CN117417867A - Enterobacter cloacae (Enterobacter cloacae) ZJUT16 and application thereof - Google Patents

Enterobacter cloacae (Enterobacter cloacae) ZJUT16 and application thereof Download PDF

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CN117417867A
CN117417867A CN202311417495.3A CN202311417495A CN117417867A CN 117417867 A CN117417867 A CN 117417867A CN 202311417495 A CN202311417495 A CN 202311417495A CN 117417867 A CN117417867 A CN 117417867A
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enterobacter cloacae
reaction
ecntr
nitroacetophenone
enzyme
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欧志敏
王娜娜
刘美
邓立霞
王延妮
戴艳梅
陶紫娟
张清宇
黄长顺
罗亮丽
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses enterobacter cloacae (Enterobacter cloacae) ZJUT16 and application thereof, wherein the enterobacter cloacae (Enterobacter cloacae) bacterial strain ZJUT16 with the capability of reducing nitro compounds (particularly m-nitroacetophenone) is obtained by screening from soil through a substrate enrichment culture strategy. The nitroreductase EcNTR contained in the preparation method can be used for reducing nitro compounds to prepare aromatic amine, and is particularly used for converting m-nitroacetophenone to prepare an important intermediate m-aminoacetophenone in synthesis of phenylephrine medicines, antitumor medicines and sedative hypnotics.

Description

Enterobacter cloacae (Enterobacter cloacae) ZJUT16 and application thereof
Technical Field
The invention relates to a nitroreductase EcNTR-producing enterobacter cloacae (Enterobacter cloacae) ZJUT16 strain from Enterobacter cloacae bacteria and application thereof in biocatalysis.
Background
meta-Aminoacetophenone (3-amino acetophenone), CAS number 99-03-6, molecular formula C 8 H 9 NO, molecular weight 135.16, density 1.1031g/ml, boiling point 289-290 ℃, water insolubility and easy solubility in organic solvents. The meta-amino acetophenone is not only an important fine organic synthesis intermediate, but also has important application in the preparation of phenylephrine medicines, anti-tumor medicines and sedative hypnotic zaleplon. The meta-aminoacetophenone downstream products also have important uses, for example: m-hydroxyacetophenone is an intermediate for synthesizing adrenomimetic drugs; the m-chloroacetophenone can be used for synthesizing new bronchodilatory drugs, anti-purpura drugs and other drugs; 3-acetamido phenethylThe ketone is an intermediate for synthesizing the sedative hypnotic indiplon. The m-aminoacetophenone can also be used for synthesizing m-dimethylaminoacetophenone and 2-bromoacetophenone. Meanwhile, the meta-aminoacetophenone can be used as an analysis reagent for the analysis of acetoacetic acid, and is also applied to the preparation of photosensitive materials as a photosensitive reagent.
Because of the importance of m-aminoacetophenone in the production of phenylephrine, antineoplastic and sedative hypnotic zaleplon, the development of a more economical and efficient method for preparing m-aminoacetophenone is particularly important. The reduction of m-nitroacetophenone can be used for preparing m-aminoacetophenone, and the current synthesis methods of m-aminoacetophenone can be divided into chemical methods and biological methods. The chemical synthesis is generally obtained by reducing m-nitroacetophenone by iron powder or hydrogen, the traditional iron powder reduction method has long history, simple process and mature technology, but the production process can produce a large amount of wastewater and iron mud which contain aromatic amine and are difficult to treat, and the environment is easy to be seriously polluted. The method for preparing the m-aminoacetophenone by using hydrogen as a reducing agent through catalytic reduction of m-nitroacetophenone has the advantages of high product yield, high purity, little pollution and simple product separation, and has obvious technical and cost advantages compared with the traditional iron powder reduction method. However, the nitro functional group, benzene ring and carbonyl in the m-nitronitroacetophenone structure can be reduced in the hydrogenation process, and the selectivity control difficulty of the product is high. In summary, the problems associated with the chemical synthesis of meta-aminoacetophenone are: complicated steps, expensive catalyst, environmental pollution, severe temperature, high equipment requirements, no compliance with the new development concept of green environmental protection, and the like. The biological method is one of the most effective methods for synthesizing the m-aminoacetophenone at present due to the characteristics of mild reaction conditions, environmental friendliness, high reaction yield, low cost and the like.
Disclosure of Invention
The invention aims to provide an NAD (P) H-dependent nitroreductase EcNTR-producing strain and application thereof, wherein the strain is obtained by screening enterobacter cloacae (Enterobacter cloacae) ZJUT16 strain from soil by a substrate enrichment culture method, a section of NAD (P) H-dependent nitroreductase EcNTR gene is extracted from the strain, and genetic engineering bacteria are recombined and constructed on the basis of the gene, so that the strain is used for preparing a pharmaceutical intermediate m-aminoacetophenone by reducing m-nitroacetophenone. And a dimethyl sulfoxide (DMSO) -water phase (phosphate buffer) system is established by rational design to optimize a reaction system, solve the problem of poor water solubility of a substrate, and further improve the stability and activity of enzyme in the medium system, thereby improving the biocatalysis efficiency and opening up a green, environment-friendly, safe and efficient biocatalysis path for preparing the m-aminoacetophenone.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a enterobacter cloacae (Enterobacter cloacae) ZJUT16, which is preserved in the China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC), with a preservation number: CGMCC No.28310, preservation date: 2023 8, 31, deposit address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
In a second aspect, the invention provides an application of the enterobacter cloacae (Enterobacter cloacae) ZJUT16 in preparing an aminobenzene derivative by reducing a nitrobenzene derivative; the nitrobenzene derivative is one or more than two of o-nitrobenzaldehyde, 3-nitrophthalic acid, m-nitroacetophenone, p-nitroacetophenone, 2-fluoronitrobenzene, 5-bromo-1, 3-difluoro-2-nitrobenzene, 2-bromo-1-chloro-3-nitrobenzene, 2-bromo-5-fluoronitrobenzene and 5-bromo-2-nitrobenzaldehyde (preferably 3-nitrophthalic acid or m-nitroacetophenone, particularly preferably m-nitroacetophenone).
In a third aspect, the invention provides a (NAD (P) H dependent) nitroreductase EcNTR derived from said Enterobacter cloacae (Enterobacter cloacae), said nitroreductase EcNTR having an amino acid sequence as set forth in SEQ ID NO: 4.
In a fourth aspect, the present invention provides a recombinant expression plasmid of nitroreductase EcNTR, wherein the amino acid sequence of the nitroreductase EcNTR is shown in SEQ ID NO: 4.
In one embodiment of the invention, the vector of the recombinant expression plasmid is a pET28a (+) vector.
Further, the nucleotide sequence of the coding gene of the (NAD (P) H dependent) nitroreductase EcNTR is shown as SEQ ID NO: 3.
Still further, the recombinant expression plasmid is prepared by combining SEQ ID NO:3 is inserted between the BamHI site and the NdeI site of the pET28a (+) vector.
Specifically, the recombinant expression plasmid is constructed as follows:
s1, performing PCR by using genome DNA of enterobacter cloacae (Enterobacter cloacae) ZJUT16 as a template through the following primers to obtain a target gene:
F：5’-GGAATTCCATATGATGGATCTTCAACTCA-3’；
R：5’-CGCGGATCCTTAAAGGATGGAA-3’。
s2: double-enzyme cutting is carried out on the target gene in the step S1 by using restriction enzymes BamHI and NdeI to obtain an insert; double-enzyme cutting is carried out on the pET28a (+) vector by using restriction enzymes BamHI and NdeI to obtain a linearization vector; and connecting the inserted gene with the linearization vector to obtain the recombinant expression plasmid.
In a fifth aspect, the invention provides a recombinant genetically engineered bacterium constructed by the recombinant expression plasmid.
In one embodiment of the invention, the host strain of the recombinant genetically engineered bacterium is E.coli BL21 (DE 3).
In a fifth aspect, the invention provides an application of the recombinant genetically engineered bacterium in preparing an aminobenzene derivative by reducing a nitrobenzene derivative; the nitrobenzene derivative is one or more than two of o-nitrobenzaldehyde, 3-nitrophthalic acid, m-nitroacetophenone, p-nitroacetophenone, 2-fluoronitrobenzene, 5-bromo-1, 3-difluoro-2-nitrobenzene, 2-bromo-1-chloro-3-nitrobenzene, 2-bromo-5-fluoronitrobenzene and 5-bromo-2-nitrobenzaldehyde (preferably 3-nitrophthalic acid or m-nitroacetophenone, particularly preferably m-nitroacetophenone).
Specifically, the application is as follows: the recombinant genetically engineered bacterium (E.coli BL21 (DE 3) -pET28a (+) -EcNTR) is subjected to induction culture to obtain wet thalli or nitro reductase EcNTR pure enzyme extracted by ultrasonic crushing, separation and purification of the wet thalli is used as a catalyst, nitrobenzene derivatives are used as substrates, organic solvents are used as auxiliary solvents, glucose is used as auxiliary substrates, NAD (P) H is used as coenzyme, buffer solution with pH of 6-10 (preferably pH 7) is used as a reaction medium to form a reaction system, the reaction is carried out for 6-30H (preferably 30 ℃ and 180rpm and 12H) at the temperature of 25-45 ℃, and the obtained reaction solution is separated and purified to obtain the aminobenzene derivatives; the nitrobenzene derivative is one or more than two of o-nitrobenzaldehyde, 3-nitrophthalic acid, m-nitroacetophenone, p-nitroacetophenone, 2-fluoronitrobenzene, 5-bromo-1, 3-difluoro-2-nitrobenzene, 2-bromo-1-chloro-3-nitrobenzene, 2-bromo-5-fluoronitrobenzene and 5-bromo-2-nitrobenzaldehyde (preferably 3-nitrophthalic acid or m-nitroacetophenone, particularly preferably m-nitroacetophenone).
Further, the separation is purified as: the reaction solution was centrifuged (8000 rpm,4 ℃ C., 10 min), the resulting supernatant was extracted with (equal volume) ethyl acetate (3 times), the organic layers were combined, anhydrous Na 2 SO 4 Drying and volatilizing the solvent at normal temperature to obtain the aminobenzene derivative.
Preferably, the wet cell is prepared as follows:
inoculating recombinant genetic engineering bacteria (preferably E.coli BL21 (DE 3) -pET28a (+) -EcNTR) containing a gene encoding nitroreductase EcNTR to LB liquid medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ and 160-200rpm (preferably 180 rpm) for 14-18h (preferably 16 h) to obtain seed liquid; inoculating the seed solution into LB liquid medium containing 50 μg/mL kanamycin at 2% -4% (preferably 3%) volume inoculation amount, culturing at 37deg.C at 160-200rpm (preferably 180 rpm) to OD 600 Adding IPTG with a final concentration of 0.4mM at 0.6-0.8, fermenting and culturing at 23 ℃ and 180rpm for 16h to obtain fermentation liquor; centrifuging (8000 rpm,4 ℃ for 10 min) to obtain bacterial precipitate, re-suspending with physiological saline, centrifuging (8000 rpm,4 ℃ for 10 min), and collecting wet bacterial.
Preferably, the buffer solution is a phosphate buffer salt solution (PB) at pH7.0, 0.1M; in the reaction system, the final concentration of the substrate is 10-50mM (preferably 20 mM), the final concentration of glucose is 0.05-0.2M (preferably 0.1M), and the final concentration of NAD (P) H is 0.2-0.8mM (preferably 0.5 mM);
when the catalyst is wet thalli, the final concentration of the catalyst in the reaction system is 50-250g/L (preferably 100 g/L) based on the mass of the wet thalli; when the catalyst is pure enzyme, the final concentration of the catalyst in the reaction system is 0.5-3g/L (preferably 2.5 g/L) calculated by pure enzyme;
the organic solvent is one or more than two of DMSO, methanol, ethanol, isopropanol and acetone (preferably DMSO), and the volume of the organic solvent is 10% of the volume of the reaction system.
Compared with the prior art, the invention has the beneficial effects that:
the invention screens and obtains the enterobacter cloacae (Enterobacter cloacae) fungus strain ZJUT16 with the capability of reducing nitro compounds (especially m-nitroacetophenone) from soil through a substrate enrichment culture strategy. The nitroreductase EcNTR contained in the preparation method can be used for reducing nitro compounds to prepare aromatic amine, and is particularly used for converting m-nitroacetophenone to prepare an important intermediate m-aminoacetophenone in synthesis of phenylephrine medicines, antitumor medicines and sedative hypnotics. On the basis, a nitroreductase EcNTR gene is obtained from a Enterobacter cloacae strain by means of genetic engineering such as PCR amplification and the like, and is constructed into pET28a (+) -EcNTR recombinant plasmid, E.coli BL21 (DE 3) -pET28a (+) -EcNTR recombinant engineering bacteria are further constructed, and the recombinant engineering bacteria are used for reducing nitro compounds to prepare aromatic amine, and are particularly used for converting m-nitroethanone to prepare an important intermediate m-aminoacetophenone in synthesis of phenylephrine medicines, antitumor medicines and sedative hypnotic medicines. The nitroreductase EcNTR has a wide substrate spectrum and can catalyze the reduction reaction of various nitro compounds.
M-nitroacetophenone is poorly soluble in water, which hampers the biocatalytic process of producing m-aminoacetophenone. In order to solve the problems that the organic substrate is difficult to dissolve in water and the mass transfer in the water phase is low, the method is low in cost and environment-friendly. According to the invention, a DMSO-water phase (phosphate buffer) system is introduced and constructed in an aqueous medium to increase the solubility of a substrate so as to improve the biocatalysis efficiency, the yield is only 12.5% under the condition of 20mM substrate concentration in a single-water phase system without DMSO, and under the same other conditions, the yield is increased to 99.9% in the DMSO-water phase (phosphate buffer) system, and the yield is increased by 7.9 times.
Drawings
FIG. 1 is a colony morphology of a plate of Enterobacter cloacae (Enterobacter cloacae) strain.
FIG. 2 shows a ZJUT16 phylogenetic tree of Enterobacter cloacae (Enterobacter cloacae).
FIG. 3 shows the electrophoresis pattern of Enterobacter cloacae strain genome nucleic acid, and Lane1 and Lane 2 are Enterobacter cloacae strain genomes.
FIG. 4 shows the PCR amplification of nucleic acid of EcNTR target gene using Enterobacter cloacae bacterial genome as template.
FIG. 5 is a diagram showing PCR nucleic acid electrophoresis of colonies obtained by picking different single colonies; each single colony sample was randomly selected from the same plate, followed by colony PCR.
FIG. 6 shows the nucleic acid electrophoresis pattern of successfully constructed pET28a (+) -EcNTR recombinant plasmid, wherein Lane1, lane 2 and Lane3 are pET28a (+) -EcNTR recombinant plasmids.
FIG. 7 shows the SDS-PAG E of sodium dodecyl sulfate polyacrylamide protein electrophoresis of nitroreductase EcNTR. The molecular weight of the nitroreductase EcNTR is 28.1kDa, and the purified protein accords with the actual molecular weight. Lane1 and Lane 2 are pET28a (+) -EcNTR crushed supernatant and crushed sediment; lane3 is a flow-through liquid; lane 4 and 5 are washing solutions 1 and 5; lane6, 7, 8 are eluents 1, 2, 3.
FIG. 8 is a schematic diagram of an EcNTR catalytic reaction system.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the enterobacter cloacae (Enterobacter cloacae) strain used in the embodiment of the invention is obtained by soil enrichment culture screening in the laboratory, and the nitroreductase EcNTR gene is extracted from the screened Enterobacter cloacae strain. The nitroreductase EcNTR used in the embodiment of the invention is prepared by engineering bacteria constructed by the laboratory according to the invention.
The LB liquid culture medium comprises the following components: 10g/L NaCl, 10g/L peptone, 5g/L yeast extract, water as solvent, pH7.0.
The LB solid culture medium comprises the following components: 10g/L NaCl, 10g/L peptone, 5g/L yeast extract, 20g/L agar, water as solvent, pH7.0.
PB buffer used in the examples of the present invention means a 0.1M phosphate buffer at pH7.0.
Example 1: screening and identification of enterobacter cloacae (Enterobacter cloacae) ZJUT16
The process for screening nitroreductase-rich strains from soil by substrate enrichment culture comprises the following steps: enrichment culture, flat plate re-screening, seed culture, fermentation culture, biotransformation, gas chromatography detection of products, acquisition of excellent strains, and strain identification, wherein the method comprises the following steps:
1. strain screening
(1) Collecting a soil sample: soil samples were collected from the areas of Hangzhou, huzhou, zhejiang, etc., and deep soil samples of about 5-10 cm below the surface were taken.
(2) Enrichment culture: 1g of soil samples in different areas are respectively mixed in 10mL of 0.9% physiological saline in a shaking way and kept stand for 1h. 1mL of the soil sample clear liquid is inoculated into 50mL of a primary screening liquid culture medium sterilized at 115 ℃ for 30min, and the culture medium contains 10mmol/L of m-nitronitroacetophenone (filtered by a 0.22 mu m filter head), and is subjected to shaking culture at a constant temperature of 180rpm at 30 ℃ for 2-3 days until the liquid culture medium is obviously turbid.
Primary screening of liquid medium: (NH) 4 ) 2 SO 4 5g/L，MgSO 4 0.25g/L，K 2 HPO 4 ·3H 2 O 1g/L，KH 2 PO 4 1g/L, water as solvent, pH7.0, and sterilizing at 115℃for 30min.
(3) And (3) re-screening: and (3) dipping a loop of the primary screening liquid culture medium bacterial liquid after the culture in the step (2) by using an inoculating loop, inoculating the primary screening liquid culture medium bacterial liquid into a re-screening solid culture medium containing 10mmol/L of m-nitroacetophenone by using a plate streaking method, and culturing in a constant temperature incubator at 30 ℃ until a large number of single colonies grow out. Single colonies are selected from the solid medium plates of the double screen, inoculated to liquid complete culture mediums with corresponding numbers respectively, and cultured in a constant temperature shaking table at 30 ℃ and 180rpm until the culture mediums are obviously turbid, and a large number of thalli grow out. And inoculating to solid complete culture medium for inclined preservation.
The formula of the rescreening solid culture medium is as follows: (NH) 4 ) 2 SO 4 5g/L，MgSO 4 0.25g/L，K 2 HPO 4 ·3H 2 O 1g/L，KH 2 PO 4 1g/L, 20g/L of agar, water as solvent, pH7.0.
The formula of the liquid complete culture medium is as follows: 30g/L glucose, 3g/L yeast extract powder, (NH) 4 ) 2 SO 4 5g/L，MgSO 4 0.25g/L，K 2 HPO 4 ·3H 2 O 1g/L，KH 2 PO 4 1g/L, water as solvent, pH7.0, and wet heat sterilizing at 115 deg.C for 30min.
The formula of the solid complete culture medium is as follows: 30g/L glucose, 3g/L yeast extract powder, (NH) 4 ) 2 SO 4 5g/L，MgSO 4 0.25g/L，K 2 HPO 4 ·3H 2 O 1g/L，KH 2 PO 4 1g/L, 20g/L of agar, water as solvent, pH7.0, and sterilizing at 115 ℃ for 30min under moist heat.
(4) Fermentation culture: and (3) picking a proper amount of thalli from the inclined plane preserved in the step (3) into 50mL of corresponding seed culture medium by using an inoculating loop, and culturing at 30 ℃ and 180rpm overnight until the culture medium is obviously turbid, wherein a large amount of thalli grow out. After the seed solution was cultured, the seed solution was inoculated to 150mL of a fermentation medium at an inoculum size of 3% by volume, and the culture was continued at 30℃and 180rpm for 24 hours until the medium became clear and cloudy, and a large amount of cells grew out. The fermentation broth was centrifuged at 8000rpm at 4℃for 10min and the supernatant was discarded. The bacterial pellet was resuspended in 20mL of physiological saline, centrifuged at 8000rpm at 4℃for 10min, and the supernatant was discarded, and the wet bacterial pellet was collected for subsequent bioconversion reactions.
Fermentation medium composition: 30g/L glucose, 3g/L yeast extract powder, (NH) 4 ) 2 SO 4 5g/L，MgSO 4 0.25g/L，K 2 HPO 4 ·3H 2 O 1g/L，KH 2 PO 4 1g/L, water as solvent, pH7.0, and wet heat sterilizing at 115 deg.C for 30min.
(5) Bioconversion reactions: adding the wet thallus of the step (4) into pH7.0,0.1M Phosphate Buffer (PB), adding DMSO as a cosolvent and adding substrate M-nitronitroacetophenone to form a 2mL reaction system, wherein the addition amount of wet bacterial precipitation is 100g/L (calculated by the volume of the buffer), the addition amount of the DMSO is 10%, and the addition amount of the substrate is 10mM; bioconversion reaction is carried out at 30 ℃ for 24 hours, after the reaction is finished, the reaction solution is centrifuged (8000 rpm,4 ℃ for 10 min), the reaction supernatant is extracted with an equal volume of ethyl acetate (3 times) and then combined, and the collected extract is subjected to anhydrous Na 2 SO 4 Drying, volatilizing ethyl acetate at normal temperature to obtain the product of m-aminoacetophenone, and detecting by gas chromatography, specifically using the method of example 4.
The excellent strain with high conversion rate is obtained by screening and is named as strain ZJUT16.
2. Identification of strains:
(1) Colony morphology
The strain ZJUT16 is inoculated to a solid complete culture medium, and is cultured for 2 days at 30 ℃, and the colony morphology is observed, so that the following observation can be obtained: the colony is large and round, the edge is neat, the surface is smooth, the center is slightly raised, the color is yellow and white, the texture of the colony is moist and sticky, the colony can be easily picked, and the observation diagram of the plate colony is shown in fig. 1.
(2) Identification of bacteria 16S
The bacterial 16S identification (the identification sequence is shown as SEQ ID NO: 1) is carried out on the strain ZJUT16 by the Hangzhou branch of the biological technology limited company of Beijing, NCBI is carried out sequence alignment, the strain ZJUT16 is identified as Enterobacter cloacae bacterial strain by combining with colony morphology, the strain ZJUT16 is named enterobacter cloacae (Enterobacter cloacae) ZJUT16 and is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC), and the preservation number is CGMCC NO:28310, 2023, 8, 31 th day of deposit address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
SEQ ID NO:1 sequence:
TGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCACAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAG。
example 2: recombinant genetically engineered bacterium E.coli BL21 (DE 3) -pET28a (+) -EcNTR
(1) The following primers F and R were designed based on the oxidoreductase Gene (Gene ID:75145039, SEQ ID NO: 2) of Enterobacter cloacae (Enterobacter cloacae) in NCBI database, and were synthesized by Hangzhou division, a biological technology Co., ltd. In Beijing, and transferred to the other.
Primer F:5'-GGAATTCCATATGATGGATCTTCAACTCA-3' the number of the individual pieces of the plastic,
primer R:5'-CGCGGATCCTTAAAGGATGGAA-3'.
SEQ ID NO:2 sequence:
ATGGATCTTCAACTCACCGGTAAGACCGCGCTGGTTACGGGCGCAACCGCAGGCATTGGTTTGGCCATCGCCCGCACGCTCGCTCAAGAAGGGGTTGCCGTCACCCTTACCGGACGCGACCCGGCAAAGCTGCAAAAAGCCGCGGCCACCATCACCGACGCGACGCCTGACGCACAGGTCTCCACCGTTGTCGTTGACCTCGGTACCATGAATGGAGCAGAAGCCCTGTTCGCCGCCTGCCCTGATACCGATATTCTGATTAATAACCTGGGGTTCTACGAAGCCAAAGCCTTCGCGGATATTAACGATGAAGACTGGCTGCGCATGTTCAACACCAATGTGATGTCCGGCGTCCGCCTCTCACGCCACTACTTCCCGCGCATGCTGGAGCGGAACTGGGGACGGGTAATTTTTATATCCAGCGAAGTGGGCGCCTTTACGCCGCCAGACATGGTGCATTACGGCGTCAGCAAATCAGCGCAGCTTGCTGTTTCGCGCGGTATGGCCGAACTGACCCGGGGAACCGGCGTGACGGTTAACAGCGTGCTGCCGTCGGCGACGCGCTCGGACGGCATTATTGAGTACCTTCGCCAGACCGCGCCTGCACCGGATATGACCGATCGGGAGATCGAAGCGCATTTCTTCCAGACCTACCGCCCCAGCTCGTTGATAGCCAGAATGATTGAGGCAGACGAGATCGCGGCGATGGTCGCTCTGTTAGCCAGCCCACTGGGCGCGGCATCCAACGGGGCGGCTGTACGCGTCGAGGGCGGCACGTTCCGTTCCATCCTTTAA。
(2) The Enterobacter cloacae ZJUT single colony selected in example 1 was inoculated into a liquid complete medium (composition same as in example 1), cultured overnight at 37℃and 180rpm, and then Enterobacter cloacae genomic DNA was extracted by referring to the bacterial genome extraction kit instructions of the company Limited of the holothurian biotechnology, and verified by 0.9% nucleic acid agarose gel electrophoresis (FIG. 2).
(3) And (3) taking the genomic DNA obtained in the step (2) as a template, carrying out PCR amplification of a target fragment (EcNTR) by adopting the primer F and the primer R in the step (1), verifying the amplified fragment by 0.9% nucleic acid agarose gel electrophoresis (figure 3), and sending the amplified fragment to Hangzhou division of Beijing qingke biotechnology Co Ltd for sequencing verification.
PCR amplification system: the total volume of the reaction was 50. Mu.L, wherein the genome template was 2. Mu.L, primer F2. Mu.L, primer R2. Mu.L, 2*Hieff PCR Master Mix 25. Mu.L, ddH 2 O 19μL。
The PCR amplification procedure was: (1) 94℃for 5min, (2) 94℃for 30s, (3) 55℃for 30s, (4) 72℃for 1min, and (5) 72℃for 10min, wherein (2) (3) (4) was cycled 35 times. 2*Hieff PCR Master Mix from Beijing Optimu Biotechnology Co.
(4) And (3) recovering and purifying the target fragment (EcNTR) of the PCR product by using a column type DNA gel recovery and extraction kit, and then carrying out double enzyme digestion on the target fragment (EcNTR) and the pET28a (+) vector by using Takara endonuclease BamHI and Takara endonuclease NdeI respectively, wherein the enzyme digestion system reacts for 3 hours at 37 ℃.
The target fragment (EcNTR) double enzyme digestion reaction system is as follows: bamHI 3. Mu.L, ndeI 3. Mu.L, 10X K6. Mu.L, ecNTR 33. Mu.L, ddH 2 O 15μL。
The pET28a (+) vector double enzyme digestion reaction system is as follows: bamHI 3. Mu.L, ndeI 3. Mu.L, 10X K18. Mu.L, pET28a (+) 156. Mu.L.
The nucleotide sequence of the target fragment EcNTR is shown as SEQ ID NO:3, shown in the following:
ATGGATCTTCAACTCACCGGTAAGACCGCGCTGGTTACGGGCGCAACCGCAGGCATTGGTTTGGCCATCGCCCGCACGCTCGCTCAAGAAGGGGTTGCCGTCACCCTTACCGGACGCGACCCGGCAAAGCTGCAAAAAGCCGCGGCCACCATCACCGACGCGACGCCTGACGCACAGGTCTCCACCGTTGTCGTTGACCTCGGTACCATGAATGGAGCAGAAGCCCTGTTCGCCGCCTGCCCTGATACCGATATTCTGATTAATAACCTGGGGTTCTACGAAGCCAAAGCCTTCGCGGATATTAACGATGAAGACTGGCTGCGCATGTTCAACACCAATGTGATGTCCGGCGTCCGCCTCTCACGCCACTACTTCCCGCGCATGCTGGAGCGGAACTGGGGACGGGTAATTTTTATATCCAGCGAAGTGGGCGCCTTTACGCCGCCAGACATGGTGCATTACGGCGTCAGCAAATCAGCGCAGCTTGCTGTTTCGCGCGGTATGGCCGAACTGACCCGGGGAACCGGCGTGACGGTTAACAGCGTGCTGCCGTCGGCGACGCGCTCGGACGGCATTATTGAGTACCTTCGCCAGACCGCGCCTGCACCGGATATGACCGATCGGGAGATCGAAGCGCATTTCTTCCAGACCTACCGCCCCAGCTCGTTGATAGCCAGAATGATTGAGGCAGACGAGATCGCGGCGATGGTCGCTCTGTTAGCCAGCCCACTGGGCGCGGCATCCAACGGGGCGGCTGTACGCGTCGAGGGCGGCACGTTCCGTTCCATCCTTTAA。
the amino acid sequence of the target fragment EcNTR encoded protein is shown as SEQ ID NO:4, as follows:
MDLQLTGKTALVTGATAGIGLAIARTLAQEGVAVTLTGRDPAKLQKAAATITDATPDAQVSTVVVDLGTMNGAEALFAACPDTDILINNLGFYEAKAFADINDEDWLRMFNTNVMSGVRLSRHYFPRMLERNWGRVIFISSEVGAFTPPDMVHYGVSKSAQLAVSRGMAELTRGTGVTVNSVLPSATRSDGIIEYLRQTAPAPDMTDREIEAHFFQTYRPSSLIARMIEADEIAAMVALLASPLGAASNGAAVRVEGGTFRSIL*。
(5) The target fragment and the vector after enzyme digestion are recovered and purified through a column type DNA gel recovery kit, then the enzyme digestion EcNTR and the pET28a (+) vector are connected according to a corresponding enzyme linked system, the connection system reacts for 30min at 25 ℃, and the obtained recombinant plasmid pET28a (+) -EcNTR is transferred into E.coli DH5 alpha competent cells (purchased from Beijing family biotechnology Co., ltd.) and coated on LB solid plates containing 50 mug/mL kanamycin, and is cultured overnight at 37 ℃.
The system for enzyme linkage of the digested target gene EcNTR and the vector pET28a (+) is as follows: 5*Quick Loading Buffer 2. Mu.L, pET28a (+) 1.7. Mu.L, ecNTR 2.8. Mu.L, T4 DNA Quick Ligase 1. Mu.L, ddH 2 O2.5. Mu.L. Wherein T4 DNA Quick Ligase and 5*Quick Loading Buffer are purchased from general Bio Inc.
The method for introducing the pET28a (+) -EcNTR recombinant plasmid into E.coli DH5 alpha competent cells comprises the following steps: after melting the E.coli DH5 alpha competent cells on ice for 20-30min, adding 10 mu L of recombinant plasmid into 100 mu L of E.coli DH5 alpha competent cells, lightly blowing and mixing, then carrying out ice bath for 20min, carrying out hot shock for 90s in a water bath at 42 ℃ and carrying out ice bath for 2min, adding 0.9mL of LB non-antibiotic liquid medium, and resuscitating for 1h at 37 ℃ and 180 rpm. After the resuscitated bacterial liquid is instantaneously separated in a centrifuge, 900 mu L of supernatant is discarded, and after 100 mu L of bacterial liquid and sediment are evenly mixed by blowing, the mixture is coated on LB solid medium containing 50 mu g/mL kanamycin, and the mixture is cultured overnight at 37 ℃.
(6) 8 single colonies were picked from the overnight plates of step (5) and individually grown on 30. Mu.L ddH corresponding to the number 2 Mixing by blowing in O, transferring 10 μl of each mixed solution into LB liquid medium containing 50 μg/mL kanamycin, culturing at 37deg.C and 180rpm overnight, boiling the rest 20 μl respectively for 10min, cooling, performing colony PCR as template, performing verification by 0.9% nucleic acid agarose gel electrophoresis (FIG. 4), selecting colony of PCR target band, and transferring to Hangzhou division of Beijing family biotechnology Co Ltd for sequencing verification.
The single colonies are randomly selected, and the colony PCR system is as follows: the total volume of the reaction was 25. Mu.L, in which 1. Mu.L of the bacterial liquid template, 1. Mu.L of the primer F, 1. Mu.L of the primer R, 2*Hieff PCR Master Mix 12.5. Mu.L, ddH were boiled 2 O9.5. Mu.L. The PCR amplification procedure was: (1) 94℃for 5min, (2) 94℃for 30s, (3) 55℃for 30s, (4) 72℃for 1min, and (5) 72℃for 10min, wherein (2) (3) (4) was cycled 30 times. 2*Hieff PCR Master Mix from Beijing Optimuno Biotechnology Co., ltd, primer was prepared in the same manner as in step (1).
(7) And (3) extracting the E.coli DH5 alpha-pET 28a (+) -EcNTR recombinant bacteria with correct sequencing verification through a Takara plasmid DNA extraction kit, performing detection verification through 0.9% nucleic acid agarose gel electrophoresis (figure 5), and transferring the E.coli DH5 alpha-pET 28a (+) -EcNTR recombinant bacteria into E.coli BL21 (DE 3) competent cells (synchronous 5), namely E.coli BL21 (DE 3) -pET28a (+) -EcNTR recombinant genetic engineering bacteria.
Example 3: e.coli BL21 (DE 3) -pET28a (+) -EcNTR wet cell culture, nitroreductase EcNTR protein induction, expression and purification
(1) Fermentation culture
10. Mu.L of E.coli BL21 (DE 3) -pET28a (+) -EcNTR recombinant genetically engineered bacterium glycerol bacteria obtained in example 2 was inoculated into 50mL of LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured overnight at 37℃and 180rpm to obtain a seed solution.
Inoculating the seed solution into 150mL LB liquid medium containing 50 μg/mL kanamycin at an inoculum size of 3% by volume, culturing at 37 ℃ at 180rpm to OD 600 Adding IPTG with a final concentration of 0.4mM at 23 ℃ for induction culture at 180rpm for 16h to obtain fermentation broth. Centrifuging the fermentation broth at 8000rpm and 4deg.C for 10min, re-suspending the obtained thallus precipitate with physiological saline, centrifuging at 8000rpm and 4deg.C for 10min, and collecting wet thallus.
(2) Crude enzyme solution
The wet thalli in the step (1) is resuspended by 0.1M pH7.0 PB buffer solution, the resuspension is placed on ice for 30min, ultrasonic crushing is carried out (the conditions are that ultrasonic power is 360W, ultrasonic is 3s, intermittent is 7s, ultrasonic is 10 min), the crushed liquid after ultrasonic is centrifuged at 8000rpm and 4 ℃ for 10min, and then sediment is removed, thus obtaining supernatant which is crude enzyme liquid.
(3) Pure enzyme
The EcNTR pure enzyme solution can be prepared by treating and purifying the crude enzyme solution according to the specification of the Biyundian His-Tag reduction-resistant chelating filler, and the specific operation is as follows:
filtering the crude enzyme solution obtained in the step (2) by a 0.45 mu m filter membrane, removing floating sediment, ultrafiltering and concentrating the crude enzyme solution volume to 4ml, and mixing the crude enzyme solution with Beyogold TM His-tag reduction-resistant chelating medium (purchased from Shanghai Biyun biotechnology Co., ltd.) in a volume ratio of 8:1 after slowly shaking in a shaker at 0℃and 40rpm for 1h, the sample was applied to Beyogold TM His-tag filling column is firstly washed by non-denaturingWashing the column with washing liquid for 5 times to remove the impurity protein, and eluting 1 column volume each time; then eluting with non-denaturing eluent for 8 times, 1 column volume each time, verifying the collected flow-through liquid, washing liquid and eluent by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (figure 6), comparing the purity and molecular weight of the enzyme, collecting all eluents containing target proteins, ultrafiltering and concentrating the eluents, and freeze-drying to obtain the pure enzyme of nitroreductase EcNTR.
Non-denaturing lysate: 50mM NaH 2 PO 4 ·2H 2 O，300mMNaCl，pH8.0；
Non-denaturing wash: 50mM NaH 2 PO 4 ·2H 2 O,300mM NaCl,2mM imidazole, pH8.0;
non-denaturing eluent: 50mM NaH 2 PO 4 ·2H 2 O,300mM NaCl,50mM imidazole, pH8.0.
(4) Enzyme activity identification
The nitroreductase activity was determined by spectrophotometry by detecting the change in absorbance at 340 nm. The method for measuring the activity of the reductase comprises the following steps: to 2mL of the reaction system (100 mM PB buffer, pH 7.0), 10mM meta-nitroacetophenone and 0.5mM ADH were added, and after 1 minute incubation at 30℃a suitable amount of the enzyme solution prepared in example 3 (200. Mu.L of 0.136mg/mL of the pure enzyme aqueous solution of the fusion enzyme) was added, followed by rapid mixing, and the change in absorbance at 340nm was detected. The calculation formula of the enzyme activity is as follows: enzyme activity (U) =ew×v×103/(6220×l); wherein EW is the change of absorbance at 340nm within 1 min; v is the volume of the reaction solution, and the unit is mL;6220 is the molar extinction coefficient of NADPH, unit L/(mol cm); l is the optical path distance in cm. Nitroreductase enzyme activity is defined as: under the above conditions, the amount of enzyme catalyzing the consumption of 1. Mu. Mol of NADPH per minute is defined as 1 enzyme activity unit. The nitroreductase enzyme activity was determined to be 0.81U.
Example 4: e.coli BL21 (DE 3) -pET28a (+) -EcNTR recombinant bacterium induced expression condition optimization
E.coli BL21 (DE 3) -pET28a (+) -EcNTR was optimized for Inducer (IPTG) concentration, and the final concentrations of IPTG in step (1) of example 3 were 0.2, 0.4, 0.6, 0.8 and 1.0mM, respectively, and the substrate m-nitroacetophenone was 25mM, and the other operations were the same, to prepare each wet cell.
The total volume of the reaction system was 2mL: the substrate m-nitroacetophenone is 25mM, glucose is 0.1M, NAD (P) H is 0.5mM, the final concentration of the added wet thalli of different concentrations of IPTG induction culture is 100g/L, the volume concentration of DMSO is 10%, and the reaction medium is 0.1M pH7.0 PB buffer solution to be complemented to 2mL. Reacting at 30deg.C and 180rpm for 12 hr, centrifuging (8000 rpm, 4deg.C and 10 min), extracting the supernatant with equal volume of ethyl acetate for 3 times, mixing, and concentrating the extractive solution with anhydrous Na 2 SO 4 Ethyl acetate was volatilized at normal temperature after drying, and the contents of the substrate and the product were measured by Gas Chromatography (GC), and the yield was calculated, and the results are shown in table 1 below.
Gas chromatography detection conditions: the instrument is Shimadzu GC2014; column CP7502 (25 m.times.0.25 mm.times.0.25 μm); sample inlet temperature 250 ℃, column temperature 130 ℃, detector 250 ℃, flow rate 2mL/min, split ratio 1:15, sample injection amount is 1 mu L.
The yield calculation formula is as follows:
in the formula (1), M S : molecular weight of the substrate; m is M P : molecular weight of the product; q: the mass of the substrate at the beginning of the reaction; p: mass of product at the end of the reaction
The results are shown in Table 1 below.
TABLE 1 catalytic effect of different concentrations of IPTG on the Induction of Wet cells
The concentration of the Inducer (IPTG) has a great influence on the expression of the target protein by engineering bacteria. When the concentration of the inducer is low, the enzyme yield is low, and the inducer has certain toxicity to cells, and when the concentration is too high, the inducer can influence the growth of cells, so that the proper concentration of the inducer is necessary to be selected. As can be seen from the results in the table, the conversion increased with increasing IPTG concentration at below 0.4mM and decreased with increasing IPTG concentration at above 0.4mM, so that the optimum inducer concentration was selected at 0.4 mM.
Example 5: influence of the reaction time on the reduction reaction
The total volume of the reaction system was 2mL: the substrate m-nitronitroethanone 25mM, NAD (P) H0.5 mM, glucose 0.1M, DMSO concentration 10% by volume, the final concentration of wet cell addition was 100g/L, and 2mL was made up with 0.1M pH7.0 PB buffer. The reaction was carried out at 30℃and 180rpm for 6h, 12h, 18h, 24h and 30h, respectively. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 Ethyl acetate was evaporated at normal temperature after drying and the results were shown in table 5 using gas chromatography GC as described in example 4.
TABLE 2 influence of different reaction times on the reduction reaction
In order to be suitable for industrial production, the time cost is lower as the reaction time required for reaching the same yield is smaller in the reaction process, so the reaction is designed for exploring the yield of the product in the reaction system under different reaction times. As can be seen from Table 2, the reaction time has a large effect on the conversion rate, and at the reaction time of 6 hours, the conversion rate reaches 88.1%, and the enzyme catalyzed reaction is very fast, but the conversion rate is still lower than that of 12 hours, so that the subsequent reaction time is still 12 hours.
Example 6: influence of the reaction temperature on the reduction reaction
The total volume of the reaction system was 2mL: the substrate m-nitronitroethanone 20mM, NAD (P) H0.5 mM, glucose 0.1M, DMSO concentration 10% by volume, the final wet cell addition concentration 100g/L, and 2mL of the buffer pH7.0 PB buffer solution 0.1M. The reaction was carried out at 25, 30, 35, 40, 45℃and 180rpm for 12h, respectively. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 DryingEthyl acetate was then evaporated at normal temperature and tested by GC as described in example 4, the results are shown in table 3.
TABLE 3 influence of different reaction temperatures on the reduction reaction
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And detecting catalytic activity of the EcNTR at different temperatures, exploring influence of different temperatures on the activity of the EcNTR, and designing a reduction reaction system. As can be seen from Table 3, the maximum catalytic activity of the enzyme was observed at 30℃reaction. Although the increase in temperature increases the number of collisions between the enzyme and the substrate molecules in the low temperature range, and thus increases the catalytic rate, when the temperature is higher than 30 ℃, the enzyme activity starts to decrease, probably because the hydrogen bonds which maintain the structure of the enzyme are destroyed due to the fact that the enzyme absorbs a large amount of energy due to the fact that the temperature is too high, the spatial morphology of the protein is changed and the effective catalytic function cannot be exerted, so that the reaction is preferably controlled at 30 ℃ to be most favorable for catalysis.
Example 7: influence of the reaction pH on the reduction reaction
The total volume of the reaction system was 2mL: the wet cells were added with final concentration of 100g/L, substrate M-nitroacetophenone 20mM, NAD (P) H0.5 mM, glucose 0.1M, DMSO concentration 10% by volume, and 2mL of the resulting mixture was supplemented with buffers (6.0, 7.0, 0.1M PB,8.0, 9.0, 0.1M Tris-HCl,10.0, 0.05M glycine-NaOH buffer) having pH values of 6.0, 7.0, 8.0, and 10.0, respectively. The reaction was carried out at 30℃and 180rpm for 24 hours, respectively. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 Ethyl acetate was evaporated at normal temperature after drying and the results were shown in table 4 using gas chromatography GC as described in example 4.
TABLE 4 influence of different pH on the reduction reaction
From Table 4, it can be seen that EcNTR is most active at pH7. Enzymes exhibit activity in the optimum pH range, and above or below the optimum pH, the activity of the enzyme is reduced. Too high or too low a pH can change the charge state of the substrate molecules and enzyme molecules, causing unfolding and exposure of internal structures, resulting in loss of enzyme activity, thereby affecting the binding of the enzyme to the substrate; in addition to the great influence on the enzyme activity, pH also has a great influence on the stability of the enzyme. Too high a pH too low may alter the conformation of the active center of the enzyme or even alter the structure of the whole enzyme molecule to denature it for inactivation. Thus, ecNTR is suitable for reaction in PB buffer at pH7.
Example 8: effect of co-solvent on reduction reaction
The total volume of the reaction system was 2mL: the substrate M-nitroacetophenone was 20mM, NAD (P) H was 0.5mM, glucose was 0.1M, and the final concentration of the wet cell was 100g/L, and 2mL was supplemented with 0.1M pH7.0 PB buffer. Respectively using DMSO, methanol, ethanol, isopropanol and acetone with volume concentration of 10% as cosolvent, and reacting for 12h at 30 ℃ and 180 rpm. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 Ethyl acetate was evaporated at normal temperature after drying and the results were shown in table 5 using gas chromatography GC as described in example 4.
TABLE 5 influence of different cosolvents on reduction reactions
Most of the catalytic reactions of carbonyl reductase are carried out in an aqueous phase, but the substrate m-nitronitroacetophenone has poor water solubility, and a cosolvent is required to be added to improve the solubility of the substrate m-nitronitroacetophenone in water. This example examined the effect of 5 organic solvents on the catalytic activity of the fusion enzyme in total. As can be seen from Table 5, acetone has a great influence on the catalytic activity of the nitroreductase EcNTR, possibly due to some toxicity of the organic solvent to the enzyme or the denaturation of its protein. The DMSO has small toxicity to the protein while promoting the dissolution of the substrate m-nitronitroacetophenone, and promotes the forward progress of the reduction reaction, so that the DMSO is selected as the most suitable cosolvent for the subsequent reaction.
Example 9: influence of substrate concentration on reduction reaction
The total volume of the reaction system was 2mL: glucose 0.1M, NAD (P) H0.5 mM, wet cell addition final concentration of 100g/L, DMSO volume concentration of 10%, substrate m-nitronitroacetophenone final concentration of 10, 20, 30, 40, 50mM respectively, reaction medium 0.1M pH7.0 PB buffer make up 2mL. The reaction was carried out at 30℃and 180rpm for 24 hours. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 Ethyl acetate was evaporated at normal temperature after drying and the results were shown in table 6 using gas chromatography GC as described in example 4.
TABLE 6 investigation of optimum substrate concentration
In order to meet the needs of industrial production, the substrate concentration in the reaction system needs to be increased as high as possible under the condition of ensuring the yield of the product and high enantioselectivity. The reaction was thus designed to explore the product conversion at different substrate concentrations. As is clear from Table 6, as the concentration of m-nitroacetophenone as the substrate increases, the concentration of m-nitroacetophenone in the solution becomes higher at a concentration of 20mM or more, which means that the higher the concentration of m-nitroacetophenone in the solution, the lower the conversion rate of m-nitroacetophenone to the substrate, which means that the higher concentration of m-nitroacetophenone has an inactivating effect on the enzyme protein, whereas when the initial concentration of m-nitroacetophenone is 20mM or less, the limitation of the substrate to the enzyme is lower, and therefore, when the conversion reaction of m-nitroacetophenone is carried out, 20mM is selected as the optimal substrate concentration.
Example 10: capability of nitroreductase EcNTR to convert different substrates
Reaction system 2mL: the final concentration of the different substrates is 20mM, glucose is 0.1M, NAD (P) H is 0.5mM, the final concentration of the wet bacterial cells is 100g/L, the volume concentration of DMSO is 10%, and the reaction medium is 0.1M pH7.0 PB buffer solution to make up 2mL. The reaction was carried out at 30℃and 180rpm for 24 hours. After the reaction was completed, the reaction solution was centrifuged (8000 rpm,4 ℃,10 min), the reaction supernatant was extracted 3 times with an equal volume of ethyl acetate, and the extracts were combined, and the extracts were taken over anhydrous Na 2 SO 4 Ethyl acetate was evaporated at normal temperature after drying and the results were shown in table 7 using gas chromatography GC as described in example 4.
TABLE 7 screening Table of the ability of nitroreductase EcNTR to convert different substrates
The present experiment examined the catalytic reduction ability of the nitroreductase EcNTR on different aromatic nitro compounds, as can be seen from Table 7, the nitroreductase EcNTR has a better reduction ability on most aromatic nitro compounds. When the meta position or para position of the benzene ring connected with the nitro is connected with electron withdrawing groups such as carbonyl, carboxyl and the like, the nitroreductase EcNTR shows stronger catalytic activity; catalytic activity decreases when a group is attached to the ortho position of the benzene ring, and it is hypothesized that when a group is attached to the ortho position, the meta-position resistance increases when the active site of nitroreductase EcNTR binds to the substrate, resulting in a decrease in conversion.

Claims (4)

1. Enterobacter cloacae (Enterobacter cloacae) ZJUT16, deposited in China general microbiological culture Collection center, accession number: CGMCC NO:28310, date of preservation: 2023 8, 31, deposit address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. The use of enterobacter cloacae (Enterobacter cloacae) ZJUT16 according to claim 1 for the preparation of aminobenzene derivatives by reduction of nitrobenzene derivatives; the nitrobenzene derivative is one or a mixture of more than two of o-nitrobenzaldehyde, 3-nitrophthalic acid, m-nitroacetophenone, p-nitroacetophenone, 2-fluoronitrobenzene, 5-bromo-1, 3-difluoro-2-nitrobenzene, 2-bromo-1-chloro-3-nitrobenzene, 2-bromo-5-fluoronitrobenzene and 5-bromo-2-nitrobenzaldehyde.
3. The use according to claim 2, wherein: the nitrobenzene derivative is 3-nitrophthalic acid or m-nitroacetophenone.
4. A use according to claim 3, wherein: the nitrobenzene derivative is m-nitroacetophenone.
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