SGNH family esterase SH2 screened in Tibetan hot spring environment and its application

Abstract

This invention discloses the SGNH family esterase SH2 screened from the hot spring environment of Tibet and its applications, belonging to the field of genetic engineering technology. This invention screened a novel SGNH family esterase gene from the hot spring environment of Tibet and achieved recombinant expression in *E. coli*. The recombinant esterase prepared by this invention is thermally stable, salt-resistant, and resistant to organic solvents, and can be safely used in teeth whitening or skin exfoliation products, avoiding damage to tooth enamel caused by traditional chemical bleaching agents. It has significant application value in the fields of daily chemicals, pharmaceuticals, and food processing.

Description

Technical Field

[0001] This invention relates to SGNH family esterase SH2 and its application in the screening of hot spring environments in Tibet, and belongs to the field of genetic engineering technology. Background Technology

[0002] SGNH family esterases, as unique members of the esterase family, belong to the SGNH-hydrolases superfamily and are important components of the bacterial GDSL lipase family. The core characteristic of this family of esterases is the presence of highly conserved serine (Ser), glycine (Gly), asparagine (Asn), and histidine (His) residues in regions I, II, III, and V of their primary sequence. Unlike the typical pentapeptide motif GxSxG possessed by most bacterial esterases, SGNH family esterases have a unique GDSL sequence motif. This structural specificity leads to significant differences in their catalytic properties and substrate recognition compared to other esterases. In terms of distribution, SGNH family esterases are widely found in various organisms from prokaryotes to eukaryotes, participating in numerous important physiological processes.

[0003] Currently, toothpaste formulations in the daily chemical industry utilize various lipases to catalyze the production of substrates such as triglycerides. This reaction mechanism provides a natural alternative for teeth whitening, significantly reducing the risk of enamel corrosion from traditional chemical bleaching agents. In toothpaste formulations, the hydrolysis efficiency of SGNH esterase directly determines the rate and concentration of hydrogen peroxide generation, thus affecting the whitening effect of the product. The specific mechanism of action is as follows: SGNH esterase recognizes and binds to triacylglycerol molecules through its hydrophobic substrate channel, forming an enzyme-substrate complex; the serine residue (Ser) at the active site initiates a nucleophilic attack on the acyl carbon of the substrate, forming a tetrahedral transition state; glycine (Gly) and asparagine (Asn) residues stabilize the transition state, and histidine (His), acting as a generalized base catalyst, accepts a proton, leading to ester bond cleavage, releasing diacylglycerol and free fatty acids, which are ultimately converted into hydrogen peroxide. This enzymatic reaction process is highly dependent on the structural integrity of the enzyme molecule and the accessibility of the catalytic active site. However, existing commercially available SGNH esterases face multiple challenges in toothpaste applications: limited diffusion in the high-viscosity matrix of toothpaste leads to reduced substrate contact efficiency; surfactants in the formulation (such as sodium lauryl sulfate) may induce conformational changes in the enzyme, reducing activity; and insufficient enzyme stability during long-term storage limits the product's shelf life. These technical bottlenecks severely restrict the performance improvement and market expansion of enzyme-based oral care products. Meanwhile, existing natural strains used to produce related esterases exhibit numerous problems, such as long fermentation cycles leading to low production efficiency; extremely low enzyme yields, making it difficult to meet the needs of large-scale industrial production; and extremely difficult enzyme purification processes, which not only increase production costs but also further limit the large-scale application of the enzymes. Therefore, obtaining heat-stable lipases can truly help products achieve their intended functions, and screening for lipases with unique characteristics from thermal environments has become an important direction for developing novel industrial enzyme preparations. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an alkaline thermostable SGNH family esterase SH2 screened from the hot spring environment in Tibet and related applications.

[0005] This invention provides a thermostable SGNH family esterase SH2 containing the amino acid sequence shown in SEQ ID NO.1.

[0006] The present invention also provides compositions containing the family esterase SH2.

[0007] In one embodiment, the composition includes, but is not limited to, enzyme preparations or daily chemical products.

[0008] In one embodiment, the composition is toothpaste.

[0009] The present invention also provides a gene encoding the alkaline thermostable SGNH family esterase SH2.

[0010] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] The present invention also provides a recombinant vector having a complete coding reading frame sequence containing the gene.

[0012] In one implementation, the term includes, but is not limited to, pET-28a.

[0013] The present invention also provides recombinant strains expressing the SGNH family esterase SH2, or containing the recombinant vector.

[0014] In one embodiment, the strain includes, but is not limited to, Escherichia coli, yeast, Bacillus, or Lactobacillus.

[0015] In one embodiment, the strain is *Escherichia coli* (E. coli). Escherichia coli BL21(DE3).

[0016] The present invention also provides a method for preparing alkaline thermostable SGNH family esterase SH2, comprising: culturing the recombinant strain, inducing the expression of SGNH family esterase SH2, collecting the bacterial cells in the fermentation broth, and separating and purifying the SGNH family esterase SH2 protein.

[0017] In one implementation, the induction is performed using IPTG.

[0018] In one embodiment, the induction is to culture the recombinant strain to OD200. 600 The concentration was 0.5~0.8, and it was induced with IPTG at a concentration of 0.5~1mM.

[0019] In one embodiment, the recombinant strain is cultured in LB medium.

[0020] In one embodiment, the recombinant strain is cultured in LB medium at 37-40°C until OD200. 600 The concentration was 0.5~0.7, and IPTG was added. The mixture was then incubated at 28~30℃ for 6~10 h.

[0021] In one embodiment, the method further includes collecting the recombinant strain cells, lysing the cells, and collecting the SGNH family esterase SH2.

[0022] This invention also provides the application of the SGNH family esterase SH2 in the preparation of pharmaceutical or daily chemical products.

[0023] In one embodiment, the daily chemical product includes oral hygiene products.

[0024] In one embodiment, the daily chemical product is toothpaste.

[0025] Beneficial effects:

[0026] This invention screened a novel SGNH family esterase gene from the hot spring environment in Tibet and recombinantly expressed the gene. The recombinant enzyme exhibits thermostability, salt tolerance, and organic solvent tolerance. Using triacetin as a substrate, the optimal temperature was 30–60°C; the optimal pH was 4.5–9.5. After incubation at 37°C for 60 min, the enzyme activity remained relatively stable. After incubation at 50°C and 80°C for 60 min, the enzyme activity remained above 50%. After treatment at 37°C for 60 min in a buffer solution with pH 3.0–11.0, the relative activity remained above 50%.

[0027] The SGNH family esterases screened in this invention overcome the technical bottlenecks of poor stability and low activity of existing oral care enzymes in toothpaste bases. These enzymes, through a natural pathway catalyzing the production of peracetic acid from triacetin, can be safely applied in teeth whitening or skin exfoliation products, avoiding damage to tooth enamel caused by traditional chemical bleaching agents. This has significant application value in the fields of daily chemicals, pharmaceuticals, and food processing. Attached Figure Description

[0028] Figure 1 Sequence homology analysis for different SGNH family esterases.

[0029] Figure 2 SDS-PAGE analysis of recombinant SGNH family esterase SH2 expressed in Escherichia coli: Lane 1: Protein Marker; Lane 2: Total protein expressed after BL21(DE3) / pET-28a-SH2 induction; Lane 3: Soluble protein expressed after induction; Lane 4: Penetrating protein during purification; Lane 5: Eluted contaminants; Lanes 6-10: Purified recombinant SGNH family esterase SH2.

[0030] Figure 3 This is a schematic diagram showing the optimal temperature for recombinant SGNH family esterase SH2.

[0031] Figure 4 A schematic diagram illustrating the thermostability of recombinant SGNH family esterase SH2.

[0032] Figure 5 This is a schematic diagram showing the optimal pH for recombinant SGNH family esterase SH2.

[0033] Figure 6 This is a schematic diagram of the pH stability of the recombinant SGNH family esterase SH2. Detailed Implementation

[0034] The present invention will be further described below with reference to embodiments.

[0035] 1. Strains and vectors:

[0036] Escherichia coli BL21(DE3) was purchased from Novagen; expression vector pET-28a was purchased from TransGen.

[0037] 2. Enzymes and other biochemical reagents: DNA polymerase and dNTPs were purchased from Novizan Pharmaceuticals; other reagents were purchased from Sinopharm Group. Triacetin (glyceryl triacetate) and acetic acid were purchased from Maclean Pharmaceuticals; other reagents were purchased from Sinopharm Group.

[0038] 3. Culture medium:

[0039] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH natural (7.0). Solid medium is prepared by adding 15 g / L agar.

[0040] 4. Assay Method: Add 500 μL of 100 mM PBS buffer (pH 6.5), 250 mM triacetin, and 500 mM hydrogen peroxide to a 1.5 mL EP tube. Add an appropriate amount of enzyme solution, and then bring the reaction volume to 1 mL with water. Incubate at 30°C and 200 rpm for 5 min. Determine the amount of peracetic acid produced using liquid chromatography. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose triacetin to produce 1 μmol / L of peracetic acid per minute under the assay conditions.

[0041] Example 1: Screening and homology comparison of SGNH family esterases

[0042] Microbial gene resources obtained from the hot spring environment in Tibet were screened based on highly conserved motifs of the SGNH family esterase donor-substrate binding. Sequence homology alignment was performed using NCBI's BLAST function, and four sequences with high sequence similarity that might possess esterase activity were selected. Amino acid sequence alignment is as follows: Figure 1 The amino acid sequences of H109, ​​H652, H036, and H002 are shown in SEQ ID NO.1 and SEQ ID NO.3~5, respectively, and the enzyme activity was determined.

[0043] Example 2: Construction of plasmids expressing SGNH family esterases

[0044] The DNA sequences encoding SGNH family esterases were synthesized by Nanjing Genscript Biotech Co., Ltd. (nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.6~8, respectively) and ligated into the pET-28a vector by homologous recombination. The H109 gene was amplified using primers H109-F and H109-R; the H652 gene was amplified using primers H652-F and H652-R; the H036 gene was amplified using primers H036-F and H036-R; and the H002 gene was amplified using primers H002-F and H002-R. The vector backbone was amplified from pET-28a using primers pET-28a-F and pET-28a-R (Table 1). The amplified gene fragments were then ligated to the vector backbone to obtain recombinant plasmids pET-28a-H109, ​​pET-28a-H652, pET-28a-H036, and pET-28a-H002, respectively.

[0045] Table 1 Primers used in this implementation case

[0046]

[0047] Example 3: Preparation and purification of recombinant SGNH family esterases

[0048] The recombinant plasmids pET-28a-H109, ​​pET-28a-H652, pET-28a-H036, and pET-28a-H002 obtained in Example 2 were transformed into BL21(DE3) competent cells and named BW1, BW2, BW3, and BW4, respectively. The resulting recombinant bacteria BW1, BW2, BW3, and BW4 were then transferred to LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 200 rpm for 8 h. They were then inoculated at a 1% (v / v) inoculation rate into test tubes containing 200 mL of LB liquid medium, and kanamycin was added to a final concentration of 50 μg / mL. The cultures were then cultured at 37 °C and 200 rpm for 3 h until OD (occurrence limit) was reached. 600 The initial concentration was 0.6, followed by the addition of 1 mM IPTG as an inducer. The cells were cultured at 30°C and 200 rpm for 8 h, then centrifuged at 5000 rpm and 4°C for 10 min to collect the cells. The precipitate was resuspended in 50 mM Tris-HCl buffer (pH 7.4) to obtain the OD. 600A 600-cell suspension of recombinant E. coli was sonicated (30% power, 30 min), centrifuged (12000 rpm, 30 min), and the supernatant was obtained. The supernatant was filtered through a 0.22 μm filter membrane to obtain 30 mL of each of the crude enzymes H109, ​​H652, H036, and H002. A HisTrap HP (16*25mm, 5mL) IMAC column (purchased from Topvan) was equilibrated with 50mL of binding buffer (50mM pH 7.4 Tris-HCl, 0.5M NaCl, 20mM imidazole). After the column was equilibrated, the crude enzyme filtrate was pushed into the Akta system through the injection loop. The target enzyme bound to the column through affinity, while other proteins were eluted. The target enzyme was then eluted from the column using elution buffer (50 mM Tris-HCl pH 7.4, 0.5M NaCl, 500 mM imidazole). The collected eluents were purified enzymes H109, ​​H652, H036, and H002, respectively. The four tubes of purified enzyme solution were desalted and concentrated using a 10 kDa ultrafiltration tube. The treatment method was to centrifuge three times at 5000 rpm for 30 min. After the first two centrifugations, 50 mM pH 7.4 PBS buffer was added to displace the high concentrations of NaCl and imidazole in the purified enzyme solution, finally yielding 1 mL of purified enzyme. Figure 2 The concentrations of four purified enzymes were determined using a BCA protein quantification kit (purchased from Sangon Biotech Co., Ltd.). The concentrations of purified enzyme H109 were 4.5 g / L, H652 was 3.5 g / L, H036 was 3.8 g / L, and H002 was 3.2 g / L.

[0049] Example 4: Activity analysis of recombinant SGNH family esterases

[0050] The recombinant SGNH family esterases purified in Example 3 were used as substrates for activity assays, and the activities of four purified enzymes were compared. 1.5 mL centrifuge tubes were numbered 1, 2, 3, 4, and 5, with tubes 1, 2, 3, and 4 serving as the experimental groups (three replicates) and tube 5 as the control group. 500 μL of 100 mM PBS buffer (pH 6.5), 250 mM triacetin, and 500 mM hydrogen peroxide were added to each well. Then, 10 μL of purified enzymes H109, ​​H652, H036, and H002 diluted 10-fold were added to tubes 1, 2, 3, and 4, respectively. An equal volume of water was added to tube 5 (control). The reaction mixture was then brought to a final volume of 1 mL with water. The reaction was carried out at 30°C and 200 rpm for 20 min. The amount of peracetic acid synthesized was determined using liquid chromatography. The specific activities of the four enzymes were calculated: H109 pure enzyme had a specific activity of 17.3 U / mg, H652 pure enzyme had an activity of 10.6 U / mg, H036 pure enzyme had an activity of 6.3 U / mg, and H002 pure enzyme had an activity of 3.4 U / mg. The pure enzyme with the highest activity, H109, ​​was named SH2.

[0051] Example 5: Determination of the optimal temperature and thermal stability of recombinant SGNH family esterase SH2

[0052] (1) Determination of optimal temperature:

[0053] The SH2 enzyme prepared in Example 3 was incubated at a concentration of 45 mg / L in 50 mM pH 6.5 PBS buffer containing 250 mM triacetin and 500 mM hydrogen peroxide at 20°C, 30°C, 37°C, 40°C, 50°C, 60°C, 65°C, 70°C, 80°C, and 90°C for 5 min, and the amount of peracetic acid synthesized was measured.

[0054] The results showed that the enzyme activity of SH2 remained stable at over 95% after reacting at 30-60℃ for 5 minutes. Figure 3 ).

[0055] (2) Determination of temperature stability:

[0056] The SGNH family esterase SH2 prepared in Example 3 was added to 50 mM pH 6.5 PBS buffer at a concentration of 45 mg / L. The mixture was incubated at 37°C, 50°C, and 80°C for 5 min, 10 min, 20 min, 30 min, and 60 min, respectively. Then, triacetin and hydrogen peroxide were added to a final concentration of 250 mM for enzymatic reaction. Untreated enzyme solution was used as a control. The reaction was carried out at 30°C and 200 rpm for 5 min, and the enzyme activity of SH2 was measured.

[0057] The results are as follows Figure 4As shown, the enzyme activity remained basically stable after incubation at 37℃ for 60 min, and after incubation at 50℃ and 80℃ for 60 min, the enzyme activity remained above 50%, indicating that the recombinant SGNH family esterase SH2 has the strongest thermostability.

[0058] Example 6: Determination of the optimal pH and pH stability of recombinant SGNH family esterase SH2

[0059] (1) Determination of the optimal pH of the enzyme:

[0060] SH2 enzyme was catalyzed at a concentration of 45 mg / L at 37 °C with 50 mM glycine-hydrochloric acid buffer at different pH values ​​(1.0, 2.0, 3.0, and 4.8), 50 mM PBS buffer at different pH values ​​(4.8, 6.0, 7.0, 8.0, and 8.5), and 50 mM borax-NaOH buffer at different pH values ​​(8.5, 10.0, 11.0, and 12.0) with a final concentration of 250 mM triacetin and 500 mM hydrogen peroxide. The results showed that... Figure 5 The SH2 enzyme has a higher activity between pH 4.5 and 9.5, maintaining above 95%.

[0061] (2) Determination of pH stability:

[0062] SH2 enzyme was incubated at a concentration of 45 mg / L at 37℃ in 50 mM citrate-citrate buffer at pH 2.0, 3.5, and 4.8; in 50 mM PBS buffer at pH 4.8, 6.4, 7.6, and 8.8; and in 50 mM borax-NaOH buffer at pH 8.8, 10.0, 11.0, and 12.0 for 60 min. Then, triacetin and catalase were added to a final concentration of 250 mM and 500 mM, respectively, to promote the enzymatic reaction for 5 min. Untreated enzyme solution was used as a control. The results showed ( Figure 6 After being treated with buffer solutions ranging from pH 3.5 to pH 11.0 at 37°C for 60 min, the SGNH family esterase SH2 maintained more than 50% of its activity, indicating that it has excellent pH stability.

[0063] The above results demonstrate that the SGNH family esterase SH2 enzyme screened from the hot spring environment of Tibet exhibits excellent thermal stability, ensuring that it will not be deactivated by subsequent processing after being added to the product. Its wide range of optimal temperature and pH makes SH2 enzymes suitable for a broader range of applications, meeting the diverse catalytic requirements of various fields such as oral care, personal care, and pharmaceuticals.

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. SGNH family esterase SH2, characterized in that, The amino acid sequence of the SGNH family esterase SH2 is shown in SEQ ID NO.

1.

2. A composition containing the SGNH family esterase SH2 as described in claim 1.

3. The gene encoding the SGNH family esterase SH2 as described in claim 1.

4. A recombinant vector, characterized in that, The vector contains the complete coding reading frame sequence of the gene described in claim 3.

5. A recombinant strain, characterized in that, It expresses the SGNH family esterase SH2 as described in claim 1, or contains the recombinant vector as described in claim 4.

6. Recombinant Escherichia coli, characterized in that, Using pET-28a as a vector, the SGNH family esterase SH2 described in claim 1 was expressed in Escherichia coli BL21(DE3).

7. A method for preparing the SGNH family esterase SH2 according to claim 1, characterized in that, The recombinant Escherichia coli of claim 6 was cultured to induce the expression of the SGNH family esterase SH2.

8. The method according to claim 7, characterized in that, The induction is performed by culturing the recombinant Escherichia coli to an OD600 of 0.5-0.8 and inducing it with IPTG at a concentration of 0.5-1 mM.

9. The method according to claim 8, characterized in that, The method also includes collecting recombinant strain cells, lysing the cells, and collecting the SGNH family esterase SH2.

10. The application of the SGNH family esterase SH2 as described in claim 1 in the preparation of pharmaceutical or daily chemical products.

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