Nitroreductase mutant, preparation method and application

Abstract

The invention provides a nitroreductase mutant as well as a preparation method and application thereof. The nitroreductase mutant is characterized in that compared with wild type nitroreductase, the nitroreductase mutant has at least one amino acid mutation in the following seven sites or functional equivalent sites: the 15th site, the 69th site, the 172nd site and the 173rd site; the wild type nitroreductase has an amino acid sequence as shown in SEQ ID NO: 1. The obtained nitroreductase mutant has higher catalytic activity, specificity and thermal stability. In industrial application, the catalytic efficiency can be remarkably improved, reagent waste is reduced, the production cost is saved, generation of redundant by-products in chemical reaction is reduced, and environmental pollution is reduced.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular, to a nitroreductase mutant, a preparation method and an application thereof. More particularly, it relates to a nitroreductase mutant and a preparation method thereof, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant strain, a method for preparing a nitroreduction product, a kit and an application thereof. Background Art

[0002] Nitroreductases are a class of cytoplasmic enzymes that are dependent on FMN or FAD and can use NAD(P)H to achieve the reduction metabolism of nitroaromatic compounds and nitroheterocyclic derivatives. Nitroreductases have a wide range of applications, including drug activation, hydroxylamine synthesis, and pollutant biometabolism. However, the current catalytic efficiency of nitroreductases is low, which seriously limits their widespread use in clinical applications.

[0003] Therefore, there is an urgent need in the art to develop a nitroreductase with higher catalytic activity. Summary of the invention

[0004] This application is completed by the inventor based on the following findings:

[0005] 1) The catalytic activity of existing nitroreductase is low.

[0006] 2) The application of nitroreductase with high catalytic activity in nitroreduction reactions can increase the yield, save reagent usage, and cause less pollution to the environment.

[0007] Therefore, one object of the present invention is to provide a nitroreductase mutant with high catalytic activity.

[0008] The present application aims to solve one of the technical problems in the related art to a certain extent.

[0009] In the first aspect of the present application, the present application proposes a nitroreductase mutant. According to an embodiment of the present application, the mutant comprises: compared with the wild-type nitroreductase, the mutant has at least one amino acid mutation in the following four sites or functionally equivalent sites: the 15th, the 69th, the 172nd, and the 173rd.

[0010] According to the embodiments of the present application, the inventors used error-prone PCR technology and site-directed mutagenesis technology to molecularly modify the wild-type nitroreductase BaNTR, thereby obtaining a nitroreductase BaNTR mutant with improved catalytic activity. The modified nitroreductase has higher catalytic activity, specificity and thermal stability. In industrial applications, it can significantly improve catalytic efficiency and reduce reagent waste, save production costs, reduce the generation of excess by-products in chemical reactions, and reduce environmental pollution.

[0011] It should be noted that the equivalent functional sites refer to the amino acid sites corresponding to the same functional domain in a nitroreductase. For example, a certain nitroreductase has two functional domains A and B with the same functions, and the sites in the B domain and the corresponding sites in the A domain are functionally equivalent sites to each other.

[0012] It should be noted that the amino acid positions in the amino acid sequence of the nitroreductase mutant described in the present application are located with reference to the amino acid positions in the amino acid sequence of the wild-type nitroreductase BaNTR.

[0013] According to an embodiment of the present application, the above-mentioned nitroreductase mutant further includes at least one of the following technical features:

[0014] According to an embodiment of the present application, the wild-type nitroreductase has an amino acid sequence as shown in SEQ ID NO:1.

[0015] MATNDNFFNVLYERTSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIAYNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVSSRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:1).

[0016] Among them, the nucleic acid encoding the wild-type nitroreductase has a nucleotide sequence as shown in SEQ ID NO:2.

[0017] (SEQ ID NO:2).

[0018] According to an embodiment of the present application, the nitroreductase mutant has at least 80% identity with the wild-type nitroreductase.

[0019] According to an embodiment of the present application, the nitroreductase mutant has at least 90% identity with the wild-type nitroreductase.

[0020] According to an embodiment of the present application, the nitroreductase mutant has at least 95% identity with the wild-type nitroreductase.

[0021] According to an embodiment of the present application, the nitroreductase mutant has at least 98% identity with the wild-type nitroreductase. Generally speaking, the higher the identity of the amino acid sequence, the more similar its structure and function are.

[0022] According to an embodiment of the present application, the mutant has the following mutations: the 15th amino acid T mutates to S or A or K or V; the 69th amino acid Y mutates to N or H or C; the 172nd amino acid S mutates to P; the 173rd amino acid S mutates to P or D.

[0023] According to an embodiment of the present application, the mutant has any one of the following combined mutations: the 15th amino acid T mutates to S or A or K or V, the 69th amino acid does not mutate, the 172nd amino acid does not mutate, and the 173rd amino acid does not mutate; or the 15th amino acid T mutates to S or A or K or V, and the following three positions have at least one amino acid mutation: the 69th, the 172nd, and the 173rd; or the 69th amino acid Y mutates to N or H or C, the 15th amino acid does not mutate, the 172nd amino acid does not mutate, and the 173rd amino acid does not mutate; or the 69th amino acid Y mutates to N or H or C, and the following three positions have at least one amino acid mutation. or the amino acid S at position 172 mutates to P, the amino acid at position 15 does not mutate, the amino acid at position 69 does not mutate, and the amino acid at position 173 does not mutate; or the amino acid S at position 172 mutates to P, and the following three positions have at least one amino acid mutation: position 15, position 69, and position 173; or the amino acid S at position 173 mutates to P or D, the amino acid at position 15 does not mutate, the amino acid at position 69 does not mutate, and the amino acid at position 172 does not mutate; or the amino acid S at position 173 mutates to P or D, and the following three positions have at least one amino acid mutation: position 15, position 69, and position 172.

[0024] According to some specific embodiments of the present invention, different mutation sites produce different catalytic activities of nitroreductases, which can provide more options for actual production needs.

[0025] According to an embodiment of the present application, the mutant has any one of the following combined mutations: the 15th amino acid T mutates to A, and the 172nd amino acid S mutates to P; or the 69th amino acid Y mutates to N; or the 69th amino acid Y mutates to H, and the 173rd amino acid S mutates to P; or the 69th amino acid Y mutates to N, and the 173rd amino acid S mutates to D; or the 15th amino acid T mutates to K, the 69th amino acid Y mutates to N, and the 173rd amino acid S mutates to D.

[0026] According to some specific embodiments of the present application, when the amino acid sequence of the nitroreductase mutant has the above mutation, the catalytic activity is stronger. In addition, the above mutation sites can also be selectively combined to obtain a nitroreductase mutant suitable for the required catalytic activity.

[0027] It should be noted that the above mutant amino acid sequences are all obtained by mutation based on the sequence of SEQ ID NO:1.

[0028] According to an embodiment of the present application, the mutant has an amino acid sequence shown in SEQ ID NO: 3-7.

[0029] MATNDNFFNVLYERASTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIAYNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVPSRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:3);

[0030] MATNDNFFNVLYERTSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIANNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVSSRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:4);

[0031] MATNDNFFNVLYERTSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIAHNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVSPRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:5);

[0032] MATNDNFFNVLYERTSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIANNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVSDRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:6);

[0033] MATNDNFFNVLYERKSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIANNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAKATGWDTCAIGGFNPQALTDEFNVSDRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:7).

[0034] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the nitroreductase mutant described in the first aspect of the present application. The nitroreductase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.

[0035] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule described in the second aspect of the present application. According to an embodiment of the present application, the expression vector can express a nitroreductase mutant in vitro.

[0036] According to an embodiment of the present application, the above expression vector may further include at least one of the following technical features:

[0037] According to an embodiment of the present application, the expression vector is selected from at least one of a pET series vector, a shuttle vector, a phage and a viral vector.

[0038] According to an embodiment of the present application, the pET series vectors include pET-3a, pET-11a, pET-15a, pET-28a, pET-30a, pET-32a and pET-22b.

[0039] According to an embodiment of the present application, the pET series vector is pET-28a. According to a specific embodiment of the present application, pET-28a is used as an expression vector, and the relative activity and volume enzyme activity of the nitroreductase mutant are higher.

[0040] In the fourth aspect of the present application, the present application provides a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the second aspect of the present application or the expression vector described in the third aspect of the present application. The recombinant cell is used to express or secrete the nitroreductase mutant described in the first aspect of the present application.

[0041] According to an embodiment of the present application, the above-mentioned recombinant cell may further include at least one of the following technical features:

[0042] According to an embodiment of the present application, the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

[0043] In the fifth aspect of the present application, the present application proposes a recombinant strain. According to an embodiment of the present application, the recombinant strain expresses the nitroreductase mutant described in the first aspect of the present application. The nitroreductase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.

[0044] In the sixth aspect of the present application, the present application proposes a method for preparing a nitroreductase mutant. According to an embodiment of the present application, the method comprises: culturing the recombinant cell described in the fourth aspect of the present application or the recombinant strain described in the fifth aspect under conditions suitable for protein expression to obtain the nitroreductase mutant. According to a specific embodiment of the present application, a large number of nitroreductase mutants with high catalytic activity can be cultivated by this method.

[0045] In the seventh aspect of the present application, the present application proposes a method for preparing a nitroreductase mutant. According to an embodiment of the present application, the method comprises: subjecting a wild-type nitroreductase to a mutation treatment so as to obtain a nitroreductase mutant library; subjecting a nitroreductase substrate to a first catalytic treatment under the condition of the presence of the nitroreductase mutant library; and subjecting the first catalytic treatment product to a screening treatment so as to obtain the nitroreductase mutant described in the first aspect of the present application. According to a specific embodiment of the present application, a nitroreductase mutant with high catalytic activity can be obtained by this method.

[0046] According to an embodiment of the present application, the method for preparing a nitroreductase mutant may further include at least one of the following technical features:

[0047] According to an embodiment of the present application, the mutation treatment is selected from at least one of random mutation and site-directed mutation. According to a specific embodiment of the present application, the mutation treatment includes but is not limited to methods such as using chemical mutagens or PCR error introduction.

[0048] According to an embodiment of the present application, the nitroreductase substrate includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts.

[0049] According to an embodiment of the present application, the nitro reduction product is 6-chloro-2-methyl-2H-indazol-5-amine.

[0050] According to an embodiment of the present application, the first catalytic treatment is carried out in the presence of NADPH.

[0051] According to an embodiment of the present application, the screening process includes obtaining a nitroreductase mutant having a relative activity higher than that of a control group.

[0052] According to an embodiment of the present application, the control group is wild-type nitroreductase.

[0053] In the eighth aspect of the present application, the present application proposes a method for preparing a nitro-reduction product. According to an embodiment of the present application, the method comprises: subjecting a nitro-reduction enzyme substrate to a second catalytic treatment in the presence of the nitro-reduction enzyme mutant described in the first aspect of the present application, the recombinant cell described in the fourth aspect, or the recombinant strain described in the fifth aspect, so as to obtain a nitro-reduction product. According to an embodiment of the present application, the nitro-reduction product is prepared using the method, with higher catalytic efficiency and lower environmental pollution.

[0054] According to an embodiment of the present application, the method for preparing a nitro-reduction product further includes at least one of the following technical features:

[0055] According to an embodiment of the present application, the nitroreductase substrate includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts.

[0056] According to an embodiment of the present application, the nitro reduction product is 6-chloro-2-methyl-2H-indazol-5-amine.

[0057] According to an embodiment of the present application, the second catalytic treatment system further includes an oxidation-reduction enzyme system.

[0058] According to an embodiment of the present application, the oxidoreductase system includes glucose dehydrogenase, glucose and coenzyme NADP.

[0059] According to an embodiment of the present application, the pH of the second catalytic treatment system is 7.5-8.

[0060] According to an embodiment of the present application, the second catalytic treatment is carried out in a 5% vanadium pentoxide aqueous solution.

[0061] According to an embodiment of the present application, the second catalytic treatment is carried out at 25-35°C.

[0062] According to an embodiment of the present application, the second catalytic treatment is carried out at 30°C.

[0063] In the ninth aspect of the present application, the present application proposes a kit. According to the embodiments of the present application, the kit comprises: the nitroreductase mutant described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the recombinant strain described in the fifth aspect. According to the specific embodiments of the present application, the kit has the advantages of low cost and convenient use.

[0064] According to an embodiment of the present application, the above-mentioned kit may further include at least one of the following technical features:

[0065] According to an embodiment of the present application, the kit further includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts.

[0066] According to an embodiment of the present application, the kit further includes at least one of a 5% vanadium pentoxide aqueous solution, glucose dehydrogenase, glucose, and coenzyme NADP.

[0067] In the tenth aspect of the present application, the present application proposes the use of the kit described in the ninth aspect in preparing a nitro-reduction product. According to a specific embodiment of the present application, the kit can be used to prepare a nitro-reduction product.

[0068] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0070] Figure 1 Schematic diagram of the synthesis of 6-chloro-2-methyl-2H-indazole-5-amine (ESTD04) from 6-chloro-2-methyl-5-nitro-2H-indazole (ESTD03) catalyzed by nitroreductase provided in the examples of the present application;

[0071] Figure 2 Schematic diagram of nucleotide gel electrophoresis results of error-prone PCR system optimization results provided in the embodiment of the present application; wherein, M: 2000bp DNA Marker; the lanes correspond to different Mn 2+ and Mg 2+ Concentration (Mn in lane 1 2+and Mg 2+ The concentrations are all 0 mM; Mn in lanes 2-4 2+ The concentrations were 0.2mM, 0.4mM and 0.6mM respectively; Mg in lanes 5-7 2+ The concentrations are 1mM, 2mM and 3mM respectively);

[0072] Figure 3 This is a schematic diagram of the nucleotide gel electrophoresis results of the saturation mutation result of the mutant Y69N provided in the example of the present application; wherein, M: 5000bp DNA Marker; the lanes correspond to the PCR products amplified using different primer pairs (lanes 1-4 correspond to the saturation mutation products of sites 15, 69, 172, and 173, respectively). DETAILED DESCRIPTION

[0073] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0074] definition

[0075] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0076] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0077] In this application, unless otherwise indicated, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0078] In this application, unless otherwise specified, the term "nitroreductase" refers to an enzyme that can catalyze the reduction reaction of nitroaromatic compounds to aromatic amines. In this application, "homology" has the conventional meaning in the art and refers to the "identity" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of the same nucleotides or amino acid residues between the two sequences to be compared after the best alignment, and the differences between the two sequences are randomly distributed over their entire length. In this application, the mutants are described according to their mutations at specific residues, and their positions are determined by comparison with the wild-type enzyme sequence SEQ ID NO:1 or the reference enzyme sequence SEQ ID NO:1.

[0079] In this application, unless otherwise indicated, the terms "mutant" and "variant" can be used interchangeably, "substitution" or "mutation" can be used interchangeably, and these expressions refer to amino acids relative to wild-type proteins, such as the wild-type sequence SEQ ID NO: 1 nitroreductase BaNTR, or derived from nitroreductase BaNTR, containing changes at one or more positions, i.e., substitutions, insertions and / or deletions, and still retaining their activity. Mutants can be obtained by various techniques known in the art, such as products of chemical synthesis, or produced from prokaryotic or eukaryotic hosts using recombinant technology. In particular, exemplary techniques for modifying DNA sequences encoding wild-type proteins for recombinant technology include, but are not limited to, directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides.

[0080] In this application, unless otherwise indicated, the term "substitution" with respect to an amino acid position or residue means that the amino acid at the specific position has been replaced by another amino acid. Substitutions may be conservative or non-conservative.

[0081] In this application, unless otherwise specified, the form of "XaY" is used to represent amino acid mutation or substitution, wherein a represents the position of the amino acid in SEQ ID NO: 1, X represents the wild-type amino acid species at position a in SEQ ID NO: 1, and Y represents the mutated amino acid species at position a in SEQ ID NO: 1. For example, "T15K" means that, compared with SEQ ID NO: 1, threonine T at position 15 of SEQ ID NO: 1 is replaced by lysine K.

[0082] In this application, unless otherwise specified, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of foreign DNA into cells.

[0083] In the present application, unless otherwise specified, the term "relative activity" refers to the enzyme activity of each nitroreductase relative to pet26b-BaNTR enzyme calculated when the enzyme activity of pet26b-BaNTR is 100%.

[0084] In this application, unless otherwise specified, the term "volumetric enzyme activity" refers to the rate of enzyme-catalyzed substrate conversion per unit time under certain conditions.

[0085] In this application, unless otherwise specified, the term "stereoisomer" refers to compounds with the same chemical constitution but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0086] In this application, unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0087] In the present application, unless otherwise specified, the term "salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.

[0088] The following is a detailed introduction to the embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. The details of some molecular cloning methods vary depending on the different vendors providing reagents, enzymes or kits, and should be performed according to the product instructions, and will not be described in detail in the embodiments.

[0089] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0090] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0091] Example 1: Construction of recombinant expression vector

[0092] The nitroreductase Escherichia coli engineered bacteria E.coli Trans 1-T1 / pET26b-BaNTR preserved in this laboratory was cultured, and the plasmid pET26b-BaNTR was extracted as a template. Subsequently, the gene fragment SEQ ID NO:2 was amplified using the following primers (Primers F and Primers R), and Nco I and Xho I restriction sites were added to both ends of SEQ ID NO:2. The amplified target fragment was recovered by gel cutting, and the amplified gene fragment was double-digested by adding restriction endonucleases Nco I and Xho I. At the same time, pet22b, pet28a, pet30a and pet32a were used as expression vectors, and these vectors were double-digested using the same restriction endonucleases Nco I and Xho I. After the digestion was completed, the target gene fragment and all expression vectors were purified by agarose gel electrophoresis, and the target fragment and the treated expression vector were recovered using an agarose gel DNA recovery kit. Subsequently, the target fragments were mixed with plasmids pet22b, pet28a, pet30a and pet32a that had been digested with Nco I and Xho I, and ligated at 16°C for 4 h under the action of T4 DNA ligase to obtain recombinant expression plasmids pET22b-BaNTR, pet28a-BaNTR, pet30a-BaNTR and pet32a-BaNTR.

[0093] The above four recombinant expression plasmids pET22b-BaNTR, pet28a-BaNTR, pet30a-BaNTR and pet32a-BaNTR were transformed into E. coli Trans 1-T1 competent cells respectively. Positive recombinants were screened on resistance plates containing kanamycin and ampicillin (medium components LB medium, peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L and agar 2%, antibiotic content 50mg / L) (pET22b and pet32a are ampicillin resistance, pet28a and pet30a are kanamycin resistance), single clones were picked, recombinant strains were cultured, and plasmids were extracted after plasmid amplification for sequencing verification.

[0094] Primers F: CATG CCATGG CCACAAACGACAACTTTTTTAACG(Nco I);

[0095] Primers R: CCG CTCGAG TTATTTTGCCCACTCGCTTACTTGCTC(Xho I).

[0096] Example 2: Construction of recombinant engineering strains, fermentation culture and enzyme activity determination

[0097] The correctly sequenced recombinant plasmids pET22b-BaNTR, pet28a-BaNTR, pet30a-BaNTR and pet32a-BaNTR constructed in Example 1 and the recombinant pet26b-BaNTR preserved in the laboratory were transformed into E. coli BL21 (DE3) competent cells, respectively, and after heat shock and recovery, they were spread on plates containing corresponding resistance, and inverted and cultured at 37°C overnight to obtain positive recombinant E. coli BL21 (DE3) / pET26b-BaNTR, E. coli BL21 (DE3) / pET22b-BaNTR, E. coli BL21 (DE3) / pET28a-BaNTR, E. coli BL21 (DE3) / pET30a-BaNTR and E. coli BL21 (DE3) / pET32a-BaNTR.

[0098] The five recombinant engineering bacteria constructed above were inoculated into LB medium containing corresponding resistance (the concentration of ampicillin and kanamycin was 50 mg / L) and cultured overnight at 37°C with shaking. The next day, the inoculation amount was 2% (v / v) and inoculated into a 250mL Erlenmeyer flask containing 60mL fermentation medium (10g / L yeast powder, 20g / L peptone, 15g / L glycerol, 10g / L NaCl, 3g / L KNO3, 1.74g / L K2HPO4, 1.36g / L KH2PO4 and 0.5g / L MgCl2), and cultured on a shaking table at 37°C and 220rpm. When the OD 600 of the culture reached 0.8, IPTG was added at a final concentration of 0.5mmol / L for induction. After induction at 22°C for 20h, the culture was centrifuged and the cells were collected.

[0099] The collected bacterial cells were resuspended in 50mM Na2HPO4-NaH2PO4 (pH=7.5), and the bacterial concentration was adjusted to 5g / L. Then ultrasonic disruption was performed. After the disruption was completed, the supernatant was centrifuged and the enzyme activity was determined using an enzyme marker. The above supernatant was used as a biocatalyst for enzyme activity determination. The determination system was: 178μL of substrate solution (1g / L ESTD03 dissolved in PBS buffer containing 0.5% Tween 80 (0.05mM, pH=7.5)) + 20μL of bacterial lysis supernatant + 2.5μL of NADPH (50mM) were mixed evenly and placed in an enzyme marker, and the absorbance values ​​of 0min and 4.5min were read at 340nm. The level of enzyme activity was determined by comparing the changes in absorbance values ​​(higher activity consumes more NADPH). The specific reaction data results are shown in Table 1 below:

[0100] Table 1 Bacterial weight and enzyme activity results corresponding to different recombinant expression plasmids

[0101]

[0102] Enzyme activity is defined as: the amount of enzyme required to consume 1 μmol / L NADPH per minute at 30°C, pH 8.0 is defined as one enzyme activity unit (U); the relative activity in the above table is the enzyme activity of each nitroreductase relative to the pet26b-BaNTR enzyme calculated when the enzyme activity of pet26b-BaNTR is 100%.

[0103] Example 3: Construction of error-prone PCR mutant library

[0104] From the data results in Table 1 of Example 2, it can be seen that the enzyme activity of E. coli BL21 (DE3) / pET28a-BaNTR is 2.4 times that of the original recombinant bacteria E. coli BL21 (DE3) / pET26b-BaNTR, and the volume enzyme activity is also 26% higher than that of the original recombinant bacteria E. coli BL21 (DE3) / pET26b-BaNTR. Therefore, E. coli BL21 (DE3) / pET28a-BaNTR was selected as the research object in the follow-up.

[0105] Although the volumetric enzyme activity and specific enzyme activity of E. coli BL21(DE3) / pET28a-BaNTR were improved, they still could not meet the requirements of industrial application production. Therefore, the strain was subjected to directed evolution modification using the error-prone PCR method.

[0106] The recombinant plasmid pET28a-BaNTR was used as a template to construct a mutation library using error-prone PCR technology. 2+ and Mn 2+ Optimize the concentration.

[0107] (1) Mn 2+ Concentration optimization (50 μL): 5 μL 10× buffer; 4 μL dNTP mix (2.5 mM); 1 μL template; 0.5 μL EX DNA Polymerase; 1 μL each of forward / reverse primers; Mn 2+ (10 mM) add 0, 1, 2, 3 μL respectively; the rest is made up to 50 μL with water.

[0108] (2)Mg 2+ Concentration optimization (50 μL): 5 μL 10× buffer; 4 μL dNTP mix (2.5 mM); 1 μL template; 0.5 μL EX DNA Polymerase; 1 μL each of forward / reverse primers (the primer pair in Example 1); Mg 2+ (50mM) add 1, 2, and 3 μL respectively; make up the rest with water to 50 μL.

[0109] The amplification procedure was: pre-denaturation at 98°C for 5 min; (denaturation at 98°C for 10 s; annealing at 58°C for 10 s; extension at 72°C for 1 min) × 30 cycles, and extension at 72°C for 10 min. After successful identification by 0.9% agarose gel electrophoresis, the gel was cut and recovered ( Figure 2 ).

[0110] Then, the pet28a vector and the amplified recovered product were double-digested with the double-digestion system of 50 μL: 30 μL recovered product; 4 μL 10× buffer; 1 μL each of QuickCutTMNco I and QuickCutTMXho I, and the rest was water. After mixing, place in a 37°C constant temperature incubator for 0.5 h.

[0111] The vector fragment and the target gene fragment were recovered by agarose gel electrophoresis and then connected. The connection system was 10 μL: 5.5 μL of the recovered product; 1.5 μL of the recovered vector fragment (pet28a); 2 μL of 5× buffer; 1 μL of T4 DNA ligase. After mixing, place it in a 25°C constant temperature incubator for 0.5 h. The connection product was then transformed into E. coli BL21 (DE3) competent cells, and after heat shock and recovery, it was coated on a plate containing kanamycin resistance and placed at 37°C for overnight culture.

[0112] Randomly pick single colonies on the plate for sequencing analysis, and finally select a system with a base mutation rate of about 2-4 (Mn 2+ and Mg 2+ The target gene fragments were obtained by amplification, restriction enzyme ligation and transformation, and error-prone PCR mutation library was constructed.

[0113] Example 4: Fermentation culture and high-throughput screening of mutants

[0114] Use a sterilized toothpick to pick the monoclonal sub-organisms on the plate into a 96-deep-well plate containing 300 μL sterile LB liquid medium (containing 50 μg / mL of kanamycin resistance). Then seal the 96-deep-well plate with a sterile Axygen sealing film and place it at 37°C and 800 rpm for 20 hours. Take out 150 μL of seed solution from the cultured 96-deep-well plate and transfer it to a new sterile 96-deep-well plate, and each well has 850 μL of LB liquid medium (containing kanamycin with a final concentration of 50 μg / mL and IPTG with a final concentration of 0.2 mM), and place it at 30°C and 800 rpm to induce protein expression. The remaining 150 μL of bacterial solution was mixed with 100 μL of 50% glycerol and stored in a -20°C refrigerator. After culturing for 20 hours, centrifuge at 3500 rpm and 20°C for 15 minutes, discard the supernatant, and collect bacterial cells for subsequent high-throughput screening.

[0115] Prepare a sodium phosphate buffer (0.05M) with a pH of 7.5, then add 5U / mL of DNase I and 1mg / mL of lysozyme to 180mL of the buffer. After mixing, dispense 400μL into each well of a 96-well plate, mix by pipetting, and incubate at 30℃ and 800rpm for 1h. Centrifuge at 3500rpm for 15min, collect the supernatant, and store at room temperature.

[0116] Weigh 0.1 g of ESTD03 sample and dissolve it in PBS buffer (0.05 mM, pH=7.5) containing 0.5% Tween 80 to prepare a sample with a concentration of 2 g / L. Ultrasonic treatment was performed for 15 min, followed by filtration using a 0.22 μm membrane, and the sample solution was used for high-throughput screening in a 96-well plate.

[0117] Mix 178 μL of substrate solution + 20 μL of bacterial lysis supernatant + 2.5 μL of NADPH (50 mM) and place in a microplate reader. Read the absorbance values ​​at 340 nm at 0 min, 2.5 min and 5.0 min. The enzyme activity can be determined by comparing the changes in absorbance values ​​(high activity consumes more NADPH). The activity calculation formula is:

[0118] Vitality = [OD 340(c) -OD 340(r) ] / 1.2173t;

[0119] Among them, OD 340(c) and OD 340(r) represents the absorbance at 340 nm at 0 min and 5.0 min of reaction, t represents the reaction time, and 1.2173 is the coefficient. The activity ratio of the mutant to BaNTR is the relative activity.

[0120] The relative activity of mutant and wild-type BaNTR was calculated as follows:

[0121] Relative activity = [OD 340(c) -OD 340(r)mutant ] / [OD 340(c) -OD 340(r)BaNTR ]*100%;

[0122] Among them, OD 340(c) Represents the absorbance value at 340nm at 0min of reaction, OD 340(r)mutant and OD 340(r)BaNTR Represent the absorbance values ​​of mutant and wild-type BaNTR at 340 nm after 5.0 min of reaction, respectively.

[0123] Based on the above-mentioned high-throughput screening method, about 5000 clones were screened, and about 50 mutants with obvious changes in activity were screened. These mutants were then screened by shaking flasks, and these single colonies were transferred to 10mL of LB medium and cultured overnight at 30°C and 220rpm. The next day, a portion of the seed liquid was transferred to 60mL of fermentation medium at a content of 2% (V / V) and cultured at 37°C and 220rpm. After culturing for 2h, IPTG (final concentration of 0.2mM) was added and cultured for 22h at 22°C and 220rpm to induce protein expression. Centrifugation (8 000rpm, 5min) was performed to collect bacterial cells and determine the enzyme activity. The enzyme activity determination method was referred to Example 2. The results of the re-screening showed that the mutants with significantly improved activity were Y69H / S173P, Y69N, and T15A / S172P, whose activities were 1.28, 1.36, and 1.45 times that of the starting strain BaNTR, respectively. Single clones were selected and sent to Shanghai Biotech Co., Ltd. for sequencing analysis. Y69N was subsequently selected as the research object for further molecular modification.

[0124] Example 5: Construction of a saturation mutation library of nitroreductase BaNTR

[0125] The mutant Y69N of BaNTR was used as the parent, and saturation mutation was performed on sites 15, 69, 172, and 173. Using NNK degenerate primers (N represents A, T, C, G; K represents G, T; M represents A, C.), the degenerate primers corresponding to the above 4 sites are as follows:

[0126] T15-F: 5′-CTATATGAACGCNNKTCTACACGTGCATTCAACCC-3′;

[0127] T15-R: 5′-CACGTGTAGAMNNGCGTTCATATAGAACGTTAAAA-3′;

[0128] Y69-F: 5′-CATCCAATAGCTNNKAACCAACAACAAATTCTTG-3′;

[0129] Y69-R: 5′-GTTGGTTMNNAGCTATTGGATGTAATCGTTTTTG-3′;

[0130] S172-F: 5′-CGAATTTAACGTTNNKCCTCGTTACGTTTCCAAT-3′;

[0131] S172-R: 5′-GTAACGAGGMNNAACGTTAAATTCGTCTGTCAGTG-3′;

[0132] S173-F: 5′-GAATTTAACGTTTCANNKCGTTACGTTCCAATTATG-3′;

[0133] S173-R: 5′-GAACGTAACGMNNTGAAACGTTAAATTCGTCTGTCAG-3′.

[0134] 50 μL amplification system: 25 μL PrimeSTAR Max PreMix, 70 ng plasmid fragment, 0.2 μM upstream / downstream primers, and the rest is water.

[0135] Amplification procedure: pre-denaturation 98°C, 3 min; (denaturation 98°C, 10 s; annealing 58°C, 5 s; extension 72°C, 90 s) × 30 cycles, and extension again at 72°C, 5 min. Figure 3 ), add 0.5 μL Dpn I to the product, mix well and place at 37°C for 30 min. Then, heat-shock transform the treated sample into BL21 (DE3) competent cells, ice bath, resuscitation, and coating. The next day, pick the transformants into 96-well plates for fermentation culture, high-throughput screening and shake flasks. For specific steps, refer to Example 4.

[0136] The best mutant obtained in this round of mutagenesis was Y69N / S173D, whose catalytic activity was 1.7 times that of the parent Y69N and 2.3 times that of the wild-type BaNTR. In addition, the volumetric enzyme activity of the mutant Y69N / S173D was 2.8 times that of the wild-type BaNTR.

[0137] Example 6: Site-directed mutagenesis of nitroreductase

[0138] Using Y69N / S173D as the parent, saturation mutation was performed on sites 15 and 172. Using NNK degenerate primers (N represents A, T, C, G; K represents G, T; M represents A, C), the degenerate primers corresponding to the above two sites are as follows:

[0139] T15-F: 5′-CTATATGAACGCNNKTCTACACGTGCATTCAACCC-3′;

[0140] T15-R: 5′-CACGTGTAGAMNNGCGTTCATATAGAACGTTAAAA-3′;

[0141] S172-F: 5′-CGAATTTAACGTTNNKGATCGTTACGTTTCCAAT-3′;

[0142] S172-R: 5′-GTAACGATCMNNAACGTTAAATTCGTCTGTCAGTG-3′.

[0143] 50 μL amplification system: 25 μL PrimeSTAR Max PreMix, 70 ng plasmid fragment, 0.2 μM upstream / downstream primers, and the rest is water.

[0144] Amplification program: pre-denaturation 98°C, 3min; (denaturation 98°C, 10s; annealing 58°C, 5s; extension 72°C, 90s) × 30 cycles, re-extension 72°C, 5min. After the PCR product is correctly detected by agarose gel electrophoresis, 0.5μL Dpn I is added to the product, mixed and placed at 37°C for 30min. The treated sample is then heat-shock transformed into BL21 (DE3) competent cells, ice-bathed, revived, and plated. The next day, the transformants are picked up in a 96-well plate for fermentation culture, high-throughput screening and shaking flasks. The specific steps can be referred to Example 4.

[0145] The mutant obtained in this round of mutagenesis is T15K / Y69N / S173D, whose catalytic activity is 1.8 times that of the wild-type BaNTR.

[0146] Example 7: Application of mutants in biocatalysis of ESTD03 to ESTD04

[0147] The mutant E. coli BL21 (DE3) / pET28a-BaNTR / Y69N / S173D obtained in Example 5 and the wet bacterial cells of the parent strain E. coli BL21 (DE3) / pET28a-BaNTR and the starting strain E. coli BL21 (DE3) / pET26b-BaNTR were used as biocatalysts to catalyze the synthesis of ESTD03 from ESTD04 ( Figure 1 ).

[0148] The reaction system is as follows: 2g glucose, 100mg NADP and 100mg GDH are added to 10mL PBS (100mM, pH 7.5), and then 2.5mL DMSO solution containing 20% ​​ESTD03 and 0.9mL 5% V2O5 aqueous solution are added after stirring. Finally, about 1.0g of bacterial powder is added. React at 30°C, and the pH is adjusted to about 7.5-7.8 by dropping saturated sodium carbonate aqueous solution. 200μL of samples are taken every 2h. The sample is processed by adding 20μL of 10% potassium borohydride aqueous solution and diluting it to 1-1.5mL with anhydrous ethanol, and then centrifuging and filtering the membrane and detecting it by HPLC. After 4 hours, the conversion rates of ESTD03 in the reaction mediated by the mutant E. coli BL21 (DE3) / pET28a-BaNTR / Y69N / S173D, the mother strain E. coli BL21 (DE3) / pET28a-BaNTR, and the starting strain E. coli BL21 (DE3) / pET26b-BaNTR were 98.6%, 51.8%, and 25.1%, respectively. It can be seen that under the same reaction conditions, the substrate spatiotemporal conversion rate of the mutant Y69N / S173D was greatly improved compared with the starting strain.

[0149] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0150] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nitroreductase mutant, characterized in that: include: Compared with the wild-type nitroreductase, the protein has at least one amino acid mutation in the following four positions or functionally equivalent positions: No. 15, No. 69, No. 172, No.

173.

2. The nitroreductase mutant according to claim 1, characterized in that The wild-type nitroreductase has an amino acid sequence as shown in SEQ ID NO: 1; Optionally, the nitroreductase mutant has at least 80% identity with the wild-type nitroreductase; preferably has more than 90% identity; more preferably has more than 95% identity; most preferably has more than 98% identity.

3. The nitroreductase mutant according to claim 1, characterized in that The mutant has the following mutations: The amino acid T at position 15 mutates to S, A, K or V; The amino acid Y at position 69 is mutated to N, H or C; The amino acid S at position 172 mutated to P; The amino acid S at position 173 was mutated to P or D.

4. The nitroreductase mutant according to claim 3, characterized in that The mutant has any of the following combined mutations: The amino acid T at position 15 is mutated to S, A, K or V, the amino acid at position 69 is not mutated, the amino acid at position 172 is not mutated, and the amino acid at position 173 is not mutated; or The amino acid T at position 15 is mutated to S, A, K or V, and at least one amino acid mutation occurs at the following three positions: position 69, position 172, and position 173; or The amino acid Y at position 69 is mutated to N, H or C, the amino acid at position 15 is not mutated, the amino acid at position 172 is not mutated, and the amino acid at position 173 is not mutated; or The amino acid Y at position 69 is mutated to N, H or C, and at least one amino acid mutation occurs at the following three positions: position 15, position 172, and position 173; or The amino acid S at position 172 is mutated to P, the amino acid at position 15 is not mutated, the amino acid at position 69 is not mutated, and the amino acid at position 173 is not mutated; or The amino acid S at position 172 is mutated to P, and at least one of the following three positions has an amino acid mutation: position 15, position 69, and position 173; or The amino acid S at position 173 is mutated to P or D, the amino acid at position 15 is not mutated, the amino acid at position 69 is not mutated, and the amino acid at position 172 is not mutated; or The amino acid S at position 173 mutated to P or D, and the following three positions had at least one amino acid mutation: position 15, position 69, and position 172.

5. The nitroreductase mutant according to claim 4, characterized in that The mutant has any of the following combined mutations: The amino acid T at position 15 mutates to A, and the amino acid S at position 172 mutates to P; or The amino acid Y at position 69 is mutated to N; or The amino acid Y at position 69 is mutated to H, and the amino acid S at position 173 is mutated to P; or The amino acid Y at position 69 is mutated to N, and the amino acid S at position 173 is mutated to D; or The amino acid T at position 15 mutated to K, the amino acid Y at position 69 mutated to N, and the amino acid S at position 173 mutated to D; Optionally, the mutant has an amino acid sequence shown in SEQ ID NOs: 3-7.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the nitroreductase mutant according to any one of claims 1 to 5.

7. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 6.

8. The expression vector according to claim 7, characterized in that The expression vector is selected from at least one of pET series vectors, shuttle vectors, phage and virus vectors; Optionally, the pET series vectors include pET-3a, pET-11a, pET-15a, pET-28a, pET-30a, pET-32a and pET-22b; Preferably, the pET series vector is pET-28a.

9. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to claim 6 or the expression vector according to any one of claims 7 to 8.

10. The recombinant cell according to claim 9, characterized in that The recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

11. A recombinant strain, characterized in that: The recombinant strain expresses the nitroreductase mutant according to any one of claims 1 to 4.

12. A method for preparing a nitroreductase mutant, characterized in that: include: The recombinant cell according to claim 9 or 10 or the recombinant strain according to claim 11 is cultured under conditions suitable for protein expression to obtain the nitroreductase mutant.

13. A method for preparing a nitroreductase mutant, characterized in that: include: Mutating a wild-type nitroreductase to obtain a nitroreductase mutant library; subjecting a nitroreductase substrate to a first catalytic treatment in the presence of the nitroreductase mutant library; The first catalytic treatment product is subjected to screening treatment to obtain the nitroreductase mutant according to any one of claims 1 to 5.

14. The method according to claim 13, characterized in that The mutation treatment is selected from at least one of random mutation and site-directed mutation.

15. The method according to claim 13, characterized in that The nitroreductase substrate includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts; Optionally, the first catalytic treatment is carried out in the presence of NADPH.

16. The method according to claim 13, characterized in that The screening process includes obtaining a nitroreductase mutant having a relative activity higher than that of a control group; Optionally, the control group is wild-type nitroreductase.

17. A method for preparing a nitro reduction product, characterized in that: include: The nitroreductase substrate is subjected to a second catalytic treatment in the presence of the nitroreductase mutant according to any one of claims 1 to 5, the recombinant cell according to claim 9 or 10, or the recombinant strain according to claim 11, so as to obtain a nitroreduction product.

18. The method according to claim 17, characterized in that The nitroreductase substrate includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts; Optionally, the nitro reduction product is 6-chloro-2-methyl-2H-indazol-5-amine.

19. The method according to claim 17, characterized in that The second catalytic treatment system further comprises an oxidoreductase system; Optionally, the oxidoreductase system comprises glucose dehydrogenase, glucose and the coenzyme NADP; Optionally, the pH of the second catalytic treatment system is 7.5-8.

20. The method according to claim 17, characterized in that The second catalytic treatment is carried out in a 5% vanadium pentoxide aqueous solution; Optionally, the second catalytic treatment is carried out at 25-35°C, preferably 30°C.

21. A kit, characterized in that: include: The nitroreductase mutant according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6, or the expression vector according to any one of claims 7 to 8, or the recombinant cell according to any one of claims 9 to 10, or the recombinant strain according to claim 11.

22. The kit according to claim 21, characterized in that The kit further includes 6-chloro-2-methyl-5-nitro-2H-indazole or a stereoisomer, tautomer and salt thereof.

23. The kit according to claim 21, characterized in that The kit further comprises at least one of a 5% vanadium pentoxide aqueous solution, glucose dehydrogenase, glucose and a coenzyme NADP.

24. Use of the kit according to any one of claims 21 to 23 in the preparation of nitro-reduction products.

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