A mutant alcohol dehydrogenase and lyophilized powder thereof and use thereof for preparing (2s,3s)-2-hydroxy-4-phenylbutane derivatives

Abstract

The application belongs to the technical field of enzyme engineering and bioengineering, and particularly relates to a mutant alcohol dehydrogenase, a freeze-dried powder thereof and application of the mutant alcohol dehydrogenase in preparation of (2S, 3S)-2-hydroxy-4-phenylbutane derivatives. The mutant alcohol dehydrogenase of the application has a reduced enzyme dosage by half for achieving the same conversion rate and stereoselectivity in catalyzing synthesis of (2S, 3S)-2-hydroxy-4-phenylbutane derivatives, and shows higher catalytic efficiency. The enzyme amorphous powder obtained by freeze-drying is easy to store and transport for a long time, and can be directly weighed and fed in use, which is convenient to operate. The preparation method of the application uses high-concentration isopropyl alcohol (60% m / V) as a cosolvent and a coenzyme regeneration system, and does not need to additionally add glucose and glucose dehydrogenase, thereby simplifying a process flow, reducing a cost, and achieving a short reaction time (16-18 h), a high conversion rate (up to 99.8% or more), and excellent stereoselectivity (de value > 99.9%), which is very suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and bioengineering technology, specifically relating to a mutant alcohol dehydrogenase and its lyophilized powder, and its application in the preparation of (2S,3S)-2-hydroxy-4-phenylbutane derivatives. Background Technology

[0002] AIDS is currently the most serious infectious disease in the world, with over 40 million people worldwide living with HIV (Human Immunodeficiency Virus). Existing antiviral drugs can be classified into nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors based on their mechanisms of action. Clinically, a treatment regimen of one or two reverse transcriptase inhibitors plus one protease inhibitor (also known as cocktail therapy) is generally used. Non-peptide HIV protease inhibitors (PIs) are the first-line drugs for HIV treatment, such as saquinavir, nelfinavir, fosanavir, and darenavir. For example, darenavir works by blocking the formation of new, mature viral particles released from the surface of infected host cells, inhibiting viral proteases. In June 2006, the FDA approved darenavir in combination with other antiretroviral drugs for the treatment of adult patients infected with HIV. In March 2007, darenavir was marketed in the 27 member states of the European Union.

[0003] The chiral (2S,3S)-2-hydroxy-4-phenylbutane derivative is an important intermediate for the aforementioned drugs, and its stereoconfiguration directly affects drug activity. This molecule has a complex structure, and its chemical synthesis is highly polluting and difficult. The structure contains two chiral centers. One chiral center originates from natural phenylalanine, and the other chiral center can reduce the pre-chiral ketone to a chiral alcohol, thereby introducing a second chiral center onto the BOC-epoxide. If the introduced second chiral center is in the R configuration, the product is (2R,3S)-N-tert-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane; if the introduced second chiral center is in the S configuration, the product is (2S,3S)-N-tert-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane.

[0004] Traditional chemical synthesis methods for (2S,3S)-N-tert-butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane suffer from problems such as cumbersome steps, low stereoselectivity, and environmental pollution. Enzymatic catalysis has become a research hotspot due to its advantages of high selectivity, mild reaction conditions, and environmental friendliness; however, existing wild-type alcohol dehydrogenases have drawbacks such as low catalytic efficiency and narrow substrate spectrum.

[0005] Therefore, developing alcohol dehydrogenase mutants with higher catalytic efficiency, better stability, and greater suitability for industrial production is of significant practical importance. Summary of the Invention

[0006] The purpose of this invention is to address existing problems by providing a mutant alcohol dehydrogenase, its lyophilized powder, and its application in the preparation of (2S,3S)-2-hydroxy-4-phenylbutane derivatives.

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

[0008] The first objective of this invention is to provide a mutant alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.1. Compared to the wild-type (SEQ ID NO.3), this sequence contains mutations at three key sites: serine (S) at position 86 is mutated to asparagine (N), leucine (L) at position 115 is mutated to alanine (A), and lysine (K) at position 158 is mutated to arginine (R). Furthermore, this invention introduces a His-tag at the N-terminus of the sequence.

[0009] The amino acid sequence shown in SEQ ID NO.1 comes from Sphingomonaspaucimobilis.

[0010] SEQ ID NO.1:

[0011] MHHHHHHKAMILDAPGGLDRLRLVDRPDPGAPGPGMIRVRLHATSLNYHDYGIVSGNMPTEDGRIPMADGAGLVEAIGDGVTELAVGDAVVNCFFPGWLEGPPRVGDFATVPGDGIDGYAAETVVAPATAFTRAPQGYDHEAAATITTAGLTAWRSLVVDGGLRAGDRIL VLGTGGVSIWALQIAKHMGASVAVTSSSGEKLERARALGADFTVNYREQEQWGRQVHDWADGGVDHVIEVGGPATLAQSIDAVRIGGHIGLIGVLTGVSGQVPTAALMAKQARLQGLIVGSRRMQQEFVRALDTSDIRPVIDRRFPLEGLVDAFRHQESGSHFGKIAVTW

[0012] SEQ ID NO.2

[0013]

[0014] The K mutation at position 158 is changed to R. Structurally, this reduces the distance between the active site and the substrate, enhancing the interaction between the active site and the substrate, allowing for better binding and catalytic reaction. The S mutation at position 86 is changed to N. Structurally, this brings the catalytic site of the protease closer to the substrate, enabling more efficient catalysis of chemical reactions. The L mutation at position 115 is changed to A. Structurally, this increases the binding pocket between the protease and the substrate, allowing for the accommodation of larger molecular substrates. Structurally, the cofactor binding region GAGLVEA remains unchanged, allowing for efficient assistance in catalytic reactions. To improve the water solubility of alcohol dehydrogenase without affecting its activity, a His-tag sequence was added to the N-terminus of SEQ ID NO.4 to form SEQ ID NO.1. The His-tag can further improve the water solubility of alcohol dehydrogenase. Figure 1 ).

[0015] A second object of the present invention is to provide a gene fragment encoding the alcohol dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0016] A third object of the present invention is to provide a recombinant plasmid comprising the aforementioned gene fragment.

[0017] Furthermore, the recombinant plasmid uses pET-28a(+) as a vector, and the gene fragment is inserted between the BamHI and XhoI restriction sites.

[0018] A fourth object of the present invention is to provide a host cell comprising the recombinant plasmid described above.

[0019] Furthermore, the host cell is *Escherichia coli* BL21 Star(DE3), which is a modified version of the commonly used BL21(DE3). Compared to the traditional BL21(DE3), the 131st arginine residue of the RNaseE gene in BL21 Star(DE3) is replaced by a histidine residue. This mutation significantly reduces RNaseE enzyme activity, thereby reducing mRNA degradation and increasing its stability. Therefore, BL21 Star(DE3) exhibits higher mRNA stability and increased expression levels. The expression level of the target protein in BL21 Star(DE3) is approximately 40% higher than that in BL21(DE3). Figure 2 ).

[0020] The fifth objective of this invention is to provide a mutant alcohol dehydrogenase lyophilized powder, which is obtained by inducing culture of the host cells, collecting the cells by centrifugation, resuspending and sonicating them, and then freeze-drying the supernatant to obtain the alcohol dehydrogenase lyophilized powder.

[0021] The sixth objective of this invention is to provide a method for preparing a (2S,3S)-2-hydroxy-4-phenylbutane derivative, comprising the following steps:

[0022] Using (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, isopropanol, magnesium sulfate, and NAD+ / NADP+ were added, and the pH was adjusted to 7.0±0.5. Then, the lyophilized powder of the alcohol dehydrogenase was added, and the enzyme was catalyzed at pH 7.5±0.5 for 12-18 h to obtain the (2S,3S)-2-hydroxy-4-phenylbutane derivative.

[0023] Furthermore, the concentration of NAD+ / NADP+ in the reaction system is 0.1-1.0 mmol / L.

[0024] Furthermore, the reaction system does not contain glucose.

[0025] Furthermore, the concentration of isopropanol in the reaction system is 60 m / v.

[0026] The present invention has the following advantages over the prior art:

[0027] Significantly improved enzyme catalytic efficiency: Compared with existing technologies (such as the alcohol dehydrogenase shown in SEQ ID NO. 5 of patent 202010230686.9), the mutant alcohol dehydrogenase of this invention requires half the amount of enzyme needed to achieve the same conversion rate and stereoselectivity in the synthesis of (2S,3S)-2-hydroxy-4-phenylbutane derivatives, demonstrating higher catalytic efficiency. This is attributed to the enhanced interaction between the active site and the substrate due to the K→R mutation at position 158; and the S→N mutation at position 86, which brings the catalytic site closer to the substrate, resulting in more efficient catalysis.

[0028] Substrate spectrum broadening: The L→A mutation at position 115 increases the enzyme's substrate binding pocket, enabling the enzyme to accommodate larger molecular substrates and improving its applicability to target macromolecular substrates.

[0029] The enzyme has good stability and is easy to store and use: the amorphous enzyme powder obtained by freeze drying is easy to store and transport for a long time, and can be directly weighed and added when used, making the operation convenient.

[0030] The preparation method is simple, efficient, and green: The preparation method of this invention uses high-concentration isopropanol (60% m / V) as a co-solvent and coenzyme regeneration system, eliminating the need for additional glucose and glucose dehydrogenase, simplifying the process, reducing costs, and having a short reaction time (16-18 h), high conversion rate (up to 99.8% or more), and excellent stereoselectivity (de value > 99.9%), making it very suitable for industrial production. Attached Figure Description

[0031] Figure 1 This is an SDS-PAGE electrophoresis image comparing the water solubility of the mutant alcohol dehydrogenase (SEQ ID NO.1) and the wild-type alcohol dehydrogenase (SEQ ID NO.3) of the present invention.

[0032] Figure 2 This is a comparison of the expression levels of the recombinant plasmid of the present invention in different host cells (BL21(DE3) and BL21 Star(DE3)). Detailed Implementation

[0033] To further explain the present invention, the following specific embodiments are described.

[0034] Example 1: Preparation of mutant alcohol dehydrogenase (SEQ ID NO.1) and its lyophilized powder

[0035] (1) Gene synthesis and mutation:

[0036] Based on the designed mutant alcohol dehydrogenase amino acid sequence (SEQ ID NO.1, i.e., based on SEQ ID NO.3, introducing S86N, L115A, K158R mutations and adding a His-tag in the N segment), the corresponding nucleotide sequence (SEQ ID NO.2) was synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0037] The His-tag sequence is HHHHHH.

[0038] (2) Construction of recombinant plasmids:

[0039] The synthesized gene fragment SEQ ID NO.2 was ligated with the pET-28a(+) plasmid (purchased from Invitrogen) after double digestion with BamHI and XhoI to construct a recombinant expression plasmid;

[0040] (3) Host cell transformation and culture:

[0041] The recombinant expression plasmid was added to Escherichia coli BL21(DE3) competent cells for transformation. After incubation on ice for 30 min, the cells were heat-shocked in a water bath at 45℃ for 9 s, followed by an ice bath for 2 min. The bacterial culture was then evenly spread on LB solid medium plates containing kanamycin resistance. After incubation at 37℃ for 18 h, single colonies were picked and transferred to 4 mL of LB liquid medium containing kanamycin resistance. The cells were then incubated at 37℃ and 220 rpm for 12 h to obtain the corresponding alcohol dehydrogenase expression strain.

[0042] (4) Induced expression:

[0043] All bacterial culture was inoculated into 600 mL of TB liquid medium and cultured at 37℃ and 180 rpm until the OD600 reached about 2. IPTG was added to a final concentration of 0.5 mM and expression was induced at 25℃ for 16 h.

[0044] (5) Collection and disruption of bacterial cells:

[0045] After induction, the culture medium was centrifuged at 5000 rpm for 20 min to collect the cells containing alcohol dehydrogenase. 30 g of the cells were resuspended in phosphate buffer (0.1 mol / L, pH 7.0), sonicated for 10 min, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of recombinant alcohol dehydrogenase.

[0046] (6) Preparation of freeze-dried powder:

[0047] The crude enzyme solution was pre-frozen overnight and then freeze-dried for 24-48 hours to obtain freeze-dried powdered recombinant alcohol dehydrogenase.

[0048] Example 2

[0049] Using (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, the recombinant alcohol dehydrogenase obtained in Example 1 was used for enzyme activity experiments. The reaction system was prepared as follows: 50 mL system: 500 mg of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone was added, followed by 60% (m / V) isopropanol. The mixture was stirred at 40 °C, then 2.4 g of magnesium sulfate and 130 mg of NAD+ were added. The volume was adjusted to 50 mL with potassium phosphate or sodium buffer (100 mmol / L, pH 8.0), and 100 mg of recombinant alcohol dehydrogenase powder was added. The reaction was allowed to proceed for 24 h, and the conversion rate and de value were detected by high performance liquid chromatography (HPLC).

[0050] The results showed a conversion rate of 99.8% and a de value of 99.9%, indicating that the recombinant alcohol dehydrogenase can convert to almost complete substrate depletion at a concentration of 60% (m / V) isopropanol, and the product has a high de value.

[0051] According to a previous report (202010230686.9), SEQ ID NO.5 can also catalyze the above reaction under the same conditions. We expressed SEQ ID NO.5 using the same method and prepared lyophilized powder. In the experiment comparing the catalytic activities of the two proteases, we found that if the same conversion rate % / de value is to be achieved, the amount of SEQ ID NO.1 required is half that of SEQ ID NO.5 (Table 1).

[0052] Table 1. Effects of different enzyme amounts on substrate conversion and De value.

[0053] Enzyme powder addition amount This invention relates to an alcohol dehydrogenase (SEQ ID NO.1). Control alcohol dehydrogenase (SEQ ID NO.5) 100 mg 99.8% / 99.9% 80.6% / 82.4% 200 mg 99.8% / 99.9% 99.8% / 99.9%

[0054] Note: Each cell in the table represents "conversion rate % / de value %".

[0055] Example 3

[0056] In a 1 L catalytic system, 80 g of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, 2.4 g of magnesium sulfate, 60 g of glucose, and 130 mg of NAD+ were added. The pH was then adjusted to 7.0 with sodium carbonate solution, and the mixture was stirred at 40°C. 20 g of glucose dehydrogenase and 2 g of the recombinant alcohol dehydrogenase powder from Example 1 were then added to initiate the reaction. The pH was maintained at approximately 7.5 using sodium carbonate solution. After 40 h of reaction, 70% of the substrate remained in the reaction system, indicating a conversion rate of only 30%.

[0057] During the conversion process, it was found that as the reaction proceeded, the conversion solution in the later stages of the system became extremely fluid due to the poor water solubility of both the substrate and product, making stirring difficult and reducing the contact area between the substrate and enzyme, which may lead to a lower conversion rate. Therefore, the addition of a cosolvent was considered. For example, 60% (m / V) of different types of isopropanol was added to the aforementioned reaction system. The pH was maintained at approximately 7.5 using sodium carbonate solution. After 24 hours of reaction, the system with added isopropanol showed the best results, with a conversion rate of 51%. However, both groups of reactions still had a significant amount of substrate remaining after 24 hours. During the conversion process, it was found that as the reaction proceeded, due to the poor water solubility of both the substrate and product, and the need to replenish sodium carbonate solution as needed to maintain pH and further expand the aqueous phase volume while diluting the cosolvent concentration, a significant amount of substrate remained. Using a high concentration of isopropanol is clearly more suitable as a cofactor for hydrogen donation than glucose.

[0058] Example 4

[0059] In a 1 L catalytic system, 100 g of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, 200 g of isopropanol were added, and the mixture was stirred at 40 °C. Then, 750 g of potassium phosphate or sodium buffer (100 mmol / L, pH 8.0), 2.4 g of magnesium sulfate, 130 mg of NAD+ or NADP+, 2 g of the recombinant alcohol dehydrogenase obtained in Example 1 above, and 60% (m / V) concentration of isopropanol were added to initiate the reaction. After 16 h of reaction, the conversion rate of the system was measured to be 99.8%, and the de value of the product was greater than 99.9%.

[0060] Example 5

[0061] In a 1 L catalytic system, 400 g of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone and 200 g of isopropanol were added. The mixture was stirred at 40 °C, and then 750 g of potassium phosphate or sodium buffer (100 mmol / L, pH 8.0), 2.4 g of magnesium sulfate, 130 mg of NAD+ or NADP+, and 8 g of the recombinant alcohol dehydrogenase from Example 1 were added to initiate the reaction. After 30 h of reaction, the conversion rate of the conversion system was measured to be 54.9%, and the de value of the product was greater than 99.9%. By comparing Examples 5 and 6, it can be seen that increasing the substrate concentration to 200 g / L significantly reduced the solubilizing effect of 20% (m / V) isopropanol, resulting in a lower substrate conversion rate.

[0062] Example 6

[0063] In a 1 L catalytic system, 400 g of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone and 600 g of isopropanol were added. The mixture was stirred at 40 °C, and then 750 g of potassium phosphate or sodium buffer (100 mmol / L, pH 8.0), 2.4 g of magnesium sulfate, 130 mg of NAD+ or NADP+, and 8 g of the recombinant alcohol dehydrogenase from Example 1 were added to initiate the reaction. After 16 h of reaction, the conversion rate of the conversion system was found to be 99.8%, and the de value of the product was greater than 99.9%. This indicates that increasing the isopropanol concentration to 60% (m / V) almost depletes the substrate.

[0064] Example 7

[0065] In a 1000 L catalytic system, 400 kg of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone and 600 kg of isopropanol were added. The mixture was stirred at 40 °C, and then 450 kg of potassium phosphate or sodium buffer (100 mmol / L, pH 8.0), 2.4 kg of magnesium sulfate, 130 g of NAD+ or NADP+, and 8 kg of the recombinant alcohol dehydrogenase obtained in Example 1 were added to initiate the reaction. After 16 h of reaction, the conversion rate of the conversion system was found to be 99.8%, and the de value of the product was greater than 99.9%.

[0066] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant alcohol dehydrogenase, characterized in that, The amino acid sequence of the mutant alcohol dehydrogenase is shown in SEQ ID NO.

1.

2. The gene fragment encoding the alcohol dehydrogenase of claim 1, characterized in that, The nucleotide sequence of the gene fragment is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, It includes the gene fragment as described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid uses pET-28a(+) as a vector, and the gene fragment is inserted between the BamHI and XhoI restriction sites.

5. A host cell, characterized in that, It includes the recombinant plasmid as described in claim 3 or 4.

6. A mutant alcohol dehydrogenase lyophilized powder, characterized in that, The host cells described in claim 5 are induced and cultured, the bacterial cells are collected by centrifugation, resuspended and ultrasonically disrupted, and the supernatant is freeze-dried to obtain the lyophilized powder of the alcohol dehydrogenase.

7. A method for preparing a (2S,3S)-2-hydroxy-4-phenylbutane derivative, characterized in that, Includes the following steps: Using (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, isopropanol, magnesium sulfate, and NAD+ / NADP+ were added, and the pH was adjusted to 7.0±0.

5. Then, the lyophilized powder of the alcohol dehydrogenase described in claim 6 was added, and the enzyme was catalyzed at pH 7.5±0.5 for 12-18 h to obtain the (2S,3S)-2-hydroxy-4-phenylbutane derivative.

8. The method for preparing a (2S,3S)-2-hydroxy-4-phenylbutane derivative according to claim 7, characterized in that, The concentration of NAD+ / NADP+ in the reaction system is 0.1-1.0 mmol / L.

9. The method for preparing a (2S,3S)-2-hydroxy-4-phenylbutane derivative according to claim 7, characterized in that, The reaction system does not contain glucose.

10. The method for preparing a (2S,3S)-2-hydroxy-4-phenylbutane derivative according to claim 7, characterized in that, The concentration of isopropanol in the reaction system is 60 m / v.

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