SAM hydrolase mutant and application thereof

Abstract

The invention discloses an SAM hydrolase mutant and application thereof, the specific activation effect of halogen anions (F <->, Cl <->, Br <-> and I <->) on SAM hydrolase (containing MJ1651 wild type and mutant) is found for the first time, and the active sequence of the SAM hydrolase is F <-> > I <-> > Br <-> = Cl <->. According to the method, 400 mM of halide salt is added into a reaction system, so that the catalytic efficiency is improved in a breakthrough manner; the yield of wild type enzyme adenosine is improved by 20-300% (optimally, an F-system); the yield of the mutant R89C is soaring up by nearly 6 times under the action of KF; the activity of the immobilized enzyme is still more than 90% after the immobilized enzyme is repeatedly used for 5 times. The method is easy and convenient to operate, green production is achieved with water as the solvent (three wastes are reduced by 90%), the comprehensive cost is reduced by 41%-80 / kg, and an efficient solution is provided for industrial biological manufacturing of antiviral drug intermediates and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a SAM hydrolase mutant and its applications. Background Technology

[0002] S-adenosyl-L-methionine (SAM), as one of the most important methyl donors in living organisms, participates in regulating more than 200 biochemical reactions, including DNA methylation, neurotransmitter synthesis, and phospholipid metabolism. Recent studies indicate that the human body produces approximately 6–8 g of SAM daily, and its metabolic abnormalities are closely related to various diseases such as depression, liver disease, and osteoarthritis. In clinical treatment, SAM has been widely used to treat liver diseases such as intrahepatic cholestasis, viral hepatitis, and alcoholic liver disease. It has also shown significant efficacy in improving depressive symptoms, and when used in combination with L-DOPA, it can effectively enhance the treatment effect of Parkinson's disease. SAM hydrolases (such as the MJ1651 enzyme derived from Methanococcus jannaschii) play a crucial role in maintaining intracellular SAM homeostasis by catalyzing the breakdown of SAM into methylthioadenosine and homoserine. MJ1651 enzyme has become an ideal model for studying SAM metabolism due to its unique thermal stability and catalytic mechanism, but its natural form faces multiple technical bottlenecks in practical applications.

[0003] Natural SAM hydrolases face three major technical bottlenecks in practical applications, which severely restrict their application value in industrial catalysis and biomedicine: (1) Generally low catalytic efficiency: The specific activity of most wild-type SAM hydrolases is less than 5 U / mg, which is far below the basic requirements for industrial catalysis (usually >20 U / mg). In contrast, the specific activity of the recently engineered halide methyltransferase BxHMT mutant M4 can reach 4.08 U / mg, but it still cannot meet the industrial needs. This low catalytic efficiency leads to a longer reaction cycle and increased production costs, which severely limits its potential for large-scale application. (2) Poor enzyme stability: The half-life at room temperature (25℃) is usually less than 8 hours, which is difficult to meet the needs of continuous production. Especially under industrial production conditions, enzyme molecules are easily deactivated by temperature, pH and mechanical shear force, requiring frequent catalyst replacement, which greatly increases production costs. In contrast, although some engineered SAM synthase mutants (such as I303V / Q22R / M255K) have improved in activity, the stability problem has not been fundamentally solved. (3) Severe substrate inhibition: When the SAM concentration exceeds 1 mM, the activity of most enzymes decreases by more than 30%, forcing the substrate concentration in the reaction system to remain at a low level, severely affecting product yield and production efficiency. This phenomenon also exists in SAM synthases; when SAM accumulation exceeds 1 mM, most SAM synthases exhibit significant product inhibition. This substrate inhibition effect is closely related to the allosteric regulation mechanism of enzymes and is one of the key factors limiting their industrial application. In addition, industrial applications place other stringent requirements on enzyme catalysis technology: reaction conditions must be compatible with large-scale production, costs must be controlled below $50 / kg of product, and process stability must meet the requirement of a coefficient of variation of <5% for 30 consecutive batches of production. Existing technologies cannot simultaneously meet these requirements, and there is an urgent need to develop innovative methods that combine high efficiency and economy.

[0004] Currently, the main technical routes for improving the activity of SAM hydrolases include rational protein design, directed evolution, reaction engineering optimization, and immobilization. However, these methods all have significant limitations: (1) Rational design strategy: Site-directed mutagenesis based on enzyme structure (such as the E56D / V195R / S281Y mutation of SAM synthase) can increase enzyme activity by 30% to 56.8%, but the improvement is limited. This strategy relies on a deep understanding of the relationship between enzyme structure and function. However, the catalytic mechanism of SAM hydrolases is complex, and the key active sites and allosteric regulatory networks have not been fully elucidated, making it difficult to select rational design targets. (2) Directed evolution technology: By constructing a random mutation library through error-prone PCR and combining it with high-throughput screening (such as the glycine oxidase / methyltransferase combined method), mutants with an 86.3% increase in SAM accumulation capacity (such as I303V / Q22R / M255K) can be obtained. However, this method is very labor-intensive, and the accumulation of beneficial mutations is limited, with an improvement of less than 15% in hydrolases activity. (3) Reaction engineering optimization: By adjusting the reaction temperature, pH, or adding cofactors (such as K+) + (3) Immobilization technology: Immobilizing enzymes on a carrier (such as SAM synthase) can partially maintain enzyme conformational stability. For example, in SAM synthesis, adding magnesium salt, potassium salt and mercaptoethanol to the reaction system and controlling the reaction temperature at 25-30℃ and pH at 7.0-8.0 can improve product yield. However, these methods have limited improvement on catalytic efficiency (usually <10%) and cannot fundamentally solve the substrate inhibition problem. (4) Immobilization technology: Immobilizing enzymes on a carrier (such as SAM synthase immobilization) can enhance operational stability and enable enzyme reuse. Studies have shown that immobilization technology can extend the half-life of enzymes by 2-3 times, but may introduce diffusion limitation, reduce apparent activity, and the immobilization process is complex and increases production costs.

[0005] It is worth noting that existing patented technologies (such as CN117384876A) mainly focus on enzyme molecule modification or immobilization carrier development, with almost no research on ionic effects in the reaction system. Particularly in the area of ​​halide ion regulation, although sporadic studies have shown that K... + It can maintain the conformational stability of certain enzymes, but regarding halogen anions (F... - Cl - ,Br - I - The mechanism by which halogenated methane methyltransferases affect SAM hydrolases has not been systematically reported. Although studies on halogenated methane methyltransferases (MCTs) have shown that halogen affinity exists at I... - >Br - >Cl - >F - The order of these sequences is fundamentally different from the catalytic mechanism of hydrolytic enzymes. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a SAM hydrolase mutant and its applications. Through an innovative mechanism, it overcomes the bottlenecks in SAM hydrolase applications. The synergistic effect of halide ion regulation technology and the mutant offers a novel research direction for the field of biocatalysis. This technology not only solves the long-standing problems of substrate inhibition and stability but also opens up a new "ion engineering" research paradigm for enzyme engineering, possessing profound scientific significance and application value.

[0007] This invention is achieved through the following technical solution:

[0008] A SAM hydrolase mutant, wherein the SAM hydrolase mutant is derived from the MJ1651 protein sequence with NCBI number MEO2116876.1 by any of the following mutations:

[0009] F19I, F19L, F19P, R89C, R89E, R89Q.

[0010] The gene encoding the above-mentioned SAM hydrolase mutant.

[0011] Recombinant vectors containing the above-mentioned genes.

[0012] Immobilized enzymes or engineered bacteria containing the above-mentioned SAM hydrolase mutants or the above-mentioned recombinant vectors.

[0013] The application of the above-mentioned SAM hydrolase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned immobilized enzyme or engineered bacteria in catalytic substrates.

[0014] Preferably, the substrate is S-adenosyl-L-methionine.

[0015] A method for producing adenosine based on halide-enhanced SAM hydrolase and its mutant activity involves adding the aforementioned SAM hydrolase mutant or an expression system containing the mutant to a reaction system containing a substrate, while simultaneously adding halide ions to carry out the reaction. The substrate is S-adenosine-L-methionine.

[0016] Preferably, the concentration of the halide ions is 400 mM.

[0017] Preferably, the halide ion is F. - Cl - ,Br - I - .

[0018] Preferably, the halide ions exist in the form of potassium halides, including KF, KCl, KBr, and KI.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention is the first to demonstrate the potent activation effect of halide anions on SAM hydrolase (MJ1651), overturning traditional understanding (previously only the cationic effect was considered), and revealing for the first time the F - >I - >Br - =Cl - The unique active sequence of F, in which F - The activation capacity for the mutant R89C was 6-fold (adenosine production increased by 600%), far exceeding the limits of existing technologies (typically <100%). This discovery also overturns the traditional theory that "halogen affinity is positively correlated with ionic radius" (as seen in MCT enzymes with I...). - >Br - >Cl - >F - This provides a new paradigm for the study of enzyme catalytic mechanisms.

[0021] (2) This invention breaks through the conventional concentration limitations of ionic additives (usually ≤100mM), and for the first time proves that a high concentration of 400mM halide salt can significantly improve enzyme activity without causing salting out, laying the foundation for industrial-grade high ionic strength reaction systems. Furthermore, this invention replaces complex enzyme modification processes with inexpensive halide salts, reducing industrialization costs by more than 50%. Experiments show that under optimal conditions (100μM enzyme, 2.8mM SAM, 400mM KCl, reaction at 30℃ for 24h), the yield of adenosine, the product of SAM hydrolysis by the wild-type enzyme, is >20% higher than that of the control group without added halide ions.

[0022] (3) Using the method of this invention, the yield of adenosine in a single batch reaches 582g (yield 88%), which is 82% higher than that of the chemical method (320g, 65%). The production cycle is shortened to 36h, which is 3.3 times faster than that of the chemical method (120h), and the capacity per unit time is increased by 450% (16.2g / h vs 2.7g / h). The method of this invention uses water as the only solvent (the chemical method requires xylene / DMF), completely eliminating VOC pollution, and the amount of waste is <5kg / batch (the chemical method is >50kg), which meets the EU REACH green standard. The total cost of this invention is $80 / kg (enzyme $15 + raw materials $40 + energy consumption $25), which is $55 lower than that of the chemical method ($135 / kg). Based on an annual production capacity of 116.4 tons, the annual cost savings are $6.4 million ($55 / kg × 116,400kg). The immobilized enzyme of this invention can be reused 5 times, and the enzyme cost accounts for only 18.75% (chemical catalysts are not recyclable). Attached Figure Description

[0023] Figure 1 The image shows the plasmid pattern of MJ1651 in Example 1;

[0024] Figure 2The structure of MJ1651 in Example 1 is compared with that of SAM hydrolase (2ZBU) containing adenosine product;

[0025] Figure 3 These are gel images of purified proteins from the wild-type and mutant MJ1651 in Example 2.

[0026] Figure 4 The effect of halide ions on adenosine production in Example 3. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0028] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Experimental materials: Plasmid pET28a was stored in our research group's laboratory. All genes used in this study were expressed with codon optimization and in the pET28a vector digested with NdeI and XhoI. The MJ1651 mutant was generated using a seamless cloning and assembly kit (Zoman, Beijing). Tryptone, yeast extract, and sodium chloride were purchased from Sinopharm Group. Trifluoroacetic acid, tris(hydroxymethyl)aminomethane (Tris), sodium dihydrogen phosphate, disodium hydrogen phosphate, and phosphate were all purchased from Maclean's Reagent Company. DpnI rapid digestion enzyme and PrimeSTAR high-fidelity enzyme were purchased from Takara.

[0031] Example 1: Construction of expression vector and design of SAM hydrolase mutant

[0032] The synthesis of the MJ1651 whole genome (NCBI ID: MEO2116876.1) was commissioned to Nanjing GenScript Biotech Co., Ltd., and the expression vector used was PET-28a with a His tag. The constructed plasmid is shown below. Figure 1 As shown. The crystal structure of MJ1651 was aligned with the crystal structure of the SAM hydrolase containing the product adenosine (PDB:2ZBU) using Pymol software. Figure 2 F19 and R89 in the active pocket were selected as mutation sites. Six mutants, F19I, F19L, F19P, R89C, R89E and R89Q, were randomly selected. The primer design is shown in Table 1 below.

[0033] Table 1 Primers used for MJ1651 mutations

[0034]

[0035] The specific process of site-directed mutation is as follows:

[0036] (1) Each 20μL system

[0037] 18 μL mixture + 2 μL primer F;

[0038] 18 μL mixture + 2 μL primer R.

[0039] After preparing the reaction mixture in both tubes, place them in a PCR instrument for reaction. The reaction procedure is as follows:

[0040] Denature at 98℃ for 1 min, (98℃, 10 s, 60℃, 15 s, 72℃, 3 min, 30 s) 3 cycles, extend at 72℃ for 10 min, store at 4℃.

[0041] (2) After the reaction is complete, the two reaction systems are mixed to form a 40 μL system, and an appropriate amount of high-fidelity polymerase (PrimeSTAR) is added. Then, the reaction is continued for 16 cycles using the above reaction procedure.

[0042] (3) Add 1 μL of DpnI fast digester and 4 μL of 10×NEB cutsmart buffer to the obtained PCR product to digest the template plasmid (the principle is that DpnI enzyme only digests methylated DNA) and place it at 37℃ for 1 h (or 2-3 h to ensure clean processing).

[0043] (4) Take 5 μL and run it on a gel to check for bands. Take the remaining portion for bacterial transformation and plate it. The transformation process is as follows: take 5 μL of plasmid and incubate it on ice for 30 min to make the plasmid adhere tightly to the cell wall. Incubate it in a 42℃ metal bath for 45 s to allow the plasmid to enter the cell. Incubate it on ice for 4 min to close the cell wall. Add 1 mL of antibiotic-free medium and incubate at 37℃, 220 rpm for 1 h to allow antibiotic expression. Centrifuge and take the supernatant to 100 μL for plate.

[0044] (5) After 12 hours, select 3 to 5 clones for sequencing.

[0045] Example 2: Protein Expression and Purification

[0046] 1. Cultivating bacteria

[0047] Inoculate the bacteria from the preservation tube into a shaker containing 3 mL of Kana resistance medium. Incubate overnight at 37°C and 200 rpm for 12 h. Then, inoculate 3 mL of bacterial culture into a 500 mL Erlenmeyer flask containing 200 mL of Kana resistance medium. Incubate at 37°C and 200 rpm for 2 h until OD = 0.6–0.8. Add 200 μL of 100 mM IPTG and continue incubation for 20 h.

[0048] 2. Collecting and crushing bacteria

[0049] The bacterial culture was collected in 50 mL centrifuge tubes at 8000 rpm and 4℃, washed twice, and 400 mL of bacterial culture was resuspended in 30 mL (pH=8.0) PBS. 50 mg of lysozyme was added and the culture was disrupted for 30 min (50 power ratio, 5 s over time and 5 s intermittent time). The disrupted bacterial culture was centrifuged at 10000 rpm and 4℃ for 1 h to precipitate organelles. The protein supernatant was filtered through a 0.45 μm membrane.

[0050] 3. Purification

[0051] Using the AKTA system, a 5 mL nickel column was first washed with 30 mL of 500 mM imidazole and then equilibrated with 30 mL of 20 mM imidazole. Protein was loaded onto the nickel column at a flow rate of 2 mL / min, leveled with 20 mM imidazole, then leveled again with 50 mM imidazole. Finally, the protein was eluted using an automated elution program at 250 mM. 40 μL of the eluted protein was added to 10 μL of 5× protein loading buffer and heated at 95°C for 10 min to allow for complete protein denaturation. 10 μL of the sample was then run on a gel for 70 min. Alternatively, a gravity column was used to pre-equilibrate 10 mL of Ni-NTA resin with 35 mL of lysis buffer, then added to the supernatant and incubated at 4°C for 30 min. The mixture was then loaded onto a gravity column and washed with 100 mL of lysis buffer containing 20 mM imidazole (pH = 8.0) and 50 mL of lysis buffer containing 200 mM imidazole (pH = 8.0). The purified protein was eluted with elution buffer (500 mM imidazole lysis buffer, pH 8.0). The elution fraction was concentrated to 2 mL using an Amicon centrifuge (Millipore, Germany), and then desalted using a PD-10 desalting column (Cytiva) with lysis buffer. The desalted protein was concentrated and stored at -80°C until further use. Protein concentration was determined using a Beyotime BCA kit, and protein levels were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 3 As shown, the molecular weight of MJ1651 and its mutant is 31 kDa, and the corresponding protein bands are between 25 kDa and 33 kDa, indicating that they were successfully expressed and purified.

[0052] Example 3 Enzyme activity reaction and activity test

[0053] 1. Preparation before the experiment

[0054] (1) Reagents and Instruments

[0055] Substrate: S-adenosyl-L-methionine (SAM, purity ≥98%, Sigma-Aldrich);

[0056] Enzyme preparation: Recombinant SAM hydrolase (MJ1651, expressed in Escherichia coli BL21(DE3), with a purified protein concentration ≥1mg / mL and a specific activity ≥10U / mg);

[0057] Buffer solution: 0.1M sodium phosphate buffer (pH = 8.0, adjusted with NaOH, calibrated with a pH meter);

[0058] Potassium halides: potassium fluoride (KF), potassium chloride (KCl), potassium bromide (KBr), and potassium iodide (KI), all of analytical grade (≥99.0%);

[0059] Terminating agent: 10% trifluoroacetic acid (TFA, v / v, prepared with ultrapure water and sterilized by filtration);

[0060] Chromatographic reagents: acetonitrile (chromatographic grade, Fisher Scientific), ultrapure water (resistivity ≥18.2 MΩ·cm);

[0061] Instruments: Thermo Scientific constant temperature shaking incubator, Beckman Coulter Avanti J-26XPI high-speed centrifuge, Shimadzu LC-30A high performance liquid chromatograph with autosampler, Shimadzu Inertsil ODS-3 column (5μm×4.6mm×150mm), electronic balance (Sartorius CP225D), pH meter (Mettler Toledo S220).

[0062] (2) Preparation of standard products

[0063] Accurately weigh 10 mg of adenosine standard (≥99.0%, Sigma-Aldrich), dissolve it in ultrapure water and dilute to 10 mL to prepare a 1 mg / mL stock solution;

[0064] Take 100 μL, 200 μL, 500 μL, 1 mL, and 2 mL of stock solution, respectively, and dilute them with ultrapure water to 10 mL to obtain standard working solutions with concentration gradients of 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL (for plotting standard curves).

[0065] 2. Preparation and incubation of the reaction system

[0066] (1) Construction of the reaction system (total volume 200 μL)

[0067] Add the following ingredients sequentially to a sterile 96-well plate:

[0068] Sodium phosphate buffer (pH=8.0): 140 μL (to maintain the acid-base stability of the reaction system);

[0069] SAM substrate: 2.8 mM (i.e., 0.28 μmol, dissolved in buffer);

[0070] Potassium halides (KF / KCl / KBr / KI): 400 mM (i.e., 0.4 μmol, dissolved in buffer);

[0071] Enzyme solution: 100 μM (i.e., 0.02 μmol, diluted with buffer to the required concentration);

[0072] Note: All reagents must be filtered through a 0.22μm filter membrane for sterilization to avoid contamination; SAM must be prepared fresh for use (as it is easily degraded) and stored at 4℃ for no more than 24 hours after preparation.

[0073] (2) Control of incubation conditions

[0074] Place the centrifuge tubes in a constant temperature shaking incubator, set the temperature to 30℃ (the optimal reaction temperature for the enzyme) and the rotation speed to 80 rpm (gentle shaking to promote contact between the substrate and the enzyme);

[0075] The incubation time was set at 24 hours (as verified by preliminary experiments, the SAM conversion rate tends to stabilize at this time, avoiding product degradation due to excessively long reaction time);

[0076] Each experiment was conducted in three biological replicates, with a blank control group (no enzyme solution added, all other components were the same) and a positive control group (with known active enzyme solution) to ensure data reliability.

[0077] 3. Reaction Termination and Sample Preparation

[0078] (1) Termination of enzyme-catalyzed reaction

[0079] After incubation, remove the centrifuge tube and immediately add an equal volume (200 μL) of 10% TFA (v / v), vortex for 30 seconds (to ensure sufficient enzyme denaturation); The role of TFA is to denature and precipitate the enzyme protein through a strongly acidic environment (pH ≤ 3.0), thereby terminating the catalytic reaction and inhibiting the spontaneous hydrolysis of the substrate or product (SAM is more stable under acidic conditions).

[0080] (2) Centrifugal separation

[0081] Transfer the terminated reaction solution to a 1.5 mL centrifuge tube (pre-cooled to 4°C) and centrifuge at 12000 g for 50 min (4°C). The purpose of centrifugation is to precipitate denatured enzymes and unreacted proteins to the bottom of the tube, and the supernatant contains the soluble product L-methionine.

[0082] (3) Supernatant treatment

[0083] Carefully aspirate 100 μL of supernatant using a pipette (avoid touching the precipitate) and transfer it to a new liquid chromatography vial (clearly labeled). If the supernatant is cloudy (it may contain incompletely precipitated enzyme), centrifuge again at 15000g for 10 min and repeat the process until the supernatant is clear.

[0084] 4. HPLC analysis and product quantification

[0085] (1) Chromatographic conditions settings

[0086] Chromatographic column: Shimadzu Inertsil ODS-3C18 column (5μm×4.6mm×150mm);

[0087] Mobile phase:

[0088] Solvent A: 0.1% (v / v) TFA aqueous solution (prepared with ultrapure water and ultrasonically degassed for 10 min);

[0089] Solvent B: 0.1% (v / v) TFA acetonitrile solution (acetonitrile:water = 99.9:0.1, ultrasonically degassed for 10 min);

[0090] Table 2 Gradient elution program (flow rate 1 mL / min)

[0091] Time (min) Solvent B percentage (%) Flow rate (mL / min) 0-1 0 1 1-10 0-4.9 1 10-15 4.9-14 1 15-19 14-40 1 19-19.01 40-95 1 19.01-21 95 1 21-26 95-0 1

[0092] Detection parameters: Detection wavelength 260nm (the maximum absorption wavelengths of SAM and L-methionine are 258nm and 261nm respectively, and 260nm is the optimal detection wavelength for the overlap between the two); column temperature 40℃ (to improve separation efficiency); injection volume 20μL (to cover the detection linear range).

[0093] (2) Plotting the standard curve

[0094] L-methionine standard working solutions (10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) were sequentially injected into the HPLC system, and the peak areas were recorded. A standard curve equation (A = kC + b, R0) was obtained by fitting the concentration (C, μg / mL) to the x-axis and the peak area (A) to the y-axis. 2 ≥0.999).

[0095] Sample detection and data processing: The processed sample supernatant was injected into HPLC, and the peak area of ​​L-methionine was detected under the conditions described above; the concentration of L-methionine in the sample (C0) was calculated based on the standard curve. sample =(A sample -b) / k).

[0096] Product yield calculation formula: Hydrolysis rate (%) = C SAM初始 ×V SAM ×Csample ×V 总 ×D×100%;

[0097] In the formula: V 总 The total reaction volume is 200 μL, D is the sample dilution factor (1 if undiluted), and C is the total reaction volume. SAM初始 The initial concentration of SAM (2.8 mM), V SAM Add 200 μL of SAM.

[0098] 5. Quality Control and Precautions

[0099] Blank control: Each experiment should include an enzyme-free blank control group (containing only buffer, SAM and potassium halide) to exclude interference from substrate spontaneous hydrolysis or TFA on the detection.

[0100] Instrument calibration: Before using HPLC, the wavelength and flow rate must be calibrated with standards to ensure a stable baseline;

[0101] Repeatability verification: Each experiment was repeated 3 times, and the mean and standard deviation of the hydrolysis rate were calculated.

[0102] 6. Experimental Results

[0103] like Figure 4 As shown, the addition of potassium halide significantly increased the adenosine production in all groups, especially in R89C, where the adenosine production increased more than sixfold after the addition of potassium fluoride.

[0104] Example 4: 50L scale-up process for producing adenosine from SAM hydrolysis using MJ1651

[0105] Adenosine is a key intermediate in nucleic acid drugs (such as the antiviral drug ribavirin). Traditional chemical synthesis methods suffer from problems such as numerous steps (≥5 steps) and heavy pollution (large amounts of organic solvents). This embodiment utilizes the MJ1651 enzyme to catalyze the hydrolysis of SAM to generate adenosine in one step, aiming to achieve: product purity ≥95%, single batch yield ≥500g, and production cost ≤$80 / kg (40% lower than the chemical method). The specific steps are as follows:

[0106] Table 3 Raw Materials and Equipment

[0107] project Specifications / Parameters Recombinant MJ1651 enzyme Specific activity ≥6 U / mg (after optimization with 400 mg MMKF) SAM raw materials Purity ≥ 98% (Shanghai Maclean Biochemical) Reactor 50L Stainless Steel Stirred Tank (with Online pH / Temperature Monitoring) Purification system Hollow fiber ultrafiltration (10 kDa) + C18 reversed-phase chromatography column

[0108] 1. Process Flow (Reaction Stage)

[0109] (1) Feeding

[0110] 40L 50mM sodium phosphate buffer (pH=8.0), 2.8kg SAM (final concentration 2.8mM, 10% excess), 1.6kg KF (final concentration 400mM), 200g MJ1651 enzyme (final concentration 100μM).

[0111] (2) Conditional control

[0112] Temperature 30±0.5℃, stirring speed 80rpm, reaction time 22h (stopped when HPLC monitoring conversion rate ≥90%), reaction termination and crude separation: add 4L 10% formic acid (final concentration 1%), plate and frame filter (0.45μm membrane) to remove precipitated protein.

[0113] (3) Product refining

[0114] Ultrafiltration: Tangential flow filtration (10kDa membrane pack) removes large molecular impurities;

[0115] Chromatography: C18 column gradient elution (water:methanol = 95:5 → 70:30);

[0116] Crystallization: Concentrate the filtrate to 5L and crystallize at 4℃ for 12h.

[0117] 2. Key Data and Results (Table 4)

[0118] Table 4 Key Data and Results

[0119] index result Comparison (Chemical Method) adenosine production 582g / batch (yield 88%) 320g / batch (65%) Purity (HPLC) 96.2％ 92.5％ Production cycle 36h 120h Solvent consumption Water-based (green process) Xylene / DMF, etc. Waste generation <5kg / batch >50kg / batch

[0120] As shown in Table 4, the process of this invention significantly improves adenosine yield and purity compared to conventional chemical methods, with a single batch adenosine yield increasing by 82%. Simultaneously, this process has a shorter production cycle, 3.3 times faster than the chemical method, and a 450% increase in capacity per unit time (16.2 g / h vs 2.7 g / h). Furthermore, this process uses water as the sole solvent (chemical methods require xylene / DMF, etc.), completely eliminating VOC pollution, with waste generation <5 kg / batch, less harmful substances generated, and safer solvent usage.

[0121] 3. Economic Analysis

[0122] (1) Cost Structure

[0123] Enzyme cost: $15 / kg (reusable 5 times after immobilization);

[0124] SAM raw material: $40 / kg (bulk purchase price);

[0125] Energy / Consumables: $25 / kg;

[0126] Total cost: $80 / kg.

[0127] In comparison, the total cost of the chemical method is $135 / kg.

[0128] (2) Annual benefits (based on 200 batches / year)

[0129] Annual output: 116.4 kg;

[0130] Cost savings (compared to chemical methods): 116.4 × (135 - 80) = $6402.

[0131] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A SAM hydrolase mutant, characterized in that, The SAM hydrolase mutant is the MJ1651 protein sequence with NCBI number MEO2116876.1 mutated by any of the following methods: F19I, F19L, F19P, R89C, R89E, R89Q.

2. The gene encoding the SAM hydrolase mutant as described in claim 1.

3. A recombinant vector containing the gene as described in claim 2.

4. An immobilized enzyme or engineered bacteria comprising the SAM hydrolase mutant as described in claim 1 or the recombinant vector as described in claim 3.

5. The application of the SAM hydrolase mutant as described in claim 1, the gene as described in claim 2, the recombinant vector as described in claim 3, or the immobilized enzyme or engineered bacteria as described in claim 4 in catalytic substrates.

6. The application according to claim 5, characterized in that, The substrate is S-adenosyl-L-methionine.

7. A method for producing adenosine based on halide-enhanced SAM hydrolase and its mutant activity, characterized in that, The SAM hydrolase mutant as described in claim 1 or an expression system containing the mutant is added to a reaction system containing a substrate, and a halide ion is added to carry out the reaction. The substrate is S-adenosyl-L-methionine.

8. A method for producing adenosine according to claim 7, characterized in that, The concentration of the halide ions is 400 mM.

9. A method for producing adenosine according to claim 7, characterized in that, The halide ion is F. - Cl - ,Br - I - .

10. A method for producing adenosine according to claim 7, characterized in that, The halide ions exist in the form of potassium halides, including KF, KCl, KBr, and KI.

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