Cysteine ​​desulfurization / desulfurization bifunctional enzymes derived from marine Streptomyces and their uses

Abstract

This invention belongs to the field of biocatalysis technology and relates to a bifunctional enzyme for cysteine ​​desulfurization / desulfurization derived from marine Streptomyces and its applications. The amino acid sequence of the enzyme is shown in SEQ ID NO.1 of the sequence listing. This bifunctional enzyme can efficiently catalyze the conversion of cysteine ​​to alanine and pyruvate under mild conditions. The resulting alanine can be used as a food additive and pharmaceutical intermediate, while pyruvate has wide applications in the pharmaceutical and nutritional supplement fields. Therefore, this enzyme has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a bifunctional enzyme for cysteine ​​desulfurization / desulfurization derived from marine Streptomyces and its uses. Background Technology

[0002] Cysteine ​​desulfurase and cysteinedesulfhydrase are two important classes of sulfur-containing amino acid metabolic enzymes. Both rely on pyridoxal phosphate (PLP) as a cofactor to participate in the transfer of sulfur atoms and the synthesis of sulfur-containing biomolecules in organisms.

[0003] Cysteine ​​desulfurases (such as NifS and IscS) primarily catalyze the breakdown of L-cysteine ​​into L-alanine. They then transfer sulfur atoms to various biosynthetic pathways through the formation of protein-bound cysteine ​​persulfate intermediates, including the synthesis of iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, tRNA-modified nucleosides, and NAD. These enzymes are widely found in bacteria, archaea, and eukaryotes, and can be divided into Group I and Group II based on sequence similarity, each with different conserved sequences and functional preferences.

[0004] On the other hand, cysteine ​​desulfurases (such as those from...) Streptococcus anginosus Icd) catalyzes the α,β-elimination reaction of L-cysteine, producing hydrogen sulfide (H2S), pyruvate, and ammonia. Besides L-cysteine, this type of enzyme can also act on compounds containing βC-S bonds, such as DL-cystathionine and S-methyl-L-cysteine, to produce corresponding thiols. The generated H2S is not only cytotoxic but can also lead to hemoglobin modification and hemolysis, which is closely related to the pathogenicity of certain pathogens.

[0005] Although considerable research has been conducted on cysteine ​​desulfurases and desulfurases from microorganisms such as Escherichia coli, nitrogen-fixing bacteria, and streptococci, no such enzymes from marine actinomycetes (such as Streptomyces) have been reported. Furthermore, reports on bifunctional enzymes possessing both highly efficient desulfurization and desulfurization activities are extremely limited. Microorganisms in the marine environment may evolve enzyme versions with unique catalytic properties, a broader substrate range, or higher stability, holding significant research and application potential. Summary of the Invention

[0006] The purpose of this invention is to identify a bifunctional enzyme for cysteine ​​desulfurization / desulfurization derived from marine Streptomyces, which converts L-cysteine ​​into L-alanine or pyruvate, NH3, and H2S at a temperature of 30°C and with pyridoxal 5'-phosphate (PLP) as a cofactor.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a bifunctional enzyme for desulfurization / desulfonation of cysteine ​​derived from marine Streptomyces, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0008] Preferably, the gene encoding a bifunctional enzyme for cysteine ​​desulfurization / desulfhydrylation has the nucleotide sequence shown in SEQ ID NO.2.

[0009] Preferably, the present invention also provides an expression vector containing the gene, wherein the expression vector is a eukaryotic vector, a prokaryotic vector, a plasmid vector, or a viral vector.

[0010] Preferably, the present invention also provides a host cell comprising the expression vector, wherein the host cell is a bacterium.

[0011] Preferably, the present invention also provides an engineered strain, wherein the engineered strain contains the aforementioned gene or the aforementioned expression vector.

[0012] Preferably, the present invention also provides the use of a bifunctional enzyme for desulfurizing / desulfurizing cysteine ​​derived from marine Streptomyces, which is used to catalyze the decomposition of L-cysteine ​​into L-alanine, or to catalyze compounds containing βC-S bonds to produce corresponding thiols, hydrogen sulfide, and ammonia.

[0013] Preferably, the present invention further provides a method for producing L-alanine or pyruvate, which uses the aforementioned cysteine ​​desulfurization / desulfurization bifunctional enzyme to convert L-cysteine ​​into L-alanine or pyruvate at 30°C with pyridoxal 5'-phosphate as a cofactor.

[0014] The bifunctional cysteine ​​desulfurization / desulfurization enzyme derived from marine Streptomyces provided by this invention exhibits high catalytic activity for L-cysteine, combining efficient desulfurization and desulfurization activities. This enzyme also possesses a broader substrate range and higher stability. Attached Figure Description

[0015] Figure 1 This is a pET28a plasmid map expressing TlnS in an embodiment of the present invention;

[0016] Figure 2 The results of SDS-PAGE analysis of purified TlnS;

[0017] Figure 3 LC-MS analysis for the detection of alanine in TlnS activity assay;

[0018] Figure 4 LC-MS analysis of pyruvate detection in TlnS activity assay. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments and accompanying drawings. More details are set forth in the following description in order to provide a full understanding of the present invention. However, the present invention can obviously be implemented in many other ways different from those described herein. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention are within the protection scope of the present invention, and the protection scope of the present invention should not be limited by the content of this specific embodiment.

[0020] This study investigated Streptomyces symbiotic with deep-sea sponges in the Antarctic polar region. streptomyces Bioinformatics analysis of the genome of sp. S6043a identified a cysteine ​​desulfurization / desulfatylase, named TlnS. This enzyme specifically converts L-cysteine ​​to L-alanine, pyruvate, NH3, and H2S. The amino acid sequence of this enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0021] The functional characteristics and catalytic activity of this enzyme were tested and verified through experiments.

[0022] To determine the novelty of the TlnS amino acid sequence, this invention used online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to search for its homologous proteins in GenBank and sorted them by sequence similarity. Table 1 lists the 10 sequences in the GenBank database that are most similar to TlnS. Among them, the protein that is most evolutionarily similar to TlnS has a similarity of 98.46% (WP_030308285.1), indicating that the full-length sequence of this protein has not been publicly disclosed before and is novel.

[0023] Table 1. Protein sequences in GenBank that are highly identical to TlnS

[0024] .

[0025] 1. tlnS Cloning of genes

[0026] by streptomyces Using the genome of sp. S6043a as a template, amplification was performed using TlnS-F and TlnS-R primers. The PCR amplification conditions were: 98℃ for 30 seconds; 98℃ for 10 seconds, 63℃ for 5 seconds, 72℃ for 20 seconds, for 30 cycles; 72℃ for 1 minute, and then cooled to 4℃ for storage.

[0027] 2. Construction of engineered strains

[0028] Will get tlnSThe fragment and the linearized pET22b vector fragment were ligated using a seamless cloning kit (2 × ClonExpress Ultra One Step Cloning Kit, Novizan, Nanjing) to form the expression vector pET28atlnS. The chromatogram is shown below. Figure 1 As shown, the strain was transformed into *E. coli* expression strain *Escherichia coli* BL21(DE3) to obtain expression strain *E. colitlnS*. Strain *E. colitlnS* expresses a fusion protein with an N-terminal His tag, and TlnS can be obtained by Ni-NTA affinity chromatography for activity detection.

[0029] 3. Expression and purification of TlnS

[0030] Genetically engineered *E. coli* strains expressing TlnS (corresponding sequence as shown in SEQ ID NO.1) were inoculated into LB medium and cultured overnight with shaking at 37°C and 220 rpm. This seed culture was then transferred to fresh LB medium at a 10% inoculation rate and cultured with shaking at 37°C and 220 rpm until the bacterial culture reached its OD value. 600 When the plasmid value reaches 0.8–1.0, add 0.2 mM IPTG and induce culture at 18°C ​​and 180 rpm for 18–24 hours to express the target protein. Appropriate antibiotics should be added to the LB medium to maintain plasmid stability.

[0031] The induced bacterial cells were centrifuged, and the precipitate was collected. The bacterial cells were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) and the cells were lysed by sonication. The lysed sample was centrifuged at 4°C and 10,000 × g to separate the supernatant from the cell debris.

[0032] The supernatant contained the target protein TlnS, and Ni was used. 2+ Purification was performed using an NTA affinity chromatography column. First, the column was washed with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) until the eluent was colorless to remove non-specifically bound proteins. Then, the target protein TlnS was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0).

[0033] For further purification, the protein solution was concentrated using an ultrafiltration tube with an appropriate molecular weight cutoff, and finally, residual imidazole was removed by passing it through a PD-10 desalting column to obtain the high-purity target protein TlnS. The SDS-PAGE results of this protein are shown below. Figure 2 As shown.

[0034] 4. Activity determination of TlnS and detection of reaction products

[0035] (1) Activity assay:

[0036] Protein reaction buffer (50 mM NaH2PO4) 4， Add 10 μM TlnS, 2 mM MPLP, 2 mM L-Cys, and 2 mM DTT to a solution containing 10% glycerol (pH 7.4) and react at 30°C for 4 hours. Then, add an appropriate amount of methanol to the reaction system to inactivate the reaction.

[0037] (2) Alanine detection:

[0038] Take the reaction solution, add an equal volume of dansyl chloride solution (5 mg / mL, dissolved in acetone) and twice the volume of sodium carbonate solution (0.1 M, pH = 11.5), and react at 60℃ in the dark for 30 minutes. After the reaction is complete, centrifuge and collect the supernatant for LC-MS analysis. The analytical results are as follows: Figure 3 As shown in the figure. LC-MS analysis showed that, compared to the negative control where TlnS was boiled, alanine was indeed produced in the reaction group, with a conversion rate of approximately 80%.

[0039] (3) Detection of pyruvate:

[0040] Take the reaction solution and add an equal volume of 4-fluorophenylhydrazine hydrochloride solution (0.1 mg / mL). React at 50℃ for 2 hours. After the reaction is complete, centrifuge and collect the supernatant for LC-MS analysis. The analytical results are as follows: Figure 4 As shown in the figure. LC-MS analysis indicated that pyruvate production was indeed detected in the reaction group compared to the negative control (TlnS was boiled), with a conversion rate of approximately 20%.

Claims

1. The use of a bifunctional enzyme for cysteine ​​desulfurization / desulfatinization derived from marine Streptomyces, characterized in that, The amino acid sequence of this enzyme is shown in SEQ ID NO.1 of the sequence listing; this enzyme is used to catalyze L-cysteine ​​to produce pyruvate, hydrogen sulfide, and ammonia.

2. A method for producing L-alanine or pyruvate, characterized in that: Using the bifunctional enzyme for cysteine ​​desulfurization / desulfurization shown in SEQ ID NO.1, L-cysteine ​​can be converted to pyruvate at 30°C with pyridoxal 5'-phosphate as a cofactor, or L-cysteine ​​can be converted to both pyruvate and L-alanine simultaneously.

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