D-pantoic acid lactone enzyme, recombinant bacteria and application thereof

Abstract

The present application relates to a kind of D-pantoic acid lactone enzyme and its coding gene and recombinant bacteria, belong to the field of genetic engineering.The nucleotide sequence of the D-pantoic acid lactone enzyme as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1.The recombinant bacteria is to construct the D-pantoic acid lactone enzyme coding gene Escherichia coli recombinant expression vector, then is imported into Escherichia coli species, and screening is obtained the recombinant bacteria of expression D-pantoic acid lactone enzyme.The D-pantoic acid lactone enzyme can hydrolyze or synthesis D-pantoic acid lactone, gamma-butyrolactone, benzofuran ketone.Hydrolyze D-pantoic acid lactone, gamma-butyrolactone and benzofuran ketone, the D-pantoic acid lactone enzyme suitable reaction temperature is 30~50 ℃, reaction pH value is 7.5±0.2;Synthesis D-pantoic acid lactone, gamma-butyrolactone and benzofuran ketone, the D-pantoic acid lactone enzyme suitable reaction temperature is 30~50 ℃, reaction pH value is 7.0 or less.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a D-pantolactone enzyme, its encoding gene and recombinant bacteria, and its application in the hydrolysis or synthesis of D-pantolactone, γ-butyrolactone or benzofuranone. Background Technology

[0002] D-Pantothenic acid lactone is a key precursor for the synthesis of D-pantothenic acid (also known as vitamin B5). The annual demand for D-pantothenic acid, a component of intracellular acetyl-CoA and acyl carrier proteins, exceeds 20,000 tons. Currently, D-pantothenic acid lactone is mainly synthesized by chemical or chemo-enzymatic methods. Starting with formaldehyde and isobutyraldehyde, reactions such as aldehyde condensation, nitrification, and lactone formation generate DL-pantothenic acid lactone, which is then selectively resolved using chiral resolution reagents or enzymatic methods to obtain D-pantothenic acid lactone. Chiral resolution of DL-pantothenic acid lactone typically involves the selective hydrolysis of D-pantothenic acid lactone by D-pantothenic acid lactone hydrolase to generate D-pantothenic acid, followed by chemical lactone formation to regenerate D-pantothenic acid lactone. The remaining L-pantothenic acid lactone is then chemically racemic to regenerate DL-pantothenic acid lactone, followed by a new round of chiral resolution. Currently, the industrial conversion of D-pantolytic acid to D-pantolytic lactone is generally achieved through acidification. This involves adding sulfuric acid or hydrochloric acid to D-pantolytic acid to adjust the pH to below 2, reacting at a higher temperature, and then adjusting the pH to 5-7 to obtain D-pantolytic lactone. Compared to chemical acidification, enzymatic synthesis offers milder reaction conditions, is more environmentally friendly, and is more suitable for the industrial synthesis of D-pantolytic lactone. Currently, there are no reports on the enzymatic lactonization of D-pantolytic acid. Using D-pantolytic lactonease for enzymatic lactonization of D-pantolytic acid will further achieve a green and efficient chiral resolution process for DL-pantolytic lactone, possessing significant industrial application value and importance. Summary of the Invention

[0003] The purpose of this invention is to provide a D-pantolactone enzyme, its encoding gene, and a recombinant bacterium.

[0004] The technical solution adopted by the present invention to achieve the above objectives is as follows: In a first aspect, the present invention provides a D-pantoacid lactonease, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0005] In a second aspect, the present invention provides a gene encoding the above-mentioned D-pantolactone enzyme, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] Thirdly, the present invention provides a recombinant expression plasmid with an inserted coding gene as shown in SEQ ID NO.1.

[0007] Preferably, the recombinant expression plasmid is obtained by inserting the coding gene shown in SEQ ID NO.1 into the pET-28a(+) vector. Specifically, the coding gene shown in SEQ ID NO.1 is inserted into the BamHI and Hind III sites of the pET-28a(+) vector to obtain the recombinant expression plasmid.

[0008] Fourthly, the present invention provides a recombinant genetically engineered bacterium containing the expression plasmid.

[0009] Specifically, the recombinant genetically engineered bacteria are obtained by transferring the recombinant expression plasmid into the host cell Escherichia coli BL21(DE3).

[0010] Fifthly, the present invention also provides a method for producing D-pantolactone, the method comprising: fermenting and culturing recombinant genetically engineered bacteria, breaking the cell wall of the cultured recombinant bacteria and separating and purifying them to obtain D-pantolactone.

[0011] Furthermore, the fermentation culture steps are as follows: the recombinant genetically engineered bacteria are inoculated into LB liquid medium containing kanamycin and cultured in a shaker at 37°C and 200 rpm. When the OD600 reaches 0.8, isopropyl thiogalactoside (IPTG) is added to make the final concentration reach 0.2 mM. The culture is continued at 20°C for 18 h. The obtained fermentation broth is centrifuged to collect the wet bacterial cells, and the cultured recombinant bacterial cells are obtained.

[0012] Furthermore, after the recombinant bacterial cells are cultured, they are separated and purified by breaking the cell wall. The preferred specific operation steps are as follows: after washing and resuspending the recombinant bacterial cells in phosphate buffer, the cell wall is broken by ultrasound under ice bath conditions until the bacterial solution is clear. The supernatant is collected by centrifugation at 10,000 r / min for 5 min. The collected supernatant is the crude enzyme solution of D-pantolactonease.

[0013] The crude enzyme solution was purified using a HisTrap™ HP nickel column: The sample was loaded onto a HisTrap™ HP nickel column, and the column was washed with starting buffer to remove contaminating proteins. Elution was performed with starting buffer containing 20 mM imidazole to remove non-specific impurities from the column, and the eluent was collected. Elution was then performed sequentially with starting buffers containing 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole, and the eluents were collected. The eluent containing the target protein was collected and passed through a desalting column to remove the imidazole from the buffer, yielding the purified enzyme solution.

[0014] The starting buffer consists of: 20 mM phosphate buffer (Na2HPO4 and NaH2PO4, pH=7.0), 200 mM NaCl, and 10% glycerol.

[0015] Sixthly, the present invention also provides the application of the D-indohydrinase in the hydrolysis of D-indohydrin, γ-butyrolactone, and benzofuranone. When hydrolyzing D-indohydrin, γ-butyrolactone, and benzofuranone, the suitable reaction temperature for the D-indohydrinase is 30–50°C, and the suitable reaction pH is 7.5 ± 0.2.

[0016] Furthermore, the preferred method for the application is as follows: using wet bacterial cells collected by fermentation of recombinant genetically engineered bacteria that produce D-pantolactone hydrolase as a biocatalyst, with D-pantolactone, γ-butyrolactone, or benzofuranone as the raw material, controlling the reaction temperature at 30-50℃ (preferably 30℃), controlling the reaction pH at 7.5±0.2, and carrying out the hydrolysis reaction to generate D-pantolactone, hydroxybutyric acid, or o-hydroxyphenylacetic acid.

[0017] In a seventh aspect, the present invention also provides the application of the aforementioned D-pantolactone enzyme in the synthesis of D-pantolactone, γ-butyrolactone, and benzofuranone. When synthesizing D-pantolactone, γ-butyrolactone, and benzofuranone, the suitable reaction temperature for the D-pantolactone enzyme is 30–50°C, and the reaction pH is below 7.0, preferably 5.5 ± 0.2.

[0018] Furthermore, the preferred method for the application is as follows: using wet bacterial cells collected by fermentation of recombinant genetically engineered bacteria that produce D-pantolytic acid lactone hydrolase as a biocatalyst, with D-pantolytic acid, hydroxybutyric acid or o-hydroxyphenylacetic acid as the raw material, controlling the reaction temperature at 30-50℃ (preferably 30℃), controlling the reaction pH at 5.5±0.2, and carrying out a lactone reaction to generate products D-pantolytic acid lactone, γ-butyrolactone or benzofuranone.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: In the existing technology for preparing D-pantolactone from DL-pantolactone via chiral resolution, a chemical method is required to lactonize D-pantolactone. Compared to this chemical method, the present invention utilizes a D-pantolactone enzyme for lactonization of D-pantolactone, which is more environmentally friendly and efficient. The reaction conditions are mild, the temperature is low, and a strongly acidic environment is not required, making it more suitable for industrial production. Furthermore, the D-pantolactone enzyme described in this invention can also achieve the hydrolysis and synthesis of some other 1,4-lactones, possessing significant industrial application value and importance. Attached Figure Description

[0020] Figure 1This is an electrophoresis result of the D-pantolactone gene sequence obtained by cloning with designed specific primers.

[0021] Figure 2 This is an SDS-PAGE protein electrophoresis image of D-pantolactone enzyme expressed by recombinant bacteria BL21(DE3). Band 1 is the cell lysate supernatant of the recombinant D-pantolactone enzyme strain; band 2 is the cell lysate precipitate of the recombinant D-pantolactone enzyme strain; band 3 is the cell lysate supernatant of the recombinant D-pantolactone enzyme strain with duplicates; band 4 is the cell lysate precipitate of the recombinant D-pantolactone enzyme strain with duplicates; bands 5 to 10 are the cell lysate supernatant and cell lysate of recombinant bacteria derived from other lactone enzyme genes of Agrobacterium tumefaciens, respectively; band 11 is the cell lysate supernatant of the control strain with empty pET-28a; and band 12 is the cell lysate precipitate of the control strain with empty pET-28a.

[0022] Figure 3 This is an SDS-PAGE protein electrophoresis image of purified D-indomethacin lactonease. Detailed Implementation

[0023] The present invention will be described in detail below with reference to the embodiments. The following embodiments do not limit the present invention.

[0024] Example 1: Construction and transformation of recombinant plasmid pET-28a encoding D-pantolactone lactonease gene (1) Culture of Agrobacterium tumefaciens: Agrobacterium tumefaciens (strain number: MCCC1A01339, China Marine Microbial Culture Collection Center) was streaked into a solid culture medium with the following composition: peptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 5 g / L, agar powder 20 g / L, and pH 7.0.

[0025] (2) Cloning of lactonease genes and construction of expression vectors Based on the nucleotide sequence of the D-pantothenic acid lactonease gene found in *Agrobacterium tumefaciens*, specific primers were designed to amplify the gene and ligate it into the expression vector pET-28a (purchased from Novagen). The gene was inserted into the BamHI and Hind III sites of the pET-28a(+) vector to obtain a recombinant plasmid, which was then introduced into *E. coli* for expression. The primers are designed as follows: Upstream primer: 5'-CCGAATTCGAGCTCCGTCGAATGAGCGAAACGATCCACGT-3' (as shown in SEQ ID NO: 3) Downstream primer: 5'-TTGTTAGCAGCCGGATCTCATCATGGCGCCCAGTATATATCC-3' (as shown in SEQ ID NO: 4).

[0026] PCR amplification conditions: 95℃ pre-denaturation for 5 min; 95℃-30 s, 58℃-30 s, 72℃-30 s, 30 cycles; final extension at 72℃ for 5 min.

[0027] Linearized primers were designed at the 5' ends of both the upstream and downstream primers for ligation into the expression vector pET-28a. The designed primers were used to amplify the DNA sequence, which is likely the D-pantolactone gene. The PCR amplification products were then detected by electrophoresis. The electrophoresis results are shown below. Figure 1 As shown in lane 1, the amplification product was purified and the target amplification fragment was recovered using a kit. Then, a one-step cloning process was performed using linearized primers and upstream and downstream primers to ligate the purified PCR amplification product with the vector pET-28a. The ligation product was transformed into *E. coli* DH5α competent cells (purchased from TAKARA), and the recombinant plasmid was screened and verified to obtain the expression plasmid. Simultaneously, the recombinant plasmid was sent to Qingke Biotechnology Co., Ltd. for sequencing analysis. The DNA sequence of the D-pantolactone enzyme gene was determined to be the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence of the D-pantolactone enzyme is shown in SEQ ID NO: 2.

[0028] Example 2: Induction, expression, and purification of D-pantolactonease The constructed recombinant expression plasmid was transformed into competent cells of *E. coli* BL21 (DE3) strain (purchased from TAKARA). Transformants were picked and cultured in LB broth containing 50 μg / ml kanamycin at 37°C and 200 rpm in a shaker. When the OD600 reached approximately 0.8, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and the cells were cultured at 20°C for another 18 h to induce expression. The fermentation broth after induction was centrifuged to collect the cells. The resulting wet cells could be used for enzyme-catalyzed hydrolysis or synthesis reactions. The wet cells were washed twice with cell suspension buffer, then resuspended in 1 / 10 volume of phosphate buffer. The cells were then sonicated under ice bath conditions until the cell wall was clear. The cells were centrifuged at 10,000 rpm for 5 min to collect the supernatant, which was the crude enzyme solution of D-pantolactone.

[0029] The HisTrap™ HP nickel column (purchased from GE) was cleaned and regenerated. After column equilibration, the crude enzyme solution was filtered through a 0.22 μm filter membrane and loaded onto the HisTrap™ HP nickel column. The column was flow-throughd twice, and the unbound portion of the protein mixture was collected and passed through the binding column. The nickel column was washed with 10 column volumes of starting buffer to remove contaminating proteins. Elution was then performed with 5 column volumes of starting buffer containing 20 mM imidazole to remove non-specific impurities from the column, and the eluent was collected. Elution was then performed sequentially with 5 column volumes of starting buffer containing 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole, and the eluent was collected for each step. The target protein band was detected by SDS-PAGE. The purified target protein was found in the eluent containing 200 mM imidazole. The imidazole in the buffer was then removed by a desalting column to obtain the purified enzyme solution. See Figure 2 and Figure 3 ,in Figure 2 This is an SDS-PAGE protein electrophoresis image of D-indomethacinase expressed by recombinant bacteria BL21(DE3). Figure 3 This is an SDS-PAGE protein electrophoresis image of purified D-indomethacin lactonease.

[0030] The starting buffer used for protein purification was: 20 mM phosphate buffer (Na2HPO4 and NaH2PO4, pH=7.0), 200 mM NaCl, and 10% glycerol.

[0031] Example 3: Experiments on the hydrolysis of D-indomethacin, γ-butyrolactone and benzofuranone by wild-type Escherichia coli.

[0032] The biocatalytic system was as follows: Wet cells collected from wild-type *E. coli* bacteria without D-pantolactone hydrolase were used as the biocatalyst. A whole-cell catalytic solution was prepared by adding PBS buffer to the wet cells, with a cell concentration of 20 g / L. 200 μL of 0.1 mol D-pantolactone, γ-butyrolactone, and benzofuranone were dissolved in 600 μL of PBS buffer and placed into three separate EP tubes. After thorough mixing, the solutions were rapidly added to a constant-temperature shaker at 200 rpm and 30°C. Then, 200 μL of the whole-cell catalytic solution was added to the EP tubes to initiate the hydrolysis reaction, maintaining the reaction temperature at approximately 30°C. The pH of the reaction was monitored online using a pH meter, and controlled at 7.5 ± 0.2 by adding sodium hydroxide. The reaction time was approximately 30 min. The reaction was terminated when the pH stabilized. The gas phase results are shown in Table 1.

[0033] Table 1 5 3.5 2

[0034] Example 4: Application of D-indomethacinase in the hydrolysis of D-indomethacin, γ-butyrolactone and benzofuranone.

[0035] The biocatalytic system was as follows: Wet cells collected from the fermentation of engineered *E. coli* bacteria producing D-pantolactone hydrolase were used as the biocatalyst. A whole-cell catalytic solution was prepared by adding PBS buffer to the wet cells, with a cell concentration of 20 g / L. Solutions of D-pantolactone, γ-butyrolactone, and benzofuranone at a final concentration of 25 mM were respectively placed into three EP tubes, stirred thoroughly, and then rapidly added to a constant-temperature shaker at 200 rpm and 30°C. 200 μL of the whole-cell catalytic solution was then added to the EP tubes to initiate the hydrolysis reaction, which was maintained at approximately 30°C. The pH of the reaction was monitored online using a pH meter, and controlled at 7.5 ± 0.2 by adding sodium hydroxide. The reaction time was approximately 30 min. The reaction was terminated when the pH stabilized. The gas phase results are shown in Table 2. The results showed that D-indohydrin hydrolase could catalyze the hydrolysis of D-indohydrin, γ-butyrolactone, and benzofuranone at pH 7.5±0.2 to prepare D-indohydrin, hydroxybutyric acid, and o-hydroxyphenylacetic acid.

[0036] Table 2 60 53 50

[0037] Example 5: Application of wild-type Escherichia coli in the synthesis of D-pantolytic acid lactone, γ-butyrolactone and benzofuranone.

[0038] The biocatalytic system was as follows: Wet cells collected from the fermentation of wild-type *E. coli* were used as the biocatalyst. A whole-cell catalytic solution was prepared by adding PBS buffer to the wet cells, with a cell concentration of 20 g / L. D-pantolytic acid, hydroxybutyric acid, and o-hydroxyphenylacetic acid, with a final concentration of 25 mM, were separately loaded into three EP tubes. After thorough mixing, the solutions were rapidly added to a constant-temperature shaker at 200 rpm and 30°C. Then, 200 μL of the whole-cell catalytic solution was added to the EP tubes to initiate the synthesis reaction, which was maintained at approximately 30°C. The pH of the reaction was monitored online using a pH meter, and controlled at 5.5 ± 0.2 by adding hydrochloric acid. The reaction time was approximately 30 min. The reaction was terminated when the pH stabilized. The gas phase results are shown in Table 3.

[0039] Table 3 0 0 0

[0040] Example 6: Application of D-indohydrinase in the synthesis of D-indohydrin, γ-butyrolactone and benzofuranone.

[0041] The biocatalytic system was as follows: Wet cells collected from the fermentation of engineered *E. coli* bacteria producing D-pantolactone hydrolase were used as the biocatalyst. A whole-cell catalytic solution was prepared by adding PBS buffer to the wet cells, with a cell concentration of 20 g / L. D-pantolactone, hydroxybutyric acid, and o-hydroxyphenylacetic acid, with a final concentration of 25 mM, were separately loaded into three EP tubes. After thorough mixing, the solutions were rapidly added to a constant-temperature shaker at 200 rpm and 30°C. Then, 200 μL of the whole-cell catalytic solution was added to the EP tubes to initiate the synthesis reaction, which was maintained at approximately 30°C. The pH of the reaction was monitored online using a pH meter, and controlled at 5.5 ± 0.2 by adding hydrochloric acid. The reaction time was approximately 30 min. The reaction was terminated when the pH stabilized. The gas phase results are shown in Table 4. The results showed that D-pantolactone hydrolase could catalyze the lactone reaction at pH 5.5 ± 0.2 and a reaction temperature of 30 °C to produce D-pantolactone, γ-butyrolactone, and benzofuranone. Compared with the existing high-temperature acidification method, the reaction conditions were milder.

[0042] Table 4 55 32 30

[0043] The GC method for detecting lactone content is as follows: Chromatographic conditions: Gas column (30×0.25mm, Durabond: CYCLOSIL-B, Size: 0.25μm, Part number: 112-6632, Serial Number: US2189343H, USA); The temperature program for the column oven was as follows: initial temperature 100℃, hold for 0.5 min, increase to 140℃ at 10℃ / min, hold for 6 min, decrease to 100℃ at 40℃ / min, hold for 0.5 min. Sample preparation: Dilute the sample with ethyl acetate to 50 mM and remove water with anhydrous sodium sulfate, keeping the lactone content between 5 mM and 10 mM; Mobile phase: Nitrogen; Data collection time: 23 min.

Claims

1. The application of D-pantolactone enzyme in the hydrolysis or synthesis of D-pantolactone, γ-butyrolactone, or benzofuranone, characterized in that, The amino acid sequence of the D-pantothenic acid lactonease is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that: The method of application is as follows: wet bacterial cells collected by fermentation of recombinant genetically engineered bacteria that produce D-pantolactone hydrolase are used as biocatalysts. The raw materials are D-pantolactone, γ-butyrolactone or benzofuranone. The reaction temperature is controlled at 30-50℃ and the reaction pH is controlled at 7.5±0.2 to carry out the hydrolysis reaction and generate D-pantolactone, hydroxybutyric acid or o-hydroxyphenylacetic acid.

3. The application as described in claim 1, characterized in that: The method of application is as follows: using wet bacterial cells collected by fermentation of recombinant genetically engineered bacteria that produce D-pantolytic acid lactone hydrolase as a biocatalyst, the raw materials are D-pantolytic acid, hydroxybutyric acid or o-hydroxyphenylacetic acid, the reaction temperature is controlled at 30-50℃, the reaction pH is controlled at 5.5±0.2, and a lactone reaction is carried out to generate D-pantolytic acid lactone, γ-butyrolactone or benzofuranone.

Images