CN108823258A - Oxidation method using whole cells as catalyst - Google Patents
Oxidation method using whole cells as catalyst Download PDFInfo
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- CN108823258A CN108823258A CN201810755903.9A CN201810755903A CN108823258A CN 108823258 A CN108823258 A CN 108823258A CN 201810755903 A CN201810755903 A CN 201810755903A CN 108823258 A CN108823258 A CN 108823258A
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- cell
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- oxidation
- bridging
- catalyst system
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- 239000012530 fluid Substances 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010020688 glycylhistidine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 108010077158 leucinyl-arginyl-tryptophan Proteins 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 108010047926 leucyl-lysyl-tyrosine Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 108010005652 splenotritin Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical class S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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Abstract
The invention discloses an oxidation method using whole cells as a catalyst, and bridged flavin as intracellular NAD (P)+Regenerated catalyst, and intracellular NAD (P) -dependent+Coupled to the redox-catalyzed oxidation reaction of (a) to form intracellular NAD (P)+And (4) regenerating a circulating system. Compared with the prior art for regulating and controlling intracellular cofactor balance based on an artificial cofactor system, the method does not need to construct a mutant enzyme libraryArtificial cofactors are not needed, membrane proteins which can generate toxicity to cells do not need to be expressed, the applicability is strong, and the artificial cofactors can be coupled with a plurality of intracellular NAD (P) + oxidoreductases to accelerate the regeneration of the intracellular NAD (P) +; bridged flavin can enter the cell interior under the condition of not changing the permeability of a cell membrane or over-expressing a membrane protein, and the regeneration of intracellular nicotinamide cofactor is accelerated.
Description
Technical field
The present invention relates to redox reactions, and in particular to a kind of using full cell as the method for oxidation of catalyst.
Background technique
Co-factor provides redox carrier for biosynthesis and decomposition reaction, be intracellular energy transmitting it is important because
Son, it can be seen that co-factor plays an important role in biochemical reaction.Co-factor NADH/NAD+And NADPH/NADP+
As redox carrier most important in cell metabolism, can not only as the electron acceptor of catalysis substrate catabolism,
Reducing power can be provided for the redox reaction of energy independent.Therefore, NADH/NAD+And NADPH/NADP+Oxidation with also
Former rate equation is the necessity for maintaining normal anabolism and catabolism balance.By rationally controlling co-factor water intracellular
It is flat, biocatalysis process can be made more efficiently to carry out.Currently, more commonly control co-factor balance strategy have it is following
Three kinds：1) pass through the endogenous co-factor balance regulation means of the methods of gene knockout or the certain genes of overexpression；2) pass through introducing
External source regenerates the external source co-factor balance regulation means of the methods of co-factor enzyme；3) by the methods of protein engineering change it is auxiliary because
The Preference of son.
Another relatively new strategy is that the artificial co-factor system of building is regulated and controled.Artificial co-factor is compared to natural
Co-factor has that stability is high, easily prepared, relative low price, and catalytic efficiency is auxiliary better than natural in certain enzymatic reactions
Thus the characteristics of factor, is regulation co-factor balance, provides a kind of more efficient and cheaper price approach.But
Even is rested on for the application of artificial co-factor, the application for participating in metabolism intracellular about artificial co-factor only has the extracellular stage
An example, Zhao Zong protect seminar and use artificial synthesized natural niacinamide cofactor analogon --- niacinamide cytimidine dinucleotides
(NCD) a kind of selective electron transport system is constructed, the nicotinamide cofactor analog of the synthesis is able to enter large intestine bar
Bacterium cell can identify that the malic dehydrogenase of the artificial co-factor and phosphorous acidohydrogenase utilize, efficiently produce after being mutated
Malic acid.But this system intracellular based on artificial co-factor, it needs to construct mutant enzyme library to identify artificial co-factor, needs
Protein called membrane transporters Ndt is overexpressed to transport artificial co-factor and enter intracellular, therefore the artificial co-factor system does not have wide spectrum
Property, same membrane channel protein of expressing can generate toxicity to cell.
Summary of the invention
Goal of the invention：In order to solve the problems, such as that easy-regulating does not influence redox reaction efficiency to intracellular co-factor level,
The present invention provides a kind of using full cell as the method for oxidation of catalyst.
Technical solution：Of the present invention a kind of using full cell as the method for oxidation of catalyst, bridging flavine is as thin
NAD (P) intracellular+Regenerated catalyst, and rely on NAD (P) into the cell+Redox enzymatic oxidation reaction coupling, formed thin
NAD (P) intracellular+Regeneration cycle system.Inventor has found that bridging flavine can not change permeability of cell membranes for the first time
Or enter cell interior in the case where being overexpressed memebrane protein, accelerate intracellular NAD (P)+Regeneration；Wherein NAD (P)+Regeneration
It needs to complete under the atmosphere of air or oxygen.
Intracellular NAD (P)+Regenerated reaction process is as follows：It is intracellular to rely on NAD (P)+Redox substrate for enzymatic activity
Carry out oxidation reaction, NAD (P)+It is reduced to NAD (P) H, using bridging flavine as NAD (P)+Regenerated catalyst aoxidizes NAD (P)
H generates NAD (P)+, form intracellular NAD (P)+Regeneration cycle system, ancillary redox enzyme continuous catalysis substrate carry out oxygen
Change reaction.NAD (P) of the present invention+For NAD+Or NADP+；NAD (P) H is NADH or NADPH.
Specifically, catalyst system includes full cell, relies on NAD (P)+The substrate of oxidoreducing enzyme, bridging flavine, buffering
Liquid, the catalyst system catalysis rely on NAD (P)+Oxidoreducing enzyme substrate carry out oxidation reaction.Preferably, oxidation reaction
Catalyst system is separated by solid-liquid separation after the completion, is added into obtained precipitating and relies on NAD (P)+Oxidoreducing enzyme substrate,
Buffer continues oxidation reaction, and the full cell and bridging flavine are reused 3-8 times.It is preferred that 5 times.
In another case, catalyst system includes full cell, bridging flavine, cell culture fluid, in the cell culture fluid
Including relying on NAD (P)+Oxidoreducing enzyme substrate, catalyst system catalysis relies on NAD (P)+Oxidoreducing enzyme bottom
Object carries out oxidation reaction.Catalyst system is separated by solid-liquid separation after the completion of oxidation reaction, cell training is added into obtained precipitating
Nutrient solution continues oxidation reaction, and the full cell and bridging flavine are reused 3-8 times.It is preferred that 5 times.
Preferably, the full cell is selected from Escherichia coli (Escherichia coli) cell, yeast
(Saccharomyce) cell, clostridium acetobutylicum (Clostridium acetobutylicum) cell, Corynebacterium glutamicum
(Corynebacterium glutamicum) cell, bacillus subtilis (Bacillus subtilis) cell, streptomycete
(Streptomycetaceae) recombinant cell of cell, aspergillus niger (Aspergillus niger) cell or above-mentioned cell.
It is highly preferred that the full cell is Escherichia coli (Escherichia coli) cell or recombinant Bacillus coli cells；More preferably
For recombination bacillus coli MtDH-BL21, GlyDH-BL21.
Preferably, the full cell be using corresponding cell need fluid nutrient medium culture to logarithmic phase, stationary phase or
The cell of decline phase；It is preferred that cultivating to logarithmic phase, the cell of stationary phase.
Preferably, the recombinant cell is to be obtained by knocking out cytogene, expression endogenous gene or expression alien gene
Recombinant cell；Preferred expression foreign gene, the recombinant cell for being overexpressed endogenous gene.
Preferably, the concentration of full cell is 0.01-100g/L, preferably 5-50g/L, more preferable 10g/ in the catalyst system
L。
The bridging flavine general structure is as follows：
Wherein, R1And R2It is independently selected from：Hydrogen, methyl, trifluoromethyl, methoxyl group, halogen atom, nitro, amino；R3
It is selected from：Hydrogen, the alkyl of C1~C5, phenyl, benzyl；X-It is selected from：Halogen ion, nitrate anion, trifluoromethanesulfonic acid radical ion.Preferably, R1
Including but not limited to hydrogen, methyl, halogen atom；R2Including but not limited to hydrogen, methyl, halogen atom, trifluoromethyl；R3Including but it is unlimited
Yu Qing, methyl；X-For halogen ion.
It is highly preferred that the bridging flavine includes but is not limited to 7- Trifluoromethyl-1,10- ethylene group isoalloxazine chlorate
The chloro- 1,10- ethylene group isoalloxazine chlorate (compound III) of (compound ii), 8- or 1,10- ethylene group isoalloxazine chlorate
(compound IV).
Most preferably, bridging flavine is 7- Trifluoromethyl-1,10- ethylene group isoalloxazine chlorate.
Bridging flavine of the present invention can voluntarily synthesize document, such as document with reference to existing disclosed document
[Tetrahedron,2001,57,4507-4522]；Or directly commercially.
The concentration of bridging flavine is 0.001-10mM, more preferable 0.05-0.5mM, more preferable 0.1- in the catalyst system
0.5mM, most preferably 0.1mM.
The dependence NAD (P)+Oxidoreducing enzyme be selected from EC1.1.1.X, EC1.2.1.X, EC1.3.1.X,
EC1.4.1.X、 EC1.5.1.X、EC1.6.1.X、EC1.7.1.X、EC1.8.1.X、EC1.10.1.X、EC1.12.1.X、
All enzymes of EC1.13.1.X, EC1.16.1.X, EC1.17.1.X, EC1.18.1.X, EC1.20.1.X, EC1.22.1.X.Bottom
Object is determined by different oxidoreducing enzyme.
Preferably, the dependence NAD (P)+Oxidoreducing enzyme be mannitol dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase
Enzyme, acyl-CoA dehydrogenase.
Preferably, NAD (P) is relied in the catalyst system+Oxidoreducing enzyme substrate initial concentration be 50-1000
mM。
Preferably, the catalyst system further includes the NAD (P) of external source addition+, the NAD (P) of the external source addition+Concentration
For 0.05-5mM.
Beneficial effect：(1) compared with the technology of the regulation co-factor balance intracellular with existing based on artificial co-factor system, this
Patent application does not need building mutant enzyme library and identifies artificial co-factor, does not need to express the memebrane protein that can generate cell toxicity,
Strong applicability, can be with a variety of dependence NAD (P) intracellular+Oxidoreducing enzyme coupling, accelerate NAD intracellular (P)+Regeneration；(2) bridging
Flavine can enter cell interior in the case where not changing permeability of cell membranes or being overexpressed memebrane protein, accelerate cigarette intracellular
Amide cofactor regeneration；(3) the full cell in present patent application catalyst system and bridging flavine can guarantee the feelings of efficiency of pcr product
It is reused 3-8 times under condition；(4) stability is had more compared with the enzymatic reaction that external bridging flavine mediates, and is eliminated brokenly
The step of chopping fine born of the same parents and purifying enzyme, it ensure that the activity of enzyme, save the cost of separation enzyme；(5) it is mediated with external bridging flavine
Enzymatic reaction it is less or without using natural nicotinamide cofactor compared to having used, saved reaction cost, and reaction efficiency
Higher than vitro reactions.
Detailed description of the invention
Fig. 1 schemes for the nucleotide alignments of the mannitol dehydrogenase gene before codon optimization and after optimization；
Fig. 2 is 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as NAD intracellular+Regenerated catalyst in large intestine
The route map for the mannitol dehydrogenase coupling reaction expressed in bacilli-cell.
Specific embodiment
The optimization of 1 mannitol dehydrogenase gene of embodiment
The analysis of rare codon：Rare codon mainly passes through software E.coli Codon Usage Analysis
2.0 analyses obtain the codon that the use relative frequency in Escherichia coli is lower than threshold value (10 ‰).
Optimize rare codon：Frequency of use is lower than threshold value according to frequency of use of the different codons in Escherichia coli
Codon optimize.Optimum results are shown in Table 1：
Codon compares before table 1 optimizes and after optimization
Present patent application optimizes mannitol dehydrogenase gene order according to the codon preference of Escherichia coli, optimization
Such as SEQ ID NO of sequence afterwards:Shown in 1.It is found after being compared by DNAMAN software, improved sequence (MtDH) and former sequence
The homology for arranging (MtDH_Ag) is 73.35%, is specifically shown in Fig. 1.
Embodiment 2 expresses the building of the recombination bacillus coli MtDH-BL21 of mannitol dehydrogenase
The mannitol dehydrogenase gene M tDH of 1 codon optimization of embodiment is cloned on pET-28a carrier and is recombinated
Plasmid pET-28a-MtDH, then by recombinant plasmid transformed into E.coli.BL21 to get the recombination of overexpression MtDH gene
Escherichia coli MtDH-BL21.
Wherein, vector plasmid pET-28a is bought from excellent precious biology；E.coli BL21 is bought from Takara company.
The recombination of gene M tDH and pET-28a carrier, then convert to E.coli.BL21, the preparation of related culture medium etc.
It is carried out according to the method for the Molecular Cloning:A Laboratory guide third edition (Huang Peitang etc. is translated, China, Science Press, 2002)；It is required to draw
Object is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.
The expression of 3 recombination bacillus coli MtDH-BL21 of embodiment
The recombination bacillus coli MtDH-BL21 that embodiment 2 constructs 1% is inoculated in equipped with 200mL LB liquid by volume
In the triangular flask of body culture medium, 37 DEG C, 200rpm is cultivated to OD600Reach 0.6-0.8, IPTG is added into culture solution, it is described
Then the final concentration of 0.6mM of IPTG induces 12-16h under the conditions of 25 DEG C, 200rpm, obtains recombination bacillus coli after centrifugation
Wet thallus.
The formula of LB liquid medium is as follows：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L.
Embodiment 4 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration of NAD+Catalyst and recombination
The coupling catalysed mannitol of Escherichia coli MtDH-BL21 prepares mannose
(1) in the pH9.5 kaliumphosphate buffer of 10mL 100mM, mannitol initial concentration is 250mM, NAD+Concentration is
1mM, 7- Trifluoromethyl-1, the recombination bacillus coli MtDH- that 10- ethylene group isoalloxazine concentration is 0.1mM, embodiment 3 obtains
BL21 weight in wet base concentration is 10g/L, and reaction solution is connected with outside air.It reacts for 24 hours under 25 DEG C of 150rpm, measures after reaction
The amount of mannose is 213mM in reaction system.
(2) system for obtaining step (1) is centrifuged, and the pH9.5 potassium phosphate of 10mL100mM is added into obtained precipitating
Buffer and mannitol, so that the initial concentration of mannitol is 250mM, reaction solution is connected with outside air, 25 DEG C of 150rpm
Lower reaction is for 24 hours；Recombination bacillus coli and 7- Trifluoromethyl-1 are reused by the way of batch feeding, 10- ethylene group is different to be coughed up
Piperazine 5 times, substrate and product are removed after each reaction, mannose yield is 72% after reusing 5 times.
Embodiment 5 is using 1,10- ethylene group isoalloxazine chlorate as regeneration of NAD+Catalyst and recombination bacillus coli
The coupling catalysed mannitol of MtDH-BL21 prepares mannose
Method is with embodiment 4, the difference is that bridging flavine is 1,10- ethylene group isoalloxazine chlorate, concentration 0.1mM.
The amount for measuring mannose in reaction system after reaction is 168mM.
Recombination bacillus coli and 1, after 10- ethylene group isoalloxazine chlorate is reused 5 times, mannose yield is 52%.
Embodiment 6 is using the chloro- 1,10- ethylene group isoalloxazine chlorate of 8- as regeneration of NAD+Catalyst and recombination large intestine bar
The coupling catalysed mannitol of bacterium MtDH-BL21 prepares mannose
Method is with embodiment 4, the difference is that bridging flavine is chloro- 1, the 10- ethylene group isoalloxazine chlorate of 8-, concentration is
0.1 mM.The amount for measuring mannose in reaction system after reaction is 173mM.
After recombination bacillus coli and chloro- 1, the 10- ethylene group isoalloxazine chlorate of 8- are reused 5 times, mannose yield is
61%.
The building of the expression of embodiment 7 glycerol dehydrogenase recombination bacillus coli GlyDH-BL21
Glycerol dehydrogenase gene GlyDH (the GenBank of E.coli K12 will be derived from:AAC43051.1 it) is cloned into
Recombinant plasmid pET-28a-GlyDH is obtained on pET-28a carrier, then by recombinant plasmid transformed into E.coli.BL21 to get
It is capable of the recombination bacillus coli GlyDH-BL21 of overexpression GlyDH gene.
Wherein, vector plasmid pET-28a is bought from excellent precious biology；E.coli BL21 is bought from Takara company.
The recombination of gene GlyDH and pET-28a carrier, then convert to E.coli.BL21, the preparation of related culture medium etc.
It is carried out according to the method for the Molecular Cloning:A Laboratory guide third edition (Huang Peitang etc. is translated, China, Science Press, 2002)；It is required to draw
Object is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.
The expression of 8 recombination bacillus coli GlyDH-BL21 of embodiment
Expression with embodiment 3, be not both recombination bacillus coli be GlyDH-BL21.
Embodiment 9 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration NAD intracellular+Catalyst with
The coupling catalysed glycerol of recombination bacillus coli GlyDH-BL21 prepares dihydroxyacetone (DHA) DHA
(1) in the pH 9.5Tris-HCl buffer of 10mL 100mM, glycerol initial concentration is 10g/L, NAD+Concentration
For 5mM, 7- Trifluoromethyl-1, the recombination bacillus coli GlyDH- that 10- ethylene group isoalloxazine concentration is 0.1mM, embodiment 8 obtains
BL21 weight in wet base concentration is 10g/L, and reaction solution is connected with outside air.2h is reacted under 30 DEG C of 150rpm, is measured after reaction
The concentration of DHA is 3.05g/L in reaction system.
(2) recombination bacillus coli GlyDH-BL21 and 7- Trifluoromethyl-1,10- are reused by the way of batch feeding
Ethylene group isoalloxazine 5 times, substrate and product are removed after each reaction, DHA yield is 27% after reusing 5 times.
Embodiment 10 expresses the building of the recombined bacillus subtilis MtDH-Bs168 of mannitol dehydrogenase
The mannitol dehydrogenase gene M tDH of 1 codon optimization of embodiment is cloned on pMA5 carrier and obtains recombinant plasmid
PMA5-MtDH, then by recombinant plasmid transformed into bacillus subtilis B.subtilis 168 to get being capable of overexpression
The recombined bacillus subtilis MtDH-Bs168 of MtDH gene.
Wherein pMA5 plasmid is purchased from biological wind Biofeng；Bacillus subtilis B.subtilis 168 is purchased from the silent winged public affairs of match
Department.
Gene GlyDH is connected with the recombination of pMA5 carrier, is then converted to the preparation of E.coli.BL21, related culture medium
It is carried out Deng according to the method for the Molecular Cloning:A Laboratory guide third edition (Huang Peitang etc. is translated, China, Science Press, 2002)；It is required
Primer is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.
The expression of 11 MtDH-BL21 recombined bacillus subtilis of embodiment
The recombined bacillus subtilis MtDH-Bs168 that embodiment 10 is constructed uses the culture of LB culture medium, then presses 1%
Inoculum concentration be forwarded to 200mL LB liquid medium, cultivated under the conditions of 37 DEG C, 200rpm for 24 hours, obtain recombinant bacillus gemma bar
The wet thallus of bacterium.
Embodiment 12 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration NAD intracellular+Catalyst
Mannose is prepared with the coupling catalysed mannitol of recombined bacillus subtilis MtDH-Bs168
In 9.5 kaliumphosphate buffer of pH of 10mL 100mM, mannitol initial concentration is 50mM, NAD+Concentration is
0.5mM, 7- Trifluoromethyl-1, the recombined bacillus subtilis that 10- ethylene group isoalloxazine concentration is 0.1mM, embodiment 11 obtains
MtDH-Bs168 weight in wet base concentration is 10g/L, and reaction solution is connected with outside air.25 DEG C, 12h, reaction knot are reacted under 150rpm
The amount that mannose in reaction system is measured after beam is 42mM.Recombined bacillus subtilis is reused by the way of batch feeding
With 7- Trifluoromethyl-1,10- ethylene group isoalloxazine 5 times removes substrate and product after each reaction, after reusing 5 times
Mannose yield is 71%.
Embodiment 13 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration NAD intracellular+Catalyst
Ethyl alcohol, butanol, 3-hydroxy-2-butanone are prepared with clostridium acetobutylicum coupling
By clostridium acetobutylicum (C.acetobutylium) 428, using P2 culture medium, stationary culture is extremely under the conditions of 37 DEG C
Logarithmic phase, by 7- Trifluoromethyl-1,10- ethylene group isoalloxazine is added in the culture solution of above-mentioned logarithmic phase (7- trifluoromethyl-
1,10- ethylene group isoalloxazine concentration is 0.5mM), 37 DEG C of standing anaerobic fermentation 72h, final ethanol production is 1g/L, butanol yield
For 12.3g/L, 3-hydroxy-2-butanone yield is 0.8g/L.
Wherein, P2 fermentation medium is divided into four parts and is respectively configured：
1, glucose 60g/L；
2, ammonium acetate 2.2g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L；
3, sodium chloride 0.01g/L, Manganous sulfate monohydrate 0.01g/L, green vitriol 0.01g/L, seven hydrated sulfuric acids
Magnesium 0.2g/L；
4, biotin 0.01mg/L, vitamin B1 1mg/L, p-aminobenzoic acid 1mg/L.
Wherein, the concentration of above-mentioned each component is the final concentration of component in P2 fermentation medium.
Solution 1 and solution 2 are individually sterilized 15min in 121 DEG C of high steam pots, solution 3 and solution 4 are separately dissolved in sterile
In ultrapure water, 0.22 μm of water system sterilised membrane filter filtering.It is calculated by 1L fermentation medium is fermentation system, by bacterium of individually having gone out
Solution 1 and solution 2 directly mix, and the solution 3 and each 50 μ L of solution 4 of degerming are added in the solution 1 and solution 2 mixed.
Clostridium acetobutylicum and 7- Trifluoromethyl-1,10- ethylene group isoalloxazine 5 are reused by the way of batch feeding
It is secondary, substrate and product are removed after each reaction, after reusing 5 times, final ethanol production is 1.3g/L, and butanol yield is
12.5g/L, 3-hydroxy-2-butanone yield are 0.76g/L.
Embodiment 14 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration NAD intracellular+Catalyst
Ethyl alcohol is prepared with saccharomycete coupling
By yeast (saccharomyce) BY4741 in complex medium, 30 DEG C, the aerobic culture of 200rpm to logarithmic phase,
Then by 7- Trifluoromethyl-1,10- ethylene group isoalloxazine is added in the culture solution of above-mentioned logarithmic phase (- Trifluoromethyl-1,10-
Ethylene group isoalloxazine concentration is 0.5mM), 32 DEG C, 200rpm aerobic fermentation 30h, final ethanol production is 11.6g/L.
Wherein complex medium is configured to be divided into：10g/L peptone, 5g/L yeast powder, 90g/L glucose, 9g/L
NaCl。
Reuse yeast and 7- Trifluoromethyl-1 by the way of batch feeding, 10- ethylene group isoalloxazine 5 times, every time
Substrate and product are removed after reaction, and after reusing 5 times, final ethanol production is 12g/L.
Comparative example 1 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration of NAD+Catalyst and sweet dew
The coupling catalysed mannitol of alcohol dehydrogenase prepares mannose
It is compared with embodiment 4, vitro reactions carry out in 9.5 kaliumphosphate buffer of pH of 10mL 100mM, sweet dew
Alcohol initial concentration is 250mM, NAD+Concentration is 1mM, 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine concentration is 0.5mM, sweet dew
Alcohol dehydrogenase dosage is 10 μM, and reaction solution is connected with outside air.25 DEG C, react for 24 hours under 150rpm, measure after reaction
The amount of mannose is 100mM in reaction system.Mannose yield is significantly lower than embodiment 4, and reaction system can not reuse.
Wherein mannitol dehydrogenase concentration is equal to that 10g/L recombination bacillus coli wet cell is broken to obtain mannitol after purification
The concentration of dehydrogenase.
Comparative example 2 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration of NAD+Catalyst and glycerol
The coupling catalysed glycerol of dehydrogenase prepares dihydroxyacetone (DHA) DHA
It is compared with embodiment 9, under in vitro conditions in the pH 9.5Tris-HCl buffer of 10mL 100mM, glycerol
Initial concentration is 10g/L, NAD+Concentration is 10mM, 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine concentration is 0.2mM, glycerol
Dehydrogenase 7 μM, reaction solution are connected with outside air.2h is reacted under 30 DEG C of 150rpm, is measured in reaction system after reaction
The concentration of DHA is 2.51g/L.DHA yield is significantly lower than embodiment 9, and reaction system can not reuse.
Wherein glycerol dehydrogenase enzyme concentration is equal to that 10g/L recombination bacillus coli wet cell is broken to obtain glycerol dehydrogenase after purification
The concentration of enzyme.
Comparative example 3 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration NAD intracellular+Catalyst with
The coupling catalysed mannitol of mannitol dehydrogenase prepares mannose
It is compared with embodiment 12, under in vitro conditions in 9.5 kaliumphosphate buffer of pH of 10mL 100mM, sweet dew
Alcohol initial concentration is 50mM, NAD+Concentration is 0.5mM, 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine concentration is 0.1mM, sweet dew
6 μM of alcohol dehydrogenase, reaction solution is connected with outside air.25 DEG C, react 12 h under 150rpm, measure reactant after reaction
The amount of mannose is 38mM in system.Sweet dew sugar yield is significantly lower than embodiment 12, and reaction system can not reuse.
Wherein mannitol dehydrogenase concentration is equal to that 10g/L recombination bacillus coli wet cell is broken to obtain mannitol after purification
The concentration of dehydrogenase.
Comparative example 4 is with 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine chlorate is as regeneration of NAD+Catalyst and password
The coupling catalysed mannitol of mannitol dehydrogenase recombination bacillus coli before son optimization prepares mannose
The building of recombination bacillus coli and expression are with embodiment 1, embodiment 2 and embodiment 3, the difference is that mannitol
Dehydrogenase gene MtDH is the MtDH_Ag before codon optimization.
It compares, is carried out in 9.5 kaliumphosphate buffer of pH of 10mL 100mM, mannitol is initially dense with embodiment 4
Degree is 250mM, NAD+Concentration is 1mM, 7- Trifluoromethyl-1, and 10- ethylene group isoalloxazine concentration is 0.5mM, comparative example 4 obtains
Recombination bacillus coli weight in wet base concentration is 10g/L, and reaction solution is connected with outside air.25 DEG C, react for 24 hours under 150rpm, reaction
After measure mannose in reaction system amount be 83mM.Mannose yield is significantly lower than embodiment 4, illustrates codon optimization
Preceding mannitol dehydrogenase recombination bacillus coli substrate tolerance is low.
Sequence table
<110>Nanjing University of Technology
<120>It is a kind of using full cell as the method for oxidation of catalyst
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1092
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gcaaaaagca gcgagatcga gcatccggtt aaagcctttg gctgggcagc acgtgatacc 60
acaggcctgc tgagtccgtt caaattcagt cgccgtgcca ccggcgaaaa ggacgttcgc 120
ctgaaggtgc tgttttgcgg cgtttgccat agcgatcacc acatgatcca caacaactgg 180
ggctttacca cctacccgat tgtgccgggt catgagatcg ttggtgtggt taccgaggtt 240
ggtagcaagg tggagaaagt gaaggtgggt gacaacgtgg gcattggctg cctggtgggt 300
agctgccgta gctgcgagag ctgctgcgat aatcgcgaga gtcactgcga gaataccatc 360
gatacctacg gtagcattta cttcgacggc accatgaccc atggtggtta tagcgacacc 420
atggtggcag acgagcattt cattctgcgc tggccgaaaa acctgccgct ggatagcggt 480
gcccctctgc tgtgtgccgg tatcaccacc tatagcccgc tgaagtacta cggcctggac 540
aaacctggca ccaaaatcgg tgtggtgggc ttaggcggtc tgggtcatgt ggccgtgaaa 600
atggccaaag cattcggcgc ccaggttacc gtgattgaca ttagcgaaag caaacgcaaa 660
gaggccctgg aaaaactggg tgcagacagc tttctgctga acagcgatca ggagcagatg 720
aaaggtgccc gtagtagcct ggatggcatc attgacaccg tgccggtgaa tcatcctctg 780
gccccgctgt tcgatctgct gaaaccgaac ggcaaactgg tgatggtggg tgcaccggaa 840
aagccgttcg aactgccggt gtttagcctg ctgaagggcc gtaagctgct gggcggtacc 900
atcaatggcg gcatcaaaga aacccaggag atgctggact ttgccgccaa acacaacatt 960
accgccgacg tggaagtgat cccgatggac tacgttaaca ccgccatgga gcgcctggtg 1020
aaaagcgatg tgcgttaccg cttcgtgatc gatatcgcca acacaatgcg caccgaagaa 1080
agcctgggtg ca 1092
<210> 2
<211> 364
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Ala Lys Ser Ser Glu Ile Glu His Pro Val Lys Ala Phe Gly Trp Ala
1 5 10 15
Ala Arg Asp Thr Thr Gly Leu Leu Ser Pro Phe Lys Phe Ser Arg Arg
20 25 30
Ala Thr Gly Glu Lys Asp Val Arg Leu Lys Val Leu Phe Cys Gly Val
35 40 45
Cys His Ser Asp His His Met Ile His Asn Asn Trp Gly Phe Thr Thr
50 55 60
Tyr Pro Ile Val Pro Gly His Glu Ile Val Gly Val Val Thr Glu Val
65 70 75 80
Gly Ser Lys Val Glu Lys Val Lys Val Gly Asp Asn Val Gly Ile Gly
85 90 95
Cys Leu Val Gly Ser Cys Arg Ser Cys Glu Ser Cys Cys Asp Asn Arg
100 105 110
Glu Ser His Cys Glu Asn Thr Ile Asp Thr Tyr Gly Ser Ile Tyr Phe
115 120 125
Asp Gly Thr Met Thr His Gly Gly Tyr Ser Asp Thr Met Val Ala Asp
130 135 140
Glu His Phe Ile Leu Arg Trp Pro Lys Asn Leu Pro Leu Asp Ser Gly
145 150 155 160
Ala Pro Leu Leu Cys Ala Gly Ile Thr Thr Tyr Ser Pro Leu Lys Tyr
165 170 175
Tyr Gly Leu Asp Lys Pro Gly Thr Lys Ile Gly Val Val Gly Leu Gly
180 185 190
Gly Leu Gly His Val Ala Val Lys Met Ala Lys Ala Phe Gly Ala Gln
195 200 205
Val Thr Val Ile Asp Ile Ser Glu Ser Lys Arg Lys Glu Ala Leu Glu
210 215 220
Lys Leu Gly Ala Asp Ser Phe Leu Leu Asn Ser Asp Gln Glu Gln Met
225 230 235 240
Lys Gly Ala Arg Ser Ser Leu Asp Gly Ile Ile Asp Thr Val Pro Val
245 250 255
Asn His Pro Leu Ala Pro Leu Phe Asp Leu Leu Lys Pro Asn Gly Lys
260 265 270
Leu Val Met Val Gly Ala Pro Glu Lys Pro Phe Glu Leu Pro Val Phe
275 280 285
Ser Leu Leu Lys Gly Arg Lys Leu Leu Gly Gly Thr Ile Asn Gly Gly
290 295 300
Ile Lys Glu Thr Gln Glu Met Leu Asp Phe Ala Ala Lys His Asn Ile
305 310 315 320
Thr Ala Asp Val Glu Val Ile Pro Met Asp Tyr Val Asn Thr Ala Met
325 330 335
Glu Arg Leu Val Lys Ser Asp Val Arg Tyr Arg Phe Val Ile Asp Ile
340 345 350
Ala Asn Thr Met Arg Thr Glu Glu Ser Leu Gly Ala
355 360
Claims (17)
1. a kind of using full cell as the method for oxidation of catalyst, which is characterized in that bridging flavine is as intracellular NAD (P)+
Regenerated catalyst, and rely on NAD (P) into the cell+Redox enzymatic oxidation reaction coupling, form intracellular NAD
(P)+Regeneration cycle system.
2. method for oxidation according to claim 1, which is characterized in that catalyst system includes full cell, relies on NAD (P)+'s
Substrate, the bridging flavine, buffer of oxidoreducing enzyme, the catalyst system catalysis rely on NAD (P)+Oxidoreducing enzyme bottom
Object carries out oxidation reaction.
3. method for oxidation according to claim 2, which is characterized in that carry out solid-liquid to catalyst system after the completion of oxidation reaction
Separation is added into obtained precipitating and relies on NAD (P)+Substrate, the buffer of oxidoreducing enzyme continue oxidation reaction,
The full cell and bridging flavine are reused 3-8 times.
4. method for oxidation according to claim 1, which is characterized in that catalyst system includes full cell, bridging flavine, cell
Culture solution includes relying on NAD (P) in the cell culture fluid+Oxidoreducing enzyme substrate, catalyst system catalysis relies on
NAD(P)+Oxidoreducing enzyme substrate carry out oxidation reaction.
5. method for oxidation according to claim 4, which is characterized in that carry out solid-liquid to catalyst system after the completion of oxidation reaction
Separation adds cell culture fluid into obtained precipitating and continues oxidation reaction, and the full cell and bridging flavine repeat to make
With 3-8 times.
6. method for oxidation described in -5 any one according to claim 1, which is characterized in that the full cell is selected from Escherichia coli
(Escherichia coli) cell, yeast (Saccharomyce) cell, clostridium acetobutylicum (Clostridium
Acetobutylicum) cell, Corynebacterium glutamicum (Corynebacterium glutamicum) cell, bacillus subtilis
(Bacillus subtilis) cell, streptomycete (Streptomycetaceae) cell, aspergillus niger (Aspergillus
Niger) the recombinant cell of cell or above-mentioned cell.
7. method for oxidation according to claim 6, which is characterized in that the full cell is cultivated to logarithmic phase, stationary phase
Or the cell of decline phase.
8. method for oxidation according to claim 6, which is characterized in that the full cell is Escherichia coli (Escherichia
Coli) cell or recombinant Bacillus coli cells.
9. method for oxidation according to claim 6, which is characterized in that the recombinant cell be by knock out cytogene,
The recombinant cell that expression endogenous gene or expression alien gene obtain.
10. according to method for oxidation described in claim 2-5 any one, which is characterized in that full cell in the catalyst system
Concentration be 0.01-100g/L.
11. method for oxidation described in -5 any one according to claim 1, which is characterized in that the bridging flavine general structure
It is as follows：
Wherein, R1And R2It is independently selected from：Hydrogen, methyl, trifluoromethyl, methoxyl group, halogen atom, nitro, amino；R3It is selected from：
Hydrogen, the alkyl of C1~C5, phenyl, benzyl；X-It is selected from：Halogen ion, nitrate anion, trifluoromethanesulfonic acid radical ion.
12. method for oxidation according to claim 11, which is characterized in that the bridging flavine is 7- Trifluoromethyl-1,10-
The chloro- 1,10- ethylene group isoalloxazine chlorate of ethylene group isoalloxazine chlorate, 8- or 1,10- ethylene group isoalloxazine chlorate.
13. according to method for oxidation described in claim 2-5 any one, which is characterized in that bridging is yellow in the catalyst system
The concentration of element is 0.001-10mM.
14. method for oxidation described in -5 any one according to claim 1, which is characterized in that the dependence NAD (P)+Oxidation
Reductase be selected from EC1.1.1.X, EC1.2.1.X, EC1.3.1.X, EC1.4.1.X, EC1.5.1.X, EC1.6.1.X,
EC1.7.1.X、EC1.8.1.X、EC1.10.1.X、EC1.12.1.X、EC1.13.1.X、EC1.16.1.X、EC1.17.1.X、
All enzymes of EC1.18.1.X, EC1.20.1.X, EC1.22.1.X.
15. method for oxidation according to claim 14, which is characterized in that the dependence NAD (P)+Oxidoreducing enzyme be it is sweet
Reveal alcohol dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, acyl-CoA dehydrogenase.
16. according to method for oxidation described in claim 2-5 any one, which is characterized in that rely on NAD in the catalyst system
(P)+Oxidoreducing enzyme substrate initial concentration be 50-1000mM.
17. according to method for oxidation described in claim 2-5 any one, which is characterized in that the catalyst system further includes outer
The NAD (P) of source addition+, the NAD (P) of the external source addition+Initial concentration be 0.05-5mM.
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| CN111826409A (en) * | 2019-04-16 | 2020-10-27 | 中国科学院大连化学物理研究所 | Method for reducing NAD analogue by using methanol |
| CN112375040A (en) * | 2020-11-27 | 2021-02-19 | 南京工业大学 | Method for preparing nitrogen-containing heterocyclic compound and derivative thereof by enzyme-chemical cascade method |
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| CN105622693A (en) * | 2016-01-08 | 2016-06-01 | 南京工业大学 | Oxidized coenzyme NAD (P)+Chemical regeneration method of |
| CN106811488A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of bioanalysis coproduction mannitol and gluconic acid or the method for gluconate |
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2018
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| CN106811488A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of bioanalysis coproduction mannitol and gluconic acid or the method for gluconate |
| CN105622693A (en) * | 2016-01-08 | 2016-06-01 | 南京工业大学 | Oxidized coenzyme NAD (P)+Chemical regeneration method of |
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|---|---|---|---|---|
| CN111826409A (en) * | 2019-04-16 | 2020-10-27 | 中国科学院大连化学物理研究所 | Method for reducing NAD analogue by using methanol |
| CN112375040A (en) * | 2020-11-27 | 2021-02-19 | 南京工业大学 | Method for preparing nitrogen-containing heterocyclic compound and derivative thereof by enzyme-chemical cascade method |
| US12024732B2 (en) | 2020-11-27 | 2024-07-02 | Nanjing Tech University | Method for preparing nitrogen-containing heterocyclic compound and derivative thereof by enzymatic-chemical cascade method |
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