Use of ω-transaminase in preparing 3-aminopropanol by means of bioenzymatic process

Abstract

Provided is a method for preparing 3-aminopropanol by means of a bioenzymatic process, which method comprises: using 3-hydroxypropanal as a substrate and ω-transaminase as a catalyst to perform a catalytic reaction on the substrate 3-hydroxypropanal, thereby obtaining 3-aminopropanol.

Description

Applications of ω-transaminase in the enzymatic preparation of 3-aminopropanol Technical Field

[0001] This application belongs to the field of 3-aminopropanol preparation, specifically involving the application of ω-transaminase in the preparation of 3-aminopropanol. Background Technology

[0002] 3-Aminopropanol (also known as 3-AP) is an important fine chemical intermediate widely used in pharmaceuticals, pesticides, and dyes, playing a crucial role in the synthesis of drugs such as cyclophosphamide and cyclophosphamide, as well as key raw materials such as vitamin B5 (panthenol). With the improvement of people's living standards, the application of panthenol in daily chemical products is increasing, especially in hair care products and specialized cosmetics, thus significantly boosting the market demand for 3-aminopropanol. Currently, the mainstream preparation process for 3-aminopropanol uses 3-hydroxypropionitrile as a raw material, employing a catalyst for hydrogenation reduction to obtain 3-aminopropanol. In addition, several other feasible processes have been reported: ① Synthesizing 3-aminopropanol from ethyl 3-aminopropionate and 2-cyanoethanol; ② Synthesizing ketoximes from methyl isobutyl ketone or cyclohexanone, followed by condensation with acrylonitrile and catalytic hydrogenation cracking to prepare 3-aminopropanol; ③ Synthesizing 3-aminopropanol from dimethyl sulfoxide (DMSO) and dichloromethane; ④ Using 1,4-butyrolactone as a starting material, sequentially adding hydrazine hydrate and sodium nitrite aqueous solution to generate acyl azide, which is then rearranged to generate 3-aminopropanol. However, the above-mentioned process routes generally suffer from problems such as poor catalyst activity, low yield of 3-aminopropanol, high selectivity of by-products, difficulty in product separation, harsh reaction conditions, low safety, and high equipment requirements.

[0003] Patent CN115819253A discloses a process route for the catalytic synthesis of 3-aminopropanol from β-alanine. Compared with the aforementioned production processes, this route is simpler, more environmentally friendly, and sustainable, with its main advantage lying in the acquisition of the raw material (β-alanine). The mainstream preparation process uses β-hydroxypropionitrile, obtained through a series of complex chemical processes from the petroleum derivative acrylic acid. In contrast, the β-alanine used in CN115819253A can be obtained through bioconversion of renewable biomass feedstocks (such as glucose and glycerol). The reaction process is mild and environmentally friendly, and the biosynthesis of β-alanine has already achieved commercial production. Although this "bioconversion + chemical catalysis" process achieves partial green synthesis of 3-aminopropanol, it still does not fundamentally solve the problems of high energy consumption, high cost, and difficulty in guaranteeing product quality in 3-aminopropanol production. Therefore, the development of a fully biological production process for 3-aminopropanol remains a challenge for the industry.

[0004] Structurally, 3-aminopropanol possesses two active functional groups: an amino group (-NH2) and a hydroxyl group (-OH). Based on the principle of bioretrosynthesis, we hypothesize that, while maintaining the hydroxyl group of the compound, an amino group can be added to the other end of a substrate with an existing hydroxyl group to obtain the target product 3-aminopropanol. After in-depth analysis, 3-hydroxypropanal (also known as 3-HPA) has become a candidate substrate. Patent CN116102437A discloses a process for producing 3-aminopropanol using 3-hydroxypropanal as a substrate via chemical catalysis. Like other chemical hydrogenation processes, this route still suffers from drawbacks such as expensive catalysts and high temperature and pressure requirements. Furthermore, 3-hydroxypropanal can be produced through microbial fermentation. In summary, using 3-hydroxypropanal as a substrate and undergoing biological transamination is key to achieving the fully biological production of 3-aminopropanol. To date, no publicly available reports have addressed this technology.

[0005] ω-transaminases are 5′-pyridoxal-dependent 5′-phosphate transferases that stereospecifically catalyze the reversible transfer of amino groups between amino donors (such as amino acids, alkylamines, and aromatic amines) and carbonyl compounds (such as aldehydes, ketones, and keto acids), with PLP as a coenzyme. Most ω-transaminases have been reported to exhibit a catalytic preference for keto acids, methyl ketones (especially cyclic or aromatic compounds), and larger aliphatic substrates (chain lengths of at least 6 carbons). In recent years, some studies have reported the application of ω-transaminases in the biosynthesis of amino alcohols from aldehydes. However, currently available ω-transaminases show a catalytic preference for cyclic or long-chain aliphatic aldehydes, with catalytic efficiency positively correlated with the carbon number of the substrate. The catalytic efficiency for short-chain aldehydes decreases significantly, limiting their application in the large-scale biosynthesis of short-chain amino alcohols such as 3-aminopropanol. Furthermore, the low catalytic efficiency and poor stability of natural ω-transaminases for characteristic substrates remain the biggest obstacles to their industrial application. Summary of the Invention

[0006] The enzymatic production of 3-aminopropanol via transamination using 3-hydroxypropanal as a substrate is an economical and environmentally friendly route. To achieve this goal, the screening of specific transaminases is crucial. This invention aims to screen for ω-transaminases with high catalytic activity towards the specific substrate 3-hydroxypropanal.

[0007] In this invention, for the biosynthesis of 3-aminopropanol, a transaminase ω-TA2 derived from Bacillus megaterium was first screened. Unexpectedly, ω-TA2 was found to exhibit excellent catalytic activity towards 3-hydroxypropanal, efficiently converting it to the target product, 3-aminopropanol. Based on ω-TA2, and using bioinformatics analysis, the applicant further screened ω-TA12 and ω-TA13, which also exhibit high catalytic activity towards 3-hydroxypropanal, providing a new approach for the industrial production of 3-aminopropanol. This invention marks the first successful bio-enzymatic synthesis of 3-aminopropanol using ω-transaminase as a catalyst and 3-hydroxypropanal as a substrate.

[0008] Specifically, this invention screened natural ω-transaminases with high catalytic activity for the specific substrate 3-hydroxypropanal through extensive wet experiments. By combining multi-omics data analysis, bioinformatics screening, computer-aided design, and experimental verification, an ω-transaminase with higher enzyme activity, stability, and industrial application potential was discovered from a wide range of biological resources. This enabled the transamination catalysis of 3-hydroxypropanal, breaking through the technical barriers to the industrial production of 3-aminopropanol from 3-hydroxypropanal and providing a new route for the bioenzymatic synthesis of 3-aminopropanol.

[0009] In this invention, the ω-transaminase involved is selected from any one of the following (a) to (r):

[0010] a. The amino acid sequence of ω-transaminase ω-TA1 with accession number WP_011135573.1 in the NCBI database is shown in SEQ ID NO:1;

[0011] b. The amino acid sequence of ω-transaminase ω-TA2 with amino acid sequence accession number 5G09_A in the PDB database is shown in SEQ ID NO:2;

[0012] c. The amino acid sequence of ω-transaminase ω-TA3 with accession number WP_011750218.1 in the NCBI database is shown in SEQ ID NO:3;

[0013] d. The amino acid sequence of ω-transaminase ω-TA4 with accession number WP_003084297.1 in the NCBI database is shown in SEQ ID NO:4;

[0014] The amino acid sequence of ω-transaminase ω-TA5 with accession number B0KJV9 in the e.Uniprot database is shown in SEQ ID NO:5.

[0015] The amino acid sequence of ω-transaminase ω-TA6 with accession number B1JX81 in the f.Uniprot database is shown in SEQ ID NO:6.

[0016] g. The amino acid sequence of ω-transaminase ω-TA7 with accession number Q9JXW0 in the Uniprot database is shown in SEQ ID NO:7;

[0017] The amino acid sequence of ω-transaminase ω-TA8 with accession number Q8D3C8 in the h.Uniprot database is shown in SEQ ID NO:8.

[0018] i. The amino acid sequence of ω-transaminase ω-TA9 with accession number A4J6H0 in the Uniprot database is shown in SEQ ID NO:9;

[0019] The amino acid sequence of ω-transaminase ω-TA10 with accession number A5ITJ2 in the j.Uniprot database is shown in SEQ ID NO:10.

[0020] The amino acid sequence of ω-transaminase ω-TA11 with accession number Q4H4F5 in the k.Uniprot database is shown in SEQ ID NO:11.

[0021] l. The amino acid sequence of ω-transaminase ω-TA12 with accession number WP_076629700.1 in the NCBI database is shown in SEQ ID NO:12;

[0022] The amino acid sequence of ω-transaminase ω-TA13 with accession number WP_127113105.1 in the m.NCBI database is shown in SEQ ID NO:13.

[0023] The amino acid sequence of ω-transaminase ω-TA14 with accession number O66998 in the n.Uniprot database is shown in SEQ ID NO:14.

[0024] The amino acid sequence of ω-transaminase ω-TA15 with accession number O66442 in the o.Uniprot database is shown in SEQ ID NO:15.

[0025] The amino acid sequence of ω-transaminase ω-TA16 with accession number C1DUY4 in the p.Uniprot database is shown in SEQ ID NO:16.

[0026] The amino acid sequence of ω-transaminase ω-TA17 with accession number Q5SJS4 in the q.Uniprot database is shown in SEQ ID NO:17.

[0027] The amino acid sequence of ω-transaminase ω-TA18 with accession number Q58131 in the r.Uniprot database is shown in SEQ ID NO:18.

[0028] In a preferred embodiment, the ω-transaminase is selected from any one of ω-TA1, ω-TA2, ω-TA3, ω-TA5, ω-TA8, ω-TA10, ω-TA12, ω-TA13, ω-TA15, or ω-TA17.

[0029] In a more preferred embodiment, the ω-transaminase is selected from any one of ω-TA2, ω-TA12, and ω-TA13.

[0030] Specifically, the present invention provides the following technical solutions:

[0031] 1. A method for preparing 3-aminopropanol by a bioenzymatic process, comprising using 3-hydroxypropanal as a substrate and ω-transaminase as a catalyst to catalyze the transamination reaction of the substrate 3-hydroxypropanal to obtain 3-aminopropanol.

[0032] 2. The method according to Project 1, wherein the sequence of the ω-transaminase is as shown in any one of SEQ ID NO:2, 12 or 13.

[0033] 3. The method according to Project 1, wherein the ω-transaminase is artificially synthesized or obtained through expression in host cells;

[0034] Optionally, the host cell includes Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

[0035] 4. The method according to item 1 or 2, wherein the ω-transaminase can synthesize 3-aminopropanol via in vitro catalysis or intracellular catalysis;

[0036] Optionally, the ω-transaminase further includes a signal peptide; optionally, the signal peptide includes, but is not limited to, signal peptides used for purification, localization, and solubilization.

[0037] 5. The method according to Project 1 or 2, wherein the pH value in the catalytic transamination reaction is 6.5-9.0, preferably 7.5.

[0038] In a specific implementation, the pH value in the catalytic transamination reaction is 7.0-9.0, 7.0-8.5, or 7.0-8.0; for example, the pH value is 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.

[0039] 6. The method according to Project 1 further includes using L-alanine as an amino donor;

[0040] Preferably, it further includes the addition of a coenzyme; more preferably, the coenzyme is pyridoxal 5′-phosphate.

[0041] 7. The method according to Project 6, wherein the molar ratio of the amino donor to the substrate is 4-6:1, preferably 5:1.

[0042] 8. According to the method described in Project 1, wherein in the catalytic transamination reaction, the reaction temperature is 35-40℃, preferably 37℃.

[0043] 9. According to the method described in Project 1, wherein in the catalytic transamination reaction, the reaction time is 20-24h, preferably 22h.

[0044] 10. Use of ω-transaminase in the preparation of 3-aminopropanol by a bioenzymatic method, wherein 3-hydroxypropanal is used as a substrate; preferably, the sequence of the ω-transaminase is as shown in any one of SEQ ID NO:2, 12 or 13.

[0045] the term

[0046] In this article, "wet experiments" refer to experiments conducted in a laboratory involving liquid samples such as chemicals, pharmaceuticals, blood, proteins, and DNA. These experiments typically require specific laboratory equipment, such as test tubes, centrifuges, microscopes, colorimeters, and PCR instruments, and must be performed in a strictly controlled environment to ensure safety and accuracy. In contrast to wet experiments are "dry experiments," which primarily involve computer simulations, data analysis, and other experiments that do not directly use liquid chemicals or biological materials.

[0047] In this paper, transaminase (TA) (EC2.6.1.X) is a class of pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze amino transfer between an amino donor and an amino acceptor (i.e., compounds containing a carbonyl functional group). Based on the position of the transferred amino group, transaminases can be broadly classified into α-transaminases (catalyzing the transfer of the amino group at the α-carbon) and ω-transaminases (where the transferred amino group is located further from the carboxyl group or the substrate does not contain a carboxyl group). Therefore, α-transaminases are commonly used for the preparation of chiral α-amino acid compounds. Because some ω-transaminases allow for the absence of a carboxyl group in the substrate molecule, their applications are more extensive; they can not only synthesize other types of amino acids but also accept various carbonyl compounds, such as aldehydes and ketones, catalyzing the biosynthesis of various chiral amines. Beneficial effects

[0048] This invention, through screening and wet experimental verification, successfully identified a natural transaminase ω-TA2 with high activity towards the short-chain substrate 3-hydroxypropanal, which can efficiently convert 3-hydroxypropanal into the target product 3-aminopropanol. Furthermore, based on ω-TA2, bioinformatics analysis revealed ω-TA12 and ω-TA13, which also exhibit high catalytic activity towards 3-hydroxypropanal, with ω-TA13 showing even higher enzyme activity and stability. This provides a new approach for the industrial production of short-chain amino alcohols such as 3-aminopropanol. Attached Figure Description

[0049] Figure 1 shows the SDS-PAGE electrophoresis diagram of ω-TA1 to ω-TA4 expression in recombinant host cells;

[0050] Among them, band 1 shows the SDS-PAGE image of E. coli before induction of ω-TA1 enzyme expression, band 2 shows the SDS-PAGE image of the supernatant of E. coli after lysis after induction of ω-TA1 enzyme expression, and band 3 shows the SDS-PAGE image of the bacterial cell precipitate of E. coli after lysis after induction of ω-TA1 enzyme expression.

[0051] Band 4 shows the SDS-PAGE image of E. coli before induction of ω-TA2 enzyme expression, Band 5 shows the SDS-PAGE image of the supernatant of E. coli after lysis after induction of ω-TA2 enzyme expression, and Band 6 shows the SDS-PAGE image of the bacterial cell precipitate of E. coli after lysis after induction of ω-TA2 enzyme expression.

[0052] Band 7 shows the SDS-PAGE image of E. coli before lysis before induction of ω-TA3 enzyme expression; Band 8 shows the SDS-PAGE image of the supernatant of E. coli after lysis after induction of ω-TA3 enzyme expression; Band 9 shows the SDS-PAGE image of the bacterial cell precipitate of E. coli after lysis after induction of ω-TA3 enzyme expression.

[0053] Band 10 shows the SDS-PAGE image of E. coli before lysis before induced expression of ω-TA4 enzyme; Band 11 shows the SDS-PAGE image of the supernatant of E. coli after lysis following induced expression of ω-TA4 enzyme; and Band 12 shows the SDS-PAGE image of the bacterial pellet of E. coli after lysis following induced expression of ω-TA4 enzyme.

[0054] Figure 2 shows the yield data of 3-aminopropanol catalyzed by ω-TA1 to ω-TA4.

[0055] Figure 3 shows the three-dimensional structure of ω-TA13 and a schematic diagram of the catalytic pocket, with darker colors representing amino acids in the catalytic pocket.

[0056] Figure 4 shows electrophoresis diagrams of ω-TA5 to ω-TA18; where "supernatant" represents the electrophoresis diagram of the supernatant after lysis of E. coli cells expressing each ω-TA enzyme, and "precipitate" represents the electrophoresis diagram of the bacterial precipitate after lysis of E. coli cells expressing each ω-TA enzyme.

[0057] Figure 5 shows a comparison of the yields of 3-aminopropanol generated by candidate ω-TA and the yields of 3-aminopropanol at different pH values. (A) shows the yield of 3-aminopropanol generated by candidate ω-TA catalysis; (B) shows the yield results of 3-aminopropanol generated by candidate ω-TA at different pH values. Detailed Implementation

[0058] The present invention is further illustrated by the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope or embodiments of the invention. The scope of the invention is limited by the appended claims. Based on this specification and general knowledge in the art, those skilled in the art can clearly understand the scope of the claims. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications and alterations to the technical solutions of the invention, and such modifications and alterations are also included within the scope of the invention.

[0059] Unless otherwise stated, the experimental methods used in the following examples are conventional methods, such as those described in Molecular Cloning: A Laboratory Manual by J. Sambrook et al.; unless otherwise stated, the reagents and materials used are commercially available.

[0060] In some implementations, the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae, etc.

[0061] The culture media and solutions involved in the following examples are shown below:

[0062] LB solid medium: 10 g·L⁻¹ tryptone, 5 g·L⁻¹ yeast extract, 10 g·L⁻¹ sodium chloride and 2 g·L⁻¹ agar powder.

[0063] LB liquid medium: 10 g·L⁻¹ tryptone, 5 g·L⁻¹ yeast extract and 10 g·L⁻¹ sodium chloride.

[0064] Preparation of kanamycin resistance solution: Accurately weigh 10 mg of kanamycin and dissolve it in 100 mL of sterile water. Filter the solution through a 0.22 μm microporous membrane in a laminar flow hood to remove bacteria. Aliquot the solution into sterile 2 mL centrifuge tubes and store at -20°C for later use.

[0065] Example 1: Construction of host cells expressing ω-TA1 to ω-TA4 and expression of ω-TA1 to ω-TA4

[0066] 1. Construction of recombinant host cells containing genes encoding ω-TA1 to ω-TA4

[0067] In this embodiment, the sources and gene sequence information of ω-transaminases were retrieved through literature search and multiple sequence alignment; the gene sequences or amino acid sequences of ω-transaminases from different sources were queried through the gene database (https: / / www.ncbi.nlm.nih.gov / genome / ), and four ω-transaminases (i.e., ω-TA1 to ω-TA4) that may efficiently catalyze 3-hydroxypropanal were finally screened out. Their accession numbers in the prior art database are shown in Table 1 below.

[0068] Table 1. Login numbers from ω-TA1 to ω-TA4

[0069] Subsequently, the coding genes of ω-TA1 to ω-TA4 were codon optimized according to the codon preference of E. coli. The codon-optimized coding genes were then synthesized by a commercial company, ligated into the vector pETDuet-1 (purchased from Hongxun Biotechnology), and transformed into host cells E. coli BL21(DE3) (purchased from Sangon Biotech) to obtain recombinant host cells.

[0070] 2. Expression of ω-TA1 to ω-TA4

[0071] Add kanamycin resistance solution to LB liquid medium to a final concentration of 100.0 μg / mL. After activating the recombinant host cells containing the coding sequences for ω-TA1 to ω-TA4 by streaking on LB agar plates, inoculate them into 5 ml of the above LB liquid medium and culture at 37°C and 220 rpm / min for 10–12 h. Then, transfer 2 ml of the seed culture to 200 ml of LB liquid medium in a 1000 ml flask. Induce culture with 0.3 mM IPTG until the optical density (OD600) at 600 nm reaches 0.70, and then culture at 25°C for another 20 h. Harvest whole *E. coli* cells by centrifugation at 4°C (6000 g, 20 min), and wash twice with phosphate-buffered saline (100 mM, pH 7.5). The obtained whole *E. coli* cells are strains capable of expressing ω-TA1 to ω-TA4 enzymes, which can be used for subsequent expression of ω-transaminase in a catalytic system for transamination catalysis.

[0072] To verify the soluble expression of ω-transaminase in E. coli, cells were collected and resuspended in PBS to prepare a 40 g / L system. The cells were placed on ice and sonicated at 400 W for 90 times with a 4-second interval. After centrifugation (12000×g, 4℃), the supernatant of the cell lysate was collected, and the precipitate was resuspended in an equal volume of PBS. The SDS-PAGE gel (see Figure 1) showed that all four transaminases could be expressed solublely, with TA2 showing the best soluble expression. This indicates that after E. coli was lysed, ω-transaminase was present in the supernatant of the cell lysate (TA1-4 enzyme sizes were 51.2, 53.1, 50, and 50.5 kDa, respectively).

[0073] Therefore, ω-TA1 to ω-TA4 enzymes can also be directly extracted from the E. coli and added to the catalytic system for transamination catalysis. In this case, the E. coli cells need to be lysed. The cells can be resuspended in PBS buffer and the suspension placed on ice. The cells are sonicated at 400W for 90 times with a 4s interval to obtain cell-free extracts. After centrifugation (12000×g, 4℃), the supernatant of the cell lysate (i.e., ω-TA1 to ω-TA4 enzymes, respectively) is collected and stored at -20℃ for later use.

[0074] Example 2: Construction of catalytic reaction systems from ω-TA1 to ω-TA4

[0075] In this embodiment, a reaction system for producing 3-aminopropanol was constructed.

[0076] In the reaction system, L-alanine is used as the amino donor, 3-hydroxypropanal is used as the reaction substrate, and ω-TA1 to ω-TA4 enzymes are used as catalysts respectively. For ω-TA1 to ω-TA4 enzymes, whole Escherichia coli cells collected in Example 2 can be directly added to the reaction system to express ω-TA1 to ω-TA4 enzymes, or the supernatant obtained by cell disruption and extraction in Example 2 (which contains ω-TA1 to ω-TA4 enzymes respectively) can be added to the reaction system.

[0077] In this embodiment, whole Escherichia coli cells collected in Example 2 were directly added to the reaction system to express ω-TA1 to ω-TA4 enzymes, and the specific conditions for the catalytic reaction are as follows.

[0078] Maintaining a molar ratio of amino donor (i.e., L-alanine) to reaction substrate (i.e., 3-hydroxypropanal) of (4-6):1, for example 5:1, place the whole Escherichia coli cells collected in Example 1 above into a 2 mL centrifuge tube for culture. After preparing the transformation system according to the table below, place the centrifuge tube in a shaker at 220 rpm and 37°C for substrate transamination reaction for 24 h.

[0079] Table 2 Enzyme Conversion Reaction System

[0080] Example 3: Yield analysis of 3-aminopropanol production from ω-TA1 to ω-TA4

[0081] In this embodiment, the aim was to determine whether ω-TA1 to ω-TA4 possessed highly efficient catalytic activity for 3-hydroxypropanol. Using the ω-TA-containing catalytic system constructed in Example 2, the content of 3-aminopropanol generated by the conversion was detected by HPLC.

[0082] The final results are shown in Figure 2. ω-TA1, ω-TA2, and ω-TA3 all exhibit catalytic activity towards the substrate 3-hydroxypropanal, with yields of 3-aminopropanol all exceeding 0.5 g / L. Among them, ω-TA2 showed the best activity towards 3-hydroxypropanal, achieving a yield of 0.72 g / L of 3-aminopropanol and a 3-aminopropanol yield of 71.11%, demonstrating potential for industrial-scale production.

[0083] Furthermore, in order to obtain ω-transaminases with higher activity and better stability, the applicant used ω-TA2 as a wild-type template and combined bioinformatics technology to further screen multiple ω-transaminases from databases known in the prior art.

[0084] Example 4: Database Establishment and Preprocessing

[0085] Construction of biological sequence database

[0086] The purpose of constructing a dedicated biological sequence database is to collect, integrate, and manage genetic information and protein structure data related to ω-transaminases, providing a comprehensive, accurate, and easily searchable data resource to support efficient screening and further research of ω-transaminases. The database construction follows the principles of data comprehensiveness, accuracy, real-time performance, and ease of use.

[0087] The data sources mainly consist of the following three parts: (1) Public databases: such as NCBI GenBank, UniProt, PDB, etc., from which the genetic sequence and protein structure information of known ω-transaminases, as well as the genomic information of microorganisms that may contain ω-transaminase activity, are extracted. (2) Literature collection: ω-transaminase sequences and related biological characteristics data reported in relevant scientific literature and research reports are systematically collected and organized. (3) Experimental data: including publicly available ω-transaminase sequences and their activity, stability, and other data in existing technologies (such as those obtained by our research team and partners through experiments).

[0088] This database covers a wide range of biological species and environmental samples, providing abundant raw materials for the discovery of ω-transaminases.

[0089] Example 5: Bioinformatics Mining

[0090] I. Sequence Alignment and Functional Domain Search

[0091] HMMER is a software suite that uses probabilistic methods to search for protein sequence similarity. It performs efficient contour hidden Markov models (HMMs) searches and can be used to search for single protein sequences, multiple protein sequence alignments, or contour HMMs against target sequence databases, as well as search for protein sequences against Pfam. Based on the results of multiple sequence alignments, a seed model for ω-transaminases was generated in HMMER, and this model was used to filter sequences in a local sequence library, yielding 3150 sequences with ω-transaminas function.

[0092] II. Three-dimensional structure prediction and simulation

[0093] 1. Three-dimensional structure prediction

[0094] AlphaFold (www.deepmind.com / research / highlightedresearch / alphafold) is an advanced artificial intelligence program developed by DeepMind specifically for predicting the three-dimensional structure of proteins. AlphaFold2 uses deep learning algorithms to predict the spatial structure of proteins, and this tool is used to model the structure of ω-TA. This method was used to batch model 3150 sequences with ω-transaminase function.

[0095] 2. Assessment of protein conformation rationality

[0096] The structural rationality of the protein models obtained in the previous steps was evaluated using the SAVESS v6.0 scoring system (accessible at https: / / saves.mbi.ucla.edu), and the results were presented in a Laplace plot.

[0097] 3. Molecular docking simulation

[0098] The AutoDock Vina (www.autodock.scripps.edu) was used to simulate the interactions between ω-transaminases and their potential substrates. Researchers can understand the localization of substrates at the enzyme's active site and key interactions, thereby guiding the screening, modification, and optimization of ω-transaminases.

[0099] Molecular docking was performed on 3150 modeled enzyme structures, among which the highest affinity was -4 kcal / mol, totaling 39 structures.

[0100] 4. Multiple sequence alignment and evolutionary analysis

[0101] Multiple sequence alignment (MSA) and evolutionary analysis are key steps in bioinformatics to explore the functions of genes and proteins and their evolutionary relationships. In particular, in the study of enzymes such as ω-transaminase, these analyses can provide a deeper understanding of their structural and functional diversity and evolutionary history.

[0102] Thirty-nine enzyme sequences screened through molecular docking simulation were subjected to multiple sequence alignment (MSA) using MUSCLE. The MSA results were then used to construct a phylogenetic tree using the maximum likelihood method in PhyML. The phylogenetic tree clearly shows that the 39 enzymes are divided into four groups. Based on affinity, 14 enzymes (their names and corresponding accession numbers are shown in Table 3) were selected from different branches for gene synthesis, to be used for subsequent expression and enzyme activity testing. Figure 3 provides an example of the three-dimensional structure and catalytic structure of TA13.

[0103] Table 3 shows the enzyme sequences screened.

[0104] Example 6: Construction of host cells expressing ω-TA5 to ω-TA18 and expression of ω-TA5 to ω-TA18

[0105] 1. Construction of recombinant host cells containing ω-TA5 to ω-TA18 encoding genes

[0106] The ω-TA5 to ω-TA18 sequences (their accession numbers in the database are shown in Table 3) were codon optimized in E. coli. The codon-optimized coding genes were then synthesized by a commercial company, ligated into the vector pETDuet-1 (purchased from Hongxun Biotechnology), and transformed into host cells E. coli BL21(DE3) (purchased from Sangon Biotech) to obtain recombinant host cells.

[0107] 2. Expression of ω-TA5 to ω-TA18

[0108] Add kanamycin resistance solution to LB liquid medium to a final concentration of 100.0 μg / mL. After activating the recombinant host cells containing the coding sequences for ω-TA5 to ω-TA18 by streaking on LB agar plates, inoculate them into 5 ml of the above LB liquid medium and culture at 37°C and 220 rpm / min for 10–12 h. Then, transfer 2 ml of the seed culture to 200 ml of LB liquid medium in a 1000 ml flask. Induce culture with 0.3 mM IPTG until the optical density (OD600) at 600 nm reaches 0.70, and then culture at 25°C for another 20 h. Harvest whole *E. coli* cells by centrifugation at 4°C (6000 g, 20 min), and wash twice with phosphate-buffered saline (100 mM, pH 7.5). The obtained whole *E. coli* cells are strains capable of expressing ω-TA5 to ω-TA18 enzymes, which can be used for subsequent expression of ω-transaminase in a catalytic system for transamination catalysis.

[0109] In addition, the expression of these enzymes in E. coli was verified by SDS-page. Figure 4 shows that the soluble expression of different enzymes varies. TA5, TA10 and TA13 have better soluble expression (the sizes of TA5-18 enzymes are 45.2, 44.9, 45.3, 48.9, 46.7, 46.4, 48.4, 50.3, 50.3, 46.4, 42, 46.8, 46 and 44.1 kDa, respectively).

[0110] Alternatively, ω-TA5 to ω-TA18 enzymes can be directly extracted from the *E. coli* and added to the catalytic system for transamination catalysis. In this case, the *E. coli* cells need to be lysed. The cells can be resuspended in the same buffer and the suspension placed on ice. The cells are sonicated at 400W for 90 times with a 4s interval to obtain a cell-free extract. After centrifugation (12000×g, 4℃), the supernatant of the cell lysate (i.e., ω-TA5 to ω-TA18 enzymes, respectively) is collected and stored at -20℃ for later use.

[0111] Example 7: Construction of catalytic reaction systems from ω-TA5 to ω-TA18

[0112] Referring to Example 2, reaction systems for the production of 3-aminopropanol were constructed using ω-TA5 to ω-TA18 catalysts, respectively. L-alanine was used as the amino donor, maintaining a molar ratio of 5:1 between the amino donor (i.e., L-alanine) and the reaction substrate (i.e., 3-hydroxypropanal). Whole cells of *E. coli* collected in Example 6 were placed in 2 mL centrifuge tubes, and the conversion reaction system was configured according to the table below. The centrifuge tubes were placed in a shaker at 220 rpm and 37°C for 24 h for substrate transamination.

[0113] Table 4 Enzyme Conversion Reaction System

[0114] Example 8: Yield analysis and optimal pH analysis of 3-aminopropanol production from ω-TA5 to ω-TA18

[0115] In this embodiment, we aim to determine the activity or stability of ω-TA5 to ω-TA18.

[0116] First, the activities of ω-TA5 to ω-TA18 in the catalytic system of Example 7 were tested, and their respective 3-aminopropanol yields are shown in Figure 5A. As can be seen from the figure, TA12 and TA13 both have high catalytic activity, and the 3-aminopropanol yield can reach above 0.7 g / L. Among them, TA12 catalyzes the production of 3-aminopropanol with a yield of 0.7 g / L and a 3-aminopropanol yield of 71.11%. TA13 has the highest activity, with a 3-aminopropanol yield of 0.96 g / L and a yield of 94.81%.

[0117] Therefore, the ω-TA2, ω-TA12, and ω-TA13 described in this invention all exhibit high catalytic activity towards the substrate 3-hydroxypropanal, and the yield of the product 3-aminopropanol reaches over 70%, thus enabling the efficient catalytic synthesis of 3-aminopropanol from the substrate 3-hydroxypropanal via a bioenzymatic method.

[0118] Furthermore, the applicant verified the stability of ω-TA5 to ω-TA18 at different pH values. A catalytic system containing ω-TA enzymes was constructed using Example 7, and a pH gradient from neutral to slightly alkaline (from pH 7.0 to pH 10.0, adjusted with PBS buffer and Tris-HCl buffer, respectively) was established within this system. The content of 3-aminopropanol obtained by ω-TA5 to ω-TA18 at different pH values ​​was detected by HPLC. The final results are shown in Figure 5B. TA13 exhibits certain pH stability, maintaining 90% yield (0.87 g / L) at pH 9, which is higher than the highest yields of ω-TA12 and ω-TA15.

[0119] In summary, this invention first pre-screened four ω-TA enzymes (ω-TA1 to ω-TA4) through bioinformatics analysis, and verified through wet experiments that the ω-TA2 enzyme can efficiently catalyze the production of 3-aminopropanol from the specific substrate 3-hydroxypropanal. Furthermore, based on information from the ω-TA2 enzyme and combined with bioinformatics analysis, the applicant screened ω-TA5 to ω-TA18, which may have high catalytic activity for the substrate 3-hydroxypropanal. Wet experiments further verified the high catalytic activity of ω-TA12 and ω-TA13, with ω-TA13 exhibiting even higher enzyme activity and stability. At pH 7.5, the yield of 3-aminopropanol reached 0.96 g / L (94.81%), and at pH 9, it still maintained a yield of 0.87 g / L (85.93%).

[0120] sequence:

[0121] SEQ ID NO.1: Protein sequence (ω-TA1) with accession number WP_011135573.1

[0122] SEQ ID NO.2: Protein sequence (ω-TA2) with accession number 5G09_A

[0123] SEQ ID NO.3: Protein sequence (ω-TA3) with accession number WP_011750218.1

[0124] SEQ ID NO.4: Protein sequence (ω-TA4) with accession number WP_003084297.1

[0125] SEQ ID NO.5: Protein sequence (ω-TA5) with accession number B0KJV9.

[0126] SEQ ID NO.6: Protein sequence (ω-TA6) with accession number B1JX81

[0127] SEQ ID NO.7: Protein sequence (ω-TA7) with accession number Q9JXW0

[0128] SEQ ID NO.8: Protein sequence (ω-TA8) with accession number Q8D3C8

[0129] SEQ ID NO.9: Protein sequence (ω-TA9) with accession number A4J6H0.

[0130] SEQ ID NO.10: Protein sequence (ω-TA10) with accession number A5ITJ2.

[0131] SEQ ID NO.11: Protein sequence (ω-TA11) with accession number Q4H4F5.

[0132] SEQ ID NO.12: Protein sequence (ω-TA12) with accession number WP_076629700.1

[0133] SEQ ID NO.13: Protein sequence (ω-TA13) with accession number WP_127113105.1

[0134] SEQ ID NO.14: Protein sequence (ω-TA14) with accession number O66998

[0135] SEQ ID NO.15: Protein sequence (ω-TA15) with accession number O66442.

[0136] SEQ ID NO.16: Protein sequence (ω-TA16) with accession number C1DUY4

[0137] SEQ ID NO.17: Protein sequence (ω-TA17) with accession number Q5SJS4

[0138] SEQ ID NO.18: Protein sequence (ω-TA18) with accession number Q58131

[0139] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing 3-aminopropanol by a bioenzymatic process, comprising using 3-hydroxypropanal as a substrate and ω-transaminase as a catalyst to catalyze the transamination reaction of the substrate 3-hydroxypropanal to obtain 3-aminopropanol.

2. The method according to claim 1, wherein, The sequence of the ω-transaminase is shown in any one of SEQ ID NO:2, 12 or 13.

3. The method according to claim 1, wherein the ω-transaminase is artificially synthesized or obtained through expression in host cells; Optionally, the host cell includes Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

4. The method according to claim 1 or 2, wherein the ω-transaminase can synthesize 3-aminopropanol via in vitro catalysis or intracellular catalysis; Optionally, the ω-transaminase further includes a signal peptide; optionally, the signal peptide includes, but is not limited to, signal peptides used for purification, localization, and solubilization.

5. The method of claim 1 or 2, wherein, The pH value in the catalytic transamination reaction is 6.5-9.0, preferably 7.

5.

6. The method of claim 1, further comprising using L-alanine as an amino donor; Preferably, it further includes the addition of a coenzyme; more preferably, the coenzyme is pyridoxal 5′-phosphate.

7. The method according to claim 6, wherein, The molar ratio of the amino donor to the substrate is 4-6:1, preferably 5:

1.

8. The method of claim 1, wherein, In the catalytic transamination reaction, the reaction temperature is 35-40℃, preferably 37℃.

9. The method of claim 1, wherein, In the catalytic transamination reaction, the reaction time is 20-24 hours, preferably 22 hours.

10. Use of ω-transaminase in the preparation of 3-aminopropanol by a bioenzymatic method, wherein 3-hydroxypropanal is used as a substrate; preferably, the sequence of the ω-transaminase is as shown in any one of SEQ ID NO:2, 12 or 13.

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