Dehydrogenase mutant, gene, engineering bacterium and application

Abstract

The invention belongs to the technical field of enzyme catalysis, and particularly relates to a dehydrogenase mutant, a gene, an engineering bacterium and application, the dehydrogenase mutant is obtained through mutation of original dehydrogenase, the sequence of the original dehydrogenase is SEQ ID NO.1, and the mutation mode is that the 344 site is mutated from A to W, K, F, S or N, or the 130 site is mutated from H to T, W or F; the dehydrogenase mutation diagram provided by the invention has higher conversion rate, stronger specificity and faster reaction speed.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis technology, specifically relating to a dehydrogenase mutant, gene, engineered bacteria, and its uses. Background Technology

[0002] Arnecota acetate dehydrogenase (CAS: 4380-55-6, pregn-1,4,9(11)-triene-17α,21-diol-3,20-dione-21-acetate) is an important steroidal synthesis intermediate that can be used to prepare corticosteroids such as budesonide, triamcinolone, fluocinolone acetonide and difluprednisolone ester.

[0003] Traditional chemical methods for catalyzing the 1(2)-dehydrogenation of 9(11)-dehydro-3-one steroids are prone to side reactions such as ring A aromatization, resulting in target product yields of only 11%-63%, and also suffer from harsh reaction conditions and severe environmental pollution. Microbial transformation has advantages such as mild reaction conditions, but during the transformation process, reactions such as 1(2)-dehydrogenation, deacetylation, 20β-reduction, and non-enzymatic acetyl migration compete with each other, making it difficult to directionally accumulate the target product.

[0004]

[0005] While there are numerous reports on microbial or enzymatic dehydrogenases targeting steroids, studies on dehydrogenases targeting anelocorta acetate as a substrate are scarce. By amplifying C1,2-position dehydrogenases from multiple bacterial species, including Nocardia and Rhodococcus, and expressing them exogenously in Escherichia coli, this study found that these enzymes exhibited low activity, short half-life, and low conversion rates with respect to anelocorta acetate.

[0006] The literature *Steroids* 68 (2003) 415-421 investigated the biotransformation pathway of anecoxib acetate by *Nocardioides simplex* VKM Ac-2033D. This study systematically elucidated the microbial transformation process of acetylated 9(11)-dehydrosteroids, clarifying the synergistic mechanism of 1(2)-dehydrogenation, deacetylation, 20β-reduction, and non-enzymatic acetyl migration. However, the study did not quantitatively analyze the kinetic parameters and substrate specificity of 1(2)-dehydrogenase and esterase. Furthermore, the control of side reactions during biotransformation was insufficient: during long-term transformation, deacetylation and 20β-reduction persisted, leading to a decrease in the accumulation of the target product over time.

[0007]

[0008] Patent CN 119265266 A describes a traditional microbial method using Nocardia to dehydrogenate anocorta acetate. However, this conversion process suffers from problems such as easy foaming and contamination by other microorganisms. Furthermore, due to the low background expression levels of C1,2-position dehydrogenases in naturally screened microorganisms and the transmembrane barrier between the substrate and the microorganism, the reaction cycle is as long as 140 hours. To improve the conversion rate, this process introduces organic solvents and a large amount of surfactants to emulsify the substrate, but this puts pressure on wastewater treatment. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a dehydrogenase mutant, gene, engineered bacteria and application, which has higher conversion rate, stronger specificity and faster reaction speed.

[0010] This invention provides a dehydrogenase mutant obtained by mutating a primitive dehydrogenase. The sequence of the primitive dehydrogenase is SEQ ID NO.1. The mutation method is as follows: the A at position 344 is mutated to W, K, F, S or N, or the H at position 130 is mutated to T, W or F.

[0011] Preferably, the mutation method is: the 344th site is mutated from A to W, K, F or N. More preferably, the mutation method is: the 344th site is mutated from A to K or F.

[0012] This invention provides a gene encoding the aforementioned dehydrogenase mutant.

[0013] This invention provides an engineered bacterium that expresses the dehydrogenase mutant or contains the gene described above.

[0014] Preferably, the host microorganism of the engineered bacteria is Escherichia coli.

[0015] This invention provides the use of the aforementioned dehydrogenase mutant, containing the aforementioned gene, or the aforementioned engineered bacteria in the preparation of anecoxetine dehydrogenase.

[0016] Preferably, in the preparation of anecoxat acetate dehydrogenase, the substrate is anecoxat acetate.

[0017] Preferably, the specific steps for preparing anocorta acetate dehydrogenase are as follows: mixing the substrate anocorta acetate, an antifoaming agent, a buffer solution, an electron carrier, and a dehydrogenase mutant; reacting the mixture; filtering and drying the mixture after the reaction is complete to obtain anocorta acetate dehydrogenase.

[0018] Preferably, the defoamer is a polyether defoamer, the buffer solution is a phosphate buffer solution, the electron carrier is phenazine sulfate methyl ester hydrochloride, and the reaction temperature is 5-35℃.

[0019] The beneficial effect of this invention is that it is effective against Nocardia (…). Nocardia simplex By directionally modifying the key active sites of KstD dehydrogenase, a dehydrogenase mutant was successfully constructed. This dehydrogenase mutant exhibits significant advantages: high conversion rate of the substrate anelocrine acetate, fast catalytic rate, and no hydrolysis side reaction occurs during the reaction, eliminating the need for additional organic solvents and greatly simplifying the process.

[0020] Compared with traditional synthetic routes for preparing aneloxetine acetate dehydrogenase, this invention offers higher yields and the product is easier to purify. Computer simulations and homology comparisons revealed that the dehydrogenase mutant exhibits excellent in vitro stability and high specificity for the specific substrate aneloxetine acetate. The enzyme demonstrates high catalytic efficiency and rapid reaction rate, allowing for substrate concentrations up to 100 g / L with a conversion time not exceeding 28 hours and a conversion rate exceeding 98.5%. Attached Figure Description

[0021] Figure 1 The results are TLC spotting results for Example 2, where 0 represents the crude enzyme solution of the original dehydrogenase, 1 represents KstD-01, and 3 represents the crude enzyme solution of KstD-03.

[0022] Figure 2 This is the liquid phase spectrum of anechostata acetate dehydrogenase. Detailed Implementation

[0023] Example 1 The original dehydrogenase originated from Nocardia simplex ( Nocardia simplex The amino acid sequence of the original dehydrogenase is SEQ ID NO.1, and the corresponding nucleotide sequence is SEQ ID NO.2.

[0024] The specific preparation method for the crude enzyme solution of the original dehydrogenase is as follows: 1) The artificially synthesized nucleotide sequence SEQ ID NO.2 was constructed into the pET-28a plasmid expression vector to obtain the recombinant plasmid.

[0025] 2) The recombinant plasmid was transferred into the host cell BL21(DE3) to obtain the engineered strain.

[0026] 3) The engineered strain was inoculated into LB medium and cultured at 37°C and 230 rpm for 4 hours. Then, 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) was added and cultured at 25°C for another 16 hours.

[0027] 4) After centrifuging to collect the bacterial cells, the bacterial concentration was adjusted to 300 g / L with 0.1 M, pH 8.0 phosphate buffer. After the bacterial cells were fully dispersed, they were ultrasonically disrupted under ice bath conditions to finally obtain crude enzyme solution.

[0028] The method for detecting enzyme activity is as follows: 1) Prepare 0.035% DCPIP (sodium 2,6-dichlorophenolindophenol) solution: accurately weigh 0.0035g DCPIP, dissolve it in deionized water and sonicate for 5 min, make up to 10 mL, and store at 4℃.

[0029] 2) Prepare 0.02% arnecrostatin acetate substrate solution: Weigh 0.02g arnecrostatin acetate, add 15mL DMSO and mix well, make up to 100mL with deionized water, sonicate for 5min and dispense, centrifuge for 1-2min before use and store at 4℃.

[0030] 3) Prepare 1.37% PMS solution: Weigh 0.0137g PMS (phenazine sulfate methyl ester hydrochloride), dilute to 1 mL with deionized water, store at 4℃, and use immediately after preparation.

[0031] Preheat the microplate reader for 30 minutes, adjust the wavelength to 600 nm, set the corresponding detection program, and add the following reagents in sequence:

[0032] Add the reagents to the 96-well plate according to the table. Record the initial absorbance (A1) at 20 seconds and the absorbance (A2) at 1 minute and 20 seconds at 600 nm. Calculate ΔA = A1 - A2 to obtain the ΔA value. Enzyme activity unit definition: Under specific reaction conditions (30℃, pH 8.0), one unit of enzyme activity is defined as the reduction of 1 mM DCPIP per minute.

[0033] Method for converting crude enzyme solution into aneloxetine acetate: Add 0.5 g of polyether defoamer, 3 g of aneloxetine acetate, 80 mL of 0.1 M pH 8.0 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask. Finally, add 20 mL of KstD crude enzyme solution. React at 30℃ and 200 rpm for 24 hours, then add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain the crude product containing bacterial residue.

[0034] Example 2 Construction method of KstD mutant: Mutations were performed on sites A344 and H130 of the original dehydrogenase to construct a library. The enzyme activity was then determined using the enzyme activity detection method described in Example 1 to screen for mutants with high activity. The dehydrogenase mutant with the highest activity was KstD-03. The mutation site of this mutant is A344F (alanine at position 344 is mutated to phenylalanine), and its protein sequence and nucleotide sequence are SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0035] The enzyme activity test results for all dehydrogenase mutants are as follows:

[0036] Following the method for converting arnecrophene acetate with crude enzyme solution in Example 1, conversion was performed using crude enzyme solution expressed by the original bacteria, as well as crude enzyme solutions of KstD-01 and KstD-03. The TLC results are as follows: Figure 1 As shown.

[0037] The crude product containing bacterial residue, converted from KstD-03 crude enzyme solution, was extracted with 8 times its volume of dichloromethane to remove the bacterial residue, then recrystallized with 10 times its volume of methanol, dried at 60°C, and sent for liquid chromatography analysis. The results showed a substrate residue rate of 0.25% and a product purity of 99.17%. The liquid chromatography chromatogram is shown below. Figure 2 As shown.

[0038] Example 3 Add 0.5 g of polyether defoamer, 3 g of anecoxetine acetate, 80 mL of 0.1 M pH 8.0 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask, and finally add 20 mL of KstD-03 crude enzyme solution. After reacting at 30℃ and 200 rpm for 24 hours, add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain the crude product containing bacterial residue. The substrate residue rate was 2.1%, and the conversion rate was 97.8%.

[0039] The NMR data are as follows: Arnicota acetate: 1H NMR (500 MHz, CDCl3) δ 5.73 (d, J = 1.7 Hz, 1H), 5.55 (dt, J = 6.1, 2.0 Hz, 1H), 4.96 (dd, J = 113.9, 17.5 Hz, 2H), 2.77 (ddd, J =15.5, 11.9, 3.9 Hz, 1H), 2.68 (dt, J = 16.8, 2.8 Hz, 1H), 2.62 – 2.41 (m,4H), 2.35 (dt, J = 14.4, 3.3 Hz, 1H), 2.23 (tdt, J = 10.1, 4.2, 2.2 Hz, 1H), 2.16 (s, 3H), 2.14 – 2.05 (m, 2H), 2.02 (ddt, J = 12.6, 4.6, 2.4 Hz, 1H), 1.98 – 1.82 (m, 3H), 1.59 (ddd, J = 15.0, 9.4, 5.4 Hz, 1H), 1.43 (qd, J =11.9, 5.4 Hz, 1H), 1.32 (s, 3H), 1.14 (dtd, J = 14.0, 12.5, 3.9 Hz, 1H), 0.64(s, 3H). 13 C NMR (126 MHz, CDCl3) δ 205.2, 199.6, 170.8, 170.0, 144.2, 124.1,118.9, 89.8, 68.0, 48.0, 46.9, 41.1, 37.8, 34.9, 34.4, 33.9, 33.0, 32.3,32.0, 26.3, 24.5, 20.7, 14.5.

[0040] Arnecota acetate dehydrogenase: 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 10.2 Hz, 1H), 6.26 (dd, J = 10.2, 1.8 Hz, 1H), 6.05 (t, J = 1.7 Hz, 1H), 5.56 (dt, J = 6.0, 2.0 Hz, 1H), 5.09 – 4.80 (m, 2H), 2.76 (ddd, J = 15.5, 11.9, 4.0 Hz, 1H), 2.71 – 2.53 (m, 3H), 2.41 (ddd, J = 13.7, 4.5, 2.4 Hz, 1H), 2.37 – 2.26 (m,1H), 2.19 (td, J = 5.0, 2.4 Hz, 1H), 2.16 (s, 3H), 2.00 – 1.76 (m, 3H), 1.59(ddd, J = 14.8, 9.4, 5.4 Hz, 1H), 1.39 (s, 4H), 1.21 (dtd, J = 13.8, 12.5, 4.5 Hz, 1H), 0.65 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 205.2, 186.6, 170.8,167.3, 155.1, 142.4, 127.3, 123.8, 120.8, 89.7, 68.0, 53.6, 48.3, 47.2, 46.1,37.0, 35.0, 34.8, 32.3, 32.1, 26.8, 24.7, 20.7, 14.4.

[0041] Example 4 Add 0.5 g of polyether defoamer, 5 g of anecoxetine acetate, 80 mL of 0.1 M pH 9.0 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask, and finally add 20 mL of KstD-03 crude enzyme solution. After reacting at 30℃ and 200 rpm for 24 hours, add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain the crude product containing bacterial residue. The substrate residue rate was 3.5%, and the conversion rate was 96.4%.

[0042] Example 5 Add 0.5 g of polyether defoamer, 10 g of anisodamine acetate, 70 mL of 0.1 M pH 8.5 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask, and finally add 30 mL of KstD-03 crude enzyme solution. React at 30℃ and 200 rpm for 24 hours, then add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain a crude product containing bacterial residue. The substrate residue was 2.3%, and the conversion rate was 98.6%.

[0043] Example 6 Add 0.5 g of polyether defoamer, 10 g of anisodamine acetate, 70 mL of 0.1 M pH 8.5 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask, and finally add 30 mL of KstD-03 crude enzyme solution. React at 35℃ and 200 rpm for 24 hours, then add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain a crude product containing bacterial residue. Substrate residue was 5.7%, and the conversion rate was 94.2%.

[0044] Example 7 Add 0.5 g of polyether defoamer, 10 g of anisodamine acetate, 75 mL of 0.1 M pH 9.0 phosphate buffer, and 0.03 g of phenazine sulfate methyl ester hydrochloride to a 500 mL shake flask, followed by 25 mL of KstD-03 crude enzyme solution. React at 30℃ and 200 rpm for 24 hours, then add 50 mL of ice water, filter, and dry under vacuum. Dry the filter cake at 60℃ for at least 8 hours to obtain a crude product containing bacterial residue. Substrate residue was 4.6%, and the conversion rate was 95.3%.

[0045] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0046] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A dehydrogenase mutant, characterized in that, It is obtained by mutating the original dehydrogenase, the sequence of which is SEQ ID NO.1, and the mutation mode is: the A at position 344 is mutated to W, K, F, S or N, or the H at position 130 is mutated to T, W or F.

2. The dehydrogenase mutant as described in claim 1, characterized in that, The mutation method is as follows: the 344th site is mutated from A to W, K, F or N.

3. The dehydrogenase mutant as described in claim 2, characterized in that, The mutation method is as follows: the 344th site is mutated from A to K or F.

4. A gene encoding a dehydrogenase mutant as described in any one of claims 1-3.

5. An engineered bacterium, characterized in that, The engineered bacteria express the dehydrogenase mutant as described in any one of claims 1-3 or contain the gene as described in claim 4.

6. The engineered bacteria as described in claim 5, characterized in that, The host microorganism of the engineered bacteria is Escherichia coli.

7. Use of a dehydrogenase mutant as described in any one of claims 1-3, containing the gene as described in claim 4, or an engineered bacterium as described in claim 5 or 6 in the preparation of anabolic acetic acid dehydrogenase.

8. The use as described in claim 7, characterized in that, In the preparation of anecoxat dehydrogenase, the substrate is anecoxat acetate.

9. The use as described in claim 8, characterized in that, The specific steps for preparing anocorta acetate dehydrogenase are as follows: mix the substrate anocorta acetate, defoamer, buffer, electron carrier and dehydrogenase mutant, react, filter and dry after the reaction is complete to obtain anocorta acetate dehydrogenase.

10. The use as described in claim 9, characterized in that, The defoamer is a polyether defoamer, the buffer solution is a phosphate buffer solution, the electron carrier is phenazine sulfate methyl ester hydrochloride, and the reaction temperature is 5-35℃.

Images