A method for asymmetric catalytic synthesis of antibiotic precursor D-HPG by double enzyme coupling

Abstract

The application provides a method for asymmetrically catalyzing synthesis of antibiotic precursor D-HPG by double enzyme coupling, and belongs to the technical field of biochemistry. The double enzyme comprises a D-hydantoinase mutant and an N-carbamoyl-D-amino acid amide hydrolase mutant, the amino acid sequence of the D-hydantoinase mutant is obtained by mutation of SEQ ID NO. 2, and the N-carbamoyl-D-amino acid amide hydrolase mutant is obtained by mutation of SEQ ID NO. 6. The application can realize efficient, simple and double enzyme coupling asymmetric catalytic synthesis of D-HPG without relying on expensive cofactors.

Description

Technical Field

[0001] This application relates to a method for the asymmetric catalytic synthesis of the antibiotic precursor D-HPG via dual-enzyme coupling, belonging to the field of biochemistry technology. Background Technology

[0002] D-4-hydroxyphenylglycine (D-HPG) is an important non-natural chiral amino acid. As a key side-chain precursor for semi-synthetic β-lactam antibiotics (such as amoxicillin, cefadroxil, and apucillin), its demand continues to rise with the growth of the antibiotic industry. In addition, D-HPG is widely used in peptide drugs, pesticides, and chiral resolution reagents. Since only the D-enantiomer possesses pharmacological activity, the green and efficient preparation of high-optical-purity (>99% ee) D-HPG has become a research hotspot in industrial biocatalysis.

[0003] Currently, the synthesis methods for D-HPG are mainly divided into chemical methods and bio-enzymatic methods. Chemical methods include routes such as the condensation of glyoxylic acid and phenol, chiral resolution, and asymmetric hydrogenation. Among these, the glyoxylic acid method is a mature process but produces a large amount of racemic mixtures, requiring chiral resolution, with a theoretical yield of less than 50%, and uses toxic solvents, resulting in significant waste. While asymmetric hydrogenation can directly generate chiral centers, it requires precious metal catalysts (such as rhodium and ruthenium) and chiral phosphine ligands, leading to high costs and strict requirements on substrate purity, making it unsuitable for large-scale fermentation coupling. Bio-enzymatic methods have attracted considerable attention due to their mild conditions and high stereoselectivity. The reported enzymatic routes mainly include: ① Hydantoin method: using DL-p-hydroxyphenylhydantoin as a substrate, D-HPG is generated through two-step catalysis by hydantoin and carbamoyl hydrolase. However, this route involves chemical synthesis precursors and has a long process. ② Transaminase method: using D-amino acid transaminase or ω-transaminase to catalyze the asymmetric amination of p-hydroxyphenylpyruvate. However, this enzyme has low affinity for the substrate and requires the coenzyme pyridoxal phosphate (PLP) and an amino donor, resulting in significant product inhibition. ③ Dehydrogenase method: using D-amino acid dehydrogenase coupled with coenzyme regeneration. However, the natural enzyme has poor activity and requires expensive NAD(P)H.

[0004] The methods described above generally suffer from drawbacks such as cumbersome steps, coenzyme dependence, low yield, or insufficient optical purity. In recent years, dual-enzyme coupling strategies, by synergistically combining the functions of two enzymes, can achieve in-situ substrate transformation, coenzyme cycling, or thermodynamic equilibrium-driven synthesis, significantly improving synthesis efficiency. However, there are few reports on dual-enzyme coupling systems for D-HPG synthesis, and there is a lack of enzyme molecule modification and system optimization specifically for this substrate. Summary of the Invention

[0005] In view of this, this application develops a highly efficient, simple, and cost-free dual-enzyme system that does not rely on expensive cofactors. By constructing a dual-enzyme cascade reaction system of D-hydantoinase (DHase) and N-carbamoyl-D-amino acid amide hydrolase (D-carbamoylase, DCase), D-HPG is synthesized asymmetrically. This addresses the problems of cumbersome steps, low optical purity, and serious pollution in existing chemical synthesis methods, as well as the low conversion rate and accumulation of intermediate products in single-enzyme methods. This enables the efficient and green synthesis of high-optical-purity D-HPG from the inexpensive substrate DL-p-hydroxyphenylhydantoin.

[0006] Specifically, this application is implemented through the following scheme: The first objective of this application is to provide a D-hydantoin mutant, wherein the amino acid sequence of the D-hydantoin mutant is obtained by mutation of SEQ ID NO.2.

[0007] Preferred: The specific setting of the mutation in SEQ ID NO.2 is: replacing tyrosine at position 120 with serine and replacing lysine at position 150 with cysteine.

[0008] The second objective of this application is to provide a gene or recombinant expression plasmid encoded by the amino acid sequence of the above-mentioned D-hyinase mutant, preferably: the vector of the expression plasmid is pET28a or pET32a, preferably pET28a.

[0009] A third objective of this application is to provide a recombinant genetically engineered bacterium containing the aforementioned gene or recombinant expression plasmid. Preferably, the host bacterium of the genetically engineered bacterium is... E. coli BL21 (DE3).

[0010] The recombinant genetically engineered bacteria were fermented to obtain wet cells: the engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured, and then inoculated into fresh LB liquid medium containing kanamycin resistance. The culture was carried out until the bacterial OD600 reached 0.6~0.8. After IPTG was added for induction culture, the culture was centrifuged, the supernatant was discarded, and the wet cells were collected to obtain the fermentation product of the recombinant genetically engineered bacteria corresponding to the D-hydantoin mutant.

[0011] More preferably: the engineered bacteria containing the D-hydantoin mutant encoding gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 9 h. Then, the bacteria are inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C and 150 rpm until the bacterial OD600 reaches 0.6~0.8. Isopropyl β-D-thiogalactoside (IPTG) is added to a final concentration of 0.1~0.5 mM, and the bacteria are induced to culture at 20~28°C for 12~16 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant is discarded, and the wet bacterial cells are collected to obtain the fermentation product of the recombinant genetically engineered bacteria corresponding to the D-hydantoin mutant.

[0012] The fourth objective of this application is to provide an immobilized enzyme obtained from the above-mentioned D-hydantoin mutant: using an epoxy resin as a carrier, an enzyme solution of the D-hydantoin mutant is prepared, the enzyme solution is mixed with the carrier in a certain proportion, and then the mixture is shaken and fixed at 25~30℃ and pH 7.0~8.0 for 12~18 hours. After the fixation is completed, the mixture is washed to obtain the immobilized D-hydantoin mutant, also known as an immobilized enzyme.

[0013] Preferred: The carrier was washed with deionized water before mixing and then filtered until dry.

[0014] The mixing ratio of the carrier to the enzyme solution is: 1 g of resin carrier corresponds to 100~160 U of enzyme solution.

[0015] The washing agent is phosphate buffer, which washes away unbound free enzyme proteins.

[0016] The fifth aspect of this application is to provide an N-carbamoyl-D-amino acid amide hydrolase mutant, wherein the amino acid sequence of the N-carbamoyl-D-amino acid amide hydrolase mutant is obtained by mutation of SEQ ID NO.6.

[0017] Preferably, the mutation is set such that phenylalanine at position 82 is replaced with alanine, and lysine at position 124 is replaced with glycine.

[0018] The sixth aspect of this application is to provide a gene or recombinant expression plasmid encoded by the amino acid sequence of the above-mentioned N-carbamoyl-D-amino acid amide hydrolase mutant, preferably: the vector of the expression plasmid is pET28a or pET32a, preferably pET28a.

[0019] The seventh objective of this application is to provide a recombinant genetically engineered bacterium containing the aforementioned gene or recombinant expression plasmid.

[0020] Preferred: The host bacteria of the genetically engineered bacteria are E. coli BL21 (DE3).

[0021] The recombinant genetically engineered bacteria were fermented to obtain wet cells: the engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance and cultured, and then inoculated into fresh LB liquid medium containing kanamycin resistance. The culture was carried out until the bacterial OD600 reached 0.6~0.8. After IPTG induction culture, the bacteria were centrifuged, the supernatant was discarded, and the wet cells were collected to obtain the fermentation product of the recombinant genetically engineered bacteria corresponding to the N-carbamoyl-D-amino acid amide hydrolase mutant.

[0022] More preferably: the engineered bacteria containing the gene encoding the N-carbamoyl-D-amino acid amide hydrolase mutant are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 9 h. Then, the bacteria are inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C and 150 rpm until the bacterial OD600 reaches 0.6~0.8. IPTG is added to a final concentration of 0.1~0.5 mM, and the bacteria are induced to grow at 20~28°C for 12~16 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant is discarded, and the wet bacterial cells are collected, which is the fermentation product of the recombinant genetically engineered bacteria corresponding to the N-carbamoyl-D-amino acid amide hydrolase mutant.

[0023] The recombinant genetically engineered bacteria of the above-mentioned D-hydantoin mutant and N-carbamoyl-D-amino acid amide hydrolase mutant can also be expressed as cell lysate of wet bacterial cells, or as pure enzyme solution separated from wet bacterial cells after cell lysate (i.e. crude enzyme solution) and protein purification.

[0024] The above-mentioned recombinant genetically engineered bacteria can express, alone or co-express, recombinant expression plasmids / genes of D-hydantoinase mutant and N-carbamoyl-D-amino acid amide hydrolase mutant: DHase and DCase genes are cloned into the pET28a vector, respectively, and transformed into... E. coli BL21(DE3) can be used to obtain two recombinant genetically engineered bacteria that express two enzymes separately; or recombinant genetically engineered bacteria that co-express two enzymes through a bicistronic vector.

[0025] The eighth objective of this application is to provide an immobilized enzyme obtained from the above-mentioned N-carbamoyl-D-amino acid amide hydrolase mutant: using an epoxy resin as a carrier, an enzyme solution of the N-carbamoyl-D-amino acid amide hydrolase mutant is prepared, the enzyme solution is mixed with the carrier in a certain proportion, and then the mixture is shaken and fixed at 25~30℃ and pH 7.0~8.0 for 12~18 hours. After the fixation is completed, the enzyme is washed to obtain the immobilized N-carbamoyl-D-amino acid amide hydrolase mutant, also known as an immobilized enzyme.

[0026] Preferred: The carrier was washed with deionized water before mixing and then filtered until dry.

[0027] The mixing ratio of the carrier to the enzyme solution is: 1 g of resin carrier corresponds to 100~160 U of enzyme solution.

[0028] The washing agent is phosphate buffer, which washes away unbound free enzyme proteins.

[0029] The ninth aspect of this application is to provide a method for the asymmetric catalytic synthesis of the antibiotic precursor D-HPG using a dual-enzyme coupling, wherein the dual enzymes comprise a D-hydantoin mutant and an N-carbamoyl-D-amino acid amide hydrolase mutant, wherein the amino acid sequence of the D-hydantoin mutant is obtained by mutation of SEQ ID NO.2, and the N-carbamoyl-D-amino acid amide hydrolase mutant is obtained by mutation of SEQ ID NO.6.

[0030] Furthermore, as a preferred option: The above-mentioned catalytic synthesis process is as follows: DL-p-hydroxyphenylhydantoin is used as the reaction substrate, with an initial concentration of 20-300 mM; D-hydantoin mutant, fermentation product (wet cell) of engineered bacteria containing its encoding gene, or its extracted free enzyme is used as the first catalyst; N-carbamoyl-D-amino acid amide hydrolase mutant, fermentation product (wet cell) of engineered bacteria containing its encoding gene, or its extracted free enzyme is used as the second catalyst; the total amount of the first and second catalysts added is 1-5 g DCW / L, based on the enzyme activity of the D-hydantoin mutant; the catalytic synthesis temperature is 20-50℃, and the pH is 7.0-9.0.

[0031] In the catalytic synthesis process, the ratio of D-hydantoin mutant to N-carbamoyl-D-amino acid amide hydrolase mutant bacterial cells is 1:0.5~1:6 (mass ratio).

[0032] The catalytic synthesis process is carried out under stirring, with a stirring speed of 100~1000 rpm, preferably 200~400 rpm.

[0033] The catalytic synthesis is carried out in a Tris-HCl buffer or a potassium phosphate buffer (pH 7.0-9.0). Preferably, the amount of reaction medium added is 50-100 mM.

[0034] The catalytic synthesis is carried out at a temperature of 30-50°C and a pH of 8.0-8.5.

[0035] The reaction time for the catalytic synthesis is 4 to 24 hours.

[0036] The amount of DHase added is 0.5~5 g DCW / L, and the amount of DCase added is 0.5~5 g DCW / L.

[0037] The DHase is activated with metal ions to obtain a first catalyst, preferably Mn. 2+ The addition amount is 0.05~1 mM, preferably 0.1~1 mM.

[0038] The initial concentration of the reaction substrate is 10~300 mM.

[0039] As a specific preferred example: In the catalytic synthesis process, the initial concentration of the reaction substrate is 100 mM, the enzyme activity ratio (dry weight ratio) of the D-hydantoinase mutant and the N-carbamoyl-D-amino acid amide hydrolase mutant is 1:4, and the reaction temperature is 30~40℃. Under these conditions, the substrate conversion rate is ≥98%, and the product... e.e. Value > 99%.

[0040] After the above catalytic synthesis reaction was completed, the insoluble matter was removed by centrifugation, the pH of the supernatant was adjusted to the isoelectric point of D-HPG (about 5.5), and the mixture was allowed to stand at 4°C to crystallize. After filtration, washing and drying, a white crystalline powder of D-p-hydroxyphenylglycine was obtained.

[0041] In the above-described catalytic synthesis process, the first and second catalysts can also participate in the catalytic synthesis in the form of immobilized enzymes. D-hydantoin mutants and N-carbamoyl-D-amino acid amide hydrolase mutants, participating in the catalytic synthesis as immobilized enzymes, maintained a relative enzyme activity of over 85% of the initial enzyme activity after 10 repeated batches.

[0042] In the above synthesis process, in addition to the immobilized enzyme form, the two enzymes can also be added to the reaction system in the form of whole cells (recombinant bacterial wet cells or frozen stem cells) or crude enzyme solution. When whole-cell catalysis is used, the amount of catalyst used is 5~20 gDCW / L based on the dry weight of the bacterial cells.

[0043] In the above synthesis process, the first catalyst catalyzes the substrate DL-p-hydroxybenzylhydantoin to generate the intermediate N-carbamoyl-D-p-hydroxyphenylglycine (this stage of the reaction takes approximately 4-6 hours), and the second catalyst further catalyzes the hydrolysis of the intermediate to generate D-p-hydroxyphenylglycine (this stage of the reaction takes approximately 6-8 hours). This two-step synergistic catalytic asymmetric synthesis of D-p-hydroxyphenylglycine achieves a highly efficient synthesis from the inexpensive precursor substrate DL-hydroxybenzylhydantoin to high optical purity D-p-hydroxyphenylglycine by constructing a two-enzyme cascade reaction pathway.

[0044] Compared with the prior art, the present invention has the following beneficial effects: (1) High stereoselectivity: Both D-hydantoinase and DCase strictly recognize D-type substrates, and the final product e.e. Value > 99%, no chiral splitting required.

[0045] (2) High conversion efficiency: dual enzyme synergistic catalysis, timely elimination of the accumulation of intermediate product (N-carbamoyl-D-HPG), relief of product inhibition, conversion rate of 100 mM reaction substrate ≥98% within 12 hours.

[0046] (3) Low cost: DL-p-hydroxyphenylhydantoin is used as the starting material, and its cost is only about RMB 50 / kg. It does not require chemical resolution or asymmetric catalysis, and the theoretical yield is 100%, which is much higher than the 50% of the chemical resolution method.

[0047] (4) Simple operation: The synthesis process of this application can be carried out by one-pot reaction, that is, two enzymes are added at the same time, or the enzyme addition method can be used: first add DHase to react, and after the intermediate N-carbamoyl-D-HPG accumulates to the maximum, DCase is added to continue the reaction; no external coenzyme or expensive metal catalyst is required, and ordinary stirred reactor can be used for large-scale production.

[0048] (5) Green and environmentally friendly: The reaction is carried out in reaction media such as Tris-HCl buffer or potassium phosphate buffer, which is an aqueous phase reaction. The by-products are only CO2 and NH3, and there is no emission of toxic organic solvents, which meets the requirements of green chemistry.

[0049] (6) Good enzyme stability: The DHase and DCase used in this invention have a half-life of more than 48 hours at 40°C. After immobilization, they can be reused more than 5 times with an activity of more than 80%. In particular, when immobilized enzymes are used in the reaction, after 10 batches of repeated use, the relative enzyme activity is still maintained at more than 85% of the initial enzyme activity. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0051] Figure 1 This is a schematic diagram illustrating the reaction principle of the dual-enzyme coupled asymmetric catalytic synthesis proposed in this application.

[0052] Figure 2 SDS-PAGE characterization of D-hydantoinase and N-carbamoyl-D-amino acid amide hydrolase and their mutants, M: marker, H1: Gs DHase, H2: Gs DHase-Y120S, H3: Gs DHase-Y120T, H4: Gs DHase-Y120C, H5: Gs DHase-Y120N, H6: Gs DHase-Y120E, H7: Gs DHase-K150S, H8: Gs DHase-K150T, H9: Gs DHase-K150C; H10: Gs DHase-K150N, H11: Gs DHase-K150E, H12: Gs DHase-Y120S / K150C; C1: As DCase, C2: As DCase-F82A, C3: As DCase-F82S, C4: As DCase-F82G, C5: As DCase-K124S, C6: As DCase-K124A, C7: As DCase-K124G, C8: As DCase-F82A / K124S, C9: As DCase-F82A / K124A, C10: As DCase--F82A / K124G.

[0053] Figure 3 D-hydantoinase Gs DHase and N-carbamoyl-D-amino acid amide hydrolase As Effect of different DHase ratios on the yield of D-p-hydroxyphenylglycine catalytic synthesis.

[0054] Figure 4 The effect of different pH conditions on the yield of D-p-hydroxyphenylglycine synthesized by two enzymes.

[0055] Figure 5 The effect of different reaction temperatures on the yield of D-p-hydroxyphenylglycine synthesized by dual enzyme catalysis.

[0056] Figure 6 The effect of different substrate concentrations of DL-p-hydroxyphenylhydantoin on the reaction progress of the two-enzyme reaction.

[0057] Figure 7 This is to assess the reusability of immobilized enzymes.

[0058] Figure 8 For different Mn 2+ Effects of concentration and stirring speed on the catalytic synthesis of D-p-hydroxyphenylglycine. Detailed Implementation

[0059] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0060] The mutants were obtained using site-directed mutagenesis, which was used to target D-hynanase. Gs Mutations were made in the DHase gene (SEQ ID NO.1) and the N-carbamoyl-D-amino acid amide hydrolase AsDHase gene (SEQ ID NO.3).

[0061] The obtained mutant plasmid was transformed into [the virus] using a 42°C heat shock method. E. coli BL21(DE3) competent cells were used to obtain recombinant plasmids, i.e. recombinant genetically engineered bacteria.

[0062] The recombinant genetically engineered bacteria were inoculated, transferred, induced, and the bacterial cells were recovered. The resulting resuspended bacterial solution was used as a catalyst to catalyze the synthesis of D-p-hydroxyphenylglycine from the substrate DL-p-hydroxyphenylhydantoin.

[0063] During inoculation, transfer, induction, and cell recovery, the culture medium can be any Escherichia coli culture medium used in the field to grow and express the target gene of this invention. In this embodiment, the specific composition of LB medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.0. There are no special restrictions on the culture method and conditions; the culture method and conditions can be appropriately selected based on factors such as host type and culture method.

[0064] Example 1

[0065] This embodiment describes the synthesis of D-hydantoin genetically engineered bacteria.

[0066] Through the NCBI database Corynebacterium sp D-hydantoin from source Cs DHase homologs were mined to obtain enzymes derived from... Geobacillus stearothermophilus SD-1, Agrobacterium tumefaciens , Lachnoanaerobaculum sp. , Corynebacterium sp. , Pseudolysinimonas sp. , Synergistaceae bacterium The D-hydantoinase genes have GenBank accession numbers MN073199.1 (amino acid sequence as shown in SEQ ID NO: 2), CAA62549.1, CAP7253333.1, CAQ9169314.1, CAR3587159.1, and CAR3233373.1 (where the reference sequence is shown in SEQ ID NO: 2). Gs The GenBank accession number for DHase is AVC04856.1. The above gene sequences were codon-optimized using *E. coli* as the host and synthesized by Qingke Biotechnology Co., Ltd. The cDNA fragments of each D-hydantoin were ligated to the pET-28a(+) site after TATACCAT (before the NcoI restriction site) and before CTCGAG (before the XhoI restriction site), respectively. The resulting plasmids were then transformed into... E. coli Recombinant strains were obtained from BL21(DE3). E. coli BL21(DE3) / pET28a(+)- Gs D-hydantoinase, E. coli BL21(DE3) / pET28a(+)- At D-hydantoinase, E. coli BL21(DE3) / pET28a(+)- Ls D-hydantoinase, E. coli BL21(DE3) / pET28a(+)- Cs D-hydantoinase,E. coli BL21(DE3) / pET28a(+)- Ps D-hydantoin and E. coli BL21(DE3) / pET28a(+)- Sb D-Hydinase.

[0067] The recombinant strains containing D-hydantoin and its mutants were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 10 h. Then, 1% (v / v) of the inoculum was added to fresh LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Next, 0.1 mM IPTG was added to the culture medium, and the culture was incubated at 28°C for 12 h. Finally, the culture was centrifuged at 4°C and 9000 xg for 10 min to obtain the corresponding wet bacterial cells. The D-hydantoin protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis results are shown below. Figure 2 Part A of the text.

[0068] Screening of bacterial cell catalytic performance: The catalytic ability of D-hydantoin enzyme in the obtained cells was tested by adding 100 mM DL-p-hydroxyphenylhydantoin as substrate. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the dry weight of D-hydantoin enzyme, and 0.05 mmol / L Mn was added. 2+ The conversion system was constructed using a pH 7.0, 50 mM Tris / HCl buffer as the reaction medium. The reaction was carried out at 30℃ and 800 rpm. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the amount of N-carbamoyl-D-HPG generated to calculate the product yield. The specific enzyme activity (U / mg) was calculated based on the protein concentration.

[0069] Liquid chromatography detection conditions: Samples were analyzed using a high-performance liquid chromatograph (HPLC) with μ-BONDAPAK. TM A C18 (280 × 4.6 mm) column with a UV detector at 254 nm was used. The mobile phase was 0.05 mol / L acetate-sodium acetate buffer (pH 4.2):methanol = 90:10; column temperature: 30℃; flow rate: 1.0 mL / min; injection volume: 10 μL. The product concentration was calculated based on the standard curve of D-p-hydroxyphenylglycine (D-HPG), and then the specific enzyme activity and yield were calculated.

[0070] Table 1: D-hynanase from different sources Gs DHase screening .

[0071] D-hydantoin from different sourcesGs The screening results of DHase are shown in Table 1: (Source: ...) Geobacillus stearothermophilus SD-1 D-hydantoin ( Gs D-hydantoinase had the highest specific enzyme activity, reaching 217.04 U / mg, and also the highest yield, at 81.23%, significantly superior to D-hydantoinase from other sources. At D-hydantoinase, Ls D-hydantoinase, Cs D-hydantoinase, Ps D-hydantoin and Sb D-hynanase). Therefore, selection Gs D-hydantoin is used as the optimal enzyme source for subsequent enzyme molecule modification and the synthesis of β-lactam antibiotic precursors.

[0072] Example 2

[0073] This embodiment describes the synthesis of N-carbamoyl-D-amino acid amide hydrolase (DCase) genetically engineered bacteria.

[0074] N-carbamoyl-D-amino acid amide hydrolase was analyzed using the NCBI database. As DCase homologs were mined to obtain enzymes derived from... Agrobacterium sp. (Also expressed as) Agrobacterium sp. strain KNK712 (GenBank accession number BAD00007.1, amino acid sequence see SEQ ID NO.6) Pseudomonas putida , Klebsiella grimontii , Pseudomonas chlororaphis , Aminobacter mesojensis The N-carbamoyl-D-amino acid amide hydrolase genes, with GenBank accession numbers AB007368.1, WP183853726.1, WP018183126.1, WP307237451.1, and WP184148164.1, were obtained. Codon optimization of each gene sequence was performed using *E. coli* as the host, and the sequences were synthesized by Qingke Biotechnology Co., Ltd. The cDNA fragments of each AsDHase were ligated to the pET-28a(+) site after TATACCAT (before the NcoI restriction site) and before CTCGAG (before the XhoI restriction site), respectively. The resulting plasmids were then transformed into... E. coli Recombinant strains were obtained from BL21(DE3). E. coli BL21(DE3) / pET28a(+)- As DCase E. coli BL21(DE3) / pET28a(+)- Pp DCase E. coliBL21(DE3) / pET28a(+)- Kg DCase E. coli BL21(DE3) / pET28a(+)- Pc DCase and E. coli BL21(DE3) / pET28a(+)- Am DCase.

[0075] The recombinant strains containing N-carbamoyl-D-amino acid amide hydrolase were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 10 h. Then, they were inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Next, 0.1 mM IPTG was added to the culture medium, and the culture was incubated at 28°C for 12 h. Finally, the culture was centrifuged at 4°C and 9000 xg for 10 min to obtain the corresponding wet bacterial cells. The N-carbamoyl-D-amino acid amide hydrolase protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis results are shown below. Figure 2 Part B of the document.

[0076] Screening of bacterial cell catalytic performance: The catalytic ability of N-carbamoyl-D-amino acid amide hydrolase in the obtained cells was tested by adding 100 mM of N-carbamoyl-D-p-hydroxyphenylglycine (N-carbamoyl-D-HPG) as substrate. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on enzyme dry weight, and 0.05 mmol / L of Mn was added. 2+ The conversion system was constructed using a pH 7.0, 50 mM Tris / HCl buffer as the reaction medium. The reaction was carried out at 30°C and 800 rpm. After the reaction was terminated, the reaction solution was subjected to liquid chromatography (the detection conditions were the same as in Example 1) to detect the amount of D-p-hydroxyphenylglycine (D-HPG) produced to calculate the product yield. The product concentration was calculated based on the standard curve of D-p-hydroxyphenylglycine (D-HPG), and the specific enzyme activity (U / mg) and yield were calculated based on the protein concentration.

[0077] Table 2: N-carbamoyl-D-amino acid amide hydrolases from different sources As DCase screening .

[0078] N-carbamoyl-D-amino acid amide hydrolases from different sources As The screening results of DCase are shown in Table 2: (Source: [Original Source]) Agrobacterium sp. N-carbamoyl-D-amino acid amide hydrolase (As DCase exhibited the highest specific enzyme activity (31.18 U / mg) and the highest yield (58.72%), significantly outperforming other N-carbamoyl-D-amino acid amide hydrolases (DCases). Pp DCase Kg DCase Pc DCase and Am DCase). Therefore, choose As DCase serves as the optimal enzyme source for subsequent enzyme molecule modification and the synthesis of β-lactam antibiotic precursors.

[0079] Example 3

[0080] This embodiment describes the construction of libraries containing D-hydantoinase and N-carbamoyl-D-amino acid amide hydrolase mutants.

[0081] Based on the strategy of increasing the polarity of the enzyme substrate pocket, the preparation of D-hynanase mutant libraries was achieved through site-directed mutagenesis. Primer design is shown in Table 3. E. coli BL21(DE3) / pET28a(+)- Gs DHase vector pET28a(+)- Gs Using DHase as a template, D-hydantoinase was introduced via site-directed mutagenesis PCR. Cs The 120th tyrosine residue of the DHase amino acid sequence was mutated to serine, threonine, cysteine, asparagine, or glutamine, respectively, and transformed into... E.coli BL21DE3 competent cells were used to obtain monoclonal mutants. The mutant with lysine at position 150 was obtained by PCR using the same method with the 150-position mutant primers listed in Table 3.

[0082] N-carbamoyl-D-amino acid amide hydrolase was subjected to site-directed mutagenesis to improve its catalytic efficiency, based on a substrate pocket enlargement strategy. Using F82 as the mutation primer for amino acid position 82 in Table 4, site-directed mutagenesis PCR was performed to modify the N-carbamoyl-D-amino acid amide hydrolase. As The phenylalanine at position 82 of the DCase amino acid sequence was mutated to alanine, serine, and glycine, and the resulting monoclonal mutant was obtained after transformation. The lysine mutant at position 124 was obtained by PCR using the same method with the 124 mutant primers listed in Table 4.

[0083] PCR reaction system (50 µL): 2 µL forward primer (10 μM), 2 µL reverse primer (10 μM), 25 µL 2×Phanta buffer, 1 µL dNTP mixture (10 mM each), 1 µL plasmid template, 1 µL DNA polymerase, and 18 µL ultrapure water. The PCR program set according to the Phanta Super-Fidelity DNA polymerase instructions was as follows: 95℃ pre-denaturation for 5 min, followed by 29 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 5 min), final extension at 72℃ for 10 min, and incubation at 16℃.

[0084] The obtained recombinant plasmid was transformed into [a specific plasmid] via a 42℃ heat shock method. E. coli BL21(DE3) competent cells were evenly spread on LB agar plates containing 50 µg / µL kanamycin and incubated at 37°C for 14 h. Single colonies were picked and transferred to 1 mL of LB liquid medium containing 50 µg / mL kanamycin. After incubation in 100 mL shake flasks at 37°C and 180 rpm for 10 h, the cells were induced, incubated at 28°C for 12 h, and then centrifuged to collect the wet cells.

[0085] The crude enzyme solution was prepared as follows: the wet bacterial cells were resuspended in a 50 mM phosphate buffer at pH 7.5 at a concentration of 100 g / L, and ultrasonically disrupted for 6 min on an ice-water mixture. The ultrasonic disruption conditions were: power of 400 W, disruption for 1 s, pause for 2 s, and the disrupted mixture was collected to obtain the crude enzyme solution.

[0086] The above crude enzyme solution was purified by ammonium sulfate fractionation: 100 mL of culture supernatant was placed in an ice bath, and 50% saturated ammonium sulfate solid powder was added while stirring until the ammonium sulfate was completely precipitated. After that, the mixture was placed in an ice bath for 1 h, and then centrifuged at 12000 rpm and 4℃ for 10 min to obtain the crude enzyme as the primary precipitate.

[0087] The N-carbamoyl-D-HPG was synthesized using the catalytic system in Example 1 (i.e., the "screening of bacterial catalytic performance" section in Example 1), and the catalytic synthesis yield of the D-hydantoin mutant was determined.

[0088] D-Hydantoinase GsSpecific enzyme activity assay of DHase mutant: 100 mM DL-p-hydroxyphenylhydantoin was added as the initial substrate. The mutant enzyme catalyst was catalyzed with purified enzyme at a final concentration of 1 mg / mL. The conversion system was constructed using a pH 7.0, 50 mM Tris / HCl buffer as the reaction medium. The reaction was carried out at 30℃ and 800 rpm for 1 h. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the amount of N-carbamoyl-D-HPG generated to calculate the specific enzyme activity of the mutant.

[0089] N-Carbamoyl-D-amino acid amide hydrolase As DCase specific enzyme activity assay: 100 mM of substrate N-carbamoyl-D-HPG was added. The mutant enzyme catalyst was catalyzed with purified enzyme at a final concentration of 1 mg / mL. The conversion system was constructed using a pH 7.0, 50 mM Tris / HCl buffer as the reaction medium. The reaction was carried out at 30℃ and 800 rpm for 1 h. After the reaction was terminated, the liquid phase of the reaction solution was analyzed to detect the amount of D-p-hydroxyphenylglycine produced to calculate the specific enzyme activity of the mutant.

[0090] Table 3: Primer design for site-directed mutagenesis of D-hydantoinase GsDHase .

[0091] Table 4: Primer Design for Site-Directed Mutagenesis of N-Carbamoyl-D-Amino Acid Amide Hydrolase AsDCase .

[0092] D-Hydantoinase Gs DHase activity unit (U) is defined as the amount of enzyme required to generate 1 micromole of N-carbamoyl-D-HPG per minute at 30°C and pH 7.0. Specific activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0093] N-Carbamoyl-D-amino acid amide hydrolase As DCase enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 micromole of D-p-hydroxyphenylglycine per minute at 30°C and pH 7.0. Specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0094] Protein concentration was determined using a diquinoline carboxylic acid protein assay kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing).

[0095] For D-hydantoinase Gs After screening, nine single mutants with increased enzyme activity were obtained from DHase, and the results are shown in Table 5.

[0096] Table 5: Gs Catalytic performance and specific enzyme activity of DHase and its single mutants .

[0097] The strains with increased enzyme activity are E. coli BL21(DE3) / pET28a(+)- Gs DHase-Y120S E. coli BL21(DE3) / pET28a(+)- Gs DHase-Y120C E. coli BL21(DE3) / pET28a(+)- Gs DHase-Y120N E. coli BL21(DE3) / pET28a(+)- Gs DHase-Y120E, BL21(DE3) / pET28a(+)-GsDHase-K150S, BL21(DE3) / pET28a(+)- Gs DHase-K150C, BL21(DE3) / pET28a(+)-GsDHase-K150N and BL21(DE3) / pET28a(+)- Gs DHase-K150E.

[0098] The catalytic synthesis product is N-carbamoyl-D-HPG. Gs The specific enzyme activity of DHase-Y120S is 741.76 U / mg; Gs The specific enzyme activity of DHase-Y120C is 276.62 U / mg; Gs The specific enzyme activity of DHase-Y120N is 262.36 U / mg; Gs The specific enzyme activity of DHase-Y120E is 301.72 U / mg; Gs The specific enzyme activity of DHase-K150S is 299.87 U / mg; Gs The specific enzyme activity of DHase-K150T is 287.75 U / mg; Gs The specific enzyme activity of DHase-K150C is 318.77 U / mg; Gs The specific enzyme activity of DHase-K150N is 279.14 U / mg; Gs The specific enzyme activity of DHase-K150E is 285.37 U / mg.

[0099] For N-carbamoyl-D-amino acid amide hydrolases AsAfter screening, five single mutants with increased enzyme activity were obtained from DCase, and the results are shown in Table 6.

[0100] Table 6: As Catalytic properties and specific enzyme activity of DCase and its single mutants .

[0101] The strains with increased enzyme activity are E. coli BL21(DE3) / pET28a(+)- As DCase-F82A E. coli BL21(DE3) / pET28a(+)- As DCase-F82S, E. coli BL21(DE3) / pET28a(+)- As DCase-F82G, E. coli BL21(DE3) / pET28a(+)- As DCase-K124A E. coli BL21(DE3) / pET28a(+)- As DCase-K124S and E. coli BL21(DE3) / pET28a(+)- As DCase-K124G.

[0102] The catalytic synthesis product is D-p-hydroxyphenylglycine. As The specific enzyme activity of DCase-F82A is 254.57 U / mg; As The specific enzyme activity of DCase-F82S is 78.35 U / mg; As The specific enzyme activity of DCase-F82G is 120.78 U / mg; As The specific enzyme activity of DCase-K124A is 58.48 U / mg; As The specific enzyme activity of DCase-K124S is 23.86 U / mg; As The specific enzyme activity of DCase-K124G is 76.88 U / mg.

[0103] Example 4

[0104] This embodiment describes the construction and screening of a combined mutant library of D-hydantoinase and N-carbamoyl-D-amino acid amide hydrolase mutants.

[0105] D-Hydantoinase Gs The preparation of DHase double mutant combined mutant libraries was achieved through site-directed saturation mutagenesis. Primer design is shown in Table 3. E. coliBL21(DE3) / pET28a(+)- Gs The vector pET28a(+)- in DHaseY120S Gs Using DHaseY120S as a template, the five pairs of primers for K150 in Table 1 were combined for mutation. The PCR system and procedure were the same as in Example 2.

[0106] Table 7: Gs Catalytic performance and specific enzyme activity of DHase and its combined mutants .

[0107] D-hynanase combination mutants were obtained through screening: Gs DHaseY120S / K150S Gs DHaseY120S / K150T, Gs DHaseY120S / K150C (nucleotide sequence see SEQ ID NO.3, amino acid sequence see SEQ ID NO.4) Gs DHaseY120S / K150N and Gs DHaseY120S / K150E were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. They were then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a 1% (v / v) inoculation rate and cultured at 37°C and 180 rpm for 2 h. Then, 0.1 mMIPTG was added to the culture medium and cultured at 28°C for 12 h. Finally, the culture was centrifuged at 4°C and 8000 xg for 10 min to obtain the corresponding wet bacterial cells.

[0108] Using the enzyme catalytic system in Example 1 (i.e., the "screening of cell catalytic performance" section in Example 1), the yield and specific activity of N-carbamoyl-D-HPG synthesized by various D-hydantoin mutants were determined, and the results are shown in Table 7: When the product synthesized by the mutant was N-carbamoyl-D-HPG, the mutant... Gs DHaseY120S / K150C achieved the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 894.34 U / mg and a yield of 96.27% for the catalytic synthesis of N-carbamoyl-D-HPG.

[0109] N-Carbamoyl-D-amino acid amide hydrolase As The preparation of DCase double mutant combinatorial mutant libraries was achieved through site-directed saturation mutagenesis. Primer design is shown in Table 4. E. coli BL21(DE3) / pET28a(+)- As DCase-F82A contains the carrier pET28a(+)- AsUsing DCase-F82A as a template, combined mutations were performed using the three pairs of primers for K124 in Table 1. The PCR system and procedure were the same as in Example 2.

[0110] Table 8: Gs Catalytic performance and specific enzyme activity of DHase and its combined mutants .

[0111] Screening yielded a combined mutant of N-carbamoyl-D-amino acid amide hydrolases: As DCase-F82A / K124A As DCase-F82A / K124S and As DCase-F82A / K124G (amino acid sequence shown in SEQ ID NO.8) were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. Then, 1% (v / v) of the inoculum was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Finally, IPTG was added to the culture medium to a final concentration of 0.1 mM and cultured at 28°C for 12 h. After that, the culture was centrifuged at 4°C and 8000 xg for 10 min to obtain the corresponding wet bacterial cells.

[0112] Using the enzyme catalytic system in Example 1 (i.e., the "screening of cell catalytic performance" section in Example 1), the yield and specific activity of various mutants of N-carbamoyl-D-amino acid amide hydrolase catalyzing the synthesis of D-p-hydroxyphenylglycine were determined. The results are shown in Table 8: When the product synthesized by the mutant was D-p-hydroxyphenylglycine, the mutant... As DCase-F82A / K124G achieved the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 266.16 U / mg and a yield of 91.86% for the catalytic synthesis of D-p-hydroxyphenylglycine.

[0113] Combining Examples 3 and 4, it can be seen that when used for the asymmetric catalytic synthesis of D-p-hydroxyphenylglycine, the enzyme activity of the DHase mutant was increased by 46.87%–312.06% compared to the original strain, and the enzyme activity of the DCase mutant was increased by 27.56%–241.65% compared to the original strain. The D-hydantoinase constructed in this application... Gs DHase mutant As DHase-Y120S and As The activity of DHase-K150C was significantly enhanced, increasing by 241.76% and 46.87% compared to the control group D-hydantoin, respectively. Further combined mutations at these two sites yielded a mutant with an increase in enzyme activity of 312.06%. AsDHase-Y120S / K150C; while N-carbamoyl-D-amino acid amide hydrolase As DCase mutant As DCase-F82A and As Compared with the control group, DCase-K124G increased the enzyme activity of N-carbamoyl-D-amino acid amide hydrolase by 716.45% and 146.57%, respectively. Further combined mutation of these two sites resulted in an enzyme activity increase of 753.61%.

[0114] Example 5

[0115] This embodiment uses Gs DHase-Y120S / K150C (amino acid sequence in SEQ ID NO.6) and AsDCase-F82A / K124G (nucleotide sequence in SEQ ID NO.7, amino acid sequence in SEQ ID NO.8) are catalysts for dual-enzyme catalytic synthesis. Experiments were conducted on the addition ratio of the two.

[0116] To achieve the efficient synthesis of D-p-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin, a dual-enzyme coupling reaction system of D-hydantoinase and N-carbamoyl-D-amino acid amide hydrolase was used. The system consisted of 50 mM Tris / HCl buffer (pH 7.0), with an initial substrate concentration of 100 mM DL-p-hydroxyphenylhydantoin. The reaction temperature was kept constant at 30°C, the stirring speed was 200 rpm, and the total reaction time was set to 12 hours. In this embodiment, the concentration of D-hydroxyphenylhydantoin was fixed at 100 mM. Gs The addition amount of DHase-Y120S / K150C was 1 g DCW / L (DCW cell dry weight), and then added according to different dry weight ratios. As The ratio of DCase-F82A / K124G examined ( Gs DHase: As DCase was prepared in ratios of 1:0.2, 1:0.5, 1:1, 1:2, 1:4 and 1:6, with three parallel samples for each ratio, and the final yield was the average value.

[0117] Experiments such as Figure 3 As shown: With As With increasing DCase ratio, the yield of D-p-hydroxyphenylglycine initially increased rapidly and then stabilized. When the ratio was 1:0.2, the yield was only 14.1%; increasing it to 1:0.5 increased the yield to 31.7%; at 1:1, the yield reached 53.7%; further increasing it to 1:2 significantly increased the yield to 68.5%; and when the ratio was adjusted to 1:4, the yield reached its highest value of 80.8%. Further increases... AsWhen the DCase ratio was adjusted to 1:6, the yield was 71.1%, showing almost no further improvement. This phenomenon indicates that the intermediate N-carbamoyl-D-p-hydroxyphenylglycine, produced by D-hydantoinase catalyzing the substrate, must be derived from... As DCase is further hydrolyzed into the final product; if the latter is insufficient, intermediate products will accumulate and limit the overall reaction rate; however, when As DCase excess (such as D-hydantoinase) Gs DHase and N-carbamoyl-D-amino acid amide hydrolase As When the DHase ratio is 1:6, due to limitations imposed by substrate concentration and enzymatic reaction equilibrium, additional hydrolase activity does not lead to a significant increase in yield. In summary, Gs DHase-Y120S / K150C and As The optimal enzyme activity ratio of DCase-F82A / K124G in this coupling system was determined to be 1:4. Under this optimal ratio, 100 mM DL-p-hydroxyphenylhydantoin could be converted to 80.8% D-p-hydroxyphenylglycine after 12 hours of reaction, achieving ideal yield and good economic efficiency. Establishing this ratio provides a crucial parameter for the scale-up and continuous operation of the subsequent two-enzyme coupling process, and also provides a reference for optimizing enzyme dosage in similar two-enzyme cascade reactions.

[0118] Example 6

[0119] This embodiment conducts a pH experiment on the dual-enzyme coupling synthesis of D-p-hydroxyphenylglycine to investigate the effect of the reaction system pH on the synthesis of D-p-hydroxyphenylglycine, so as to determine the optimal pH conditions for the dual-enzyme coupling process.

[0120] With D-hynanase mutant Gs DHase-Y120S / K150C and N-carbamoyl-D-amino acid amide hydrolase mutants As The DCase-F82A / K124G mutant cells were used as catalysts, and the calculation method for the catalytic performance of the mutants at different pH values ​​was the same as in Example 1.

[0121] Since the activities of both enzymes are pH-sensitive, and their recent pH values ​​may differ, the optimal pH window needs to be comprehensively evaluated based on the overall yield of the coupling reaction. The initial concentration of the substrate DL-p-hydroxyphenylhydantoin was 100 mM, and the amount of both enzymes added was: Gs DHase-Y120S / K150C is 1 g DCW / L. AsDCase-F82A / K124G was prepared at a concentration of 4 g DCW / L. The reaction temperature was 30℃, the stirring speed was 200 rpm, and the reaction time was 12 hours. Buffer systems with different pH values ​​were prepared, including seven gradients: pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. 50 mM phosphate buffer was used for pH 6.0–7.0, 50 mM Tris-HCl buffer for pH 7.5–8.5, and 50 mM glycine-NaOH buffer for pH 9.0, to ensure sufficient buffering capacity at each pH point. All reactions were carried out in a constant-temperature shaker, with three replicates per group. Samples were taken immediately after the reaction, and after appropriate dilution and termination of the reaction, the amount of D-p-hydroxyphenylglycine produced was determined by high-performance liquid chromatography (HPLC), and the yield was calculated.

[0122] The results are as follows Figure 4 As shown, the pH of the reaction system has a significant impact on the yield of the dual-enzyme coupling. At pH 6.0, the yield is only 24.5%, mainly because the acidic environment severely inhibits the activity of both enzymes, especially D-hydantoinase, which almost completely loses its activity at this pH. As the pH gradually increases, the yield shows a clear upward trend: 30.0% at pH 6.5, 46.8% at pH 7.0, and rapidly increases to 69.7% at pH 7.5. The yield reaches its highest value of 83.6% at pH 8.0, indicating the optimal synergistic efficiency of the two enzymes and the lowest accumulation of the intermediate product N-carbamoyl-D-p-hydroxyphenylglycine. This suggests that both GsDHase and AsDCase maintain high catalytic activity at this pH, and that the substrate solubility and product stability are also good. When the pH was increased further to 8.5, the yield decreased slightly to 75.8%, and further decreased to 65.4% at pH 9.0. This may be because the excessive alkalinity caused changes in the active site structure of AsDCase, and high pH may also accelerate the degradation of substrate or product.

[0123] Based on the above results, the optimal pH for the dual-enzyme coupled catalytic synthesis reaction is 8.0. Under this pH condition, not only is the final yield the highest (83.6%), but the reaction process is also stable with less accumulation of intermediate products, indicating that pH 8.0 can well balance the catalytic characteristics of the two enzymes as well as the mass transfer and equilibrium requirements of the entire cascade reaction.

[0124] This embodiment clarifies the optimal pH parameters for the dual-enzyme coupling process, providing crucial acid-base conditions for subsequent scale-up reactions and industrial applications. It also indicates that in actual operation, the pH of the reaction system must be strictly controlled around 8.0 to ensure process stability and high conversion rate.

[0125] Example 7

[0126] This embodiment investigates the reaction temperature of the dual-enzyme coupling synthesis of D-p-hydroxyphenylglycine to examine its effect on the synthesis of D-p-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin via dual-enzyme coupling, thereby determining the optimal reaction temperature for this dual-enzyme cascade process.

[0127] Since the two enzymes originate from different microorganisms, their thermal stability and optimal catalytic temperature may differ. Too low a temperature can lead to insufficient enzyme activity and a decreased reaction rate, while too high a temperature may cause enzyme protein denaturation and inactivation. Therefore, the temperature window needs to be comprehensively evaluated by considering the final yield and process rate of the coupling reaction. The initial concentration of the immobilized substrate DL-p-hydroxyphenylhydantoin was 100 mM, and the amount of the two enzymes added was... Gs DHase-Y120S / K150C 1 g DCW / L, As The reaction mixture consisted of DCase-F82A / K124G (4 g DCW / L), with a reaction buffer of 50 mM Tris-HCl (pH 8.0), a stirring speed of 200 rpm, and a total reaction time of 12 hours. Seven temperature gradients were established: 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, with three replicates for each gradient. After the reaction, the amount of D-p-hydroxyphenylglycine produced was determined by high-performance liquid chromatography (HPLC) to calculate the yield. Simultaneously, samples were taken at different time points to monitor the reaction progress and observe the effect of temperature on the reaction rate.

[0128] Figure 5 The results showed that the reaction temperature significantly affected the catalytic efficiency of the dual-enzyme coupling system, with the yield exhibiting a bell-shaped curve that first increased and then decreased with temperature. At a reaction temperature of 20℃, the yield was only 29.7%, mainly due to the slower molecular motion and reduced effective collision frequency between the enzyme and substrate at low temperatures, leading to a decrease in the rate at which D-hydantoin catalyzes the ring-opening of the substrate to N-carbamoyl-D-p-hydroxyphenylglycine. As The rate of further hydrolysis of intermediates by DCase was significantly inhibited, and equilibrium was not reached after 12 hours of reaction. Increasing the temperature to 25°C increased the yield to 42.4%, indicating that enzyme activity gradually recovered with increasing temperature. At 30°C, the yield reached 83.6%, with both enzymes exhibiting high catalytic activity at this temperature. The reaction was stable with minimal intermediate accumulation, indicating that 30°C effectively balanced the activity requirements of both enzymes with the rate matching of the entire cascade reaction. Further increasing the temperature to 35°C slightly increased the yield to 85.6%, close to the result at 30°C, indicating that this temperature range (30-35°C) was within an acceptable operating range. However, when the temperature continued to rise to 40°C, the yield significantly decreased to 78.4%, which may be due to… AsDCase is highly temperature-sensitive. At 40°C, its active site begins to undergo irreversible conformational changes, leading to a decrease in its ability to hydrolyze intermediates. This causes intermediates to accumulate and slows down the overall reaction rate. At 45°C, the yield further decreases to 61.3%, and at 50°C, the yield is only 42.6%. At this point, both enzymes have undergone varying degrees of inactivation due to prolonged exposure to high temperatures. Especially at 50°C, almost no product is generated in the later stages of the reaction, indicating that the enzyme protein has been largely denatured.

[0129] Based on the above results, the optimal reaction temperature for this dual-enzyme coupling system is 35℃, with a yield of 85.6%. Moreover, the reaction can be completed within 12 hours with minimal enzyme activity loss, demonstrating good reproducibility and promising prospects for industrial application. This provides clear operating parameters for temperature control in subsequent scale-up processes.

[0130] Example 8

[0131] This embodiment conducts experiments on the substrate concentration of D-p-hydroxyphenylglycine synthesized by dual-enzyme coupling, and analyzes the effect of different concentrations of substrate DL-p-hydroxyphenylhydantoin on the reaction process of D-p-hydroxyphenylglycine synthesis, so as to clarify the substrate tolerance range and the optimal feed concentration of the process.

[0132] This embodiment fixes two enzymes. Gs DHase-Y120S / K150C and As The addition amounts of DCase-F82A / K124G were 1 g DCW / L and 4 g DCW / L, respectively. The buffer solution was 50 mM Tris-HCl (pH 8.0, 30℃), the stirring speed was 200 rpm, and the total reaction time was 24 hours. Initial concentrations of the substrate DL-p-hydroxyphenylhydantoin were 25, 50, 100, 150, and 200 mM. Samples were taken at different time points, and the yield of D-p-hydroxyphenylglycine was determined by high-performance liquid chromatography (HPLC). The accumulation of intermediate products was also monitored.

[0133] The results are as follows Figure 6As shown: When the initial concentration of the substrate was 25 mM, the product yield stopped increasing after 12 hours, reaching equilibrium with a yield of 97.5%, but the absolute product concentration was low. When the initial concentration of the substrate was 50 mM, equilibrium was reached after 10 hours with a yield of 96.1%. When the initial concentration of the substrate was 100 mM, a small amount of intermediate product accumulated in the first 4 hours before rapid conversion, reaching a yield of 95.3% after 12 hours and maintaining it until 24 hours, achieving both high conversion and high volumetric yield. When the initial concentration of the substrate was 150 mM, although the initial rate was fast, the accumulation of intermediate product was significant, and the conversion slowed down after 8 hours, with a final yield of 85.5% after 24 hours. It is speculated that the high concentration of substrate or intermediate product inhibited D-hydantoinase, while the hydrolase was relatively insufficient. When the initial concentration of the substrate was 200 mM, substrate precipitation occurred in the early stages of the reaction, mass transfer was hindered, and the final yield was only 73.4%.

[0134] Based on the combined concentration curves, 100 mM is the optimal substrate concentration, under which the final yield is high and the reaction time is moderate. The safe operating window is 25-100 mM, while concentrations exceeding 150 mM significantly affect efficiency.

[0135] This embodiment provides a key basis for substrate feeding strategies in scale-up processes.

[0136] Example 9

[0137] In this embodiment, the catalyst was used for the dual-enzyme coupled catalytic synthesis of D-p-hydroxyphenylglycine in an immobilized enzyme manner. The catalytic synthesis effect of the immobilized dual-enzyme catalyst and the reusability of the catalyst were investigated.

[0138] The immobilization carrier was an epoxy-based resin (TJS type epoxy-based resin). An appropriate amount of epoxy-based resin carrier was weighed, thoroughly washed with deionized water, and then filtered until dry. Separate preparations were then made. Gs DHase-Y120S / K150C and As The enzyme solution of DCase-F82A / K124G was adjusted to a protein concentration of 0.35 mg / mL. The enzyme solution was mixed with the pretreated resin carrier at a ratio of approximately 133 U of enzyme solution to 1 g of resin carrier, and the mixture was fixed at 28°C and pH 7.5 with shaking for 15 hours. After fixation, the immobilized enzyme was repeatedly washed with phosphate buffer (pH 7.0) to remove unbound free enzyme protein, yielding the immobilized enzyme. Gs DHase-Y120S / K150C and Immobilization As DCase-F82A / K124G was stored at 4℃ for later use. The immobilized enzyme activity was determined to be 58.5 U, the protein immobilization rate to be 97.4%, and the enzyme activity recovery rate to be 49.3%.

[0139] The two immobilized enzymes were added simultaneously to the reaction system at an enzyme activity ratio of 1:4 (immobilized GsDHase-Y120S / K150C 10 U / mL, immobilized AsDCase-F82A / K124G 40 U / mL). The initial concentration of the substrate DL-p-hydroxyphenylhydantoin was 100 mM, the buffer was 50 mM Tris-HCl (pH 8.0), the reaction temperature was 30℃, the stirring speed was 200 rpm, and the total reaction time was 12 hours. After the reaction, the immobilized enzymes were separated from the reaction solution by filtration or centrifugation, and the yield of the product D-p-hydroxyphenylglycine was determined by high performance liquid chromatography.

[0140] The results showed that the immobilized two-enzyme catalytic system achieved a yield of 92.4% within 12 hours, which was slightly lower than that of the free two-enzyme system (95.3%), but still maintained high catalytic activity. The loss of enzyme activity was mainly due to the slight change in enzyme molecular conformation during immobilization and the mass transfer resistance between the substrate and the immobilized enzyme.

[0141] The isolated and recovered immobilized dual enzymes were washed with buffer and directly added to the next batch of reaction. Ten batches of catalytic reactions were carried out continuously under the same conditions. The results are as follows: Figure 7 As shown, after 10 cycles, the relative enzyme activity of the immobilized dual enzyme remained above 87% of the initial enzyme activity, the product yield remained above 85%, and the half-life reached more than 26 batches. Compared with the free enzyme system, the immobilized dual enzyme significantly improved the enzyme's thermostability and operational stability, avoiding the problems of difficulty in recovery and reuse of free enzymes after the reaction, and greatly reducing the cost of enzyme preparations. In addition, the immobilized dual enzyme can be quickly separated from the product by simple filtration after the reaction, simplifying the subsequent product purification process.

[0142] This embodiment demonstrates that using epoxy resin as a carrier for... Gs DHase-Y120S / K150C and As The DCase-F82A / K124G was immobilized, and the resulting immobilized dual enzymes exhibited good catalytic activity and excellent reusability, providing a feasible enzyme immobilization scheme for the industrial continuous production of this dual-enzyme coupling process.

[0143] Example 10

[0144] This embodiment involves the metal ion Mn. 2+ The effects of concentration and stirring speed during the reaction on the dual-enzyme coupling catalysis of D-p-hydroxyphenylglycine immobilization. 2+It has a significant promoting effect on the activity of D-hydantoinase and N-carbamoyl-D-amino acid amide hydrolase. The stirring speed directly affects the mass transfer efficiency of the reaction system and the contact frequency between the substrate and enzyme molecules, thus affecting the overall catalytic rate.

[0145] The initial concentration of the immobilized substrate DL-p-hydroxyphenylhydantoin was 100 mM, and the amount of the two enzymes added was... Gs DHase-Y120S / K150C 1 g DCW / L, As DCase-F82A / K124G 4 g DCW / L, reaction buffer 50 mM Tris-HCl (pH 8.0), reaction temperature 30℃, total reaction time 12 hours.

[0146] (1) In Mn 2+ In the concentration optimization experiment, MnCl2·4H2O was added to the reaction system at final concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 mmol / L, respectively. 2+ The stirring speed was kept constant at 200 rpm. Results showed that without the addition of Mn... 2+ The yield was 70.4% when the final concentration was 0; when Mn 2+ The yield increased to 89.5% at a concentration of 0.05 mmol / L; the yield reached its highest value of 95.4% at 0.1 mmol / L, an increase of approximately 25 percentage points compared to the unsupplemented group; the yield decreased slightly to 92.2% at 0.2 mmol / L; the yield decreased to 89.5% at 0.5 mmol / L; the yield decreased significantly to 85.3% at 1.0 mmol / L, and further decreased to 75.3% at 2.0 mmol / L (see...). Figure 8 (Part A of the text), suggesting excessively high concentrations of Mn 2+ It may competitively inhibit the enzyme's active site or cause conformational changes in the enzyme protein. Therefore, an appropriate amount of Mn... 2+ It has a significant activating effect on the dual-enzyme coupling reaction, and the optimal addition concentration is 0.1 mmol / L.

[0147] (2) In the experiment to optimize the stirring speed, Mn was fixed. 2+The concentration was 0.1 mmol / L, and stirring speeds were set at 50, 100, 150, 200, 250, 300, 350, 400, and 450 rpm. The results showed that at 50 rpm, the mass transfer efficiency of the reaction system was insufficient, the substrate and product distribution was uneven, and the yield after 12 hours was only 62.6%. The yield reached 79.3% at 100 rpm; 87.5% at 150 rpm; and the highest yield of 95.4% at 200 rpm, where the substrate-enzyme contact was sufficient and mass transfer resistance was low. Further increasing the speed to 250 rpm resulted in a yield that was essentially the same as at 200 rpm. However, when the speed reached 300 rpm and above, the yield decreased slightly (92.3% at 300 rpm, 90.4% at 350 rpm, 89.5% at 400 rpm, and 83.5% at 450 rpm) (see [link to relevant documentation]). Figure 8 (Part B of the text) The shear force generated by excessively high rotation speed may cause some damage to the enzyme molecular structure or lead to the physical detachment of the immobilized enzyme.

[0148] Based on the above results, the optimal Mn is determined. 2+ The optimal concentration for addition was 0.1 mmol / L, and the optimal stirring speed was 200 rpm.

[0149] Under optimal reaction conditions (initial substrate concentration of 100 mM, catalyst used) As DHase-Y120S / K150C: As DCase-F82A / K124A=1:4, Mn 2+ The highest yield of the immobilized dual-enzyme coupling reaction was 95.4% with a purity of 99% (HPLC), achieved by adding 0.1 mmol / L at a reaction concentration of 8.0, reaction pH of 30℃, reaction temperature of 30℃, stirring speed of 200 rpm, and a total reaction time of 12 h. e.e. The yield is greater than 99%. Scaled up to a 5 L immobilized bioreactor, with a substrate concentration increased to 300 mM, the yield still reaches 90.5%. Compared to chemical resolution, production costs are reduced by approximately 40%, and waste is reduced by more than 80%.

[0150] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A D-hynanase mutant, characterized in that: The amino acid sequence of the D-hydantoin mutant was obtained by mutating SEQ ID NO:

2. The mutation was set to replace tyrosine at position 120 with serine and lysine at position 150 with cysteine.

2. A gene encoded by the amino acid sequence of the D-hyinase mutant of claim 1.

3. A recombinant genetically engineered bacterium containing the gene of claim 2.

4. A dual-enzyme coupled catalyst, characterized in that: The invention comprises an N-carbamoyl-D-amino acid amide hydrolase mutant and the D-hydantoin mutant of claim 1, wherein the amino acid sequence of the N-carbamoyl-D-amino acid amide hydrolase mutant is obtained by mutating SEQ ID NO:6, and the mutation is set as follows: phenylalanine at position 82 is replaced with alanine, and lysine at position 124 is replaced with glycine.

5. A gene encoded by the amino acid sequence of the N-carbamoyl-D-amino acid amide hydrolase mutant of claim 4.

6. A recombinant genetically engineered bacterium comprising the gene of claim 5.

7. A method for the asymmetric synthesis of the antibiotic precursor D-HPG via dual-enzyme coupling, characterized in that: The dual enzymes include a D-hydantoin mutant and an N-carbamoyl-D-amino acid amide hydrolase mutant. The amino acid sequence of the D-hydantoin mutant is obtained by mutating SEQ ID NO:2, and the N-carbamoyl-D-amino acid amide hydrolase mutant is obtained by mutating SEQ ID NO:

6.

8. The method for the dual-enzyme coupled asymmetric catalytic synthesis of the antibiotic precursor D-HPG according to claim 7, characterized in that: DL-p-hydroxyphenylhydantoin was used as the reaction substrate, with an initial concentration of 20–300 mM. The first catalyst was a D-hydantoin mutant, the fermentation product of an engineered bacterium containing its encoding gene, or its extracted free enzyme. The second catalyst was an N-carbamoyl-D-amino acid amide hydrolase mutant, the fermentation product of an engineered bacterium containing its encoding gene, or its extracted free enzyme. The total amount of the first and second catalysts added, based on the enzyme activity of the D-hydantoin mutant, was 1–5 g DCW / L. The catalytic synthesis was carried out at a temperature of 20–50 °C and a pH of 7.0–9.

0.

9. The method for the dual-enzyme coupled asymmetric catalytic synthesis of antibiotic precursor D-HPG according to claim 7, characterized in that: D-hydantoin mutants and N-carbamoyl-D-amino acid amide hydrolase mutants participate in catalytic synthesis in the form of immobilized enzymes.

10. A method for the dual-enzyme coupled asymmetric catalytic synthesis of the antibiotic precursor D-HPG according to any one of claims 7 to 9, characterized in that: Using epoxy resin as a carrier, enzyme solutions of D-hydantoin mutant and N-carbamoyl-D-amino acid amide hydrolase mutant were prepared respectively. The enzyme solutions were mixed with the carrier and fixed by shaking at 25-30℃ and pH 7.0-8.0 for 12-18 hours. After fixation, the mixture was washed to obtain immobilized D-hydantoin mutant and immobilized N-carbamoyl-D-amino acid amide hydrolase mutant. The immobilized D-hydantoin mutant was used as the first catalyst, and the immobilized N-carbamoyl-D-amino acid amide hydrolase mutant was used as the second catalyst.

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