Sulfur transferase egt-st3 for ergothioneine synthesis and use thereof

Abstract

This invention discloses a thiotransferase EGT-ST3 for ergothioneine synthesis and its applications. The amino acid sequence of the enzyme is shown in SEQ ID NO:1. It catalyzes the production of ergothioneine using histidine betaine as a substrate and inorganic polysulfides as sulfur donors. This invention also provides a nucleic acid molecule encoding the enzyme, a recombinant expression vector containing the nucleic acid molecule, and a recombinant host cell. Based on this, a method for preparing a soluble recombinant expression biocatalyst is established, including steps such as constructing a fusion tag expression vector, transforming the host cell, expressing the fusion tag, preparing resting cells, preparing the immobilized enzyme, fermentation induction, cell collection, and catalyst preparation. This thiotransferase, nucleic acid molecule, expression vector, or host cell can be applied to the enzymatic synthesis of ergothioneine.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and recombinant protein expression technology, specifically to a thiotransferase EGT-ST3 for ergothionein synthesis and its applications. Background Technology

[0002] Ergothioneine is a naturally occurring sulfur-containing imidazole amino acid derivative with potent antioxidant, cell-protective, anti-inflammatory, and UV-protective functions, showing broad application prospects in functional foods, skincare cosmetics, pharmaceuticals, and dietary supplements. Since the human body cannot synthesize ergothioneine and it must be obtained through exogenous intake, developing efficient, low-cost, and industrially viable ergothioneine preparation technologies has significant industrial value.

[0003] Thiotransferases are key catalytic enzymes in the ergothioneine biosynthesis pathway, specifically using histidine betaine (HER) as a substrate and inorganic polysulfides as sulfur donors to catalyze the one-step production of ergothioneine. These enzymes can serve as core biocatalysts in in vitro enzymatic reaction systems or whole-cell catalytic systems, and are essential components of the ergothioneine biomanufacturing route. However, current technologies for thiotransferase expression in heterologous recombination generally suffer from low soluble expression levels, high inclusion body ratios, and low soluble protein yields. This not only leads to low enzyme yields but also significantly increases the cost of downstream purification processes, limiting their industrial-scale application.

[0004] For thiotransferases used in ergothioneine synthesis, simply increasing the total protein expression level cannot meet the demands of industrial applications. If the target protein exists in large quantities as insoluble inclusion bodies, a complex denaturation-renaturation process is required to recover the active protein, which results in severe enzyme activity loss and significantly increases production costs. If the expression level is increased by raising the induction temperature, increasing the concentration of the inducer, or extending the induction time, it will increase the metabolic burden on the host cell, leading to cell aging, lysis, and an increase in the proportion of misfolded proteins, thus reducing the final yield of the active enzyme.

[0005] Soluble fusion tags are a common technique for improving the soluble expression of recombinant proteins. MBP tags have strong solubilizing abilities and are widely used in E. coli expression systems. SlyD tags have chaperone-like functions, assisting in the correct folding of some target proteins. SUMO tags have a small molecular weight, also assisting in folding, and can be precisely cleaved by specific proteases. Differences in the molecular weight, spatial conformation, and relative position to the active site of the target enzyme among different tags can affect the specific activity, catalytic stability, and subsequent purification efficiency of the target enzyme to varying degrees. Therefore, for thiotransferases used in ergothioneine synthesis, a balance needs to be struck between soluble expression levels, enzyme activity per unit mass, catalytic stability, and preparation costs; currently, there is no mature, systematic solution.

[0006] Furthermore, in the preparation of resting cell catalysts, high soluble expression must be coordinated with cell integrity and physiological state. Over-induction or excessively long induction time can lead to decreased cell membrane integrity, loss of intracellular enzyme activity, and release of byproducts, ultimately reducing the efficiency of whole-cell catalysis and product purity.

[0007] In summary, there is currently a lack of systematic soluble expression and catalyst preparation processes for thiotransferases used in ergothioneine synthesis. This makes it impossible to simultaneously achieve high soluble expression, high catalytic activity, good cell condition, and low preparation cost, thus hindering the large-scale biomanufacturing of ergothioneine. Summary of the Invention

[0008] To address the shortcomings of the existing technologies, this invention aims to provide a thiotransferase EGT-ST3 for ergothionein synthesis and its application, solving problems such as low solubility, numerous inclusion bodies, significant enzyme activity loss after tag fusion, difficulty in balancing induction process with cell physiological state, and low downstream purification yield when existing thiotransferases are recombinantly expressed.

[0009] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a thiotransferase EGT-ST3 for the synthesis of ergothionein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] Furthermore, the thiotransferase EGT-ST3 uses histidine betaine as a substrate and inorganic polysulfides as sulfur donors to catalyze the production of ergothionein.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the thiotransferase EGT-ST3, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0012] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecule, wherein the 5' end of the nucleic acid molecule is operatively linked with a coding sequence of a soluble tag; the soluble tag is used to enhance the soluble expression level of thiotransferase EGT-ST3 in host cells.

[0013] Fourthly, the present invention provides a recombinant host cell, wherein the recombinant host cell comprises the recombinant expression vector described above; or the genome of the recombinant host cell is integrated with the nucleic acid molecule described above, and the nucleic acid molecule is expressed under the regulation of a host endogenous promoter.

[0014] Furthermore, the host cell is either Escherichia coli BL21(DE3) or Bacillus licheniformis; when the host cell is Escherichia coli BL21(DE3), it carries the recombinant expression vector; when the host cell is Bacillus licheniformis, the nucleic acid molecule is integrated into its genome.

[0015] Fifthly, this invention provides a method for preparing a soluble recombinant expression of ergothioneine biocatalyst, comprising the following steps: a) Construction of recombinant expression vector: The coding sequence of a soluble tag is ligated to the 5' end of the nucleic acid molecule encoding the thiotransferase EGT-ST3, and cloned into the expression vector to obtain the recombinant expression vector; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2; b) Transformation into the host cell: The recombinant expression vector obtained in step a) is transformed into the host cell to obtain recombinant engineered bacteria; c) Fermentation-induced culture: The recombinant engineered bacteria were cultured and IPTG-induced to express the bacteria. d) Collection of bacterial cells: After induction for 16-18 hours, collect the recombinant engineered bacterial cells; e) Catalyst preparation: The bacterial cells obtained in step d) are prepared into free enzyme preparations, resting cell catalysts, or immobilized enzyme preparations.

[0016] Further, in step a), the soluble tag is a SUMO, MBP, or SlyD tag, and the SUMO tag is linked to the thiotransferase EGT-ST3 via a (GGGGS)3 flexible linker peptide.

[0017] Sixthly, the present invention provides a method for the enzymatic synthesis of ergothionein, using the aforementioned thiotransferase EGT-ST3 or the aforementioned recombinant host cell or the biocatalyst prepared according to the method as the catalytic element, histidine betaine as the reaction substrate, and inorganic polysulfides as sulfur donors, to generate ergothionein under catalytic reaction conditions.

[0018] In a seventh aspect, the present invention provides the application of the aforementioned thiotransferase EGT-ST3, the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, or the aforementioned recombinant host cell in the biosynthesis of ergothionein.

[0019] The beneficial effects of this invention are as follows: Addressing the challenges of low solubility, numerous inclusion bodies, significant enzyme activity loss after tag fusion, and low downstream purification yield in heterologous expression of thiotransferases, this invention screens a fusion expression strategy combining a SUMO tag with a (GGGGS)3 flexible linker peptide. This strategy increases the soluble protein concentration (approximately 21% higher than the tagless control) while only decreasing the specific activity by about 3%, and shortens the induction time to 18 hours, achieving a total enzyme activity comparable to the tagless control after 27 hours of induction. Affinity chromatography purification yield is significantly increased from 30% to 85.7%, significantly reducing the cost of industrial enzyme preparation. This fusion enzyme exhibits excellent catalytic stability in high substrate loading reactions, retaining over 90% of its activity after 36 hours, with an ergothioneine titer of 48.3 g / L and a conversion rate of 91.7%, superior to MBP and SlyD tags. Combined with a resting cell catalyst subjected to low-temperature short-time induction and permeabilization treatment, cell integrity can be effectively maintained, improving product efflux efficiency. Furthermore, the immobilized enzyme preparation retains over 70% of its activity after 10 consecutive batches of use, demonstrating excellent operational stability. This invention provides a complete, efficient, and low-cost technical solution for the large-scale biomanufacturing of ergothioneine. Attached Figure Description

[0020] Figure 1 This is a diagram of the His6-EGT-ST3 plasmid structure.

[0021] Figure 2 The structure diagram of pET-28a (+)-MBP-EGT-ST3 plasmid.

[0022] Figure 3 The structure diagram of pET-28a (+)-SlyD-EGT-ST3 plasmid.

[0023] Figure 4 This is a diagram of the structure of the pET-SUMO-EGT-ST3 plasmid.

[0024] Figure 5 This is the HPLC chromatogram of ergothionein. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments.

[0026] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0027] Example 1: Effects of different fusion tags on the soluble expression and catalytic activity of thiotransferase EGT-ST3 This embodiment compares the effects of different soluble fusion tags on the expression level, soluble ratio, and catalytic activity of the thiotransferase EGT-ST3 in Escherichia coli, and uses wild-type EanB derived from Chlorobium limicola as a cross-sectional control to evaluate the expression performance and catalytic performance of EGT-ST3 relative to the wild-type enzyme.

[0028] 1. Construction of recombinant expression strains 1.1 EGT-ST3 encoding sequence The amino acid sequence of thiotransferase EGT-ST3 is shown in SEQ ID NO:1, and its encoding nucleotide sequence is shown in SEQ ID NO:2.

[0029] The amino acid sequence of thiotransferase EGT-ST3 (SEQ ID NO:1): MTEFREASTEDLRAKKNKNWCIIDVRKSLLGKPDVVIIDVRSTEAYFGYRDNGEPRGRIKGAKSFPRRLLKDPDRLKIVESLGIKPDDTIILYGATPEESLEMASFLRE HGYTKVYIYPKYLPEWTGNPDNPLEKVPNFERNIPASWLEGIINGKEVPYYDGTDYRVVHAYYRNESAYLDGHIPGSIPLDLTDLVSPETRRPKSPEELEAALKKHGITAD TTVILYGTNRNPDLSEPNPLSRAGWMVAVRAALVLLAAGVKDVRILNGGYQAWVDAGFPVSTENVPRKPVEDFGIEIFANPELLVDRERALAMLKDPGAALVDVVPEAVW VGEESGSPWLTRRRGRIPGALHFPRGRDAYTVDNLLTLPGSFADPQLIAERAAAVGITPDKTLAFYITGISGSVAAWQALLLGWPNVAVWVGGLYAWFADPSNPYLTGPPV.

[0030] The nucleotide sequence encoding the thiotransferase EGT-ST3 (SEQ ID NO:2):

[0031] 1.2 Expression vectors and strain grouping Using Escherichia coli BL21(DE3) as the expression host, four recombinant strains expressing EGT-ST3 were constructed, and one control strain expressing wild-type EanB was also constructed.

[0032] All expression constructs were created using prokaryotic expression vectors with a T7 promoter and were uniformly tagged with an N-terminal hexahistine (His6 tag) for purification using immobilized metal ion affinity chromatography.

[0033] The details for each group are as follows.

[0034] (1) The EGT-ST3 group without fusion tag, hereinafter referred to as the "EGT-ST3 control group". The EGT-ST3 coding sequence shown in SEQ ID NO:2 was inserted into the pET-28a(+) expression vector to obtain the recombinant expression vector pET-28a(+)-EGT-ST3. The target protein expressed by this vector is EGT-ST3 with an N-terminal His6 tag, i.e., His6-EGT-ST3. Figure 1 As shown. The sequence is (SEQ ID NO:5):

[0035] (2) MBP Fusion Group, hereinafter referred to as "MBP Group" The nucleotide sequence encoding the maltose-binding protein MBP was ligated to the 5′ end of the EGT-ST3 coding sequence to construct the recombinant expression vector pET-28a(+)-MBP-EGT-ST3, the structural diagram of which is shown below. Figure 2 As shown. This vector expresses the fusion protein His6-MBP-EGT-ST3, which has a His6 tag and an MBP tag at its N-terminus. The sequence is (SEQ ID NO:6):

[0036] (3) SlyD Fusion Group, hereinafter referred to as "SlyD Group" The nucleotide sequence encoding the SlyD protein was ligated to the 5′ end of the EGT-ST3 coding sequence to construct the recombinant expression vector pET-28a(+)-SlyD-EGT-ST3, the structural diagram of which is shown below. Figure 3 As shown. This vector expresses the fusion protein His6-SlyD-EGT-ST3, which has a His6 tag and a SlyD tag at its N-terminus. The sequence is (SEQ ID NO:7):

[0037] 4) SUMO Fusion Group, hereinafter referred to as "SUMO Group" The nucleotide sequence encoding the SUMO tag was linked to the 5′ end of the EGT-ST3 coding sequence, and a flexible linker peptide consisting of three GGGGS repeat units, namely the (GGGGS)3 flexible linker peptide, was placed between the SUMO tag and EGT-ST3 to construct the recombinant expression vector pET-SUMO-EGT-ST3. Its structural diagram is shown below. Figure 4 As shown.

[0038] The structure of the fusion protein expressed by this vector is as follows: His6-SUMO-(GGGGS)3-EGT-ST3.

[0039] (GGGGS)3 sequence: GGTGGCGGTGGCTCTGGCGGTGGCGGATCGGGAGGCGGTGGTTCA.

[0040] Setting the (GGGGS)3 flexible linker peptide helps reduce the steric hindrance between the SUMO tag and EGT-ST3, allowing the EGT-ST3 domain to fold relatively independently. The sequence is (SEQ ID NO:8):

[0041] (5) Wild-type EanB group, hereinafter referred to as "WT group" Select from Chlorobium limicola Wild-type EanB was used as a cross-sectional control. The wild-type EanB is designated as Cl-EanB-WT, with NCBI accession number WP_012467399.1 and an amino acid sequence length of 457 amino acids, as shown in SEQ ID NO:3.

[0042] Wild-type EanB is derived from Chlorobium limicola, and its amino acid sequence is as follows (SEQ ID NO:3): MQNKNFRAPQSEAIGILYKLIETGSKHKNMYDHTEITTDSLLALLGSEKVKIIDVRSADAYNGWRMRGEVRGGHIKGAKSLPAKWLTDPEWLNIVRFKQIRPEDAIVLYGYTPE ECEQTATRFKENGYNNVSVFHRFHPDWTGNDAFPMDRLEQYNRLVPAEWVNGLISGEEIPEYDNDTFIVCHAHYRNRDAYLSGHIPGATDMDTLALESPETWNRRTPEELKKALE EHGITASTTVVLYGKFMHPDNADEFPGSAAGHIGAIRLAFIMMYAGVEDVRVLNGGYQSWTDAGFAISKDDVPKTTVPEFGAPIPSRPEFAVDIDEAKEMLQSEDSDLVCVRSY PEYIGEVSGYNYIKKKGRIPGAIFAECGSDAYHMENYRNHDHTTREYHEIEDIWAKSGIIPKKHLAFYCGTGWRGSEAWFNALLMGWPRVSVYDGGWFEWSNDPENPYETGVPK.

[0043] The nucleotide sequence encoding the wild-type EanB amino acid sequence is as follows (SEQ ID NO:4):

[0044] The Cl-EanB-WT coding sequence (SEQ 4) was inserted into the NdeI and XhoI sites of the pET-28a(+) expression vector to obtain the recombinant expression vector pET-28a(+)-Cl-EanB-WT. This vector does not contain an additional soluble fusion tag, and the expression product is wild-type EanB with a His6 tag at the N-terminus, i.e., His6-Cl-EanB-WT.

[0045] Each of the above recombinant expression vectors was transformed into Escherichia coli BL21(DE3) competent cells. After resistance screening, colony PCR and sequencing verification, the corresponding recombinant expression strains were obtained and the indicators were verified, as shown in Table 1.

[0046] Table 1 Comparison of indicators between EGT-ST3 and wild-type control

[0047] 2. Fermentation culture and induced expression 2.1 Fermentation culture of EGT-ST3 and fusion protein Single colonies from the EGT-ST3 control group, MBP group, SlyD group and SUMO group were picked and inoculated into seed culture medium containing the corresponding antibiotics to obtain seed liquid.

[0048] The seed culture was inoculated into 5 L fermenters containing modified TB medium for fed-batch culture. During fermentation, the pH of the culture system was maintained at 6.45 and the oxygen content was controlled at 35%.

[0049] When the bacterial cell density OD 600 When the concentration reaches 20, the culture temperature is lowered to 25°C, and isopropyl-β-D-thiogalactoside (IPTG) is added to a final concentration of 0.5 mmol / L to induce the expression of the target protein.

[0050] in: Bacterial cells were collected after 27 h of induction culture in the EGT-ST3 control group. Bacterial cells were collected after 18 h of induction culture in the MBP, SlyD, and SUMO groups.

[0051] The above settings were used to compare the effects of each fusion tag on the expression level and catalytic activity of EGT-ST3 under shortened induction time conditions.

[0052] 2.2 Fermentation culture of wild-type EanB The WT group was fermented using the same culture system and induction conditions as the EGT-ST3 control group, including the same 5 L fermenter, modified TB medium, initial cell density, induction temperature and IPTG concentration.

[0053] Specifically, when the OD of the WT group culture medium 600 When the concentration reaches 20, the culture temperature is lowered to 25°C, IPTG is added to a final concentration of 0.5 mmol / L, and the cells are collected after 27 h of induction culture.

[0054] 3. Cell processing and soluble protein preparation Centrifuge each group of fermentation broth and collect the bacterial cells. Resuspend the bacterial cells in buffer solution with the same mass-to-volume ratio and perform cell disruption under the same conditions.

[0055] After centrifugation, the supernatant and precipitate were collected separately. The supernatant was used as the soluble protein sample, and the precipitate was resuspended in an equal volume of buffer as the insoluble protein sample.

[0056] The target protein bands in the supernatant and precipitate fractions were detected by SDS-PAGE, and the soluble proportion of the target protein was evaluated by band grayscale analysis.

[0057] The fusion proteins in the supernatants of each group could be purified using a His Trap FF affinity chromatography column. Since each construct contains a His6 tag, the same affinity chromatography process could be used for purification, reducing experimental bias caused by different purification methods.

[0058] 4. Detection Method The cell density (OD) of each group of recombinant strains at the end of induction was measured separately. 600 The concentration of soluble protein in the supernatant, the total enzyme activity per unit volume of fermentation broth, the specific activity of purified protein, and the proportion of soluble protein in SDS-PAGE were measured.

[0059] Total enzyme activity and specific activity were measured according to the thiotransferase activity assay method described in this instruction manual. All samples used the same substrate system, buffer system, reaction temperature, reaction time, and termination conditions.

[0060] Total enzyme activity is expressed as the enzyme activity of the enzyme preparation obtained per unit volume of fermentation broth, in U / L; specific activity is expressed as the enzyme activity per unit mass of protein, in U / mg.

[0061] Each experiment should be performed in at least three parallel measurements.

[0062] 5. Effects of different fusion tags on EGT-ST3 expression and activity Table 1. Effects of different fusion tags on the soluble expression and catalytic activity of EGT-ST3

[0063] As shown in Table 1, under the fermentation conditions used, the OD600 at the end of induction for each group was 148.6–152.0, and the cell density was basically the same, indicating that different fusion tags did not have a significant adverse effect on the growth of the host bacteria.

[0064] After 27 h of induction in the EGT-ST3 control group, the OD600 was 147.7, the soluble protein concentration was 5.9 g / L, the total enzyme activity was 8351.6 U / L, and the specific activity was 150 U / mg. SDS-PAGE results showed that the target protein band corresponding to His6-EGT-ST3 was present in both the lysate and the precipitate, indicating that EGT-ST3 without the soluble tag could achieve soluble expression, but some target protein still existed in an insoluble form.

[0065] After 18 h of induction, the soluble protein concentration in the MBP group reached 6.9 g / L, an increase of approximately 23.2% compared to the EGT-ST3 control group. However, the specific activity of the MBP fusion protein was 62 U / mg, only about 41.3% of that in the EGT-ST3 control group, and the total enzyme activity was 4300 U / L. These results indicate that although the MBP tag can increase the soluble expression level of the fusion protein, the catalytic activity per unit mass of the fusion protein may be significantly reduced, possibly due to the large molecular weight of the tag or its steric influence on the active conformation of EGT-ST3.

[0066] After 18 h of induction with SlyD, the soluble protein concentration was 6.5 g / L, an increase of approximately 16.1% compared to the EGT-ST3 control group; its specific activity was 132 U / mg, approximately 88.0% of the EGT-ST3 control group; and the total enzyme activity reached 7900 U / L. This indicates that the SlyD tag can improve the soluble expression of EGT-ST3 and maintain its catalytic activity to a large extent.

[0067] OD 600 The concentration of soluble protein reached 6.8 g / L, which was 152.0, an increase of about 21.4% compared with the EGT-ST3 control group; its total enzyme activity was 8200 U / L, which was about 98.3% of the total enzyme activity obtained by the EGT-ST3 control group after 27 h of induction; its specific activity was 145 U / mg, which was about 96.7% of the EGT-ST3 control group, a decrease of only about 3.3%.

[0068] Therefore, the combination of the SUMO tag and the (GGGGS)3 flexible linker peptide can improve the soluble expression level of EGT-ST3 while maintaining its catalytic activity. Compared with EGT-ST3 without the fusion tag, the SUMO fusion construct achieves essentially the same total enzyme activity with a 9-hour reduction in induction time (approximately 33.3%), thus offering advantages in shortening the fermentation cycle and improving production efficiency.

[0069] 6. Comparison of expression performance between EGT-ST3 and wild-type Cl-EanB-WT To evaluate the technical effects of EGT-ST3 compared to wild-type EanB, parallel culture and induction were performed on the EGT-ST3 control group and WT group under the same fermentation conditions.

[0070] The test results are shown in Table 2.

[0071] Table 2 Comparison of expression and catalytic performance of EGT-ST3 and wild-type Cl-EanB-WT

[0072] As shown in Table 2, under the same host, expression vector and fermentation induction conditions, the cell density at the end of induction in the EGT-ST3 group and the WT group was basically the same, indicating that the cell growth levels of the two groups were similar during the fermentation process.

[0073] However, the soluble protein concentration in the supernatant of the WT group was less than 0.10 g / L, with the vast majority of the target protein located in the insoluble precipitate, and the soluble proportion was less than 5%. In contrast, the soluble protein concentration in the EGT-ST3 group reached 5.9 g / L, and the soluble proportion in SDS-PAGE was approximately 65%. The soluble protein concentration of EGT-ST3 was more than 56 times that of Cl-EanB-WT.

[0074] The total enzyme activity of the EGT-ST3 group was 8351.6 U / L, while that of the WT group was only 36±8 U / L, meaning the total enzyme activity of EGT-ST3 was approximately 232 times that of the WT group. After affinity chromatography purification, the specific activity of EGT-ST3 was 150 U / mg, while that of the WT group was 28±3 U / mg, meaning the specific activity of EGT-ST3 was approximately 5.4 times that of the WT group.

[0075] Furthermore, the purification yield of EGT-ST3 was approximately 30%, while the purification yield of the WT group was less than 5%. These results indicate that EGT-ST3 not only significantly improved the soluble expression performance of the protein but also increased the catalytic activity per unit mass of enzyme protein, thereby significantly increasing the total enzyme activity obtained per unit fermentation volume.

[0076] 7. HPLC Detection of Ergothioneine Synthesis Catalyzed by EGT-ST3 and Cl-EanB-WT 7.1 Catalytic Reaction The purified His6-EGT-ST3 and His6-Cl-EanB-WT were used for the catalytic reaction, respectively.

[0077] The reaction system contained 15 g / L histidine betaine and 20 mmol / L sodium polysulfide (Na₂S₅), and the pH of the reaction system was 7.5. The reaction system was placed at 30℃ for 24 h. After the reaction was complete, insoluble substances were removed by centrifugation or filtration, and the supernatant of the reaction solution was analyzed by HPLC.

[0078] The amount of enzyme, reaction volume, substrate concentration, reaction temperature, pH, and reaction time used in each group of reactions were kept consistent.

[0079] 7.2 HPLC detection The reaction products were detected using a C18 reversed-phase column at a wavelength of 260 nm.

[0080] Ergothioneine standards with a purity of not less than 99% were used as qualitative and quantitative references. The standard could be Sigma-Aldrich ergothioneine standard E7521.

[0081] Under the chromatographic conditions used, the characteristic retention time of the ergothioneine standard was approximately 3.4 min. The reaction product was qualitatively identified by comparing the retention times of the sample peak and the standard peak; the concentration of ergothioneine in the reaction solution was calculated using the ergothioneine standard curve.

[0082] HPLC detection results are as follows Figure 5 As shown. Figure 5 The chromatograms include those of ergothioneine standard, Cl-EanB-WT reaction sample, and EGT-ST3 reaction sample.

[0083] Ergothioneine standard showed a characteristic chromatographic peak at approximately 3.4 min.

[0084] The Cl-EanB-WT reaction solution showed only a weak chromatographic peak at approximately 3.4 min. Based on the standard curve, its ergothioneine concentration was calculated to be 3.1 ± 0.4 g / L.

[0085] The EGT-ST3 reaction solution exhibited a distinct chromatographic peak at 3.4 ± 0.05 min. This peak was sharp and symmetrical, and its retention time was consistent with that of the ergothioneine standard. Based on the standard curve, the concentration of ergothioneine in the EGT-ST3 reaction solution reached 14.5 ± 0.8 g / L, approximately 4.7 times that of the Cl-EanB-WT group.

[0086] Example 2: Effects of expression culture conditions and permeabilization treatment on the catalytic performance of Bacillus licheniformis resting cells This embodiment investigates the effects of expression culture temperature, culture time, and Triton X-100 permeation treatment on the integrity of resting Bacillus licheniformis cells, the ability to maintain intracellular enzyme activity, and the efficiency of product efflux.

[0087] 1. Construction of engineered Bacillus licheniformis strains Bacillus licheniformis ( Bacillus licheniformis ATCC 14580 was the starting strain, and its genomic amyE site was selected as the integration site for the target gene.

[0088] The expression cassette for genome integration, along the 5' to 3' direction, comprises: an approximately 800 bp upstream homologous arm of amyE, a constitutive promoter of PamyE, a ribosome binding site, an EGT-ST3 coding sequence, an rrnB T1 / T2 tandem transcription terminator, chloramphenicol resistance selection markers with loxP sites at both ends, and an approximately 800 bp downstream homologous arm of amyE. The EGT-ST3 coding sequence, as shown in SEQ ID NO:2, is 1323 bp in length; the ribosome binding site sequence is AAAGGAGGA, separated from the start codon ATG by 8 nucleotides.

[0089] The expression cassette described above was cloned into the thermosensitive shuttle vector pE194-ts to obtain the integrative plasmid pE194-amyE-EGT-ST3. The integrative plasmid was introduced into Bacillus licheniformis ATCC 14580 using PEG-mediated protoplast transformation. Chloramphenicol-resistant transformants were screened at 30°C, followed by 5–6 passages at 45°C to promote the integration of the target expression cassette into the amyE site via a double exchange mechanism.

[0090] Integration of the target fragment was confirmed by PCR amplification and sequencing across the amyE integration site, and the starch hydrolysis phenotype of the strain was detected by iodine staining. The strain correctly identified by PCR and lacking the starch hydrolysis zone was identified as the engineered bacterium B. licheniformis ΔamyE::EGT-ST3.

[0091] If necessary, the Cre recombinase expression vector can be further introduced to remove the chloramphenicol resistance gene located between the two loxP sites, thereby obtaining an engineered strain without resistance selection markers.

[0092] 2. Seed culture The engineered bacterial culture was taken from a -80℃ glycerol storage tube and streaked onto LB solid medium containing 5 μg / mL chloramphenicol. The culture was incubated at 37℃ for 16 h. A single colony was picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium (containing 5 μg / mL chloramphenicol). The flask was incubated at 37℃ and 220 rpm for 14–16 h until the OD600 reached approximately 4.0, yielding the primary seed culture.

[0093] For engineered strains with removed chloramphenicol resistance markers, chloramphenicol is not added to the culture medium.

[0094] 3. Expression culture and grouping The primary seed culture was inoculated into the expression medium at a volume ratio of 5%. Each liter of the expression medium contained: 10 g glucose, 20 g tryptone, 10 g yeast extract, 5 g NaCl, 0.5 g MgSO4, and 0.01 g MnSO4, with an initial pH of 7.2. The glucose solution was sterilized separately at 115°C before being added to the remaining medium components.

[0095] The inoculated culture medium was cultured under the following conditions, with 3 replicates per group: Group A: Incubated at 28℃ and 220 rpm for 24 h; Group B: Incubated at 25℃ and 220 rpm for 18 h; Group C: Cultured at 25℃ and 220 rpm for 18 h, and then the bacterial cells were collected and permeabilized. Group D: Cultured at 28℃ and 220 rpm for 24 h, and then the bacterial cells were collected and permeabilized.

[0096] Since the EGT-ST3 in the engineered bacteria is expressed by a constitutive promoter, no IPTG or other small molecule inducers are added during the culture process. The "expression culture conditions" referred to in this embodiment refer to the temperature and culture time used during the expression phase of the target enzyme.

[0097] After cultivation, the fermentation broth of each group was centrifuged at 4℃ and 6000×g for 15 min, and the bacterial cells were collected. The bacterial cells were washed twice with an equal volume of pre-cooled PBS buffer. The PBS buffer contained 50 mmol / L phosphate and 150 mmol / L NaCl, and the pH was 7.4.

[0098] Group A and Group B cells were resuspended directly in PBS buffer. Group C and Group D cells were resuspended in PBS buffer containing 0.1% Triton X-100 (v / v) and gently stirred at 30°C for 30 min to perform cell permeabilization. After permeabilization, the cells were collected by centrifugation again and washed once with PBS buffer to remove residual Triton X-100.

[0099] Each group of bacterial cells was resuspended in PBS buffer and adjusted to OD600 = 80. Using a pre-established conversion between wet cell weight and OD600, the corresponding wet cell concentration was approximately 28.8 g / L. The conversion is as follows: Wet cell concentration (g / L) = 0.36 × OD600, R 2 =0.996.

[0100] 4. Resting cell catalytic reactions Establish a catalytic reaction system with a total volume of 100 mL in a 250 mL anaerobic reaction flask. The reaction system contains: 50 mmol / L HEPES buffer, pH 7.50±0.05; 15 g / L histidine betaine; 20 mmol / L sodium polysulfide; 25 mL of resting cell suspension with OD600 = 80.

[0101] Resting cell suspension accounted for 25% of the total reaction volume, corresponding to a wet bacterial cell concentration of approximately 7.2 g / L in the reaction system.

[0102] Sodium polysulfide stock solution was prepared under anaerobic conditions and added to the reaction system all at once before use. Before adding resting cells, nitrogen gas was introduced into the reaction solution at a flow rate of 0.1 L / min for 20 min, then the reaction flask was sealed, and a slight positive nitrogen pressure was maintained during the reaction.

[0103] The catalytic reaction was carried out at 35℃ and 220 rpm for 36 h. Samples were taken at 0, 4, 8, 12, 16, 20, 24, 28, 32 and 36 h of the reaction, with 1 mL samples taken each time. The samples were immediately centrifuged at 4℃ and 12000×g for 3 min, and the supernatant and bacterial precipitate were collected separately for the detection of product concentration, extracellular LDH activity and residual intracellular enzyme activity.

[0104] 5. Ergothioneine content detection The ergothioneine content in the reaction solution was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: The chromatographic column was a C18 reversed-phase column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm; the mobile phase was a 1% (v / v) methanol-water solution with isocratic elution; the detection wavelength was 260 nm; the flow rate was 0.7 mL / min; the injection volume was 5 μL; the column temperature was 30℃; and the single run time was 20 min.

[0105] The reaction supernatant was collected and the reaction was terminated by boiling in a water bath for 5 min. After cooling, it was centrifuged at 12000×g for 3 min. The supernatant was filtered through a 0.45 μm filter membrane before analysis. An external standard curve was established using ergothioneine standard, and the concentration of ergothioneine in the sample was calculated based on the peak area.

[0106] 6. Detection of extracellular LDH activity LDH activity in the reaction supernatant was detected using a lactate dehydrogenase assay kit. 50 μL of reaction supernatant was added to 50 μL of assay reagent, and the mixture was incubated at room temperature in the dark for 30 min. The absorbance was then measured at 490 nm.

[0107] A fully lysed cell suspension of the same concentration was used as a positive control for complete lysis. The positive control LDH activity was approximately 850 U / L. The lower the extracellular LDH activity in the experimental group, the better the cell membrane integrity. When the extracellular LDH activity was less than 2% of that in the positive control, i.e., less than 17 U / L, the resting cells were considered to have maintained good membrane integrity.

[0108] 7. Determination of intracellular enzyme activity retention rate Bacterial pellets from each time point were collected, resuspended in an equal volume of PBS buffer, and sonicated. After centrifugation, the supernatant was collected, and EGT-ST3 activity was measured in a standard enzyme activity assay system.

[0109] Taking the intracellular enzyme activity of resting cells in each group at the start of the reaction as 100%, the retention rate of intracellular enzyme activity after 36 h of reaction was calculated according to the following formula: Intracellular enzyme activity retention rate (%) = Total intracellular enzyme activity after 36 h of reaction ÷ Total intracellular enzyme activity at the start of the reaction × 100%.

[0110] 8. Experimental Results After 36 h of reaction, the extracellular LDH activity, intracellular enzyme activity retention rate and ergothionein efflux concentration of each group are shown in Table 3.

[0111] Table 3 Catalytic properties of resting cells under different expression culture and permeabilization conditions

[0112] Note: Results are expressed as mean ± standard deviation, n = 3.

[0113] Compared with group A, the extracellular LDH activity in group B decreased from 150 U / L to 50 U / L, the intracellular enzyme activity retention rate increased to 75%, and the ergothioneine concentration increased from 4.5 g / L to 6.8 g / L, indicating that short-term expression culture at 25℃ can reduce cell damage and improve the catalytic capacity of resting cells.

[0114] Compared with group B, after mild permeabilization treatment, the extracellular LDH activity in group C did not increase, but decreased to 12 U / L, while the intracellular enzyme activity retention rate reached 87%, and the ergothioneine concentration increased to 9.2 g / L. This indicates that under good cell physiological conditions, moderate permeabilization can reduce cell membrane mass transfer resistance and promote substrate entry and product excretion.

[0115] Although group D underwent the same permeabilization treatment, its extracellular LDH activity increased to 180 U / L, and its intracellular enzyme activity retention rate was less than 20%, indicating that when cells are already damaged due to unsuitable expression culture conditions, further permeabilization may exacerbate membrane structure damage and intracellular enzyme leakage.

[0116] Therefore, culturing at 25℃ for 18 h combined with mild permeabilization treatment with 0.1% Triton X-100 can improve the production and efflux efficiency of ergothionein while maintaining the integrity of resting cells and intracellular enzyme activity.

[0117] Example 3: Effect of SUMO fusion expression on recombinase purification yield This embodiment compares the enzyme activity recovery rates of tagless EGT-ST3 and SUMO-EGT-ST3 under the same affinity chromatography conditions to evaluate the effect of SUMO fusion expression on the efficiency of soluble recombinant enzyme preparation.

[0118] 1. Bacterial source Take 4 L of induction culture medium each of the EGT-ST3 expression strain without fusion tag and the SUMO-EGT-ST3 expression strain obtained in Example 1.

[0119] The non-fusion-labeled control group was BL21(DE3) / pET-28a(+)-EGT-ST3; the SUMO fusion group was BL21(DE3) / pET-SUMO-EGT-ST3.

[0120] The culture medium was centrifuged at 4℃ and 6000×g for 15 min, and the bacterial cells were collected. Approximately 215 g of wet bacterial cells were obtained from the control group without fusion tag, and approximately 220 g of wet bacterial cells were obtained from the SUMO fusion group.

[0121] 2. Cell disruption and preparation of crude enzyme solution The bacterial cells were resuspended at a ratio of 8 mL of pre-cooled binding buffer per gram of wet bacterial cells. The binding buffer contained 50 mmol / L Tris-HCl, 300 mmol / L NaCl, and 20 mmol / L imidazole, with a pH of 8.0.

[0122] The material was cyclically crushed three times at 800 bar using a high-pressure homogenizer. During the crushing process, an external cooling device was used to control the discharge temperature below 10°C.

[0123] The lysate was centrifuged at 4°C and 12000×g for 35 min, and the supernatant was collected as the lysate supernatant. Approximately 1.72 L of lysate supernatant was obtained from the untagged control group and approximately 1.76 L of lysate supernatant was obtained from the SUMO fusion group.

[0124] The fragmented supernatant was filtered through a 0.45 μm filter membrane to remove residual particles and then used for affinity chromatography purification.

[0125] 3. Nickel ion affinity chromatography purification Purification was performed using a chromatography column loaded with nickel ion affinity packing material. Before purification, the column was washed with 5 column volumes of deionized water and then equilibrated with 10 column volumes of binding buffer until the 280 nm UV absorbance baseline stabilized.

[0126] Since the volume of the fragmented supernatant was larger than the recommended single loading volume, each group of samples was purified by batch loading and repeated chromatography, and the target protein eluents obtained from each batch were combined.

[0127] The specific purification steps are as follows: The supernatant was loaded at a flow rate of 3.0 mL / min; After loading, wash with 10 column volumes of binding buffer; Wash 10 column volumes with washing buffer containing 50 mmol / L imidazole; Gradient elution was performed by linearly increasing the imidazole concentration from 50 mmol / L to 500 mmol / L, with a gradient volume of 5 column volumes and a flow rate of 3.0 mL / min. Collect the eluent in 1 mL fractions per tube.

[0128] Each collected fraction was analyzed by SDS-PAGE. The apparent molecular weight of the untagged EGT-ST3 was approximately 50 kDa, and that of SUMO-EGT-ST3 was approximately 62–65 kDa. The eluted fractions that showed a distinct single band at the target molecular weight position and had a purity of at least 90% according to gel grayscale analysis were combined.

[0129] The combined eluent was desalted, and the buffer system was replaced with a storage buffer containing 50 mmol / L Tris-HCl, 100 mmol / L NaCl, and 10% glycerol (v / v), at pH 8.0. The solution was then concentrated to a protein concentration of approximately 15 mg / mL using an ultrafiltration device with a 30 kDa molecular weight cutoff. After filtration through a 0.22 μm filter, the solution was aliquoted and stored at -80°C.

[0130] 4. Enzyme activity assay The standard enzyme activity assay system had a total volume of 1 mL and contained 13 g / L histidine betaine, 10 mmol / L sodium polysulfide, and 50 mmol / L HEPES buffer, with a pH of 7.5. The reaction temperature was 30℃, and the reaction time was 10 min.

[0131] After the reaction was completed, the amount of ergothioneine produced was determined by high-performance liquid chromatography. The amount of enzyme required to produce 1 μmol of ergothioneine per minute under the above conditions was defined as 1 enzyme activity unit, or 1 U.

[0132] The total enzyme activity of the lysed supernatant is calculated according to the following formula: Total enzyme activity in the supernatant = enzyme activity concentration in the supernatant × total volume of the supernatant.

[0133] The total enzyme activity after purification is calculated according to the following formula: Total enzyme activity after purification = enzyme activity concentration of combined eluent × total volume of combined eluent.

[0134] Purification yield is calculated using the following formula: Purification yield (%) = Total enzyme activity after purification ÷ Total enzyme activity of the lysed supernatant × 100%.

[0135] 5. Protein concentration determination Protein concentration was determined using the Bradford method, with bovine serum albumin as the standard. The standard curve range was 0.1–1.0 mg / mL.

[0136] Take 10 μL of appropriately diluted protein sample, add 200 μL of Bradford working solution, react at room temperature for 5 min, measure the absorbance at 595 nm, and calculate the protein concentration according to the standard curve.

[0137] 6. Experimental Results The enzyme activity concentration of the supernatant from the non-fusion-tagged control group was approximately 18.6 U / mL, with a total volume of approximately 1720 mL, corresponding to a total enzyme activity of approximately 32000 U. After affinity chromatography, approximately 8 mL of eluent was obtained, with an enzyme activity concentration of approximately 1200 U / mL, corresponding to a total purified enzyme activity of approximately 9600 U, and a purification yield of 30.0%.

[0138] The enzyme activity concentration of the supernatant from the SUMO fusion group was approximately 19.9 U / mL, with a total volume of approximately 1760 mL, corresponding to a total enzyme activity of approximately 35000 U. After affinity chromatography, approximately 10 mL of eluent was obtained, with an enzyme activity concentration of approximately 3000 U / mL, corresponding to a total purified enzyme activity of approximately 30000 U, and a purification yield of 85.7%. The results are shown in Table 4.

[0139] Table 4. Affinity chromatography purification yields of different EGT-ST3 expression forms

[0140] The above results indicate that the total enzyme activity in the supernatants of the two groups was similar, but the enzyme activity recovery rate after affinity chromatography in the SUMO fusion group was significantly higher than that in the control group without the fusion tag. SUMO fusion expression increased the purification yield from 30.0% to 85.7%, indicating that the SUMO tag helps EGT-ST3 maintain a soluble, stable, and recyclable active conformation, thereby reducing aggregation, inactivation, and irreversible adsorption during the purification process.

[0141] Therefore, SUMO fusion expression can significantly improve the purification and recovery rate of EGT-ST3 recombinase, which is beneficial to reducing the unit preparation cost of high-activity recombinase preparations.

[0142] Example 4: Effect of different soluble fusion tags on the catalytic stability of EGT-ST3 with high substrate loading In this embodiment, EGT-ST3 fusion enzyme was prepared using three soluble fusion tags: MBP, SlyD, and SUMO, and its long-term catalytic stability was compared under high concentration of histidine betaine conditions.

[0143] 1. Preparation of fusion enzyme preparations MBP-EGT-ST3, SlyD-EGT-ST3 and SUMO-EGT-ST3 fusion proteins were constructed and expressed, respectively.

[0144] The MBP fusion group used the BL21(DE3) / pET-28a(+)-MBP-EGT-ST3 strain. When the OD600 of the fermentation broth reached approximately 20, the culture temperature was adjusted to 25℃, and IPTG was added at a final concentration of 0.5 mmol / L for induction culture for 18 h.

[0145] The SlyD fusion group used the BL21(DE3) / pET-28a(+)-SlyD-EGT-ST3 strain, and its fermentation, induction, and purification conditions were the same as those of the MBP fusion group.

[0146] The SUMO fusion group used the BL21(DE3) / pET-SUMO-EGT-ST3 strain. When the OD600 of the fermentation broth reached approximately 20, the culture temperature was adjusted to 25℃, and IPTG was added at a final concentration of 0.5 mmol / L for induction culture for 18 h.

[0147] All bacterial cells were homogenized three times under high pressure at 800 bar and purified using nickel affinity chromatography. The purified fusion proteins were desalted and concentrated, and SDS-PAGE analysis showed a purity of at least 92%.

[0148] According to standard enzyme activity assays, the specific activities of MBP-EGT-ST3, SlyD-EGT-ST3, and SUMO-EGT-ST3 were 62 U / mg, 132 U / mg, and 145 U / mg, respectively.

[0149] Before the reaction, the three enzyme solutions were adjusted to a protein concentration of 1.0 mg / mL and a pH of 7.5 using 50 mmol / L HEPES buffer. The enzyme solutions were stored on ice and used within 4 h.

[0150] 2. High substrate loading catalytic reactions A reaction system with a total volume of 1 L was established in a 2 L jacketed glass reactor. The reaction system contained 45 g / L histidine betaine and 50 mmol / L HEPES buffer, with an initial pH of 7.50 ± 0.05.

[0151] Add 10 mL of MBP-EGT-ST3, SlyD-EGT-ST3 or SUMO-EGT-ST3 enzyme solution with a protein concentration of 1.0 mg / mL to each reaction system, i.e., add 10 mg of fusion protein per liter of reaction system, and the final concentration of enzyme protein is 10 μg / mL.

[0152] Based on the specific activities of each fusion enzyme, the initial enzyme activity additions for the MBP, SlyD, and SUMO fusion groups were approximately 620 U / L, 1320 U / L, and 1450 U / L, respectively. This example uses equal protein amounts for comparison to simultaneously evaluate the combined effects of different fusion tags on the specific activity and long-term stability of EGT-ST3.

[0153] Sodium polysulfide was prepared in 1 mol / L stock solution under anaerobic conditions. During the reaction, sodium polysulfide was added in pulses using an automatic syringe pump, and the addition amount was adjusted based on the free sulfide detection results to maintain the effective polysulfide concentration in the reaction system at 15 ± 3 mmol / L.

[0154] Before the reaction, nitrogen gas filtered through a 0.22 μm filter membrane was introduced into the reaction system at a flow rate of 0.2 L / min for 30 min. The reactor was then sealed, and a slight positive nitrogen pressure was maintained during the reaction.

[0155] The reaction temperature was 30±0.5℃, the stirring speed was 200 rpm, and the reaction time was 36 h. Samples were taken at 0, 4, 8, 12, 16, 20, 24, 30, and 36 h.

[0156] 3. Ergothioneine concentration detection Take 0.5 mL of the reaction solution and immediately add 0.5 mL of a stop solution consisting of 1 mol / L hydrochloric acid and 50% acetonitrile (v / v). Vortex to mix and centrifuge at 12000×g for 3 min. Filter the supernatant through a 0.45 μm filter membrane and determine the ergothioneine concentration using high-performance liquid chromatography (HPLC).

[0157] Substrate molar conversion is calculated using the following formula: Molar conversion rate (%) = ergothioneine molar concentration ÷ initial histidine betaine molar concentration × 100%.

[0158] 4. Residual enzyme activity assay Take another 0.2 mL of the reaction solution without termination treatment and immediately add it to the standard enzyme activity assay system. The final concentration of histidine betaine in the standard system is 2 g / L, and the final concentration of sodium polysulfide is 5 mmol / L. React at 30℃ for 5 min.

[0159] The amount of ergothionein generated in a short time was determined by high performance liquid chromatography, and the specific activity retention rate at different reaction time points was calculated with the enzyme activity measured at the beginning of the reaction as 100%.

[0160] 5. Experimental Results The catalytic results of the three fusion enzymes under high substrate loading conditions are shown in Table 5.

[0161] Table 5. Catalytic performance of three fusion-labeled EGT-ST3 catalysts at high substrate loading.

[0162] Note: Results are expressed as mean ± standard deviation, n = 3; molar conversion rate is calculated based on the molar amounts of ergothioneine and histidine betaine.

[0163] The specific activity retention rates of the SUMO fusion group at 12, 24 and 36 h were 95%, 92% and 90%, respectively. After 36 h of reaction, the ergothioneine concentration reached 48.3 g / L and the substrate molar conversion rate was 91.7%.

[0164] The SlyD fusion group retained 72% of its specific activity after 36 h of reaction, with an ergothioneine concentration of 38.6 g / L. The enzyme activity of this group decreased significantly at 24 h, followed by a slight recovery. This change may be related to sampling errors, changes in the reversible enzyme aggregation state in the system, or fluctuations in polysulfide concentration. However, its overall catalytic stability remained lower than that of the SUMO fusion group.

[0165] The MBP fusion group exhibited a specific activity retention rate of less than 40% after 36 h of reaction, with an ergothioneine concentration of 35.0 g / L, indicating relatively low stability under long-term, high substrate, and polysulfide environments.

[0166] The above results indicate that, under the condition of equal protein addition, SUMO-EGT-ST3 possesses both high initial specific activity and good long-term activity retention, and its catalytic performance with high substrate loading is significantly better than that of MBP-EGT-ST3 and SlyD-EGT-ST3.

[0167] The SUMO tag has a small molecular weight and a relatively independent structure. After fusion, it causes minimal steric interference with the EGT-ST3 catalytic conformation and substrate channels, which is beneficial for the target enzyme to maintain a stable active conformation under high substrate and polysulfide conditions. Therefore, SUMO fusion expression is suitable for preparing stable EGT-ST3 enzyme preparations required for high-concentration substrate conversion processes.

[0168] Example 5: Preparation and reusability of immobilized SUMO-EGT-ST3 enzyme preparation In this embodiment, SUMO-EGT-ST3 prepared in Example 3 was covalently immobilized using epoxy activated agarose gel as a carrier, and the immobilization efficiency and repeat batch catalytic performance of the obtained immobilized enzyme preparation were evaluated.

[0169] 1. Immobilization materials The immobilization carrier was an epoxy-activated agarose gel with an epoxy group density of 20–40 μmol / mL.

[0170] The enzyme source was the SUMO-EGT-ST3 enzyme solution purified in Example 3. The protein concentration of the enzyme solution was 12.5 mg / mL, the specific activity was 145 U / mg, and it was stored in a buffer solution containing 0.1 mol / L sodium phosphate and 0.5 mol / L NaCl at pH 8.0.

[0171] 2. Carrier Pretreatment Take 10 mL of epoxy-activated agarose gel and place it in a sintered glass funnel. Wash it 5 times with 20 mL of deionized water each time to remove ethanol and other additives from the carrier preservation solution.

[0172] The solution was then washed and equilibrated twice with a coupling buffer containing 0.1 mol / L sodium phosphate and 0.5 mol / L NaCl, using 50 mL of buffer each time. The pH of the coupling buffer was 8.0.

[0173] 3. Enzyme coupling Dilute the SUMO-EGT-ST3 enzyme solution to 5 mg / mL with coupling buffer to prepare 20 mL of enzyme solution containing 100 mg of SUMO-EGT-ST3 protein, corresponding to an initial total enzyme activity of approximately 14,500 U.

[0174] Mix 10 mL of the equilibrated gel with 20 mL of enzyme solution to achieve a gel sedimentation volume to enzyme solution volume ratio of 1:2. The final NaCl concentration in the mixture is 0.5 mol / L.

[0175] Seal the reaction vessel and place it on a rotary mixer. Mix gently at 10 rpm at 4–10°C for 12 hours to allow the amino groups on the surface of the enzyme protein to covalently couple with the epoxy groups of the carrier.

[0176] After coupling, allow the gel to stand for 5 minutes to settle naturally. Collect the supernatant for determination of uncoupled protein content.

[0177] 4. Blocking of active groups and washing of immobilized enzymes Add 10 mL of 1 mol / L ethanolamine solution to the coupled gel, adjust the pH to 8.0, and gently mix at 20–25 °C for 4 h to block unreacted epoxy groups on the support.

[0178] 1. After blocking, wash the immobilized enzyme sequentially with the following buffer solutions: Wash three times with 0.1 mol / L Tris-HCl buffer containing 1 mol / L NaCl, pH 8.0; Wash twice with 0.1 mol / L sodium acetate buffer at pH 4.0; Wash three times with 20 mmol / L Tris-HCl buffer at pH 7.5.

[0179] Use 10 mL of washing buffer each time. Alternating washes with high salt and different pH conditions are used to remove non-covalently adsorbed proteins. The final wash with Tris-HCl buffer is used to equilibrate the immobilized enzyme to the neutral conditions required for the catalytic reaction.

[0180] The washed immobilized enzyme gel can be used directly for catalytic reactions, or resuspended in an equal volume of 20 mmol / L Tris-HCl buffer containing 20% ​​ethanol (v / v) for short-term storage at 4°C. Before use, it should be thoroughly washed to remove ethanol from the storage solution.

[0181] 5. Immobilization efficiency determination The protein content in the enzyme solution before coupling, the supernatant after coupling, and each washing solution was determined using the Bradford method.

[0182] The protein coupling rate is calculated using the following formula: Protein coupling rate (%) = (Initial amount of protein added - residual amount of protein in supernatant and washing solution) ÷ Initial amount of protein added × 100%.

[0183] In this embodiment, the protein coupling rate is approximately 85%, that is, approximately 85 mg of SUMO-EGT-ST3 is co-coupled in 10 mL of gel, and the average enzyme loading is approximately 8.5 mg protein / mL gel.

[0184] The total enzyme activity of the free enzyme before immobilization and the apparent total enzyme activity of the immobilized enzyme were determined using standard enzyme activity assays. The apparent activity retention rate of the immobilized enzyme was calculated using the following formula: Apparent activity retention rate (%) = total apparent enzyme activity of immobilized enzyme ÷ total enzyme activity of initially added free enzyme × 100%.

[0185] The apparent total enzyme activity of the immobilized enzyme was approximately 9425 U / 10 mL gel, with an apparent activity retention rate of approximately 65%. The decrease in apparent enzyme activity after immobilization was mainly related to the mass transfer resistance within the carrier, enzyme molecule orientation, and spatial obstruction of some active regions.

[0186] 6. Repeated batch catalytic testing Each batch of the reaction had a total volume of 50 mL. The reaction system contained 30 g / L histidine betaine, 15 mmol / L sodium polysulfide and 50 mmol / L HEPES buffer, with a pH of 7.5.

[0187] Add 5 mL of immobilized enzyme gel to each batch of reaction system. The immobilized enzyme gel contains approximately 42.5 mg of immobilized protein, corresponding to an apparent enzyme activity of approximately 4700 U.

[0188] Before the reaction, the air in the reaction flask was replaced with nitrogen and the flask was sealed. The reaction was carried out at 30°C, with stirring at 100 rpm, and under anaerobic conditions for 24 h.

[0189] After each batch of reaction, stirring was stopped, and the immobilized enzyme gel was allowed to settle naturally for 5 minutes. The supernatant was collected for ergothionein content detection. Subsequently, the immobilized enzyme gel was washed twice with 50 mL of pre-cooled 20 mmol / L Tris-HCl buffer (pH 7.5). Stirring was performed for 5 minutes each time, followed by allowing the gel to settle.

[0190] After washing, add an equal volume of fresh reaction solution to begin the next batch of catalytic reaction. Repeat the above steps for 10 consecutive batches.

[0191] After filtering the reaction supernatant from each batch through a 0.45 μm filter membrane, the ergothioneine concentration was determined by high-performance liquid chromatography (HPLC). The relative enzyme activity of subsequent batches was calculated with the ergothioneine production of the first batch as 100%.

[0192] 7. Results of reuse The results of 10 consecutive batches of immobilized SUMO-EGT-ST3 are shown in Table 6.

[0193] Table 6. Repeated batch catalytic performance of immobilized SUMO-EGT-ST3

[0194] Note: Results are expressed as mean ± standard deviation, n = 3; relative enzyme activity is calculated with the ergothioneine production of the first batch as 100%.

[0195] After 10 consecutive batches of use, the immobilized SUMO-EGT-ST3 still retained 70.1% of the apparent catalytic activity of the first batch, indicating that the immobilized enzyme has good operational stability.

[0196] Each batch of reaction solution had a volume of 50 mL and a histidine betaine concentration of 30 g / L. Therefore, the total amount of histidine betaine treated in 10 batches was 15 g. Based on the ergothioneine concentration in each batch, the total amount of ergothioneine generated in 10 batches was approximately 12.0 g.

[0197] Based on the 42.5 mg of immobilized protein used per reaction, the cumulative ergothioneine production per unit of immobilized protein was approximately 282 g / g. This result indicates that immobilization treatment can significantly extend the effective lifespan of SUMO-EGT-ST3 and reduce the amount of enzyme preparation required per unit of product.

[0198] Therefore, covalent immobilization of SUMO-EGT-ST3 using epoxy-activated agarose gel can yield an immobilized biocatalyst with high enzyme loading, good activity retention, and reusability. The immobilized enzyme preparation is suitable for repeated batch catalysis and can also be further applied to packed bed, fixed bed, or continuous flow catalytic systems depending on the reactor configuration.

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A thiotransferase EGT-ST3 for ergothionein synthesis, characterized in that, The amino acid sequence of the thiotransferase EGT-ST3 is shown in SEQ ID NO:

1.

2. The thiotransferase EGT-ST3 according to claim 1, characterized in that, The thiotransferase EGT-ST3 uses histidine betaine as a substrate and inorganic polysulfides as sulfur donors to catalyze the production of ergothionein.

3. A nucleic acid molecule encoding the thiotransferase EGT-ST3 of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 3, and the 5' end of the nucleic acid molecule is operatively linked with a coding sequence of a soluble tag; the soluble tag is used to increase the soluble expression level of thiotransferase EGT-ST3 in host cells.

5. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector of claim 4; or the genome of the recombinant host cell integrates the nucleic acid molecule of claim 3, and the expression of the nucleic acid molecule is regulated by a host endogenous promoter.

6. The recombinant host cell according to claim 5, characterized in that, The host cell is Escherichia coli BL21(DE3) or Bacillus licheniformis; when the host cell is Escherichia coli BL21(DE3), it carries the recombinant expression vector of claim 4; when the host cell is Bacillus licheniformis, its genome integrates the nucleic acid molecule of claim 3.

7. A method for preparing a soluble recombinant expression of ergothioneine biocatalyst, characterized in that, Includes the following steps: a) Construction of recombinant expression vector: The coding sequence of a soluble tag is ligated to the 5' end of the nucleic acid molecule encoding the thiotransferase EGT-ST3, and cloned into the expression vector to obtain the recombinant expression vector; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2; b) Transformation into the host cell: The recombinant expression vector obtained in step a) is transformed into the host cell to obtain recombinant engineered bacteria; c) Fermentation-induced culture: The recombinant engineered bacteria were cultured and IPTG-induced to express the bacteria. d) Collect bacterial cells: After induction for 16-27 hours, preferably 16-18 hours, collect the recombinant engineered bacterial cells; e) Catalyst preparation: The bacterial cells obtained in step d) are prepared into free enzyme preparations, resting cell catalysts, or immobilized enzyme preparations.

8. The preparation method according to claim 7, characterized in that, In step a), the soluble tag is a SUMO, MBP, or SlyD tag, and the SUMO tag is linked to the thiotransferase EGT-ST3 via a (GGGGS)3 flexible linker peptide.

9. A method for the enzymatic synthesis of ergothioneine, characterized in that, Using the thiotransferase EGT-ST3 of claim 1, the recombinant host cell of claim 5, or the biocatalyst prepared according to the method of claim 7 or 8 as the catalytic element, histidine betaine as the reaction substrate, and inorganic polysulfides as sulfur donors, ergothionein is generated under catalytic reaction conditions.

10. The use of the thiotransferase EGT-ST3 of claim 1, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, or the recombinant host cell of claim 5 in the biosynthesis of ergothionein.

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