catalytic microenvironment to NADH regeneration and formic acid synthesis caused by biological decarboxylation, has mild reaction conditions, high selectivity and environment-friendly process, and avoids the carbon loss in the biological decarboxylation process and the inhibition of rapid alkalization of microenvironment to enzyme activity on the premise of not influencing the biological decarboxylation.
Claims (10)
1. The multi-enzyme modularized reactor for synchronously synthesizing the pentanediamine and the formic acid is characterized by comprising a first modularized reaction unit and a second modularized reaction unit which are sequentially connected according to a catalytic sequence, wherein lysine decarboxylase and carbonic anhydrase are synchronously fixed in the first modularized reaction unit, and the mass ratio of the lysine decarboxylase to the carbonic anhydrase is (0.1-10): 1; and the formate dehydrogenase and the glutamate dehydrogenase are synchronously fixed in the second modularized reaction unit, and the mass ratio of the formate dehydrogenase to the glutamate dehydrogenase is (0.1-10): 1.
2. The multi-enzyme modular reactor for simultaneous synthesis of pentylene diamine and formic acid according to claim 1, wherein the mass ratio of lysine decarboxylase to carbonic anhydrase is (0.3-3): 1; the mass ratio of the formate dehydrogenase to the glutamate dehydrogenase is (0.3-3): 1.
3. A method of preparing the multi-enzyme modular reactor of claim 1 or 2, comprising the steps of:
s1: preparation of enzyme granules of lysine decarboxylase and carbonic anhydrase
Mixing lysine decarboxylase and carbonic anhydrase according to the mass ratio, dissolving in water, adding an imidazole compound and a metal ion source, stirring uniformly, adding an organic amine, kong Xiushi agents and a hydrophilic reagent to form an enzyme mixed solution, stirring and loading, centrifuging, washing and drying to obtain enzyme particles containing the lysine decarboxylase and the carbonic anhydrase;
s2: preparation of enzyme granules of formate dehydrogenase and glutamate dehydrogenase
Dissolving formate dehydrogenase and glutamate dehydrogenase in water according to the mass ratio, adding an imidazole compound and a metal ion source, stirring uniformly, adding organic amine and a pore modifier to form an enzyme mixed solution, stirring and loading, centrifuging, washing and drying to obtain enzyme particles containing formate dehydrogenase and glutamate dehydrogenase;
s3: preparation of a Multi-enzyme Modular reactor
Taking a reaction column with a hollow channel with the inner diameter of 0.1-100 mm as a reaction container of the modularized reaction unit; mixing the enzyme particles prepared in the steps S1 and S2 with a carrier material to prepare immobilized enzyme particles, and filling the immobilized enzyme particles into hollow channels of a reaction column to prepare a first modularized reaction unit and a second modularized reaction unit respectively; the first modular reaction unit and the second modular reaction unit are connected in series according to a catalytic sequence to form the multi-enzyme modular reactor.
4. The method for preparing the multi-enzyme modularized reactor according to claim 3, wherein the imidazole compound comprises at least one of dimethyl imidazole, mercapto imidazole, ethyl imidazole and carboxyl imidazole; the metal ion source comprises at least one of a cobalt ion source, a copper ion source and a zinc ion source; the organic amine is polyacrylamide or polyethyleneimine, the Kong Xiushi agent is cetyl trimethyl ammonium bromide or polyvinylpyrrolidone, and the hydrophilic agent is polyethylene glycol.
5. A method of preparing a multi-enzyme modular reactor according to claim 3, wherein the carrier material in step S3 is prepared by:
dispersing sodium deoxycholate and a zinc ion source in water to form a mixed solution containing 50-200 mM sodium deoxycholate and 20-50 mM zinc ion source, stirring and mixing the mixed solution uniformly, and freeze-drying to prepare the carrier material.
6. The method for preparing a multi-enzyme modular reactor according to claim 3, wherein the concentration of lysine decarboxylase in the enzyme mixture in step S1 is 1-3 mg/mL, and the concentration of carbonic anhydrase is 1-3 mg/mL; the concentration of the imidazole compound is 50-200 mM; the concentration of the metal ion source is 20-100 mM; the concentration of the Kong Xiushi agent is 5-30 mg/mL; the concentration of the hydrophilic reagent is 10-50 mg/mL.
7. The method for preparing a multi-enzyme modular reactor according to claim 3, wherein the concentration of formate dehydrogenase in the enzyme mixture in the step S2 is 1-3 mg/mL, and the concentration of glutamate dehydrogenase is 1-3 mg/mL; the concentration of the imidazole compound is 50-200 mM; the concentration of the metal ion source is 20-100 mM, and the concentration of the organic amine is 10-50 mg/mL; the concentration of Kong Xiushi agent is 25-45 mg/mL.
8. The method for preparing a multi-enzyme modular reactor according to claim 3, wherein the weight ratio of enzyme particles to carrier material in the immobilized enzyme particles of step S3 is 1-3:1.
9. A method for synchronously synthesizing pentanediamine and formic acid by using the multi-enzyme modularized reactor as claimed in claim 1, which is characterized by comprising the following steps:
introducing a substrate solution containing 50-200 g/L L-lysine salt solution into the beginning end of the multi-enzyme modularized reactor according to claim 1 at a rate of 1-100 mL/min, and performing enzymatic reaction under the reaction conditions of 30-40 ℃ and pH of 4.5-7.5 and reaction pressure of 1-3 atmospheres to synchronously synthesize the pentanediamine and the formic acid.
10. The method for simultaneous synthesis of pentylene diamine and formic acid according to claim 9, wherein said substrate solution further contains 5-20 mM NADH and 0-1 mM PLP, said pentylene diamine yield being up to 95%; the yield of the formic acid is up to 67%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410004796.1A CN117721010A (en) | 2024-01-03 | 2024-01-03 | Multi-enzyme modularized reactor, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410004796.1A CN117721010A (en) | 2024-01-03 | 2024-01-03 | Multi-enzyme modularized reactor, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117721010A true CN117721010A (en) | 2024-03-19 |
Family
ID=90210718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410004796.1A Pending CN117721010A (en) | 2024-01-03 | 2024-01-03 | Multi-enzyme modularized reactor, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117721010A (en) |
-
2024
- 2024-01-03 CN CN202410004796.1A patent/CN117721010A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105950604B (en) | Enzyme immobilization method | |
| Campbell et al. | Enzymatic recycling of coenzymes by a multi-enzyme system immobilized within semipermeable collodion microcapsules | |
| Guoqiang et al. | Evaluation of alginate-immobilized Lactobacillus casei for lactate production | |
| CN109576256B (en) | Method for encapsulating double enzymes by magnetic DNA hydrogel | |
| CN106400466B (en) | A kind of application of modified carbon fiber in immobilization | |
| CN113061158A (en) | Method for purifying and immobilizing protein by using divalent metal ions and histidine tag and application thereof | |
| Senuma et al. | Industrial production of d-aspartic acid and l-alanine from dl-aspartic acid using a pressurized column reactor containing immobilized Pseudomonas dacunhae cells | |
| CN117721010A (en) | Multi-enzyme modularized reactor, preparation method and application thereof | |
| CN110129387B (en) | Method for preparing nicotinamide by composite material immobilized corynebacterium propionate | |
| CN105907742A (en) | Carboxyl magnetic bead immobilized glutamic acid decarboxylase and preparation method and application thereof | |
| CN113136381B (en) | Preparation method of multienzyme conjoined nano reactor and application thereof in synchronous synthesis | |
| CN106636294B (en) | Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction | |
| Pollak et al. | Immobilization of synthetically useful enzymes by condensation polymerization | |
| Kawabata et al. | New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium‐type resin | |
| CN106148319B (en) | Method for preparing immobilized enzyme based on reaction adsorption method | |
| JPS6331538A (en) | Immobilizing carrier | |
| CN109628436B (en) | Preparation method of fixed recombinant human arginase | |
| CN113621603A (en) | Co-immobilization continuous catalysis method for cofactor and cofactor-dependent enzyme | |
| CN110294824B (en) | Preparation method and application of efficient cationic polymer | |
| CN111647591A (en) | Method for preparing statin intermediate by using immobilized enzyme | |
| CN111607584A (en) | Method for immobilizing marine cyclodextrin glucosyltransferase by resin | |
| CN101525603A (en) | Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof | |
| CN113373137B (en) | Construction method of photoactive artificial cell and application of photoactive artificial cell in synchronous green synthesis of pentanediamine and formic acid | |
| Passarinho et al. | Effect of carrier matrix on fermentative production of ethanol by surface immobilised yeast cells | |
| Gorrebeeck et al. | Permeability studies on horse liver alcohol dehydrogenase (HLAD) in polyacrylamide gel beads |
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 |