2,3-Butanediol dehydrogenase mutants with enhanced enzyme activity and thermostability and their applications

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, and specifically relates to a 2,3-butanediol dehydrogenase mutant with enhanced enzyme activity and thermostability, and its applications. This invention... Sm The BDH sequence underwent rational mutation modification, which improved its oxidase activity and protein thermostability. Compared with the wild type, the dual-site combined mutant involved in this invention showed the most significant increase in activity, a substantial improvement in thermostability, and a significantly higher Tm value. This invention effectively improves the oxidative capacity and thermostability of 2,3-butanediol dehydrogenase, addressing the problem of easy inactivation under low temperature conditions. It helps solve the problem of low efficiency in enzymatic synthesis of acetoin in industrial applications, alleviating the problems of high cost, severe pollution, and cumbersome procedures associated with chemical synthesis, and thus facilitating the industrial application of biological synthesis of acetoin.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and specifically relates to 2,3-butanediol dehydrogenase mutants with improved enzyme activity and thermal stability and their applications. Background Technology

[0002] Acetoin (3-hydroxy-2-butanone) is an important natural flavor compound and platform chemical widely used in food, flavorings, pharmaceuticals, and fine chemicals. 2,3-Butanediol dehydrogenase (BDH, EC1.1.1.4) catalyzes the reversible redox reaction between meso-2,3-butanediol and acetoin, and is a key enzyme in the biocatalytic production of acetoin. For industrial biocatalysis, the thermal stability of an enzyme is a crucial indicator of its industrial application value. Higher thermal stability not only improves the catalytic efficiency of the enzyme under high-temperature conditions but also helps to increase substrate solubility, reduce system viscosity, decrease the risk of microbial contamination, and extend the enzyme's lifespan. However, naturally derived 2,3-butanediol dehydrogenases generally suffer from insufficient thermal stability, easily undergoing conformational changes, instability of the active site, or even irreversible inactivation at higher temperatures, leading to decreased catalytic efficiency and limiting their industrial applications.

[0003] In recent years, with the development of enzyme engineering technology, researchers have begun to modify 2,3-butanediol dehydrogenases using strategies such as rational design, directed evolution, and semi-rational design to improve their catalytic performance. Most of these studies focus on improving substrate specificity, enantioselectivity, and catalytic activity, while research on enzyme thermostability is relatively limited. Currently, the thermostability modification of 2,3-butanediol dehydrogenases mainly employs methods such as random mutagenesis, directed evolution, and rational design based on structure analysis. Although some studies have reported mutants with improved thermostability, the improvement is usually limited and often accompanied by a decrease in enzyme activity. Because there is often a trade-off between enzyme activity and thermostability, maintaining or even enhancing catalytic activity while improving thermostability remains a significant challenge in the field of enzyme engineering.

[0004] Particularly in 2,3-butanediol dehydrogenases, there is currently a lack of an effective modification strategy that can simultaneously improve catalytic activity and thermal stability. The synergistic relationship between activity-enhancing and thermal stability-enhancing mutations remains unclear, limiting the development of high-performance industrial enzyme preparations. Therefore, developing 2,3-butanediol dehydrogenase mutants with both high catalytic activity and high thermal stability is of great significance for promoting the green biomanufacturing of α-hydroxy ketones such as acetoin. Summary of the Invention

[0005] This application is derived from Serratia marcescens (Serratia marcescens) Serratia marcescens Starting with 2,3-butanediol dehydrogenase (BDH, EC 1.1.1.4) derived from [source missing], several single-site and double-site combined mutants with both high catalytic activity and high thermal stability were obtained through site-directed mutagenesis. Compared with the wild-type enzyme, the obtained excellent double-site combined mutants showed significantly improved ability to catalyze the oxidation of meso-2,3-butanediol to acetoin, while also exhibiting higher thermal stability. The Tm value of some mutants was about 25℃ higher than that of the wild type, showing good potential for industrial application.

[0006] In this regard, the present invention includes, but is not limited to, the following technical solutions: In one aspect, the present invention provides a 2,3 Butylene glycol dehydrogenase mutant, derived from wild-type 2,3-butanediol dehydrogenase as shown in SEQ ID NO: 1. Butanediol dehydrogenase was obtained through the following mutation: (1) The amino acid at position 106 was mutated from arginine (Arg) to leucine (Leu) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys). (2) The amino acid at position 191 was mutated from glycine (Gly) to serine (Ser) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys). (3) The amino acid at position 197 is mutated from arginine (Arg) to histidine (His) and the amino acid at position 245 is mutated from aspartic acid (Asp) to cysteine ​​(Cys); or (4) The amino acid at position 209 was mutated from alanine (Ala) to proline (Pro) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys).

[0007] In one aspect, the present invention provides a polynucleotide encoding the 2,3 described herein. Butanediol dehydrogenase mutant.

[0008] In one aspect, the present invention provides a recombinant expression vector comprising the polynucleotides described herein.

[0009] In one aspect, the recombinant expression vector of the present invention is selected from pET-28a.

[0010] In one aspect, the present invention provides a host cell comprising the recombinant expression vector described herein, or having the polynucleotides described herein integrated into its genome.

[0011] In one aspect, the host cell described in this invention is a fungal cell, a bacterial cell, or a plant cell.

[0012] In one aspect, the host cell of the present invention is a bacterial cell, preferably an Escherichia coli cell.

[0013] It should be noted that the plant cells described in this invention are not intended to protect any specific plant, nor does this invention disclose any method for developing plant cells into complete plants. The plant cells described in this invention are only intended for use as engineered cells expressing the enzyme mutants of this invention.

[0014] In one aspect, the host cell described in this invention is specifically Escherichia coli BL21(DE3).

[0015] In one aspect, the present invention provides the 2,3 described herein. Butanediol dehydrogenase mutants, the polynucleotides described in this invention, the recombinant expression vectors described in this invention, or the host cells described in this invention in catalyzing meso-2,3 Applications of butanediol oxidation.

[0016] In one aspect, the present invention provides the 2,3 described herein. The application of butanediol dehydrogenase mutants, the polynucleotides described in this invention, the recombinant expression vectors described in this invention, or the host cells described in this invention in catalyzing acetoin reduction.

[0017] In one aspect, the present invention provides a method for producing the 2,3 described herein. Methods for developing butanediol dehydrogenase mutants.

[0018] In one aspect, the method of the present invention includes: in a suitable environment, as described in 2, 3 The host cells described in this invention were cultured under conditions of butanediol dehydrogenase mutant expression, and the 2,3... Butanediol dehydrogenase mutant.

[0019] The beneficial technical effects of this application include at least the following: This invention relates to Sm The BDH sequence underwent rational mutation modification, which improved its oxidase activity and protein thermostability. Compared with the wild type, the mutant involved in this invention, R106L / D245C, showed the most significant increase in activity, with an oxidase activity of 151.74 U / mg, which is 9.0 times that of the wild type (16.86 U / mg). Its protein thermostability was also greatly improved compared with the wild type, specifically, after treatment at 60°C for 6 hours, its residual enzyme activity reached 78.56%, while the residual enzyme activity of the wild type was almost 0. Furthermore, the Tm value of this mutant reached 76.5°C, which is 24.2°C higher than that of the wild type (52.3°C).

[0020] This invention effectively improves the oxidation capacity and thermal stability of 2,3-butanediol dehydrogenase, addressing the problem of easy inactivation under low temperature. It helps solve the problem of low efficiency in enzymatic synthesis of acetoin in industrial applications, and alleviates the problems of high cost, severe pollution, and cumbersome procedures associated with chemical synthesis methods, thus facilitating the industrial application of biological synthesis of acetoin. Attached Figure Description

[0021] Figure 1 for Sm Results of Tm value determination for BDH two-point combination mutants. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to embodiments thereof, but this should not be construed as limiting the scope of the invention to the following embodiments. 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. Unless otherwise specified, the experimental methods in the present invention are conventional methods.

[0023] Reagents used in upstream genetic engineering: The genome extraction kit and DpnI used in the embodiments of this invention were purchased from TaKaRa, Takara Bio Engineering (Dalian) Co., Ltd.; the ExnaseII seamless cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the plasmid extraction kit and DNA recovery kit were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21 (DE3) was purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose gel electrophoresis reagent were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis, sequence sequencing, and whole plasmid pET28a- Sm BDH was completed by Qingke Biotechnology Co., Ltd.

[0024] The present invention will now be explained in detail with reference to the embodiments and accompanying drawings.

[0025] Example 1 Sm Construction of BDH mutant E. coli expression vector and protein expression and purification 1. Sm Construction of BDH two-point combination mutant (1) BDH whole plasmid PCR: First, use plasmids containing wild type Sm Using a plasmid with the BDH coding sequence as a template, PCR was performed using the primers in Table 1 to obtain a plasmid containing the coding sequence of a single point mutant. Then, using the obtained plasmid (for example, a plasmid containing the coding sequence of the D245C mutant) as a template, PCR was performed again using the mutant primer sequences in Table 1 to obtain plasmids containing the coding sequence of a two-site combination mutant.

[0026] Table 1. Primer sequences for mutants

[0027] The PCR amplification system is shown in Table 2 below: Table 2 PCR amplification system

[0028] PCR amplification conditions are shown in Table 3 below: a total of 30 cycles: Table 3 PCR amplification program

[0029] (2) Template digestion: The PCR products were analyzed by agarose gel electrophoresis to determine the band size. Once the band size was confirmed to be correct, the PCR products were purified using a PCR product purification kit. The remaining plasmid template was then digested with DpnI enzyme. The digestion system is shown in Table 4 below. Table 4 Dpn I. Digestive System

[0030] Digestion conditions: 1 h at 37℃, 15 min at 70℃.

[0031] (3) Transformation and screening of competent cells: After the digestion products were verified by agarose gel electrophoresis, they were transformed using the heat shock method. E. coli BL21(DE3) competent cells, the specific method is as follows: 1) Remove competent cells from -80℃ and thaw them on ice for 2 min; 2) Take 10 μL of the ligation product and... E. coli Mix BL21(DE3) competent cells thoroughly and incubate on ice for 30 min; 3) After incubation for 30 min, heat shock the centrifuge tubes in a 42℃ water bath for 1 min, and immediately remove them and incubate on ice for 3 min.

[0032] 4) In a sterile operating table, add 600 μL of LB medium preheated at 37℃ to competent cells, mix well, and incubate at 37℃ for 60 min at 200 rpm to revive the cells; 5) After reviving the bacteria, centrifuge at 6000 rpm for 30 s at room temperature. In a clean bench, discard part of the supernatant, mix the remaining liquid (approximately 100 μL) with the bacteria, and then evenly spread it onto LB agar containing Kans antibiotic using a disposable spreader. After the liquid is completely absorbed, invert the plate and incubate at 37°C for approximately 16 h. Pick 3-4 single colonies from each plate for sequencing verification to obtain the mutants obtained through sequencing.

[0033] 2. Sm Protein expression and purification of BDH mutants.

[0034] After the mutant strain successfully sequenced in Example 1 was activated by streak plating, a single colony was picked and placed into 5 mL of LB liquid medium containing 100 µg / mL kanamycin, and cultured at 37°C and 180 rpm for 16 h to prepare recombinant engineered bacterial culture. Transfer 2 mL of bacterial seed culture to 200 mL of LB liquid medium containing 100 µg / mL kanamycin (1% inoculum) and continue culturing at 37°C in a shaker at 220 rpm until OD. 600 =0.6-0.8 (approximately 3-4 hours); Add 200 µL of 1 mol / L IPTG (working concentration 1 mmol / L) that has been sterilized by filtration through a 0.22 μm pinhole membrane, and induce culture at 18℃ and 100 rpm for 16 h. After induction, the bacterial culture was transferred to a 100 mL centrifuge cup and centrifuged at 4℃ and 6000 rpm for 20 min. The supernatant was discarded, and the bacterial cells were resuspended in 30 mL of sterile PBS buffer (pH 7.4) in the centrifuge cup. The suspension was centrifuged at 6000 rpm for 20 min at 4℃, the supernatant was discarded, and 60 mL of sterilized PBS buffer was added to the centrifuge cup, which was then transferred to a clean beaker. The beaker was placed on ice to prevent heat generation during sonication from inactivating the protein. The amplitude transformer was adjusted to the optimal position, and repeated sonication was performed under the following conditions: total sonication time 20 min, working time 2 s, interval time 6 s, and sonication power 60%. Centrifuge the disrupted bacterial culture at 10,000 rpm for 10 min at 4°C, discard the precipitate, and repeat once. The final supernatant is the crude enzyme solution, and the precipitate is cell debris. Use the supernatant for immediate further purification or temporarily store at 4°C. Take 30 mL of crude enzyme solution, add 1 mL of affinity Ni-NTA resin filler, and add imidazole to make the final concentration 3 mmol / L. Incubate slowly on ice at 60 rpm to allow nickel ions to fully bind to the 6×His tag of the target protein. After incubation for 1-2 hours, purify the protein using an empty chromatography column and collect the filtrate (Flow-through, FT). The permeate can be repeated 2-3 times to improve the protein yield. Add 10-20 times the column bed volume of washing buffer to repeatedly wash the packing and collect the waste, denoted as W, Wash. After washing with washing buffer, elute the target protein with approximately 3 mL of Elution buffer (3 column bed volumes). Collect the eluent and denote it as E, Elution (add the Elution buffer along the tube wall to prevent the packing material from being flushed up). After elution, add excess Elution buffer to clean the packing material, then add ddH2O to wash away excess Elution buffer, add 20% ethanol to rinse the packing material, recover the packing material, and store at 4℃. The purified sample eluent was desalted in Tris-HCl (pH 8.0) using an ultrafiltration tube, centrifuged at 4000 rpm for 30 min at 4°C, and buffer was added to wash and replace imidazole ions until the final concentration of imidazole in the protein was less than 10 mM.

[0035] Washing buffer (20 mmol / L imidazole): Take 20 mL of Tris-HCl (pH 8.0) stock solution, 100 mL of NaCl stock solution, and 20 mL of 1 mol / L imidazole, and bring the volume to 1 L with ddH2O.

[0036] Eluent buffer (containing 250 mmol / L imidazole): Take 20 mL of Tris-HCl (pH 8.0) stock solution, 10 mL of 3 mol / L NaCl stock solution, and 500 mL of 1 mol / L imidazole, and bring the volume to 1 L with ddH2O.

[0037] Example 2 Sm Determination of enzyme activity, residual enzyme activity, and Tm value of BDH and its mutants.

[0038] BDH enzyme activity definition: The unit of enzyme activity is defined as the reduction of 1 µmol of NAD per minute at room temperature. + The required amount of enzyme is one unit of enzyme activity, U. Specific enzyme activity is defined as enzyme activity per unit protein, U / mg.

[0039] BDH enzyme activity assay: The enzyme reaction system is 200 µL, containing 12.5 mM substrate meso-2,3-butanediol and 0.56 mM NAD. +The enzymatic reaction begins immediately after the addition of a certain amount of enzyme solution. The concentration of NADH is calculated based on the change in absorbance of the reaction solution at 340 nm, and the enzyme activity is also calculated.

[0040] The obtained proteins were subjected to oxidase activity assays using a microplate reader, and the results are shown in Table 5. Table 5 shows the difference between the proteins and the wild-type protein. Sm Compared to BDH, several mutants showed significantly increased enzyme activity. The R106L / D245C, A209P / D245C, and G191S / D245C combination mutants showed the most significant increase, with oxidase activities of 151.74 U / mg. p <0.0001), 143.31 U / mg ( p <0.0001), 134.88 U / mg ( p <0.0001), which are 9.0 times, 8.5 times, and 8.0 times the wild-type enzyme activity (16.86 U / mg), respectively.

[0041] BDH and its mutant residual enzyme activity assay: Since some mutants have strong thermal stability, this invention uses residual enzyme activity after treatment at 60℃ for 10 min and 6 h to evaluate the stability of the mutants. Sm BDH and mutant proteins were uniformly diluted to 0.5 mg / mL. A portion was used for enzyme activity assay as the enzyme activity of the protein before heat treatment, and the remaining portion was incubated at 60℃ for 10 min and 6 h, respectively. After incubation, the protein was placed on ice for 5 min, and the enzyme activity of the ice-incubated protein was measured again. The ratio of enzyme activity before and after heat treatment is the residual enzyme activity. The oxidative enzyme activity of the heat-treated protein was measured using a microplate reader, and the results are shown in Table 5. Table 5 shows that the residual enzyme activity of several mutants is higher than that of the backbone mutant D245C, especially R106L / D245C, A209P / D245C, and G191S / D245C. Among them, the R106L / D245C and G191S / D245C combination mutants showed the most significant improvement in thermostability, with their residual enzyme activities reaching 119.11 U / mg (78.5%). p <0.0001), 108.34 U / mg (75.6%, p <0.0001), the wild type has almost zero residual enzyme activity.

[0042] Tm value determination of BDH and its mutants: In this invention, the mutant with the best overall performance, i.e., the mutant with the most significant improvement in activity and thermal stability, is selected for Tm evaluation. The purified BDH and its mutants are then used for Tm evaluation. SmThe concentrations of BDH and mutant proteins were adjusted to 0.5 mg / mL, and then the Tm values ​​were determined using a Prometheus protein stability analyzer. The measurement program was set as follows: the measurement range was set to 20-95℃, the heating rate was 1℃ / min, and values ​​at 350 nm and 330 nm were collected. Data analysis was performed using the corresponding software, Nano Temper. The Tm value measurement results are as follows: Figure 1 As shown. From Figure 1 It can be seen that the Tm value of the mutants is significantly increased compared with the wild type, and is higher than that of the single-point mutation D245C. The Tm values ​​of the combined mutants R106L / D245C, G191S / D245C, and R197H / D245C are 76.5℃, 77.1℃, and 77.2℃, respectively, which are 24.2℃, 24.8℃, and 24.9℃ higher than the wild type (52.3℃).

[0043] Table 5. Oxidative activity and residual enzyme activity at heat (10 min, 6 h) of SmBDH two-point combination mutants

[0044] ND: Not detected.

[0045] sequence list SEQ ID NO: 1 Wild type Sm BDH amino acid sequence MRFDNKVVVITGAGNGMGEAAARRFSAEGAIVVLADWAKEAVDKVAASLPKGRAMAVHIDVSDHVAVEKMMNEVAEKLGRIDVLLNNAGVHVAGSVLETSIDDWRRIAGVDIDGVVFCSKFALPHL LKTKGCIVNTASVSGLGGDWGAAYYCAAKGAVVNLTRAMALDHGGDGVRINSVCPSLVKTNMTNGWPQEIRDKFNERIALGRAAEPEEVAAVMAFLASDDASFINGANIPVDGGATASDGQPKIV; SEQ ID NO: 2 Wild type Sm BDH encoded sequence ATGCGCTTTGACAACAAAGTTGTTGTTATTACCGGGGCCGGGAACGGGATGGGTGAAGCAGCCGCCCGCCGCTTTAGCGCCGAAGGTGCAATTGTTGTTCTGGCCGACTGGGCAAAAGAAGCAGTGGATAAAGTGGCAGCAAGTCTGCCGAAAGGTCGTGCAATGGCGGTTCATATTGATGTTTCAGATCATGTTGCAGTGGAGAAAATGATGAATGAAGTAGCAGAAAAACTGGGACGTATTGATGTGCTGCTGAATAATGCTGGTGTGCATGTTGCAGGGAGTGTGCTGGAAACGAGCATTGATGATTGGCGTCGTATTGCAGGGGTTGATATTGATGGTGTGGTGTTTTGTAGTAAATTTGCTCTGCCGCATCTGCTGAAAACCAAAGGTTGTATTGTTAATACAGCATCTGTTAGCGGTCTGGGTGGTGATTGGGGTGCAGCGTATTATTGTGCTGCCAAAGGTGCGGTTGTTAATCTGACTCGTGCAATGGCACTGGATCATGGTGGTGATGGGGTTCGTATTAATAGTGTTTGTCCGTCTCTGGTTAAAACGAATATGACCAATGGTTGGCCGCAGGAAATTCGTGATAAATTTAATGAACGTATCGCACTGGGGCGTGCAGCAGAACCGGAGGAAGTTGCAGCAGTTATGGCATTTCTGGCAAGCGACGACGCAAGTTTTATTAATGGTGCAAATATTCCTGTGGATGGTGGTGCAACCGCGAGCGATGGACAACCGAAAATTGTA。

Claims

1. A type of 2,3 Butanediol dehydrogenase mutant, characterized in that, From the wild type 2,3 shown in SEQ ID NO: 1 Butanediol dehydrogenase was obtained through the following mutation: (1) The amino acid at position 106 was mutated from arginine (Arg) to leucine (Leu) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys). (2) The amino acid at position 191 was mutated from glycine (Gly) to serine (Ser) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys). (3) The amino acid at position 197 is mutated from arginine (Arg) to histidine (His) and the amino acid at position 245 is mutated from aspartic acid (Asp) to cysteine ​​(Cys); or (4) The amino acid at position 209 was mutated from alanine (Ala) to proline (Pro) and the amino acid at position 245 was mutated from aspartic acid (Asp) to cysteine ​​(Cys).

2. A polynucleotide, characterized in that, Encoding according to claim 1, 2, 3 Butanediol dehydrogenase mutant.

3. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It comprises the recombinant expression vector according to claim 3, or the genome thereof having the polynucleotide according to claim 2 integrated therein.

5. The host cell according to claim 4, characterized in that, The host cell is a bacterial cell.

6. The host cell according to claim 5, characterized in that, The host cell is an Escherichia coli cell.

7. The 2, 3 as described in claim 1 Butanediol dehydrogenase mutant, the polynucleotide according to claim 2, the recombinant expression vector according to claim 3, or the host cell according to any one of claims 4-6 in catalyzing meso-2,3 Applications of butanediol oxidation.

8. The 2, 3 as described in claim 1 The application of butanediol dehydrogenase mutant, the polynucleotide according to claim 2, the recombinant expression vector according to claim 3, or the host cell according to any one of claims 4-6 in catalyzing acetoin reduction.

9. A method for producing the 2,3 according to claim 1 Methods for developing butanediol dehydrogenase mutants.

10. The method according to claim 9, characterized in that, include: In accordance with the appropriate 2, 3 Host cells according to any one of claims 4-6 were cultured under conditions of butanediol dehydrogenase mutant expression, and the 2,3 Butanediol dehydrogenase mutant.

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