A microbial strain for preparing sitagliptin compounds and its application

Abstract

The present invention provides a microbial strain for preparing sitagliptin compounds and its application, belonging to the field of bioengineering technology. A microbial strain for preparing sitagliptin compounds, wherein the microbial strain is Pseudomonas aeruginosa NESita-5, and the Pseudomonas aeruginosa NESita-5 is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit date being June 21, 2024, and the deposit number being CCTCC NO: M 20241336. The present invention uses a simple and visual result analysis method, uses the culture solution containing microbial cells as a catalyst, observes the color depth of the reaction solution, and quickly judges whether there is enzyme activity and the level of enzyme activity. Through this method, a microbial strain containing transaminase is obtained, which can be used for catalytic production of the drug sitagliptin and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular, to a microbial strain for preparing sitagliptin compounds and its application. Background Art

[0002] Sitagliptin (MK-0431) is the first dipeptidyl peptidase-IV (DPP-IV) inhibitor approved by the US FDA for the treatment of type II diabetes. DPP-IV is a multifunctional enzyme that exists in the form of a homodimer on the cell membrane. It can cleave various peptide hormones, including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which are released in response to dietary intake. These two hormones are closely related to type II diabetes. GLP-1 and GIP can increase insulin synthesis and release from pancreatic β-cells through intracellular signaling pathways, and reduce glucagon secretion from pancreatic α-cells, thereby reducing hepatic glucose production. However, both GLP-1 and GIP are rapidly metabolized by DPP-4, resulting in the loss of their insulinotropic effects. Therefore, DPP-IV inhibitors improve postprandial blood glucose by inhibiting DPP-IV to reduce the degradation of these two hormones and increase their concentrations in plasma. In addition, sitagliptin increases insulin secretion in a glucose-dependent manner, has a moderate hypoglycemic effect, does not cause hypoglycemia, and has no side effects such as weight gain, nausea, and vomiting. In recent years, it has been widely used and sold.

[0003] Currently, most synthetic methods on the market require the use of catalysts, which can be divided into two categories: metal-catalyzed synthesis and enzyme-catalyzed synthesis. For metal-catalyzed synthesis, the metal catalysts used include ruthenium, platinum, palladium, etc. These catalysts are expensive, and it is also difficult to remove or recover these metal catalysts from the product. At the same time, the chiral purity of the chiral amino intermediates generated by the process using metal catalysts is not high enough and needs to be further processed by methods such as recrystallization to meet the requirements of the pharmacopoeia. The enzyme-catalyzed methods all use ω-transaminase (ω-ATA) to achieve the synthesis, and are all realized through the artificial modification of different wild-type transaminases. These enzymes use an amino donor, take the sitagliptin precursor ketone as the substrate, and use pyridoxal phosphate (PLP) as the coenzyme to catalyze the formation of specific chiral sitagliptin. Compared with metal catalysts, enzyme-catalyzed synthesis of chiral amino intermediates has high stereoselectivity, and the ee value of the transamination product can reach 99% or above, and the reaction conditions are mild. However, the publicly available data shows that due to the special chemical structure of the sitagliptin chiral amino intermediate, wild-type transaminases existing in nature either cannot catalyze the synthesis reaction of this chiral amino intermediate or have very low catalytic activity and cannot be used for industrial production.

[0004] In the invention, through the application of an amine donor (o-xylenediamine dihydrochloride), which has the dual function of effectively substituting the adverse reaction equilibrium to form a product and simultaneously providing a high-throughput colorimetric screening method independent of the substrate to detect the activity of the desired transaminase-containing microbial strains. The invention develops a transaminase-containing microorganism with high catalytic activity, good enantioselectivity, and good substrate tolerance, which can be used for the catalytic synthesis of aromatic isoindoles, especially for the catalytic synthesis of sitagliptin. Summary of the Invention

[0005] To make up for the above deficiencies, the present invention provides a microbial strain for preparing sitagliptin compounds and its application, aiming to improve the deficiencies in the prior art.

[0006] The present invention is implemented as follows:

[0007] The present invention provides a microbial strain for preparing sitagliptin compounds, characterized in that the microbial strain is Pseudomonas aeruginosa NESita-5, and the Pseudomonas aeruginosa NESita-5 is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan, China, Wuhan University, and the deposit date is June 21, 2024, and the deposit number is CCTCC NO: M 20241336.

[0008] A method for preparing a microbial strain for preparing sitagliptin compounds, comprising the following steps:

[0009] Step 1: Soil sampling and microbial isolation;

[0010] Step 2: Culturing the obtained microorganisms;

[0011] Step 3: Preparing a mixed culture and a crude enzyme solution;

[0012] Step 4: Screening the positive crude enzyme solution using a reaction system.

[0013] Preferably, the screening method for screening the positive crude enzyme solution using the reaction system is as follows:

[0014] Using o-xylenediamine dihydrochloride as the amino donor for the catalytic reaction, and judging the activity intensity according to the depth of the color;

[0015] The catalytic reaction includes a reaction system mother liquor and a crude enzyme solution. The volume of the reaction system mother liquor is 80-160 μL, and the crude enzyme solution is mixed with the reaction system mother liquor in an equal volume.

[0016] Preferably, the mother liquor of the reaction system comprises 0.1 - 0.3 mM of tris(hydroxymethyl)aminomethane, 4 - 12 g / L of donor o - phenylenediamine dihydrochloride, 0.8 - 2.4 g / L of substrate sitagliptin precursor ketone, 0.1 - 0.3 mM of coenzyme pyridoxal phosphate, and methanol with a final concentration of 20% (v / v) - 60% (v / v), and the pH value of the mother liquor of the reaction system is 6.0 - 10.0.

[0017] Use of a microbial strain for preparing a sitagliptin compound, and the preparation method of the sitagliptin compound is as follows:

[0018] Using sitagliptin precursor ketone as a substrate, methanol as a cosolvent, pyridoxal phosphate as a coenzyme, tris(hydroxymethyl)aminomethane as a buffer medium, and adding isopropylamine as a reaction system to prepare sitagliptin by adding crude enzyme solution.

[0019] Preferably, the reaction solvent in the reaction system comprises water, methanol, ethanol, propanol, isopropanol, isopropyl acetate, dimethyl sulfoxide or dimethylformamide.

[0020] Preferably, the conversion temperature in the reaction system is 20°C - 50°C.

[0021] Preferably, the pH value in the reaction system is 6.0 - 10.0.

[0022] Preferably, the volume of the crude enzyme solution is 200 - 400 μL, the isopropylamine is 2 - 4 g / L, the concentration of tris(hydroxymethyl)aminomethane is 0.1 - 0.2 mM, the concentration of the sitagliptin precursor ketone substrate is 0.4 - 0.8 g / L, 0.1 - 0.2 mM of coenzyme pyridoxal phosphate, and the final concentration of methanol is 10 - 20%.

[0023] The beneficial effects of the present invention are:

[0024] By using a simple and visual result analysis method, using the culture solution containing microbial cells as a catalyst, observing the color depth of the reaction solution, quickly judging whether there is enzyme activity and the level of enzyme activity, a microbial strain containing transaminase is obtained by this method, which can be used for catalytic production of the drug sitagliptin and has good application prospects. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It is a reaction route diagram for the preparation of sitagliptin by using a microbial strain for the preparation of sitagliptin compounds provided by an embodiment of the present invention;

[0027] Figure 2 It is a reaction result diagram of the cells that can catalyze the conversion of sitagliptin by using a microbial strain for the preparation of sitagliptin compounds provided by an embodiment of the present invention.

[0028] In the figure: 1, sitagliptin precursor ketone; 2, sitagliptin; 3, o-xylenediamine dihydrochloride; 4, cyclic imine; 5, isoindole. Specific embodiments

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0030] Example 1: A microbial strain for the preparation of sitagliptin compounds, which is Pseudomonas aeruginosa NESita-5. The Pseudomonas aeruginosa NESita-5 is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit date being June 21, 2024, and the deposit number being CCTCC NO: M 20241336.

[0031] Example 2: A preparation method for a microbial strain for the preparation of sitagliptin compounds, comprising the following steps:

[0032] Step 1: Soil sampling and microbial isolation;

[0033] Select an uncontaminated sampling site and dig the soil 5-10 cm below the surface; collect the soil sample using a sterile tool and place it in a sterile sampling bag or bottle; air-dry and sieve the soil sample to remove large particles and debris; weigh 1 g of the soil sample and put it into a bottle containing 99 ml of sterile physiological saline (0.85% NaCl), and shake vigorously for 5-10 minutes to obtain a soil suspension;

[0034] Prepare a series of dilution bottles with a 10-fold increase, each containing 9 ml of sterile physiological saline; take 1 ml of the soil suspension and add it to the first dilution bottle, and mix well; take 1 ml of the suspension from the first dilution bottle and add it to the second dilution bottle, and so on until the required dilution factor;

[0035] Prepare culture media suitable for the growth of different microorganisms; the purpose of selecting culture media with different nutrient components is to obtain various different types of microorganisms as much as possible, that is, to obtain microorganisms containing transaminase with the greatest possibility; the components of various different culture media are as follows:

[0036] Medium No. 1. **Nutrient Agar**

[0037] - Beef extract: 3 g

[0038] - Peptone: 5 g

[0039] - Sodium chloride: 5 g

[0040] - Agar: 15 g

[0041] - Distilled water: 1000 ml

[0042] - Adjust the pH to 7.0 - 7.2

[0043] Medium No. 2. **Potato Dextrose Agar (PDA)**

[0044] - Potato: 200 g (filtrate after cooking)

[0045] - Glucose: 20 g

[0046] - Agar: 15 - 20 g

[0047] - Distilled water: 1000 ml

[0048] - Adjust the pH to 5.6 (suitable for fungi)

[0049] Medium No. 3. **Sabouraud Dextrose Agar (SDA)**

[0050] - Glucose: 40 g

[0051] - Trypticase: 10 g

[0052] - Agar: 15 - 20 g

[0053] - Distilled water: 1000 ml

[0054] - Adjust the pH to 5.6 (suitable for fungi)

[0055] Medium No. 4. **MacConkey Agar**

[0056] - Trypticase: 17 g

[0057] - Trypsin peptone: 3 g

[0058] - Bile salt mixture: 1.5 g

[0059] - Sodium chloride: 5 g

[0060] - Lactose: 10 g

[0061] - Neutral red: 0.03 g

[0062] - Eosine: 0.001 g

[0063] - Agar: 13.5 g

[0064] - Distilled water: 1000 ml

[0065] - Adjust the pH to 7.1 (selectively cultivate intestinal bacteria)

[0066] Medium No. 5. **Mannitol Salt Agar**

[0067] - Tryptone: 10 g

[0068] - Beef extract: 1 g

[0069] - Sodium chloride: 75 g

[0070] - Mannitol: 10 g

[0071] - Phenol red: 0.025 g

[0072] - Agar: 15 g

[0073] - Distilled water: 1000 ml

[0074] - Adjust the pH to 7.4 (selectively cultivate salt-tolerant bacteria)

[0075] Take 0.1 ml of soil suspensions with different dilution factors and evenly spread them on the corresponding culture medium plates; inoculate at least three plates for each dilution factor to ensure the reliability of the results; invert the plates and culture them at 37 °C for 24 to 48 hours to obtain colonies with different morphologies, colors and sizes; pick single colonies with a sterile inoculation loop and inoculate them onto new culture medium plates for purification culture; repeat the purification process until pure strains are obtained;

[0076] Step 2: Cultivate the obtained microorganisms;

[0077] After the above cultivation is completed, mix and cultivate the microorganisms obtained from different culture medium plates with a liquid culture medium of the same composition, and inoculate them with 10 single colonies as a 96-well deep well plate culture unit;

[0078] Step 3: Prepare the mixed culture and crude enzyme solution;

[0079] The detailed steps for culturing bacteria, harvesting cells, and lysing cells with lysozyme to release enzyme solution in a 96-well deep-well plate are as follows:

[0080] Inoculate bacteria into a 96-well deep-well plate containing culture medium; usually add 200 - 300 µL of culture medium to each well; place the deep-well plate in a shaker incubator, set the temperature to 25 degrees Celsius; adjust the shaker speed to 250 rpm to ensure sufficient gas exchange in the culture medium; usually culture for 18 - 36 hours until the culture medium becomes turbid, indicating that the bacteria have grown sufficiently;

[0081] Centrifuge the 96-well deep-well plate at a speed of 4000 - 5000 g for 10 - 15 minutes to precipitate the bacterial cells;

[0082] After centrifugation, carefully remove the supernatant, trying to avoid disturbing the precipitated bacterial cells;

[0083] Add an appropriate amount of lysis buffer containing lysozyme to each well, 50 - 100 µL per well; the concentration of lysozyme is generally 1 mg / mL, 50 - 100 µL per well;

[0084] Place the 96-well plate on ice for 15 - 30 minutes to allow lysozyme to act on the cell wall;

[0085] Then, place the 96-well plate in a 37°C water bath or incubator, gently shake to further promote cell lysis for 30 minutes;

[0086] Finally, centrifuge the lysed mixture again to separate the soluble components in the supernatant, and these components are the released enzyme solution;

[0087] Centrifuge at a speed of 12000 - 14000 rpm at 4 degrees Celsius for 10 - 15 minutes to precipitate the insoluble cell debris;

[0088] Carefully pipette the supernatant, which is the solution containing the target enzyme to be screened;

[0089] Step 4: Screen the positive crude enzyme solution using the reaction system;

[0090] Prepare the reaction system mother liquor (prepare and use immediately): Use 0.1 mM tris(hydroxymethyl)aminomethane as the buffer medium, the concentration of the donor o-phenylenediamine dihydrochloride is 4 g / L, the concentration of the substrate sitagliptin precursor ketone is 0.8 g / L, the coenzyme PLP is 0.2 mM, the methanol concentration is 40% (v / v), and the pH is 8.5;

[0091] The solution of the target enzyme to be screened prepared in Step 3 is directly used for the screening reaction; in a 96-well reaction plate, 80 μL of the crude enzyme solution and 80 μL of the reaction mother liquor are added, and the reaction is carried out in a shaker at 45 °C and 200 rpm. After 60 minutes, observe and analyze the color change situation;

[0092] Collect and count the well positions of the plates with color changes, and select the top 5 well positions with the darkest color for rescreening confirmation and strain tracing;

[0093] Examples 3 to 7 are respectively preparation methods of microbial strains for preparing sitagliptin compounds. The operation steps are the same as those in Example 2, except that the parameters such as the volume of the reaction system mother liquor and the crude enzyme solution, the culture time, the culture temperature, and the centrifugation speed are different. See the following table for details:

[0094]

[0095] Rescreening method: The suspected target enzyme solution that has developed color is diluted to one-half and one-fourth of the original concentration. In the same reaction system and conditions, three replicates are carried out for rescreening in the same system. The final color development situation is as Figure 2 shown ( Figure 2 The color development shown in is the color development result caused by the self-polymerization of isatin itself). The results show that the degree of color development increases with the increase of the catalytic enzyme activity. Therefore, it can be confirmed that this microorganism is very likely to contain the transaminase required for the production of sitagliptin.

[0096] Example 8: Application of a microbial strain for preparing a sitagliptin compound. The preparation method of the sitagliptin compound is as follows:

[0097] Using sitagliptin precursor ketone as the substrate, methanol as the co-solvent, pyridoxal phosphate as the coenzyme, and tris(hydroxymethyl)aminomethane as the buffer medium as the reaction system, sitagliptin is prepared by adding the crude enzyme solution; the reaction solvent in the reaction system includes water, methanol, ethanol, propanol, isopropanol, isopropyl acetate, dimethyl sulfoxide or dimethylformamide. Refer to Figure 1(In the figure: 1 is sitagliptin precursor ketone; 2 is sitagliptin; 3 is o - xylylenediamine dihydrochloride; 4 is cyclic imine; 5 is isoindole; the reaction process is that sitagliptin precursor ketone is catalytically converted into sitagliptin under the action of a microbial strain containing transaminase with the preservation number CCTCC NO: M 20241336; and in this process, o - xylylenediamine dihydrochloride is catalytically converted into benzaldehyde o - imine hydrochloride and HCl under the action of a microbial strain containing transaminase with the preservation number CCTCC NO: M 20241336, and then the generated benzaldehyde o - imine hydrochloride undergoes a self - cyclization reaction to form cyclic imine (it should be noted that the generated benzaldehyde o - imine hydrochloride exists for a very short time and will quickly undergo a self - cyclization reaction to form cyclic imine, and the generated benzaldehyde o - imine hydrochloride is the transition state of cyclic imine), the cyclic imine undergoes tautomerization to form isoindole, and the isoindole undergoes a polymerization reaction by itself to produce a black precipitate, which is Figure 2 the color change in

[0098] The catalytic reaction of the target molecule is carried out with the target crude enzyme solution prepared in Step Four; the reaction system is: 200 μL of the target crude enzyme solution, 2 g / L of isopropylamine, the concentration of tris(hydroxymethyl)aminomethane is 0.1 mM, the concentration of the substrate sitagliptin precursor ketone is 0.4 g / L, 0.1 mM of pyridoxal phosphate, the final concentration of methanol is 10%, and the pH of the reaction solution is 8.5; after 24 - hour conversion at 20 °C, after the reaction is completed, an equal volume of acetonitrile to the reaction system after the conversion reaction is added to inactivate, and then centrifuged (4000 rpm, 30 min), the supernatant after centrifugation is taken, and high - performance liquid chromatography analysis is carried out according to the following analysis method and the conversion rate from the substrate to the product is calculated; after calculation, the target crude enzyme solution achieved a substrate conversion rate of more than 95% in 24 hours, indicating that the transaminase produced by this strain can effectively catalyze the reaction of sitagliptin precursor ketone to sitagliptin.

[0099] Examples 9 to 13 are respectively the applications of a microbial strain for preparing sitagliptin compounds. The operation steps are the same as those in Example 8, only the parameters such as the volume of the crude enzyme solution, the concentrations of sitagliptin precursor ketone, methanol, pyridoxal phosphate, tris(hydroxymethyl)aminomethane and isopropylamine, the conversion time, the conversion temperature, the centrifugation speed, and the centrifugation time are different. See the following table for details:

[0100]

[0101] The analytical method was high performance liquid chromatography, and the conditions were as follows: the chromatographic column was Agilent Eclipse XDB-C8 (4.6 X 150 mm / 5 μm), the eluent was 10 mM NH4Ac / MeCN, the flow rate was 1.5 ml / min, and the column temperature was 40 °C. Retention times: ketoamide substrate 1.7 minutes, sitagliptin 1.4 minutes. The ketoamide substrate and product in the eluate were determined as the peak areas at 210 nm or 286 nm.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A microbial strain for preparing sitagliptin compound, characterized in that, The microbial strain is Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), NESita-5. The Pseudomonas aeruginosa NESita-5 is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is June 21, 2024, and the deposit number is CCTCC NO: M 20241336.

2. Use of Pseudomonas aeruginosa NESita-5 as described in claim 1 in the preparation of sitagliptin compounds, characterized in that, Sitagliptin is prepared by adding crude enzyme solution to a reaction system using sitagliptin precursor ketone as the substrate, methanol as the cosolvent, pyridoxal phosphate as the coenzyme, tris(hydroxymethyl)aminomethane as the buffer medium, and adding isopropylamine.

3. The application according to claim 2, wherein The reaction solvent in the reaction system includes water, methanol, ethanol, propanol, isopropanol, isopropyl acetate, dimethyl sulfoxide or dimethylformamide.

4. The application according to claim 2, characterized in that, The conversion temperature in the reaction system is 20°C - 50°C.

5. The application according to claim 2, characterized in that, The pH value in the reaction system is 6.0 - 10.

0.

6. The application according to claim 2, characterized in that, The volume of the crude enzyme solution is 200 - 400 μL, the isopropylamine is 2 - 4 g / L, the concentration of tris(hydroxymethyl)aminomethane is 0.1 - 0.2 mM, the concentration of the sitagliptin precursor ketone substrate is 0.4 - 0.8 g / L, 0.1 - 0.2 mM of coenzyme pyridoxal phosphate, and the final concentration of methanol is 10 - 20%.

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