Mutant enzymes having halogenating activity, enzyme preparations, encoding genes, recombinant vectors, recombinant strains and uses thereof

Abstract

This invention relates to the field of enzyme engineering, and discloses a mutant enzyme with halogenation activity, its enzyme preparation, encoding gene, recombinant vector, recombinant strain, and applications. The mutant enzyme is an S-adenosyl-L-methionine hydrolase with an amino acid sequence as shown in SEQ ID NO.1, comprising at least one mutation at the F19, P83, and R89 sites; wherein the mutation at the F19 site is selected from at least one of F19P, F19C, and F19L; the mutation at the P83 site is selected from at least one of P83F, P83H, P83E, P83D, P83C, P83L, P83K, and P83I; and the mutation at the R89 site is selected from at least one of R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D. All of the above mutations enable the enzyme to acquire the new function of catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine, exhibiting excellent iodination catalytic efficiency. Its activity exceeds that of wild-type enzymes and is superior to known natural halogenases, thus solving the technical problems of scarce natural halogenase resources and severe chemical halogenation pollution.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to a mutant enzyme with halogenation activity, its enzyme preparation, encoding gene, recombinant vector, recombinant strain, and applications. Background Technology

[0002] The introduction of halogen atoms is a core strategy in modern molecular functionalization, crucial for enhancing the bioactivity, metabolic stability, and physicochemical properties of compounds. Halogenated compounds are particularly prevalent in the pharmaceutical and pesticide fields—approximately 27% of clinical drugs contain halogen atoms, and this proportion exceeds 80% in high-efficiency pesticides. However, the currently mainstream chemical halogenation methods require highly toxic and corrosive reagents such as chlorine, bromine, and iodine, resulting in poor reaction selectivity, numerous byproducts, and the generation of large quantities of difficult-to-treat halogen-containing waste, posing serious challenges to the environment and safe production.

[0003] To address the shortcomings of chemical halogenation, biocatalytic halogenation is considered an ideal green alternative. Among them, S-adenosyl-L-methionine (SAM)-dependent halogenases have attracted much attention due to their ability to catalyze the synthesis of 5'-halodeoxyadenosine, which has significant anticancer activity. However, natural SAM-dependent halogenases are extremely rare in nature, and the discovered species suffer from bottlenecks such as low catalytic efficiency, narrow substrate spectrum, and difficulty in heterologous expression, making it difficult to meet the requirements of industrial biocatalysis for efficient, stable, and scalable enzyme catalysts.

[0004] Based on the high structural homology between natural SAM-dependent halogenases and SAM hydrolases, the technology of modifying SAM hydrolases into halogenases has been recognized in the field. The core logic lies in utilizing the abundant natural resources, excellent expression characteristics, and similar substrate-binding pocket structures of hydrolases to overcome the scarcity of natural halogenases. However, existing technologies still face several key challenges: First, the hydrolysis and halogenation reaction mechanisms are fundamentally different, and the functional regulatory sites of the two types of enzymes are not yet clear, leading to a lack of accuracy in predicting modification targets; second, traditional directed evolution methods suffer from low library construction efficiency and a large screening workload, making it difficult to quickly obtain highly active mutants; third, existing modification attempts are mostly limited to hydrolases from specific sources, failing to form a generalizable strategy and hindering cross-enzyme applications of the modification technology.

[0005] Therefore, developing a method for halogenating SAM hydrolases based on rational design and supported by efficient screening, in order to obtain high-performance halogenated enzymes and apply them to green biosynthesis, has become a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of scarce natural halogenating enzyme resources, serious pollution from chemical halogenation methods, and lack of rational strategies for efficiently modifying SAM hydrolases into halogenating enzymes in the existing technology. This invention provides a mutant enzyme with halogenation activity, its enzyme preparation, encoding gene, recombinant vector, recombinant strain, and application. This mutant enzyme is obtained through precise site-directed mutagenesis and can efficiently catalyze the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine.

[0007] To achieve the above objectives, the present invention provides a mutant enzyme with halogenation activity, wherein the mutant enzyme is formed by mutating at least one of the F19, P83, and R89 sites of S-adenosyl-L-methionine hydrolase with an amino acid sequence as shown in SEQ ID NO. 1; wherein the mutation at the F19 site is selected from at least one of F19P, F19C, and F19L; the mutation at the P83 site is selected from at least one of P83F, P83H, P83E, P83D, P83C, P83L, P83K, and P83I; and the mutation at the R89 site is selected from at least one of R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D.

[0008] A second aspect of the present invention provides an enzyme preparation comprising the mutant enzyme as described above.

[0009] A third aspect of the present invention provides a gene encoding a mutant enzyme with halogenation activity, said gene being a nucleotide sequence encoding the mutant enzyme as described above.

[0010] A fourth aspect of the present invention provides a recombinant vector containing the gene as described above.

[0011] A fifth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0012] The sixth aspect of the present invention provides the use of at least one of the mutant enzyme, enzyme preparation, gene, recombinant vector, and recombinant strain described above in catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine.

[0013] Through the above technical solution, this invention, for the first time, successfully transformed the naturally occurring S-adenosine-L-methionine hydrolase, which possesses only hydrolytic activity, into an artificial enzyme with highly efficient halogenation function through precise site-directed mutagenesis. Specifically, at least one mutation was performed at the F19, P83, and R89 sites shown in SEQ ID NO. 1, involving F19P, F19C, F19L, P83F, P83H, P83E, P83D, P83C, P83L, P83K, P83I, R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D. All of these mutations enable the enzyme to acquire the novel function of catalyzing the conversion of S-adenosine-L-methionine to 5'-halodeoxyadenosine, exhibiting superior iodination catalytic efficiency. Its activity exceeds that of the wild-type enzyme and is superior to known natural halogenating enzymes, thus solving the technical challenges of scarce natural halogenating enzyme resources and severe chemical halogenation pollution. Attached Figure Description

[0014] Figure 1 In Example 1 Methanocaldococcus jannaschii A schematic diagram of the molecular docking conformation of the S-adenosine-L-methionine hydrolase MJ1651 with the substrate SAM. Figure 2 This is a schematic diagram of the initial screening HPLC analysis results of the mixed mutant library targeting F19, P83 and R89 sites in Example 1 in a reaction system containing different potassium halides (KI, KF, KCl, KBr); Figure 3 This is the "mutation-activity" relationship map obtained from the deep mutation scan in Example 2; Figure 4 This is a comparison diagram of the iodination activities of the combined mutant and the wild-type enzyme in Example 4. Detailed Implementation

[0015] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0016] The first aspect of the present invention provides a mutant enzyme with halogenation activity, wherein the mutant enzyme is formed by mutating at least one of the F19, P83 and R89 sites of S-adenosyl-L-methionine hydrolase with an amino acid sequence as shown in SEQ ID NO. 1; wherein the mutation at the F19 site is selected from at least one of F19P, F19C, and F19L; the mutation at the P83 site is selected from at least one of P83F, P83H, P83E, P83D, P83C, P83L, P83K, and P83I; and the mutation at the R89 site is selected from at least one of R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D.

[0017] This invention, for the first time, successfully modifies the naturally occurring S-adenosine-L-methionine hydrolase, which possesses only hydrolytic activity, into an artificial enzyme with highly efficient halogenation function through precise site-directed mutagenesis. Specifically, at least one mutation from the following three sites—F19P, F19C, F19L, P83F, P83H, P83E, P83D, P83C, P83L, P83K, P83I, R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D—is performed at the F19, P83, and R89 sites. Each of these mutations endows the enzyme with the novel function of catalyzing the conversion of S-adenosine-L-methionine to 5'-halodeoxyadenosine, exhibiting superior iodination catalytic efficiency. Its activity exceeds that of the wild-type enzyme and is superior to known natural halogenating enzymes, thus solving the technical challenges of scarce natural halogenating enzyme resources and severe chemical halogenation pollution.

[0018] According to the present invention, preferably, the mutation is selected from any combination of F19C / R89A, F19C / R89E, P83C / R89A, P83C / R89E, P83F / R89A, P83F / R89E, and P83K / R89E. The inventors have found that this combination mutation can synergistically enhance the iodination catalytic activity and substrate binding specificity of the enzyme, further improving the catalytic efficiency of 5'-IDA formation compared to a single point mutation, and further enhancing reaction selectivity while effectively reducing byproduct formation.

[0019] More preferably, the mutation is an F19C / R89A combined mutation. The inventors discovered that this combined mutation achieves an optimal balance between activity and stability, with a more significant improvement in 5'-IDA generation efficiency compared to the optimal single-point mutation. It can be adapted for industrial continuous production without complex processes, greatly reducing the cost of biocatalytic synthesis.

[0020] A second aspect of this invention provides an enzyme preparation comprising the mutant enzyme as described above. In this invention, the mutant enzyme can be formulated into a corresponding enzyme preparation, specifically, the enzyme preparation can exist in solid, semi-solid, or liquid form. The enzyme preparation can be prepared into a solid form by freeze-drying or spray-drying, or into a liquid / semi-solid form by mixing with buffer solutions and protective agents (such as glycerol or sucrose), wherein the content of the protective agent is 5-20% by weight of the total weight of the enzyme preparation.

[0021] A third aspect of the present invention provides a gene encoding a mutant enzyme with halogenation activity, said gene being a nucleotide sequence encoding the mutant enzyme as described above.

[0022] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. Typically, the obtained nucleotide sequence is cloned into a vector, then transformed into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods. Alternatively, the relevant nucleotide sequence can also be synthesized using known artificial chemical synthesis methods.

[0023] A fourth aspect of the present invention provides a recombinant vector containing the gene as described above.

[0024] In this invention, the "vector" used in the recombinant vector can be any vector known in the art, such as various commercially available plasmids, granules, bacteriophages and retroviruses, etc. The preferred expression vector of this invention is pET-28a.

[0025] A fifth aspect of this invention provides a recombinant bacterial strain containing the gene or the recombinant vector described above. In this invention, the recombinant vector can be transformed, transduced, or transfected into a host cell (strain) using conventional methods in the art, such as chemical transformation using calcium chloride or high-voltage electroporation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably *Escherichia coli* or *Bacillus subtilis*, and more preferably, the host cell is *Escherichia coli*, for example, *Escherichia coli*. E. coli BL21(DE3). The inventors discovered that this recombinant strain can stably and efficiently express the S-adenosyl-L-methionine hydrolase mutant, providing a reliable vector for the large-scale preparation of the S-adenosyl-L-methionine hydrolase mutant.

[0026] The sixth aspect of the present invention provides the use of at least one of the mutant enzyme, enzyme preparation, gene, recombinant vector, and recombinant strain described above in catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine.

[0027] In this invention, the method for catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine may include: mixing at least one of the mutant enzyme, enzyme preparation, gene, recombinant vector, and recombinant strain described above with S-adenosine and reacting the mixture.

[0028] According to the present invention, preferably, the catalytic reaction is carried out in a buffer solution containing iodide ions. The inventors have found that this preferred condition can efficiently activate the iodination activity of the modified halogenase, not only increasing the conversion rate of the substrate SAM to over 99%, but also greatly improving the selectivity and stability of the target product 5'-IDA, while avoiding interference from non-specific hydrolysis side reactions.

[0029] According to the present invention, preferably, the buffer solution is a sodium phosphate buffer solution. This buffer system has good buffering capacity under neutral to weakly alkaline conditions, can maintain the stability of the reaction system, and provides a suitable environment for enzyme catalysis. At the same time, sodium phosphate buffer solution is simple to prepare, inexpensive, and suitable for large-scale production applications.

[0030] According to the present invention, preferably, the concentration of iodide ions in the catalytic reaction system is 100-500 mM. This preferred concentration range provides a sufficient halogen source to ensure that the reaction equilibrium shifts towards the halogenation products, while avoiding enzyme activity inhibition that may be caused by excessively high ion concentrations, thus achieving a balance between catalytic efficiency and reaction economy.

[0031] The present invention will be described in detail below through examples. In the following examples, potassium halides (KI, KBr, KCl, KF) were analytical grade reagents produced by Sinopharm Chemical Reagent Co., Ltd.; expression vector pET-28a was purchased from Novagen; Escherichia coli BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; other raw materials and reagents were all conventional commercial products.

[0032] LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) was used for cell culture and protein expression.

[0033] Protein purification: All His-tagged proteins were purified using a Ni-NTA affinity chromatography column (HisTrap HP, Cytiva) on an AKTA pure system. Buffer A: 20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8; Buffer B: 20 ​​mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8. Linear gradient elution was used, with the target protein eluted at approximately 250 mM imidazole.

[0034] Activity assay: The standard reaction system consisted of 200 μL containing 50 mM sodium phosphate buffer (pH 8), 4 mM SAM, 500 mM potassium halide (KF, KCl, KBr, or KI), and 200 μM purified enzyme. After incubation at 30°C for 24 hours, the reaction was terminated by adding 20 μL of 10% formic acid.

[0035] Product analysis: Analysis was performed using an HPLC system equipped with a C18 reversed-phase column (5 μm, 4.6 × 150 mm) and a DAD detector (detection wavelength 260 nm). Mobile phase: A was 0.1 v / v formic acid aqueous solution, and B was 0.1 v / v formic acid acetonitrile solution. Gradient elution program: 0–5 min, 1 wt% B; 5–15 min, 1–10 wt% B; 15–20 min, 10–100 wt% B; 20–25 min, 100 wt% B; 25–30 min, 100–1 wt% B; 30–35 min, 1 wt% B. Flow rate: 1 mL / min. Qualitative analysis was performed by comparing retention times with standards, and quantification was performed using the external standard method.

[0036] Example 1: Rational Design of Mutation Sites and Construction of a Saturated Mutation Library 1. Selection and prediction of mutation sites From Methanocaldococcus jannaschii S-adenosine-L-methionine hydrolase MJ1651 (protein structure database PDB: 2F4N, its amino acid sequence is shown in SEQ ID NO. 1) was used as the starting enzyme. The substrate SAM was docked into the enzyme's active pocket using AutoDock Tools molecular docking software to obtain a stable enzyme-substrate complex conformation. Figure 1 Based on this conformation, all amino acid residues within a 5 Å range of any atom in the SAM molecule were selected, along with all amino acid residues within a 5 Å range of the water molecule closest to SAM in the active pocket. The intersection of these two sets was then used to successfully identify 10 potential key functional sites: D18, F19, P83, R89, R139, D177, F179, N181, Y221, and S241 (aspartic acid at position 18, phenylalanine at position 19, proline at position 83, arginine at position 89, arginine at position 139, aspartic acid at position 177, phenylalanine at position 179, asparagine at position 181, tyrosine at position 221, and serine at position 241). The spatial positions of these sites in the three-dimensional structure are shown below. Figure 1 As shown in the spheres, they are distributed around the SAM and may be directly involved in the regulation of substrate recognition or catalytic processes.

[0037] 2. Construction of a saturated mutation library For F19, P83, and R89 at the aforementioned sites (based on their key positions in the active pocket), the 20C-Tang site-directed saturation mutagenesis method was used. Degenerate primers were designed (primer sequences are shown in Tables 1, 2, 3, 4, and 5) to mutate each site to all 20 natural amino acids. The specific operation procedure is as follows: Using the pET-28a plasmid containing the MJ1651 gene as a template, two rounds of PCR amplification were performed. First, two 25 μL reaction mixtures containing DNA polymerase, plasmid template, specific primers (upstream or downstream), and water were prepared in two PCR tubes and placed in a PCR instrument. The reaction program was as follows: 98℃ pre-denaturation for 1 min; followed by three cycles: 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 3 min 30 s; and a final extension at 72℃ for 10 min. The products were stored at 4℃.

[0038] After the first round of reaction was completed, the two reaction systems were mixed to a total volume of 50 μL, an appropriate amount of DNA polymerase was added, and the reaction was continued for 15 cycles using the same procedure to fully amplify the mutant product.

[0039] After PCR, 2 μL of DpnI rapid endonuclease was added to the product, and the mixture was incubated at 37°C for 25 min to completely digest the template plasmid and avoid false positive transformations. 5 μL of the product was then subjected to agarose gel electrophoresis to verify whether the expected band size was obtained.

[0040] A suitable amount of the validated PCR product was used for bacterial transformation: 10 μL of the product was mixed with competent E. coli BL21(DE3) cells and incubated on ice for 30 min to allow DNA adsorption to the cell wall; a heat shock at 42°C for 50 s was performed to promote plasmid entry into the cells; the cells were immediately incubated on ice for 3 min to restore their state; 1 mL of antibiotic-free LB medium was added, and the cells were cultured at 37°C with shaking at 220 rpm for 1 h to induce expression of the resistance gene; after centrifugation, most of the supernatant was discarded, and approximately 100 μL of the bacterial cells were resuspended and plated on LB agar plates containing the corresponding antibiotic. After culturing at 37°C for 12 h, at least 96 single clones were randomly selected from each locus for sequencing verification to ensure the acquisition of a complete single-point mutant library covering all 20 amino acids at that locus.

[0041] For the few mutants that could not be obtained through the above saturation mutagenesis method, conventional site-directed mutagenesis technology was used to supplement them. The primers used are shown in Table 1. The specific PCR and transformation process is similar to the above procedure.

[0042] Through the complete and reproducible experimental procedure described above, a saturated mutant library covering the key sites of F19, P83, and R89 was successfully constructed, laying a solid foundation for subsequent functional screening.

[0043] 3. "Dichotomy" hybrid screening strategy and initial screening For each target site (F19, P83, R89), 20 single-point mutants were divided into two mixed libraries based on amino acid properties: Mixed library A: A mutant containing alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), and leucine (L).

[0044] Mixed library B: Mutants containing methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

[0045] Equal volumes of protein expression supernatants from multiple mutants in each hybrid library were mixed and tested in different reaction systems. The reaction systems included: a hydrolysis system (50 mM sodium phosphate buffer, pH 8, 4 mM SAM) and halogenation reaction systems supplemented with 500 mM KI, KF, KCl, and KBr, respectively. After incubation at 30°C for 24 hours, the products were detected by HPLC at 260 nm.

[0046] Results: The initial screening results are as follows Figure 2 As shown in the figure. HPLC analysis indicated that wild-type MJ1651 produced only the hydrolysis product adenosine in all systems. Among all 10 sites tested, only the mutant mixtures at F19, P83, and R89 sites showed a significant 5'-iododeoxyadenosine (5'-IDA) production signal in the KI system, while no corresponding fluorinated, chlorinated, or brominated products were detected in the corresponding KF, KCl, and KBr systems. This result indicates that mutations at these three specific positions specifically endow this hydrolase with a novel function in catalyzing the iodination reaction.

[0047] Table 1

[0048] Example 2: Deep Mutation Scanning and Confirmation of Highly Active Mutants Based on the positive signals obtained in the initial screening, all 20 single-point mutants at the F19, P83 and R89 sites were expressed, purified and precisely quantified one by one, and a complete "mutation-activity" relationship map was drawn.

[0049] 1. Expression and purification of mutant proteins Each single-point mutant plasmid, verified by sequencing, was transformed into *E. coli* BL21(DE3) and cultured at a scale of 1 L. After IPTG induction, the bacterial cells were collected, sonicated, and purified to high purity protein by Ni-NTA affinity chromatography using a standard method.

[0050] 2. Precise quantification of halogenation and hydrolysis activities Purified single-point mutant proteins were used to test their iodination activity in parallel under standard reaction conditions, while their hydrolytic activity under KI-free conditions was also tested. The amounts of adenosine and 5'-IDA produced were quantified by HPLC, and relative enzyme activity was calculated. The hydrolytic activity of wild-type MJ1651 was used as a baseline.

[0051] 3. Results Deep mutation scan results as follows Figure 3 As shown, the following mutants were found to exhibit iodination activity higher than the background level: F19 site: F19P, F19C and F19L mutants exhibit iodination activity above background.

[0052] P83 site: P83F, P83H, P83E, P83D, P83C, P83L, P83K and P83I mutants all acquired significant iodination function.

[0053] The R89 site mutation has the most significant impact on function, with the R89A, R89C, and R89E mutants exhibiting extremely superior iodination catalytic performance. The R89A mutant's efficiency in catalyzing the formation of 5'-IDA is more than four times that of the wild-type enzyme's own hydrolysis of SAM. Furthermore, the R89D, R89F, R89S, R89T, R89Q, and R89H mutants also exhibit moderate to good activity. The iodination efficiency of the R89A mutant not only far exceeds that of its wild-type precursor but also surpasses the catalytic capacity of all previously reported natural SAM-dependent halogenases.

[0054] Example 3: Activity characterization and efficient iodination function confirmation of mutant R89A 1. Expression and purification of R89A mutant protein The recombinant plasmid pET-28a-MJ1651-R89A, verified by sequencing, was transformed into the *E. coli* expression host BL21(DE3). Single colonies were picked and inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin, and cultured overnight (14 hours) at 37°C with shaking at 220 rpm as the seed culture. The seed culture was then transferred 1 L of LB broth containing the same antibiotic at a 1:100 ratio and cultured at 37°C with shaking at 220 rpm for expansion. When the OD of the culture medium... 600When the pH reached 0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture flask to a final concentration of 1 mM to induce target protein expression. The culture temperature was rapidly lowered to 30°C, and the culture was induced with shaking at 150 rpm for 16 hours to achieve efficient expression of soluble proteins. After induction, the culture medium was centrifuged at 8,000×g for 10 minutes at 4°C, the supernatant was discarded, and the bacterial cells were collected. The bacterial pellet was resuspended in 40 mL of pre-cooled lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8). The bacterial suspension was lysed using a Scientz-IID ultrasonic cell disruptor under ice-water bath conditions. The operating parameters were set as follows: power 300W, sonication for 3 seconds, interval for 5 seconds, and total sonication time 30 minutes. The lysate was centrifuged at 12,000×g for 50 minutes at 4°C, and the supernatant (i.e., crude enzyme solution) was carefully aspirated and the volume was recorded. Using the AKTA pure protein purification system, the crude enzyme solution was loaded at a flow rate of 2.5 mL / min onto a 5 mL HisTrap HP nickel affinity chromatography column pre-equilibrated with binding buffer (same as lysis buffer). After loading, non-specifically bound proteins were washed away with 10–15 column volumes of wash buffer A (lysis buffer containing 20 mM imidazole, pH 8). Washing was then continued with 5–10 CV of wash buffer 12% by weight B (lysis buffer containing 80 mM imidazole, pH 8) to further remove weakly bound impurities. Finally, a step gradient elution was performed with 50% by weight B elution buffer (lysis buffer containing 250 mM imidazole, pH 8.0), collecting the eluent fraction with a significant absorption peak at 280 nm. The eluent fractions containing the target protein were combined and transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and concentrated to approximately 2 mL by centrifugation at 4 °C and 5,000 × g. The concentrated protein solution was transferred to imidazole-free storage buffer (20 mM Tris-HCl, pH 8) using a PD-10 desalting column. The protein was then concentrated again using an ultrafiltration centrifuge tube to a final concentration of approximately 10 mg / mL (measured using a BCA protein assay kit). A small sample was analyzed by SDS-PAGE to confirm a single pure band and correct molecular weight (approximately 32 kDa). The high-purity protein was aliquoted and stored at -80°C for long-term storage. The solution was then transferred to storage buffer again using a PD-10 desalting column and concentrated to approximately 10 mg / mL, and stored at -80°C.

[0055] 2. Iodination activity detection and analysis Set up four 200 μL reaction systems: Experimental group (R89A+KX): 50 mM sodium phosphate buffer (pH 8), 4 mM SAM, 500 mM potassium halide (KX), 200 μM R89A purified enzyme.

[0056] Negative control 1 (WT + KX): Same as above, except the enzyme was replaced with wild-type MJ1651.

[0057] Negative control 2 (R89A, without KX): KX not present, otherwise the same as the experimental group.

[0058] Blank control (No Enzyme): No enzyme present.

[0059] After vortexing all reaction systems, the mixtures were incubated in a constant-temperature mixer at 30°C and 500 rpm for 24 hours. Immediately after incubation, 50 μL of 2% (v / v) formic acid was added to each reaction tube to terminate the reaction and precipitate the protein. The terminated reaction solution was centrifuged at 12,000 × g for 60 minutes at 4°C. The supernatant was filtered through a 0.22 μm aqueous syringe filter and transferred to a HPLC vial for analysis. An Agilent 1260 Infinity II HPLC system equipped with a ZORBAX SB-C18 column (5 μm, 4.6 × 150 mm) was used. Column temperature: 30°C. Detection wavelength: 260 nm. Injection volume: 10 μL. The mobile phase and gradient program were as described previously (general method). First, standard solutions of adenosine and 5′-iododeoxyadenosine (5′-IDA) were injected to determine their retention times (approximately 12.5 minutes for adenosine and approximately 18.2 minutes for 5′-IDA). Each reaction sample was then analyzed sequentially. Standard curves (peak area versus concentration) were constructed using different concentrations of adenosine and 5′-IDA standards. Based on the standard curves, the amounts of adenosine and 5′-IDA generated in the experimental groups were calculated. The results confirmed that the R89A mutant exhibited excellent efficiency in catalyzing the formation of 5′-IDA in the KI system. Figure 3 As shown, it is confirmed that it has a highly efficient iodination function, and the iodination activity of the R89A mutant is 4 times that of the wild type hydrolysis activity.

[0060] Example 4 Construction and high activity verification of combined mutants 1. Construction of combined mutant plasmids Using the single-point mutant plasmids (F19C, P83C, P83F, P83K, R89A, R89E) constructed in Example 1 as templates, combined mutant plasmids were constructed using overlap extension PCR technology.

[0061] Primer design: For each pair of combined mutation sites, overlapping primers containing the corresponding mutation sequences were designed (see Table 2).

[0062] PCR amplification: In the first round of PCR, fragment 1 containing the upstream mutation site and fragment 2 containing the downstream mutation site were amplified separately. In the second round of PCR, fragments 1 and 2 were used as a mixed template, and the complete combined mutant gene was obtained by extension through the overlapping region. The nucleotide sequences of the gene encoding the F19C / R89A combined mutation are shown in SEQ ID NO. 147, F19C / R89E combined mutation, P83C / R89A combined mutation, P83C / R89E combined mutation, P83F / R89A combined mutation, P83F / R89E combined mutation, and P83K / R89E combined mutation are shown in SEQ ID NO. 153.

[0063] Vector construction: The PCR product was digested with DpnI enzyme and ligated into the pET-28a expression vector, transformed into E. coli DH5α competent cells, and single clones were selected for sequencing verification to ensure that both mutation sites were correctly introduced and that there were no other random mutations.

[0064] 2. Expression and purification of combinatorial mutant proteins The seven correctly sequenced combined mutant plasmids and the wild-type (WT) plasmid were transformed into E. coli BL21(DE3) competent cells, and the expression and purification procedures were the same as in Example 3. Seed culture: 5 mL LB medium (containing 50 μg / mL kanamycin), cultured at 37℃ and 220 rpm for 14 hours; Expanded culture: Transfer to 1 L LB medium at a ratio of 1:100 and incubate at 37°C until OD. 600 =0.8, add 1 mMIPTG, induce at 30℃ and 150 rpm for 16 hours; Protein purification: After ultrasonic disruption of bacterial cells, the protein was purified by Ni-NTA affinity chromatography and desalted by PD-10 desalting column, concentrated to a protein concentration of approximately 10 mg / mL, and the purity was verified by SDS-PAGE (single pure band, molecular weight approximately 32 kDa). The protein was then stored at -80℃ for later use.

[0065] 3. Precise detection and comparison of iodination activity (1) Reaction system setup Referring to the standard system of Example 3, each group was configured with 3 parallel replicates, with wild-type MJ1651 as a negative control: Reaction system (200 μL): 50 mM sodium phosphate buffer (pH 8), 4 mM SAM, 400 mM KI, 200 μM purified combined mutant enzyme; Reaction conditions: Incubate at 30℃ and 500 rpm for 24 hours, add 50 μL of 10% formic acid to terminate the reaction, centrifuge at 4℃ and 12,000×g for 60 minutes, and filter the supernatant for analysis.

[0066] (2) Product analysis and activity calculation The amount of 5'-IDA generated was quantitatively analyzed using an HPLC system (C18 column, detection wavelength 260 nm). The relative iodination activity of each mutant combination was calculated using the hydrolytic activity of the wild-type enzyme as a baseline (set to 100). The results are as follows: Figure 4 As shown, the iodination activities of all seven combined mutants were significantly higher than those of the wild-type enzyme (WT has no iodination activity), and all were superior to the optimal single-point mutant R89A, confirming the synergistic enhancement effect of combined mutations on enzyme activity; among them, the F19C / R89A combined mutant had the best activity.

[0067] Example 5: From laboratory preparation to industrial-scale production of 5'-iododeoxyadenosine This embodiment details how to scale up the halogenase mutant (R89A as an example) obtained by this invention from laboratory gram-scale preparation to a synthetic process with potential industrial applications. The process is divided into two parts: Part A is the complete laboratory-scale (gram-scale) preparation and characterization process; Part B is the design of an industrial application scale-up scheme based on the laboratory process.

[0068] Part A: Laboratory-scale (gram-level) preparation and characterization The reaction was carried out in a 100 mL glass flask. 50 mL of 50 mM sodium phosphate buffer (pH 8) and 10 g of S-adenosylmethionine (SAM) were added sequentially to a final concentration of 400 mM (to improve substrate loading; preliminary experiments confirmed that R89A maintained high activity at this concentration). 132 g of potassium iodide was added to a final concentration of 4 M (high concentrations of halide ions favor shifting the reaction equilibrium towards halogenation). The mixture was then brought to a final volume of 80 mL with buffer solution and preheated in a 30°C water bath with stirring. 20 mL of storage buffer containing 2 g (approximately 62.5 μmol) of the purified R89A mutant protein prepared in Example 2 was added to a final reaction volume of 100 mL, with an enzyme concentration of approximately 8 mg / mL (200 μM). Timing was started immediately. The reaction flask was placed in a 30°C constant-temperature shaker and gently shaken at 200 rpm. At 6, 12, 24, and 36 hours of reaction, 100 μL of the reaction solution was taken out, and 20 μL of 2% (w / w) formic acid was added to terminate the reaction. After centrifugation, the changes in the contents of SAM, adenosine, and 5'-IDA were monitored by analytical HPLC to determine the reaction endpoint. In this experiment, the SAM conversion rate exceeded 99% and the 5'-IDA selectivity was >95% after 36 hours.

[0069] After the reaction, the reaction solution was heated in an 80°C water bath for 10 minutes to completely denature and inactivate the enzyme. The reaction solution was cooled to room temperature and then centrifuged at 10,000×g for 30 minutes at 4°C to remove precipitated protein and possible impurities. The supernatant was collected, and the volume was recorded (approximately 95 mL). At this point, the supernatant contained the target product 5'-IDA, the byproduct adenosine, excess KI, and buffer salts.

[0070] First, a tangential flow filtration system was used with an ultrafiltration membrane containing a molecular weight cutoff of 1 kDa. The sample was dialyzed with deionized water to remove most of the KI and phosphate, while simultaneously concentrating the sample volume to approximately 10 mL. The concentrate was then filtered through a 0.22 μm filter and loaded onto a preparative reversed-phase C18 column (Waters SunFire™ Prep C18 OBD). TM The chromatogram showed a diameter of 5 μm and a diameter of 19 × 150 mm. Mobile phase A was 0.1 vol% formic acid aqueous solution, and mobile phase B was acetonitrile. The flow gradient was: 0–5 min, 5 wt% B; 5–25 min, 5–25 wt% B; 25–30 min, 25–100 wt% B. The flow rate was 15 mL / min. The detection wavelength was 260 nm. Fractions corresponding to the main product peak (retention time 18–20 min) were collected based on the chromatogram. The collected product fractions were combined, pre-frozen at -80°C, and then freeze-dried in a freeze dryer for 36–48 hours to obtain a white to off-white fluffy solid.

[0071] The total weight of the lyophilized solid was 2.85 g. The purity was determined to be 98.5% by HPLC area normalization. Therefore, the actual yield of 5'-IDA was 2.85 g × 98.5% = 2.81 g. Based on the input of 10 mmol SAM, the molar yield was 66% (5'-IDA molecular weight 428.1 g / mol).

[0072] Mass spectrometry (ESI-MS) measurements [M+H] + m / z = 428, and C 10 H 12 This matches the theoretical value of IN5O4, which is 427.0.

[0073] 1H NMR spectrum ( 1 ¹H NMR (DMSO-d6): δ 8.35 (s, 1H, H-8), 8.14 (s, 1H, H-2), 5.90 (d, J = 6.0 Hz, 1H, H-1'), 5.45 (d, J = 5.5 Hz, 1H, 2'-OH), 5.20 (t, J = 5.5 Hz, 1H, 3'-OH), 4.55 (q, J = 5.5 Hz, 1H, H-2'), 4.15 (m, 1H, H-3'), 3.95 (m, 1H, H-4'), 3.65 (m, 2H, H-5'a, 5'b). All data are consistent with literature reports (…). Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase 5'-IDA spectrum Figure 1 HPLC analysis showed that the adenosine content was <0.5%, and no other halogenated byproducts (such as 5'-ClDA) were detected, demonstrating the high selectivity of enzyme catalysis.

[0074] Part B: Industrial Application Example Solution Design Based on the successful laboratory process described above, a continuous / semi-continuous biocatalytic process for industrial-scale (kilogram-level) production is designed, highlighting its green, efficient, and economical advantages.

[0075] To improve enzyme stability, enable reusability, and facilitate separation, the R89A mutant was immobilized using an epoxy resin carrier. The purified R89A enzyme solution was then immobilized with an epoxy-activated porous resin such as ReliZyme. TMEP403 was mixed in phosphate buffer and reacted with gentle stirring at 25°C for 20 hours. The immobilization efficiency (>80%) was calculated by measuring the enzyme activity of the supernatant before and after immobilization. The immobilized enzyme particles exhibited good mechanical strength and could be packed into column reactors. Its half-life at 50°C was more than 10 times longer than that of the free enzyme, and it retained more than 80% of its initial activity after 10 batches of repeated use.

[0076] Design of large-scale reaction systems: Recommended process: continuous production using a packed bed reactor.

[0077] System composition: Two tandem immobilized enzyme packed bed reactors (PBRs), each with a volume of 50 L. Equipped with precision metering pumps, preheaters, and an online pH / temperature monitoring system.

[0078] Feed solution: The substrate SAM (dissolved in 50 mM sodium phosphate buffer, pH 8) and high concentration KI solution were prepared separately, mixed online and preheated to 30°C, and then pumped into the first PBR at a certain space velocity (SV).

[0079] Reaction process: The material flows through the first PBR, where most of the SAM is converted to 5'-IDA. The effluent enters the second PBR for a "polishing" reaction to ensure complete SAM conversion. The entire system operates at 30°C and atmospheric pressure.

[0080] Advantages: Continuous flow operation is easy to automate, produces stable product quality, and has a much higher production efficiency than batch reactions. Immobilized enzymes can be used for a long time, significantly reducing catalyst costs.

[0081] Industrial-grade downstream separation and purification solutions: The reaction solution flowing from the PBR first passes through a heat exchanger, where it is rapidly heated to 80°C and maintained for a period of time to inactivate any trace amounts of free enzyme that may leak out. Then, it passes through a plate and frame filter to remove all solid impurities. The filtrate enters a continuous ion exchange system and a reverse osmosis / nanofiltration membrane system for efficient removal of inorganic salts (KI, phosphate) and simultaneous concentration of the product solution. Utilizing the difference in solubility of 5'-IDA in water and ethanol, a cooling crystallization process is employed. The concentrate is adjusted to a suitable concentration, a specific proportion of ethanol is added, and crystallization is performed using a programmed cooling process. The crystals are separated by centrifugation, and the mother liquor can be recycled. The wet crystals are washed with a small amount of cold ethanol and then dried in a vacuum belt dryer or spray dryer to obtain pharmaceutical-grade 5'-IDA raw material, which is then packaged.

[0082] Preliminary economic and technical assessment: Compared with traditional chemical synthesis methods: This biocatalytic method generates 5'-IDA without protection / deprotection steps, avoiding the complex regioisomer separation challenges of chemical methods and resulting in higher product purity. The reaction is carried out in a mild (30°C, pH 8, normal pressure) aqueous phase, eliminating the need for toxic organic solvents, heavy metal catalysts, or highly corrosive halogenating agents (such as elemental iodine or N-iodosuccinimide). Wastewater is primarily an aqueous solution containing potassium salts and trace amounts of buffer salts, making treatment simple, environmentally friendly, and extremely safe. The main costs come from the substrate SAM and the immobilized enzyme. With the decreasing cost of SAM bio-fermentation and the long lifespan and high reusability of immobilized enzymes, the overall production cost of this process is competitive with chemical methods, especially in the production of high-value-added pharmaceutical intermediates.

[0083] Conclusion: This embodiment fully demonstrates the feasibility and superiority of synthesizing 5'-IDA using the R89A halogenase mutant, from laboratory-scale preparation to industrial-scale production. The process route is clear, environmentally friendly, and highly selective, providing a promising new biomanufacturing pathway for the industrial production of 5'-IDA and its analogues, fully reflecting the significant application value of this invention.

[0084] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A mutant enzyme with halogenation activity, characterized in that, The mutant enzyme is an S-adenosyl-L-methionine hydrolase with an amino acid sequence as shown in SEQ ID NO. 1, wherein at least one of the F19, P83 and R89 sites is mutated; wherein the mutation at the F19 site is selected from at least one of F19P, F19C, and F19L; the mutation at the P83 site is selected from at least one of P83F, P83H, P83E, P83D, P83C, P83L, P83K, and P83I; and the mutation at the R89 site is selected from at least one of R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D.

2. The mutant enzyme according to claim 1, characterized in that, The mutation is selected from any combination of mutations among F19C / R89A, F19C / R89E, P83C / R89A, P83C / R89E, P83F / R89A, P83F / R89E, and P83K / R89E; Preferably, the mutation is an F19C / R89A combined mutation.

3. An enzyme preparation, characterized in that, The enzyme preparation comprises the mutant enzyme as described in claim 1 or 2.

4. A gene encoding a mutant enzyme with halogenation activity, characterized in that, The gene is a nucleotide sequence encoding the mutant enzyme of claim 1 or 2.

5. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 4.

6. The recombinant vector according to claim 5, characterized in that, The expression vector for the recombinant vector is pET-28a.

7. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene described in claim 4 or the recombinant vector described in claim 5 or 6.

8. The recombinant strain according to claim 7, characterized in that, The recombinant strain originated from Escherichia coli BL21(DE3).

9. The use of at least one of the mutant enzyme of claim 1 or 2, the enzyme preparation of claim 3, the gene of claim 4, the recombinant vector of claim 5 or 6, or the recombinant strain of claim 7 or 8 in catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine.

10. The application according to claim 9, characterized in that, The catalytic reaction is carried out in a buffer solution containing iodide ions; Preferably, the buffer solution is a sodium phosphate buffer solution; Preferably, in the catalytic reaction system, the concentration of the iodide ions is 100-500 mM.

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