CN108823258B

CN108823258B - Oxidation method using whole cells as catalyst

Info

Publication number: CN108823258B
Application number: CN201810755903.9A
Authority: CN
Inventors: 朱晨杰; 曹阳; 应汉杰; 谭卓涛; 陈勇; 李明; 庄伟�
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2018-07-11
Filing date: 2018-07-11
Publication date: 2022-02-11
Anticipated expiration: 2038-07-11
Also published as: CN108823258A

Abstract

The invention discloses an oxidation method using whole cells as a catalyst, and bridged flavin is used as intracellular NAD (P)⁺Regenerated catalyst, and intracellular NAD (P) -dependent⁺Coupled to the redox-enzyme-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 the balance of intracellular cofactors based on an artificial cofactor system, the method does not need to construct a mutant enzyme library to identify the artificial cofactors, does not need to express membrane proteins which can generate toxicity to cells, has strong applicability, can be coupled with a plurality of intracellular oxidoreductase depending on NAD (P) +, and accelerates the regeneration of 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

Oxidation method using whole cells as catalyst

Technical Field

The invention relates to redox reactions, in particular to an oxidation method using whole cells as a catalyst.

Background

The cofactor provides a redox carrier for biosynthesis and decomposition reactions, and is an important factor for energy transfer in cells, so that the cofactor has an important role in biochemical reactions. Cofactor NADH/NAD⁺And NADPH/NADP⁺As the most important redox carrier in cell metabolism, it can be used not only as an electron acceptor for catalyzing substrate catabolism but also as an electron acceptorThe amount-dependent redox reaction provides the reducing power. Thus, NADH/NAD⁺And NADPH/NADP⁺The balance of oxidation and reduction rates of (a) is a necessary requirement to maintain a normal anabolic and catabolic balance. By reasonably controlling the level of the intracellular cofactor, the biocatalysis process can be carried out more efficiently. Currently, there are three more commonly used strategies for controlling cofactor balance: 1) endogenous cofactor balance regulation means by methods of gene knockout or overexpression and the like; 2) an exogenous cofactor balance regulation and control means by introducing exogenous regenerated cofactor enzyme and other methods; 3) the preference of the cofactor is changed by methods such as protein engineering.

Another novel strategy is to construct an artificial cofactor system for regulation. Compared with natural cofactors, the artificial cofactors have the characteristics of high stability, easy preparation and relatively low price, and the catalytic efficiency is superior to that of the natural cofactors in certain enzymatic reactions, thereby providing a more efficient and lower-price way for regulating and controlling the balance of the cofactors. However, the application of artificial cofactors is still in the extracellular phase, and only one example of the application of artificial cofactors participating in intracellular metabolism is shown, and the group of Zhao Zong K.has constructed a selective electron transport system using Nicotinamide Cytosine Dinucleotide (NCD), an artificially synthesized natural nicotinamide cofactor analog, which can enter E.coli cells and be used by malate dehydrogenase and phosphite dehydrogenase that are mutated to recognize the artificial cofactors, to efficiently produce malate. However, such an artificial cofactor-based intracellular system requires the construction of a mutant enzyme library for identifying the artificial cofactor and the overexpression of the membrane transporter Ndt for transporting the artificial cofactor into the cell, and thus the artificial cofactor system has no broad spectrum and also the expression of the membrane channel protein causes toxicity to the cell.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problem that the level of a cofactor in cells is not easy to regulate and control and influences the efficiency of redox reaction, the invention provides an oxidation method by using whole cells as a catalyst.

Technical methodA scheme: the invention relates to an oxidation method using whole cells as a catalyst, and bridged flavin is used as intracellular NAD (P)⁺Regenerated catalyst, and intracellular NAD (P) -dependent⁺Coupled to the redox-enzyme-catalyzed oxidation reaction of (a) to form intracellular NAD (P)⁺And (4) regenerating a circulating system. The inventor firstly discovers that bridged flavin can enter the interior of a cell under the condition of not changing the permeability of a cell membrane or over-expressing a membrane protein, and accelerates the NAD (P) in the cell⁺Regeneration; wherein NAD (P)⁺The regeneration of (a) needs to be accomplished under an atmosphere of air or oxygen.

Intracellular NAD (P)⁺The reaction process for regeneration is as follows: intracellular dependence on NAD (P)⁺The redox enzyme of (2) catalyzes the oxidation of the substrate, NAD (P)⁺Reduced to NAD (P) H with bridged flavins as NAD (P)⁺Regenerating the catalyst, oxidizing NAD (P) H to NAD (P)⁺Formation of intracellular NAD (P)⁺And a circulating system is regenerated, and the redox enzyme is assisted to continuously catalyze the substrate to carry out oxidation reaction. NAD (P) according to the invention⁺Is NAD⁺Or NADP⁺(ii) a The NAD (P) H is NADH or NADPH.

In particular, the catalytic system comprises whole cells, NAD (P) -dependent⁺The oxidoreductase substrate of (2), bridged flavins, buffer, the catalytic system being dependent on NAD (P)⁺The substrate of the oxidoreductase (2) undergoes an oxidation reaction. Preferably, after the oxidation reaction is finished, the solid-liquid separation is carried out on the catalytic system, and NAD (P) -dependent) is supplemented into the obtained precipitate⁺The oxidation reaction is continued by using the substrate of the oxidoreductase and the buffer solution, and the whole cells and bridged flavin are reused for 3 to 8 times. Preferably 5 times.

In another case, the catalytic system comprises whole cells, bridged flavins, a cell culture fluid comprising NAD (P) -dependent⁺The catalytic system catalyzes the dependence on NAD (P)⁺The substrate of the oxidoreductase (2) undergoes an oxidation reaction. After the oxidation reaction is finished, carrying out solid-liquid separation on the catalytic system, supplementing cell culture solution into the obtained precipitate, and continuing the oxidation reaction, wherein the whole cells and the bridged flavin are repeatedIt is applied 3-8 times. Preferably 5 times.

Preferably, the whole cell is selected from an Escherichia coli (Escherichia coli) cell, a yeast (saccharomyces) cell, a Clostridium acetobutylicum (Clostridium acetobutylicum) cell, a Corynebacterium glutamicum (Corynebacterium glutamicum) cell, a Bacillus subtilis (Bacillus subtilis) cell, a streptomyces (streptomyces cerevisiae) cell, an Aspergillus niger (Aspergillus niger) cell, or a recombinant cell of the above cells. More preferably, the whole cell is an Escherichia coli (Escherichia coli) cell or a recombinant Escherichia coli cell; more preferably recombinant E.coli MtDH-BL21 and GlyDH-BL 21.

Preferably, the whole cells are cells cultured to a logarithmic phase, a stationary phase or a decline phase using a liquid medium required for the respective cells; cells cultured to the log phase, stationary phase are preferred.

Preferably, the recombinant cell is a recombinant cell obtained by knocking out a cellular gene, expressing an endogenous gene or expressing an exogenous gene; recombinant cells expressing exogenous genes, overexpressing endogenous genes are preferred.

Preferably, the concentration of whole cells in the catalytic system is 0.01-100g/L, preferably 5-50g/L, more preferably 10 g/L.

The bridged flavin has the following structural general formula:

wherein R is₁And R₂Independently of one another, from: hydrogen, methyl, trifluoromethyl, methoxy, halogen atom, nitro and amino; r₃Selected from: hydrogen, C1-C5 alkyl, phenyl and benzyl; x^-Selected from: halide ion, nitrate radical, triflate ion. Preferably, R₁Including but not limited to hydrogen, methyl, halogen atoms; r₂Including but not limited to hydrogen, methyl, halogen, trifluoromethyl; r₃Including but not limited to hydrogen, methyl; x^-Is a halide ion.

More preferably, the bridged flavins include, but are not limited to, 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride (compound II), 8-chloro-1, 10-ethylideneisoalloxazine chloride (compound III), or 1, 10-ethylideneisoalloxazine chloride (compound IV).

Most preferably, the bridged flavin is 7-trifluoromethyl-1, 10-ethylene isoalloxazine chloride.

The bridged flavins of the present invention may be synthesized by themselves with reference to the literature published at present, for example the literature [ Tetrahedron,2001,57, 4507-; or purchased directly from the market.

The concentration of bridged flavins in the catalytic system is 0.001-10mM, more preferably 0.05-0.5mM, more preferably 0.1-0.5mM, most preferably 0.1 mM.

Said dependence on NAD (P)⁺The oxidoreductase of (a) is selected from all enzymes of 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, EC1.18.1.X, EC1.20.1.X, and EC1.22.1. X. The substrates are determined by different oxidoreductases.

Preferably, said dependence on NAD (P)⁺The oxidoreductase of (2) is mannitol dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, or acetyl-CoA dehydrogenase.

Preferably, the catalytic system is NAD (P) -dependent⁺The initial concentration of substrate for the oxidoreductase of (3) is 50-1000 mM.

Preferably, the catalytic system further comprises an exogenously added NAD (P)⁺Said exogenously added NAD (P)⁺Is in a concentration of 0.05-5 mM.

Has the advantages that: (1) compared with the prior art for regulating and controlling the balance of intracellular cofactors based on an artificial cofactor system, the method does not need to construct a mutant enzyme library to identify the artificial cofactors, does not need to express membrane proteins which can generate toxicity to cells, has strong applicability, and can be used for regulating and controlling intracellular cofactor balance depending on NAD (P)⁺The oxidoreductase coupling of (2) accelerates intracellular NAD (P)⁺Regeneration; (2) the bridged flavin can enter the cell under the condition of not changing the permeability of a cell membrane or over-expressing a membrane protein, so that the regeneration of intracellular nicotinamide cofactor is accelerated; (3) the whole cells and bridged flavin in the catalytic system of the patent application can be recycled for 3-8 times under the condition of ensuring the product yield; (4) compared with in vitro bridged flavin mediated enzymatic reaction, the method has higher stability, and the steps of cell breaking and enzyme purification are omitted, so that the activity of the enzyme is ensured, and the cost for separating the enzyme is saved; (5) compared with the in vitro bridged flavin mediated enzymatic reaction, the method uses less or no natural nicotinamide cofactor, saves the reaction cost and has higher reaction efficiency than the in vitro reaction.

Drawings

FIG. 1 is a nucleotide sequence alignment chart of mannitol dehydrogenase genes before and after codon optimization;

FIG. 2 shows the use of 7-trifluoromethyl-1, 10-ethylene isoalloxazine chloride salt as intracellular NAD⁺Scheme for coupling reaction of regenerated catalyst with mannitol dehydrogenase expressed in E.coli cells.

Detailed Description

Example 1 optimization of mannitol dehydrogenase Gene

Analysis of rare codons: rare codons were mainly analyzed by the software e.coli Codon Usage Analysis 2.0 to obtain codons used with relative frequency below the threshold (10 ‰) in e.

Rare codons were optimized: codons with a frequency of usage below the threshold are optimized according to the frequency of usage of the different codons in e. The optimization results are shown in table 1:

TABLE 1 Pre-and post-optimization codon comparison

The application optimizes the mannitol dehydrogenase gene sequence according to the codon preference of escherichia coli, and the optimized sequence is shown as SEQ ID NO. 1. After comparison with DNMAN software, the homology between the modified sequence (MtDH) and the original sequence (MtDH _ Ag) was 73.35%, as shown in FIG. 1.

EXAMPLE 2 construction of recombinant E.coli MtDH-BL21 expressing mannitol dehydrogenase

The mannitol dehydrogenase gene MtDH optimized by the codon in the embodiment 1 is cloned to a pET-28a vector to obtain a recombinant plasmid pET-28a-MtDH, and then the recombinant plasmid is transformed into E.coli BL21 to obtain the recombinant Escherichia coli MtDH-BL21 of the over-expression MtDH gene.

Wherein, the vector plasmid pET-28a is purchased from Youbao organisms; coli BL21 was purchased from Takara corporation.

Gene MtDH and pET-28a vectors were recombined and then transformed into e.coli.bl21, preparation of relevant media, etc. according to the method of the third edition of molecular cloning experimental guidelines (huang peitang et al, china, scientific press, 2002); the primers required were all synthesized by Suzhou Jinweizhi Biotechnology, Inc.

Example 3 expression of recombinant E.coli MtDH-BL21

The recombinant E.coli MtDH-BL21 constructed in example 2 was inoculated in a volume ratio of 1% into a flask containing 200mL of LB liquid medium, cultured at 37 ℃ and 200rpm to OD₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.6mM into the culture solution, inducing at 25 deg.C and 200rpm for 12-16h, and centrifuging to obtain wet thallus of recombinant Escherichia coli.

The formula of the LB liquid culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride.

Example 4 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt as regenerated NAD⁺The catalyst and recombinant Escherichia coli MtDH-BL21 are coupled to catalyze mannitol to prepare mannose

(1) Mannitol in 10mL of 100mM potassium phosphate buffer, pH9.5, at an initial concentration of 250mM, NAD⁺The recombinant Large intestine obtained in example 3 at a concentration of 1mM and a concentration of 0.1mM for 7-trifluoromethyl-1, 10-ethylideneisoalloxazineThe wet weight concentration of the bacillus MtDH-BL21 is 10g/L, and the reaction liquid is communicated with the outside air. The reaction was carried out at 25 ℃ and 150rpm for 24 hours, and the amount of mannose in the reaction system was measured to be 213mM after the completion of the reaction.

(2) Centrifuging the system obtained in the step (1), adding 10mL of 100mM potassium phosphate buffer solution with pH of 9.5 and mannitol into the obtained precipitate to make the initial concentration of the mannitol be 250mM, communicating the reaction solution with the outside air, and reacting at 25 ℃ and 150rpm for 24 h; the recombinant escherichia coli and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times in a fed-batch mode, a substrate and a product are removed after each reaction is finished, and the mannose yield is 72% after the recombinant escherichia coli and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times.

Example 51, 10-Ethyleneisoalloxazine chloride salt as regenerated NAD⁺The catalyst and recombinant Escherichia coli MtDH-BL21 are coupled to catalyze mannitol to prepare mannose

The procedure is as in example 4, except that bridged flavins are 1, 10-ethylene isoalloxazine chloride, at a concentration of 0.1 mM. The amount of mannose in the reaction system was measured at 168mM after the end of the reaction.

After the recombinant escherichia coli and the 1, 10-ethylene isoalloxazine chloride are repeatedly used for 5 times, the yield of mannose is 52 percent.

Example 6 8-chloro-1, 10-ethylene-based isoalloxazine chloride salt as regenerated NAD⁺The catalyst and recombinant Escherichia coli MtDH-BL21 are coupled to catalyze mannitol to prepare mannose

The procedure is as in example 4, except that bridged flavin is 8-chloro-1, 10-ethylene isoalloxazine chloride, at a concentration of 0.1 mM. The amount of mannose in the reaction system was 173mM as measured after the completion of the reaction.

After the recombinant escherichia coli and the 8-chloro-1, 10-ethylene isoalloxazine chloride are repeatedly used for 5 times, the yield of mannose is 61%.

Example 7 construction of recombinant E.coli GlyDH-BL21 expressing Glycerol dehydrogenase

Cloning a glycerol dehydrogenase gene GlyDH (GenBank: AAC43051.1) derived from E.coli K12 to a pET-28a vector to obtain a recombinant plasmid pET-28a-GlyDH, and then transforming the recombinant plasmid to E.coli BL21 to obtain the recombinant Escherichia coli GlyDH-BL21 capable of over-expressing the GlyDH gene.

Gene GlyDH and pET-28a vector, then transformed into e.coli.bl21, preparation of relevant media, etc. according to the method of the third edition of molecular cloning protocols (yellow banking, et al, china, scientific press, 2002); the primers required were all synthesized by Suzhou Jinweizhi Biotechnology, Inc.

Example 8 expression of recombinant E.coli GlyDH-BL21

The expression method is the same as example 3, except that the recombinant Escherichia coli is GlyDH-BL 21.

Example 9 regeneration of intracellular NAD with 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt⁺The catalyst is coupled with recombinant Escherichia coli GlyDH-BL21 to catalyze glycerol to prepare dihydroxyacetone DHA

(1) In 10mL of 100mM Tris-HCl buffer, pH9.5, the initial concentration of glycerol was 10g/L, NAD⁺The concentration was 5mM, the concentration of 7-trifluoromethyl-1, 10-ethylideneisoalloxazine was 0.1mM, the wet weight concentration of the recombinant E.coli GlyDH-BL21 obtained in example 8 was 10g/L, and the reaction solution was connected to the outside air. Reacting at 30 ℃ and 150rpm for 2 hours, and measuring the concentration of DHA in the reaction system to be 3.05g/L after the reaction is finished.

(2) The recombinant Escherichia coli GlyDH-BL21 and 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times in a fed-batch mode, a substrate and a product are removed after each reaction is finished, and the DHA yield is 27% after the recombinant Escherichia coli GlyDH-BL21 and 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times.

Example 10 construction of recombinant Bacillus subtilis MtDH-Bs168 expressing mannitol dehydrogenase

The mannitol dehydrogenase gene MtDH optimized by the codon in the embodiment 1 is cloned to a pMA5 vector to obtain a recombinant plasmid pMA5-MtDH, and then the recombinant plasmid is transformed into Bacillus subtilis B.subtilis 168 to obtain the recombinant Bacillus subtilis MtDH-Bs168 capable of over-expressing the MtDH gene.

Wherein the pMA5 plasmid was purchased from Biofeng Biofeng; bacillus subtilis 168 was purchased from zemer flies.

Recombinant ligation of gene GlyDH and pMA5 vector, transformation to E.coli.BL21, preparation of related culture medium, etc. was performed according to the method of the third edition of molecular cloning protocols (Huang Petang et al, China, science publishers, 2002); the primers required were all synthesized by Suzhou Jinweizhi Biotechnology, Inc.

Example 11 expression of MtDH-BL21 recombinant Bacillus subtilis

The recombinant Bacillus subtilis MtDH-Bs168 constructed in example 10 was cultured in LB medium, then inoculated into 200mL of liquid LB medium at an inoculum size of 1%, and cultured at 37 ℃ and 200rpm for 24 hours to obtain wet cells of recombinant Bacillus subtilis.

Example 12 regeneration of intracellular NAD with 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt⁺The catalyst and the recombinant bacillus subtilis MtDH-Bs168 are coupled to catalyze mannitol to prepare mannose

Mannitol initial concentration 50mM, NAD in 10mL100mM pH9.5 potassium phosphate buffer⁺The concentration was 0.5mM, the concentration of 7-trifluoromethyl-1, 10-ethylene-based isoalloxazine was 0.1mM, the wet weight concentration of the recombinant Bacillus subtilis MtDH-Bs168 obtained in example 11 was 10g/L, and the reaction solution was communicated with the outside air. The reaction was carried out at 25 ℃ and 150rpm for 12 hours, and the amount of mannose in the reaction system was measured to be 42mM after the completion of the reaction. The recombinant bacillus subtilis and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times in a fed-batch mode, a substrate and a product are removed after each reaction is finished, and the mannose yield is 71% after the recombinant bacillus subtilis and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times.

Example 13 regeneration of intracellular NAD with 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt⁺The catalyst is coupled with clostridium acetobutylicum to prepare ethanol, butanol and acetoin

The acetone butanol clostridium (C.acetobutylium)428 is statically cultured to a logarithmic phase at 37 ℃ by using a P2 culture medium, 7-trifluoromethyl-1, 10-ethylene base isoalloxazine is added into a culture solution of the logarithmic phase (the concentration of the 7-trifluoromethyl-1, 10-ethylene base isoalloxazine is 0.5mM), and the mixture is statically and anaerobically fermented for 72 hours at 37 ℃, so that the final ethanol yield is 1g/L, the butanol yield is 12.3g/L, and the acetoin yield is 0.8 g/L.

Wherein, the P2 fermentation medium is divided into four parts which are respectively configured:

1. glucose 60 g/L;

2. 2.2g/L of ammonium acetate, 0.5g/L of monopotassium phosphate and 0.5g/L of dipotassium phosphate;

3. 0.01g/L of sodium chloride, 0.01g/L of manganese sulfate monohydrate, 0.01g/L of ferrous sulfate heptahydrate and 0.2g/L of magnesium sulfate heptahydrate;

4. biotin 0.01mg/L, vitamin B11 mg/L, p-aminobenzoic acid 1 mg/L.

Wherein the concentration of each component is the final concentration of the component in the P2 fermentation medium.

Separately sterilizing solution 1 and solution 2 at 121 deg.C under high pressure steam for 15min, separately dissolving solution 3 and solution 4 in sterile ultrapure water, and filtering with 0.22 μm water system sterile filter membrane. Directly mixing the separately sterilized solution 1 and solution 2 based on 1L fermentation medium as a fermentation system, and adding 50 μ L each of sterilized solution 3 and sterilized solution 4 into the mixed solution 1 and solution 2.

And (2) repeatedly using clostridium acetobutylicum and 7-trifluoromethyl-1, 10-ethylene isoalloxazine for 5 times in a fed-batch mode, removing a substrate and a product after each reaction is finished, and finally obtaining 1.3g/L of ethanol, 12.5g/L of butanol and 0.76g/L of acetoin after 5 times of repeated use.

Example 14 regeneration of intracellular NAD with 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt⁺The catalyst is coupled with yeast to prepare the ethanol

Yeast (Saccharomyces) BY4741 is aerobically cultured in a composite culture medium at 30 ℃ and 200rpm until the logarithmic phase, then 7-trifluoromethyl-1, 10-ethylene base isoalloxazine is added into the culture solution at the logarithmic phase (-the concentration of the trifluoromethyl-1, 10-ethylene base isoalloxazine is 0.5mM), aerobically fermented at 32 ℃ and 200rpm for 30h, and the final ethanol yield is 11.6 g/L.

The compound culture medium comprises the following components: 10g/L peptone, 5g/L yeast powder, 90g/L glucose and 9g/L NaCl.

The yeast and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times in a fed-batch mode, substrates and products are removed after each reaction is finished, and after the yeast and the 7-trifluoromethyl-1, 10-ethylene isoalloxazine are repeatedly used for 5 times, the final ethanol yield is 12 g/L.

Comparative example 1 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt as regenerated NAD⁺The catalyst and mannitol dehydrogenase are coupled to catalyze mannitol to prepare mannose

In contrast to example 4, the in vitro reaction was carried out in 10mL of 100mM potassium phosphate buffer pH9.5, with an initial concentration of 250mM mannitol, NAD⁺The concentration is 1mM, the concentration of 7-trifluoromethyl-1, 10-ethylene isoalloxazine is 0.5mM, the dosage of mannitol dehydrogenase is 10 MuM, and the reaction liquid is communicated with the outside air. The reaction was carried out at 25 ℃ and 150rpm for 24 hours, and the amount of mannose in the reaction system was measured to be 100mM after the completion of the reaction. The yield of mannose is significantly lower than that of example 4, and the reaction system cannot be reused.

Wherein the concentration of the mannitol dehydrogenase is equal to the concentration of the mannitol dehydrogenase obtained after 10g/L wet cell disruption and purification of the recombinant Escherichia coli.

Comparative example 2 use of 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt as regenerated NAD⁺The catalyst and glycerol dehydrogenase are coupled to catalyze glycerol to prepare dihydroxyacetone DHA

In contrast to example 9, the initial concentration of glycerol was 10g/L, NAD in 10mL of 100mM Tris-HCl buffer pH9.5 under in vitro conditions⁺The concentration is 10mM, the concentration of 7-trifluoromethyl-1, 10-ethylene isoalloxazine is 0.2mM, glycerol dehydrogenase is 7 mu M, and the reaction liquid is communicated with the outside air. Reacting at 30 ℃ and 150rpm for 2 hours, and measuring the concentration of DHA in the reaction system to be 2.51g/L after the reaction is finished. The DHA yield is significantly lower than that of example 9, and the reaction system cannot be reused.

Wherein the concentration of the glycerol dehydrogenase is equal to the concentration of the glycerol dehydrogenase obtained after 10g/L of recombinant Escherichia coli wet cell disruption and purification.

Comparative example 3 regeneration of intracellular NAD with 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt⁺The catalyst and mannitol dehydrogenase are coupled to catalyze mannitol to prepare mannose

In contrast to example 12, in 10mL of 100mM potassium phosphate buffer pH9.5 under in vitro conditions,mannitol initial concentration 50mM, NAD⁺The concentration is 0.5mM, the concentration of 7-trifluoromethyl-1, 10-ethylene isoalloxazine is 0.1mM, and the mannitol dehydrogenase is 6 mu M, and the reaction liquid is communicated with the outside air. The reaction was carried out at 25 ℃ and 150rpm for 12 hours, and the amount of mannose in the reaction system was measured to be 38mM after the completion of the reaction. The yield of mannose was significantly lower than that of example 12, and the reaction system could not be reused.

Comparative example 4 use of 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride salt as regenerated NAD⁺The catalyst and mannitol dehydrogenase recombinant escherichia coli before codon optimization are coupled to catalyze mannitol to prepare mannose

The construction and expression methods of recombinant E.coli were the same as in examples 1, 2 and 3, except that the mannitol dehydrogenase gene MtDH was MtDH _ Ag before codon optimization.

Comparison with example 4, in 10mL of 100mM potassium phosphate buffer pH9.5, with an initial concentration of 250mM mannitol, NAD⁺The concentration is 1mM, the concentration of 7-trifluoromethyl-1, 10-ethylene base isoalloxazine is 0.5mM, the wet weight concentration of the recombinant escherichia coli obtained in comparative example 4 is 10g/L, and the reaction liquid is communicated with the outside air. The reaction was carried out at 25 ℃ and 150rpm for 24 hours, and the amount of mannose in the reaction system was 83mM as measured after the completion of the reaction. The mannose yield is obviously lower than that of example 4, which shows that the mannitol dehydrogenase recombinant Escherichia coli substrate tolerance before codon optimization is low.

Sequence listing

<110> Nanjing university of industry

<120> an oxidation method using whole cells as a 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

1. An oxidation method using whole cells as a catalyst, characterized in that bridged flavin is used as intracellular NAD (P)⁺Regenerated catalyst, and intracellular NAD (P) -dependent⁺Coupled to the redox-enzyme-catalyzed oxidation reaction of (a) to form intracellular NAD (P)⁺Regenerating the circulating system;

the catalytic system comprises whole cells, depends on NAD (P)⁺The oxidoreductase substrate, bridged flavin and buffer solution, and the catalytic system also comprises externally added NAD (P)⁺Said exogenously added NAD (P)⁺Is 0.05-5 mM; the catalytic system is dependent on NAD (P)⁺The substrate of the oxidoreductase is subjected to oxidation reaction, after the oxidation reaction is finished, solid-liquid separation is carried out on a catalytic system, and NAD (P) -dependent precipitates are supplemented into the obtained precipitates⁺The substrate and the buffer solution of the oxidoreductase are used for oxidation reaction, and the whole cells and the bridged flavin are reused for 3-8 times; or the catalytic system comprises whole cells, bridged flavin, and cell culture fluid, wherein the cell culture fluid comprises NAD (P) -dependent⁺The catalytic system catalyzes the dependence on NAD (P)⁺Carrying out oxidation reaction on a substrate of the oxidoreductase, carrying out solid-liquid separation on a catalytic system after the oxidation reaction is finished, supplementing a cell culture solution into the obtained precipitate, and continuing the oxidation reaction, wherein the whole cells and bridged flavin are repeatedly used for 3-8 times;

the bridged flavin has the following structural general formula:

wherein R is₁And R₂Independently of one another, from: hydrogen, methyl, trifluoromethyl, methoxy, halogen atom, nitro and amino; r₃Selected from: hydrogen, C1-C5 alkyl, phenyl and benzyl; x^-Selected from: halide ion, nitrate radical, triflate ion.

2. An oxidation process according to claim 1, wherein the whole cells are selected from Escherichia coli (Escherichia coli) cells, yeast (saccharomyces) cells, Clostridium acetobutylicum (Clostridium acetobutylicum) cells, Corynebacterium glutamicum (Corynebacterium glutamicum) cells, Bacillus subtilis (Bacillus subtilis) cells, streptomyces (streptomycetetaceae) cells, Aspergillus niger (Aspergillus niger) cells, or recombinant cells of the above cells.

3. The oxidation process according to claim 2, wherein the whole cells are cells cultured to a log phase, a stationary phase or a decline phase.

4. An oxidation process according to claim 2, wherein the whole cells are E.coli (Escherichia coli) cells or recombinant E.coli cells.

5. The oxidation process according to claim 2, wherein the recombinant cell is a recombinant cell obtained by knocking out a cellular gene, expressing an endogenous gene or expressing an exogenous gene.

6. The oxidation process of claim 1, wherein the concentration of whole cells in the catalytic system is from 0.01 to 100 g/L.

7. An oxidation process according to claim 1, wherein the bridged flavin is 7-trifluoromethyl-1, 10-ethylideneisoalloxazine chloride, 8-chloro-1, 10-ethylideneisoalloxazine chloride or 1, 10-ethylideneisoalloxazine chloride.

8. An oxidation process according to claim 1, wherein the bridged flavins are present in the catalytic system in a concentration of 0.001-10 mM.

9. The oxidation process of claim 1, wherein the NAD (P) -dependent oxidation is carried out⁺The oxidoreductase of (a) is selected from all enzymes of 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, EC1.18.1.X, EC1.20.1.X, and EC1.22.1. X.

10. The oxidation process of claim 9, wherein the NAD (P) -dependent oxidation is carried out⁺The oxidoreductase of (2) is mannitol dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, or acetyl-CoA dehydrogenase.

11. An oxidation process according to claim 1, wherein the catalytic system is NAD (P) -dependent⁺The initial concentration of substrate for the oxidoreductase of (3) is 50-1000 mM.