L-pantoic acid lactone dehydrogenase mutant and application thereof

Abstract

The invention discloses an L-pantoic acid lactone dehydrogenase mutant and an application of the L-pantoic acid lactone dehydrogenase mutant. The L-pantoic acid lactone dehydrogenase mutant is subjected to any one or a combination of at least two of the following mutations on the basis of an amino acid sequence SEQ ID NO.1: T271D, A27S, T163K, V240I or L253I. The L-pantoic acid lactone dehydrogenase mutant is constructed on the basis of the L-pantoic acid lactone dehydrogenase derived from amycolatopsis methyloralis, the mutant shows excellent catalytic activity, the cell apparent activity of the mutant is improved by more than 10 times compared with that of a wild type, and the mutant provides a new biocatalyst for efficient preparation of D-pantoic acid lactone and derivatives thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and biocatalysis, and relates to a kind of L-pantoic acid lactone dehydrogenase mutant and its application. BACKGROUND

[0002] D-pantoic acid lactone is a key chiral intermediate for the synthesis of D-pantothenic acid and its derivatives. Vitamin B5, as the core precursor of coenzyme A synthesis, plays an irreplaceable role in the metabolism of carbohydrates, lipids and proteins in organisms, and has a huge market demand in the pharmaceutical, food additive and animal feed industries.

[0003] In the existing industrial technology, the preparation of D-pantoic acid lactone mainly adopts chemical resolution or enzymatic resolution method to treat racemic substrate. Among them, although the kinetic resolution process of D-lactone hydrolase is widely used, it is limited by the inherent thermodynamic equilibrium principle of kinetic resolution, and the highest theoretical yield of single pass is only 50%. In this process, a large amount of L-pantoic acid lactone, which is not hydrolyzed and utilized, remains as a byproduct. In order to reduce production cost, the residual L-pantoic acid lactone must be recovered, which usually needs to go through a series of cumbersome post-treatment steps such as acidification extraction, high-temperature chemical racemization and re-lactonization. This process not only leads to the extension of process flow and significant increase of energy consumption, but also has the technical defects of low atom utilization rate and large environmental pollution load.

[0004] In order to overcome the limitations of existing resolution process, the desymmetrization strategy based on multi-enzyme cascade catalysis has become a research hotspot in the field. This technical route uses oxidoreductase system to specifically oxidize and dehydrogenate L-pantoic acid lactone in the racemic mixture to generate achiral intermediate ketopantoic acid lactone, and then uses ketopantoic acid lactone reductase to asymmetrically reduce the intermediate to the target product D-pantoic acid lactone. Through this cascade reaction, cheap racemic raw materials can be theoretically converted to single configuration D-pantoic acid lactone with high optical purity at 100% conversion rate, thereby avoiding the problems of byproduct separation and chemical racemization, and having significant economic advantage.

[0005] In the construction of D-pantoic acid lactone biosynthesis pathway, L-pantoic acid lactone dehydrogenase is responsible for catalyzing the oxidation and dehydrogenation of L-pantoic acid lactone, and the efficiency of this step directly restricts the overall flux of the cascade reaction. However, most of the reported LPLDH (L-pantoic acid lactone dehydrogenase) is derived from Rhodococcus or Nocardia, and its natural property is mostly membrane-bound protein. When expressed heterologously in Escherichia coli, due to the existence of hydrophobic transmembrane region and the difference in folding kinetics, such enzymes are prone to aggregation in the intracellular inclusion body to form inactive inclusion body, resulting in low expression amount of soluble active protein. In addition, the natural LPLDH often has low activity, which becomes the rate-limiting bottleneck of the efficiency of the whole cell catalyst.

[0006] Therefore, there is an urgent need to provide an L-indolactone dehydrogenase with high catalytic efficiency for the substrate L-indolactone, so as to realize the green and low-cost manufacturing of D-indolactone. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides an L-indohydrin dehydrogenase mutant and its application. The mutant obtained by this invention exhibits more than 10 times higher whole-cell epigenetic catalytic activity than the wild type, which greatly enhances substrate conversion efficiency.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an L-pantolactone dehydrogenase mutant, wherein the L-pantolactone dehydrogenase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or a combination of at least two of the following mutations: T271D, A27S, T163K, V240I or L253I.

[0010] The L-pantolactone dehydrogenase (AmeLPLDH) in this invention is derived from *Amycolatopsismethanolica* 239 and exhibits strict stereoselectivity. This invention employs a computer-aided rational design strategy based on bioinformatics. First, motif scanning is performed using the NCBI conserved domain database to identify the TIM barrel domain and potential active pocket regions of the target protein. Next, multiple sequence alignment (MSA) analysis is used to compare the target sequence with homologous sequences in the database. Based on evolutionary conservation analysis, key non-conserved residues located near the substrate-binding pocket and active site are identified and selected as mutation hotspots for semi-rational design.

[0011] SEQ ID NO.1:

[0012] MSNGWFETVAEAQRRARKRLPKSVYGALVAGSERGITVDDNIAAAFAELGFAPHPVAGLSDKRELGTTVMGQPISLPVVISPTGQVAVHPDGEVAVARAAARRGTANGLSSFASKSIEEVAAANPQFFFQMYWGSRDVLVQRMERARAAGAVGLIMTLDWSFSTGRDWGSPVIPEPKLDLKAMARFAPEGITRPKWW DFAKTRKLPDLTTPNLTPPGGTAPTFFGAYGEWMQTLPLTWEDVAWLREQWGGPFMLKGVMRVDDAKRAVDAGYTAISVSNHGGNLDGTPAPIRALPAIDAVGGDVEVLLDGGIRRGSDVVKAIALGAKAVLIGRAYLWGLGAANGQAGVENVIDLRRGGIDSAVLGLGKTSIHELTRDDVVIPPGFERALGVPKS.

[0013] In a second aspect, the present invention provides a nucleic acid molecule that encodes the L-indohydrin dehydrogenase mutant described in the first aspect.

[0014] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0015] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0016] Fifthly, the present invention provides a genetically engineered bacterium containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0017] Sixthly, the present invention provides a method for preparing the L-pantolactone dehydrogenase mutant described in the first aspect, the method comprising the following steps:

[0018] (1) Based on bioinformatics, mutation sites were selected, and mutation primers were designed using the L-pantolactone dehydrogenase encoding gene SEQ ID NO.2 as a template;

[0019] (2) Site-directed mutagenesis was performed using a plasmid carrying the gene encoding L-indolactone dehydrogenase SEQ ID NO.2 as a template;

[0020] (3) The mutant plasmid was transformed into the host bacteria, cultured and purified to obtain the L-pantolactone dehydrogenase mutant.

[0021] SEQ ID NO.2:

[0022]

[0023] Preferably, the host bacterium includes Escherichia coli.

[0024] In a seventh aspect, the present invention provides the application of the L-indolactone dehydrogenase mutant described in the first aspect in a catalytic oxidative dehydrogenation reaction, wherein the substrate of the oxidative dehydrogenation reaction includes L-indolactone.

[0025] Eighthly, the present invention provides the application of the L-pantolactone dehydrogenase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the recombinant cell described in the fourth aspect, or the genetically engineered bacteria described in the fifth aspect in the preparation of D-pantolactone and / or ketopantolactone.

[0026] In a ninth aspect, the present invention provides a method for preparing D-pantolactone and / or ketopantolactone, the method comprising:

[0027] L-indophosphatidylcholine is mixed with the L-indophosphatidylcholine dehydrogenase mutant described in the first aspect, and the mixture is reacted. After the reaction is completed, the product is purified to obtain D-indophosphatidylcholine and / or ketoindophosphatidylcholine.

[0028] Preferably, the reaction temperature is 25-35°C (e.g., 25°C, 30°C or 35°C), the reaction pH is 6-7 (e.g., 6 or 7), and the reaction speed is 100-500 rpm (e.g., 100 rpm, 300 rpm or 500 rpm).

[0029] In one embodiment of the present invention, the host cell is Escherichia coli BL21(DE3).

[0030] This invention also relates to the expression of a recombinant vector containing the L-pantolactone dehydrogenase encoding gene in *Escherichia coli*, specifically as follows: A seed culture is prepared, and the recombinant engineered bacteria carrying the target gene are inoculated into LB liquid medium containing 30-100 μg / mL (e.g., 30 μg / mL, 50 μg / mL, or 100 μg / mL) of kanamycin, and incubated at 35-38°C (e.g., 35°C, 37°C, or 38°C) for 5-15 h (e.g., 5 h, 10 h, or 15 h). Subsequently, the resulting seed culture is transferred to fresh LB liquid medium containing the same concentration of kanamycin at an inoculation rate of 1.0%-3% (v / v), and cultured with shaking at 35-38°C (e.g., 35°C, 37°C, or 38°C) and 150-300 rpm (e.g., 150 rpm, 200 rpm, or 300 rpm). The absorbance value (OD) of the bacterial culture is then measured. 600When the bacterial cell count reaches the 0.6-0.8 mM range, add isopropyl thiogalactoside (IPTG) to the system to a final concentration of 0.5-1 mM (e.g., 0.5 mM or 1 mM) to initiate induction. After overnight induction at 25-35°C (e.g., 25°C, 30°C, or 35°C), centrifuge at 2-8°C (e.g., 2°C, 4°C, or 8°C) and 5000-10000 rpm (e.g., 5000 rpm, 8000 rpm, or 10000 rpm) for 15-25 min (e.g., 15 min, 20 min, or 25 min), and collect the precipitate to obtain wet bacterial cells containing the target enzyme.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The L-pantolactone dehydrogenase mutant of this invention has excellent catalytic activity, with its cellular epigenetic activity being more than 10 times higher than that of the wild type, showing broad development potential and application prospects in the field of green biomanufacturing of chiral drug precursors and their derivatives. Attached Figure Description

[0033] Figure 1 The standard curve of L-indohydrin;

[0034] Figure 2 This is a comparison chart of mutant activity.

[0035] Figure 3 This is a graph showing the molecular dynamics simulation results of AmeLPLDH and its mutants. Detailed Implementation

[0036] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0037] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0038] In the following embodiments, the names and specific nucleotide sequences of the site-directed mutagenesis primers used to prepare the mutants are shown in Table 1.

[0039] Table 1

[0040]

[0041] Gene origin: The wild-type AmeLPLDH gene involved in this invention is derived from Amycolatopsismethanolica 239 (UniProtKB: A0A076MU88).

[0042] Example 1

[0043] Construction and screening of recombinant expression plasmid pRSFDuet-1-AmeLPLDH.

[0044] 1. Acquisition and amplification of the target gene

[0045] The target gene, L-pantolactone dehydrogenase (AmeLPLDH), was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd., based on its nucleotide sequence (SEQ ID NO.2). Using the synthesized gene fragment as a template, PCR amplification was performed on SF-AF and SF-AR using specific primers to obtain the target gene fragment with homologous recombination arms at both ends. The PCR amplification products were detected by 1% agarose gel electrophoresis, excised, and purified for later use.

[0046] 2. Construction of recombinant expression vectors

[0047] The plasmid pRSFDuet-1 was selected and linearized by double digestion with BamHI and HindIII. Using a homologous recombination kit, the purified and recovered AmeLPLDH gene fragment was mixed with the linearized pRSFDuet-1 vector (vector to insert mass ratio of 5:1), and ligation was performed at 50℃ for 30 min to construct the recombinant plasmid pRSFDuet-1-AmeLPLDH.

[0048] 3. Conversion and Screening

[0049] The recombinant plasmid pRSFDuet-1-AmeLPLDH constructed above was transformed into Escherichia coli BL21(DE3) competent cells. The transformed bacterial culture was plated on solid LB agar plates containing 50 μg / mL kanamycin and incubated upside down in a 37°C incubator for 12 h.

[0050] The PCR amplification system is shown in Table 2 below, with a total volume of 50 μL.

[0051] Table 2

[0052]

[0053] PCR conditions: initial denaturation at 95℃ for 5 min, followed by cycling: denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, and after 30 cycles, extension at 72℃ for 5 min.

[0054] Induction of expression in the strain: Single colonies of the mutant strain were picked and inoculated into 50 mL LB medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 The concentration was approximately 0.6. IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was incubated overnight at 30°C and 200 rpm.

[0055] Example 2

[0056] The standard curve of L-pantolactone was plotted and a quantitative analysis method was established.

[0057] Accurately weigh L-indophosphatidyl lactone standard, dissolve and dilute with deionized water to prepare a series of standard working solutions with mass concentrations of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, and 10 g / L. Detect these standard solutions of different concentrations using a Shimadzu LC-20A high-performance liquid chromatograph, and record the response values ​​for each concentration. Perform linear regression fitting with the mass concentration of L-indophosphatidyl lactone (g / L) on the x-axis and the corresponding response values ​​on the y-axis to plot the standard curve as shown below. Figure 1 As shown, the linear regression equation and correlation coefficient R of the standard curve were calculated and obtained. 2 The results showed that, within the concentration range of 1 g / L to 10 g / L, the concentration of L-pantolactone exhibited a good linear relationship with the detection response value, and could be used for subsequent quantitative analysis of samples.

[0058] The specific detection conditions are as follows: mobile phase A is a 0.02 mol / L potassium dihydrogen phosphate solution at pH=3, and mobile phase B is acetonitrile. The injection volume is 10 μL, the UV detector wavelength is 230 nm, the flow rate is 0.8 mL / min, and the retention time of L-pantolactone is 8.9 min.

[0059] Example 3

[0060] Enzyme activity assay.

[0061] Whole-cell epigenetic catalytic activity: Under conditions of pH 6.5 and temperature 30℃, the number of micromoles of LPL (L-indohydrin lactone) consumed per gram of bacterial sludge per hour.

[0062] The formula for calculating the activity (U, μmol / (h·g)) of L-pantolactone dehydrogenase is shown in Equation (1);

[0063] Equation (1).

[0064] The specific implementation method is as follows: Weigh 0.1 g of wet bacterial mud, resuspend it in 5 mL of PBS buffer solution to prepare a cell suspension. At the same time, dissolve 0.1 g of LPL in 5 mL of PBS to prepare an LPL stock solution. Take 4 mL of the above cell suspension and 1 mL of LPL stock solution to construct a reaction system with a total volume of 5 mL. After the system is reacted in a shaker at 30℃ for 2 h, it is immediately placed in boiling water for inactivation, centrifuged and then detected by HPLC. The reaction process is shown in formula (2);

[0065] Equation (2).

[0066] Example 4

[0067] Mining and rational design of key functional sites of AmeLPLDH based on structural bioinformatics analysis.

[0068] 1. Protein domain architecture and functional annotation

[0069] To elucidate the catalytic mechanism of AmeLPLDH and define its functional regions, this embodiment first used the NCBI CD-Search algorithm to perform a deep scan and annotation of the amino acid sequence of AmeLPLDH. Bioinformatics analysis results showed that AmeLPLDH belongs to the FMN-dependent group. The FMN-dependent alpha-hydroxy-acid oxidizing protein superfamily exhibits a three-dimensional spatial fold containing a typical TIM barrel structure. This highly conserved domain forms the catalytic core of the enzyme. Further analysis revealed specific FMN cofactor binding pockets and substrate binding grooves within the sequence, providing a structural basis for subsequent active site localization.

[0070] 2. Evolutionary conservation analysis and homologous sequence alignment

[0071] Given that the evolutionary conservation of protein sequences is generally positively correlated with their functional importance, this invention selected RhoLPLDH, a homologous protein from Rhodococcus sp. with relatively detailed functional studies, as a reference template, and performed multiple sequence alignment analysis using sequences from other family sources. The alignment was performed using the ClustalW algorithm and visualized using ESPript 3.0. The results revealed that AmeLPLDH and RhoLPLDH share extremely high sequence identity in the catalytic active site region.

[0072] 3. Structural mapping and mutation strategies at key sites

[0073] Based on the sequence-structure-function correlation principle, this invention identifies key amino acid sites in RhoLPLDH that affect substrate specificity, thermal stability, and catalytic efficiency, and precisely maps these sites to the sequence coordinates of AmeLPLDH. To endow AmeLPLDH with novel catalytic properties, this invention, based on the aforementioned homology analysis results, implements a rational design strategy, targeting sites 27, 163, 240, 253, and 271 of AmeLPLDH as mutation targets. The aim is to construct a mutant library with superior performance by altering these key sites.

[0074] Example 5

[0075] Construction and screening of mutant pRSFDuet-1-AmeLPLDH-A27S.

[0076] Using the pRSFDuet-1-AmeLPLDH plasmid as a template, PCR amplification was performed on SF-AF, SF-AR, A27S-F, and A27S-R using specific primers. The PCR products were detected by 1% agarose gel electrophoresis, and the gel was excised and purified for later use. The pRSFDuet-1 plasmid was selected and linearized by double digestion with BamHI and HindIII. The digestion products were purified by gel extraction to obtain a linearized vector backbone with sticky ends. The purified gene fragments were mixed with the linearized pRSFDuet-1 vector in a specific ratio, and ligation was performed at a specific temperature to transform the recombinant ligation product into *E. coli* DH5α. In competent cells, the transformed bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted. After single colonies grew, single clones were randomly selected and sent to a sequencing company for Sanger sequencing. The sequencing results were compared with the expected sequence, confirming that the 27th amino acid was successfully mutated from alanine (Ala) to serine (Ser), and there were no other non-specific mutations, thus obtaining the correct mutant plasmid.

[0077] Example 6

[0078] Construction and screening of mutant pRSFDuet-1-AmeLPLDH-T163K.

[0079] Using the pRSFDuet-1-AmeLPLDH plasmid as a template, PCR amplification was performed on SF-AF, SF-AR, T163K-F, and T163K-R using specific primers. The PCR products were detected by 1% agarose gel electrophoresis, and the gel was excised and purified for later use. The pRSFDuet-1 plasmid was selected and linearized by double digestion with BamHI and HindIII. The digestion products were purified by gel extraction to obtain a linearized vector backbone with sticky ends. The purified gene fragments were mixed with the linearized pRSFDuet-1 vector in a specific ratio, and ligation was performed at a specific temperature to transform the recombinant ligation product into *E. coli* DH5α. In competent cells, the transformed bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted position. After single colonies grew, single clones were randomly selected and sent to a sequencing company for Sanger sequencing. The sequencing results were compared with the expected sequence, confirming that the amino acid at position 163 was successfully mutated from threonine (Thr) to lysine (Lys), with no other non-specific mutations, thus obtaining the correct mutant plasmid.

[0080] Example 7

[0081] Construction and screening of mutant pRSFDuet-1-AmeLPLDH-V240I.

[0082] Using the pRSFDuet-1-AmeLPLDH plasmid as a template, PCR amplification was performed on SF-AF, SF-AR, V240I-F, and V240I-R using specific primers. The PCR products were detected by 1% agarose gel electrophoresis, and the gel was excised and purified for later use. The pRSFDuet-1 plasmid was selected and linearized by double digestion with BamHI and HindIII. The digestion products were purified by gel extraction to obtain a linearized vector backbone with sticky ends. The purified gene fragment was mixed with the linearized pRSFDuet-1 vector in a specific ratio, and ligation was performed at a specific temperature to transform the recombinant ligation product into *E. coli* DH5α. In competent cells, the transformed bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. After single colonies emerged, individual clones were randomly selected and sent to a sequencing company for Sanger sequencing. Alignment of the sequencing results with the expected sequence confirmed that amino acid position 240 was successfully mutated from valine (Val) to isoleucine (Ile), with no other non-specific mutations, thus obtaining the correct mutant plasmid.

[0083] Example 8

[0084] Construction and screening of mutant pRSFDuet-1-AmeLPLDH-L253I.

[0085] Using the pRSFDuet-1-AmeLPLDH plasmid as a template, PCR amplification was performed on SF-AF, SF-AR, L253I-F, and L253I-R using specific primers. The PCR products were detected by 1% agarose gel electrophoresis, and the gel was excised and purified for later use. The pRSFDuet-1 plasmid was selected and linearized by double digestion with BamHI and HindIII. The digestion products were purified by gel extraction to obtain a linearized vector backbone with sticky ends. The purified gene fragments were mixed with the linearized pRSFDuet-1 vector in a specific ratio, and ligation was performed at a specific temperature to transform the recombinant ligation product into *E. coli* DH5α. In competent cells, the transformed bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted. After single colonies grew, single clones were randomly selected and sent to a sequencing company for Sanger sequencing. The sequencing results were compared with the expected sequence, confirming that the amino acid at position 253 was successfully mutated from leucine (Leu) to isoleucine (Ile), with no other non-specific mutations, thus obtaining the correct mutant plasmid.

[0086] Example 9

[0087] Construction and screening of mutant pRSFDuet-1-AmeLPLDH-T271D.

[0088] Using the pRSFDuet-1-AmeLPLDH plasmid as a template, PCR amplification was performed on SF-AF, SF-AR, T271D-F, and T271D-R using specific primers. The PCR products were detected by 1% agarose gel electrophoresis, and the gel was excised and purified for later use. The pRSFDuet-1 plasmid was selected and linearized by double digestion with BamHI and HindIII. The digestion products were purified by gel extraction to obtain a linearized vector backbone with sticky ends. The purified gene fragments were mixed with the linearized pRSFDuet-1 vector in a specific ratio, and ligation was performed at a specific temperature to transform the recombinant ligation product into *E. coli* DH5α. In competent cells, the transformed bacterial culture was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. After single colonies emerged, single clones were randomly selected and sent to a sequencing company for Sanger sequencing. Alignment of the sequencing results with the expected sequence confirmed that the amino acid at position 271, threonine (Thr), was successfully mutated to aspartic acid (Asp), with no other non-specific mutations, thus obtaining the correct mutant plasmid. The enzyme activity of the mutants was measured according to Example 3. The relative catalytic activities of all tested single-point mutants are shown in Table 3. The relative activity results are as follows: Figure 2 As shown.

[0089] Table 3

[0090]

[0091] From Table 3 and Figure 2 The data shows that the enzyme activity of the L-indo-lactone dehydrogenase mutant prepared by this invention is more than 10 times that of the wild-type L-indo-lactone dehydrogenase AmeLPLDH, and the optimal mutant T271D has an enzyme activity that is 10.36 times that of the wild-type L-indo-lactone dehydrogenase AmeLPLDH.

[0092] Example 10

[0093] Molecular dynamics simulation analysis of L-indohydrin dehydrogenase mutant T271D and wild-type L-indohydrin dehydrogenase AmeLPLDH.

[0094] This embodiment uses the GROMACS 2020.6 software package to perform all-atom molecular dynamics simulations. For force field parameter settings, the ligand small molecules are described using the GAFF2 universal force field, while the biomacromolecules are parameterized using the AMBER14SB combined with the parmbsc1 force field. In the system construction phase, the target complex is placed at the geometric center of a cubic simulation box, and the TIP3P explicit water model is introduced to construct the solvent environment. The minimum distance threshold from the solute surface to the simulation box boundary is set to 1.0 nm. Subsequently, the appropriate molar amount of Na⁺ counterions is calculated and added to achieve charge neutrality in the simulation system.

[0095] The simulation process begins by employing the steepest descent method to minimize the energy of the initial structure, thereby eliminating spatial conflicts between atoms. This is followed by an equilibrium phase, with pre-equilibrium simulations of 100 ps each conducted under isothermal and isochoric ensembles and isothermal and isobaric ensembles. Finally, the system is ensured to reach a stable thermodynamic equilibrium under isothermal conditions of 300 K and isobaric conditions.

[0096] Subsequently, long-term molecular dynamics sampling of 50 ns was performed. Root mean square deviation (RMSD) was used as a key indicator to monitor and quantify the spatial displacement of the protein-ligand complex relative to the initial coordinates and the overall conformational fluctuations. Typically, a low plateau in the RMSD indicates that the simulated system has achieved good structural convergence and stability. Comparison of trajectory data throughout the entire simulation period revealed that the T271D system exhibited a more compact conformation, with a significantly smaller structural shift than the wild type. Figure 3 Numerical statistics show that the root mean square deviation (RMS) of the WT group was 0.194 ± 0.033 nm, while that of the T271D group was only 0.183 ± 0.022 nm. Simulation results indicate that this mutation reduces the overall flexibility of the enzyme, enhances its rigidity, and thus improves its structural stability.

[0097] The radius of gyration was used to monitor changes in the overall compactness of the complex. In a 50 ns kinetic simulation, the T271D mutant exhibited volume characteristics completely consistent with the wild type, with both having an average radius of gyration of 2.069 ± 0.007 nm, indicating that the mutation did not alter the overall spatial folding state of the complex. Nevertheless, root mean square fluctuation analysis revealed specific differences in local conformational flexibility between the two, with the root mean square fluctuation distribution pattern of the mutant showing a significant shift compared to the wild type. By comparing the fluctuation spectra of different segments (… Figure 3 As can be seen, the T271D system exhibits extensive structural stabilization characteristics. Except for the C-terminal tail region, the root-mean-square fluctuation values ​​of the mutant chain are generally lower than those of the wild type, with this difference being particularly pronounced in the residue cluster at positions 163-177. This reduced volatility in this specific segment directly confirms that the mutation site induces local conformational rigidity. This microscopic kinetic characteristic reasonably explains the low root-mean-square bias and compact radius of gyration exhibited by the complex in macroscopic simulation indicators, further corroborating the thermodynamic stability of the system.

[0098] To elucidate the binding pattern between ligands and receptors, the number of hydrogen bonds in the simulated trajectory was counted. Figure 3 Data shows that the WT system has an average of 10.21 ± 1.41 hydrogen bonds, while this number decreases to 8.30 ± 1.56 in the T271D mutant. This statistically significant difference indicates that the mutation leads to a weakening of interfacial polar interactions. However, further interaction spectroscopy analysis revealed a significant increase in π-alkyl interactions in the mutant. This enhanced hydrophobic contact forms an effective compensatory mechanism, filling the binding energy gap caused by the reduction of hydrogen bonds. Overall, T271D maintains or even enhances the overall thermodynamic stability of the complex by replacing some polar connections with a binding mode dominated by hydrophobic interactions.

[0099] The solvent-exposed surface properties of biomacromolecules were evaluated using SASA analysis. The results are as follows: Figure 3 As shown, the T271D variant did not exhibit abnormal surface area expansion or contraction, and its SASA trajectory showed a synchronous steady state with the WT control group. This conservation of surface area parameter indicates that, despite the introduction of the mutation, the overall spatial extensibility and compactness of the protein-ligand complex were unaffected, and the structural folding state remained intact.

[0100] In summary, this invention constructs an L-indo-lactone dehydrogenase mutant based on α-indo-lactone dehydrogenase derived from *Amylopectinus mulliganus*. The mutant exhibits excellent catalytic activity, with its cellular epigenetic activity being 4-10 times higher than that of the wild type. This mutant shows broad development potential and application prospects in the field of green biomanufacturing of chiral drug precursors and their derivatives.

[0101] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An L-pantolactone dehydrogenase mutant, characterized in that, The L-pantothenic acid lactone dehydrogenase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or a combination of at least two of the following mutations: T271D, A27S, T163K, V240I, or L253I.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the L-indohydrin dehydrogenase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 2 or the recombinant vector of claim 3.

5. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the nucleic acid molecule as described in claim 2 or the recombinant vector as described in claim 3.

6. A method for preparing the L-pantolactone dehydrogenase mutant of claim 1, characterized in that, The method includes the following steps: (1) Based on bioinformatics, mutation sites were selected, and mutation primers were designed using the L-pantolactone dehydrogenase encoding gene SEQ ID NO.2 as a template; (2) Site-directed mutagenesis was performed using a plasmid carrying the gene encoding L-indolactone dehydrogenase SEQ ID NO.2 as a template; (3) The mutant plasmid was transformed into the host bacteria, cultured and purified to obtain the L-pantolactone dehydrogenase mutant.

7. The method for preparing L-pantolactone dehydrogenase mutant according to claim 6, characterized in that, The host bacteria include Escherichia coli.

8. The application of the L-indolactone dehydrogenase mutant of claim 1 in catalytic oxidative dehydrogenation reaction, wherein the substrate of the oxidative dehydrogenation reaction includes L-indolactone.

9. The use of the L-pantolactone dehydrogenase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, or the genetically engineered bacteria of claim 5 in the preparation of D-pantolactone and / or ketopantolactone.

10. A method for preparing D-pantolactone and / or ketopantolactone, characterized in that, The preparation method includes: L-indophosphatidyl lactone was mixed with the L-indophosphatidyl lactone dehydrogenase mutant of claim 1 and reacted. After the reaction was completed, the product was purified to obtain D-indophosphatidyl lactone and / or ketoindophosphatidyl lactone. Preferably, the reaction temperature is 25-35°C, the reaction pH is 6-7, and the reaction speed is 100-500 rpm.

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