Esterase for preparing sucralose, gene encoding esterase, recombinant vector containing gene, and engineered bacterium

Abstract

Disclosed are an esterase for preparing sucralose and a use thereof. The amino acid sequence of the esterase is as shown in SEQ ID NO. 1, and the esterase can be used to catalyze the hydrolysis of sucralose-6-ethyl ester to prepare sucralose. The present invention further provides a gene encoding the esterase, a recombinant vector containing the gene, and a genetically engineered bacterium obtained by transforming the recombinant vector. The present invention is the first to apply the esterase having the amino acid sequence as shown in SEQ ID NO. 1 to the hydrolysis of sucralose-6-ethyl ester for preparing sucralose, and achieves excellent results, having an enzyme activity up to 5427 U / g, a substrate yield up to 100%, fewer by-products, a short reaction cycle, and a simple process, thereby greatly reducing production costs.

Description

An esterase for preparing sucralose, a gene encoding the esterase, a recombinant vector containing the gene, and engineered bacteria. Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of an esterase, and more particularly to the application of an esterase in the preparation of sucralose using sucralose-6-ethyl ester as a substrate hydrolysis reaction. Background Technology

[0002] Sucralose (TGS) is a highly concentrated sweetener. Developed and patented in 1976 by Tyrell & Co. and University College London, it entered the market in 1988. It is the only functional sweetener made from sucrose, achieving a sweetness approximately 600 times (400-800 times) that of sucrose. Sucralose is characterized by being calorie-free, highly sweet, having a pure sweet taste, and being very safe, making it one of the most ideal sweeteners. The deacetylation of sucralose-6-ethyl ester to produce sucralose is one of the key steps in its preparation.

[0003] The deacetylation of sucralose-6-ethyl ester to produce sucralose is generally achieved through chemical methods. While methods exist that achieve high conversion rates and continuous deacetylation, these require methanol-based reactants, placing high demands on the construction standards of subsequent production facilities and increasing safety and production costs. Furthermore, obtaining relatively pure sucralose necessitates complex subsequent separation and purification processes. Enzymes, with their high specificity and safety, are environmentally friendly, and enzymatic synthesis can significantly avoid these problems. Currently, there are some research reports on the use of biological methods to catalyze the hydrolysis of sucralose-6-ethyl ester to produce sucralose.

[0004] In 2007, RATNAM et al. first reported the enzymatic deacylation process for the preparation of sucralose, achieving yields of over 96% using both free and immobilized enzymes (Aspergillus oryzae ATCC 26850 lipase, B. lichenformis Alcalase (Novozymes)). However, this process is quite expensive and time-consuming.

[0005] In 2013, CHAUBRY et al. proposed using a Bacillus subtilis immobilized cell reactor to catalyze deacylation, achieving a 100% single-step hydrolysis rate. Furthermore, sucralose could be directly concentrated and crystallized to achieve 100% purity without purification. Moreover, the bioconversion rate remained at 100% even after more than three consecutive cycles of continuous use of the immobilized cell reactor. This study also demonstrated that endogenous Arthrobacter sp. lipase (ABL), B. subtilis RRL-1789 lipase, as well as commercial enzymes Pig Liver Esterase (PLE) and Amano AS, could all achieve conversion rates exceeding 90%.

[0006] In 2016, Sun Jie et al. used Bacillus amyloliquefaciens WZS01 to hydrolyze sucralose-6-ethyl ester to obtain sucralose. Under the reaction conditions of substrate concentration of 75 mM, reaction system of 20% methanol and 80% phosphate buffer (25 mM; pH 7.1), and temperature of 36℃, after hydrolyzing sucralose-6-acetate with 1% stem cells for 24 hours, the yield of sucralose reached more than 99% and the purity reached 98.0%. However, as the substrate concentration increased, the yield of sucralose decreased.

[0007] In summary, existing enzyme-catalyzed deacetylation techniques for the preparation of sucralose from 6-ethyl sucralose have problems such as large enzyme addition amounts, long processing time, low enzyme catalytic efficiency, and high costs, which cannot meet the needs of industrial production.

[0008] This invention innovatively employs gene recombination technology to construct an engineered *Escherichia coli* strain capable of expressing sucralose-6-ethyl ester deacetylation esterase. The enzyme produced by this strain exhibits highly efficient deacetylation capabilities of sucralose-6-ethyl ester, with hydrolysis efficiency meeting the requirements for industrial production. Currently, there are no reports, either domestically or internationally, of this enzyme using sucralose-6-ethyl ester as a hydrolysis substrate. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide an application of a highly efficient biological deacetylesterase in the preparation of sucralose.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] An esterase for preparing sucralose, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] The application of esterase in the preparation of sucralose: using the esterase with the amino acid sequence shown in SEQ ID NO.1 as a catalyst, sucralose-6-ethyl ester as a substrate, and PBS solution as a pH adjuster, sucralose was prepared by hydrolysis.

[0013] The application of esterase in the preparation of sucralose involves adding wet bacterial cells to a PBS solution containing sucralose-6-ethyl ester to obtain a mixture containing sucralose.

[0014] To apply esterase to sucralose, weigh 0.1g of wet bacterial cells and add them to 300-600ml of PBS solution containing 1-5% β-glucose. React at 30-40℃ for 10-60min.

[0015] A gene encoding the esterase.

[0016] A recombinant vector containing the above-mentioned genes.

[0017] A genetically engineered bacterium obtained by transformation of the above-mentioned recombinant vector.

[0018] A genetically engineered bacterium, wherein the genetically engineered bacterium is obtained by transforming the positive plasmid pET30a into Escherichia coli BL21(DE3) competent cells.

[0019] Using IPTG as an inducer, high-density fermentation was carried out in the fermentation medium to induce expression, and the recombinant bacterial cells with the expressed amino acid sequence as shown in SEQ ID NO.1 were obtained.

[0020] In the high-density fermentation-induced expression process, IPTG is added to induce expression when the optical density (OD) of the bacterial cells reaches 20-25.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention marks the first application of a deacetylesterase (CcDac) with the amino acid sequence shown in SEQ ID NO.1 to the hydrolysis of sucralose-6-ethyl ester to prepare sucralose, achieving excellent results with an enzyme activity as high as 5427 U / g. The deacetylesterase with the amino acid sequence shown in SEQ ID NO.1 exhibits high conversion rate (approximately 100%) of the substrate sucralose-6-ethyl ester, high catalytic efficiency, thorough reaction, and a simple induction process for protein preparation, significantly reducing production costs. The sequence in SEQ ID NO.1 is artificially synthesized. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1. Construction diagram of deacetylesterase expression plasmid;

[0025] Figure 2 Electrophoresis diagram of deacetylesterase-expressing proteins;

[0026] Lane M is the protein marker. Lanes 1 and 2 are the protein supernatants after the host bacteria BL21(DE3) transformed with the empty vector pET-30(+) and the host bacteria BL21(DE3) transformed with the recombinant plasmid pET-30(+)-CcDac, respectively, after inducing expression of the protein supernatant of the lysed bacterial cells.

[0027] Figure 3. IPTG-induced protease activity at different concentrations;

[0028] Figure 4. Growth and enzyme activity curves of high-density fermentation culture in a 100L tank;

[0029] Figure 5. Liquid chromatography chromatograms of sucralose and sucralose-6-ethyl ester standards;

[0030] Figure 6. Liquid chromatography spectrum of ammonium acetate standard;

[0031] Figure 7. Liquid phase diagram of the deacetylation enzymatic reaction. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] During the production process, for ease of communication, sucralose-6-ethyl ester is referred to as B sugar, and sucralose is referred to as C sugar.

[0034] The method for detecting sucralose content in the following examples is as follows: HPLC detection is used, specifically:

[0035] Column: ShimNex UP C18 (4.6*150mm, 5μm);

[0036] Mobile phase: A: water, B: methanol;

[0037] Evaporative light scattering detector (ELSD);

[0038] Column temperature: 40℃;

[0039] Flow rate: 1.0 ml / min;

[0040] Drift tube temperature: 45℃;

[0041] Injection volume: 10 μL;

[0042] Gradient elution program: T / min (B%): 0 (40), 8 (90), 8.01 (40), 15 (40).

[0043] Molar conversion rate calculation formula: Molar conversion rate = (Molar amount of sucralose generated / Initial molar amount of sucralose-6-ethyl ester added) × 100%.

[0044] Enzyme activity assay method:

[0045] Definition: One unit of enzyme activity (U) is the amount of enzyme required to generate / consume 1 μmol of C / B sugar in 1 minute at a reaction temperature of 38℃.

[0046] (1) Enzyme-catalyzed reaction

[0047] Weigh 0.1% (0.1 g) of wet bacterial cells into a shake flask, add 100 ml of PBS (0.15 M, pH 7.4) solution containing 2% B sugar, react at 37 °C and 220 rpm for 3 min, take 250 μl of reaction solution and mix with 750 μl of methanol, and detect the amount of C sugar generated by liquid chromatography.

[0048] (2) Formula for calculating enzyme activity:

[0049] Enzyme activity = C-glucose production (umol) ÷ reaction time (min) ÷ wet cell weight (g)

[0050] The application of the deacetylesterase of the present invention in the hydrolysis preparation of sucralose specifically includes the following steps:

[0051] Engineered *E. coli* strains expressing deacetylesterase were induced, centrifuged at 8000 rpm for 10 min, and the supernatant was discarded to obtain wet cells of the engineered *E. coli* strains expressing deacetylesterase. Sucralose-6-ethyl ester was dissolved in 0.15 M PBS buffer (pH 7.4) and sonicated until clear to obtain mixture 1. The bio-enzyme was added to mixture 1 to obtain mixture 2. Mixture 2 was placed in a shaker at 38℃ and 220 rpm for 2 hours. After the reaction, a sample was taken, diluted 4-fold with pure methanol, and filtered through a 0.22 μm organic filter to obtain the sucralose liquid chromatography sample.

[0052] See Figure 1 for the main steps of the deacetylesterase expression plasmid. Figure 1 includes the following steps:

[0053] 1. Synthesize the CcDac gene.

[0054] 2. PCR amplification of the CcDac gene fragment.

[0055] 3. Double digestion with NdeI and XhoI. The amplified CcDac gene fragment is double-digested with two restriction endonucleases, NdeI and XhoI, to generate corresponding sticky ends. Although NdeI and XhoI enzymes have different recognition sequences, the sticky ends generated after digestion are complementary, thus achieving efficient ligation of the gene fragment to the vector.

[0056] 4. T4 ligase ligation. The digested CcDac gene fragment is ligated to the vector pET-30a(+). The ligation process uses T4 ligase, usually performed at room temperature or overnight. T4 ligase catalyzes the formation of phosphodiester bonds between DNA strands and is a commonly used ligase in gene cloning.

[0057] 5. Construct the plasmid backbone. pET-30a(+) was chosen as a commonly used expression vector containing multiple functional elements, such as the T7 lac promoter, multiple cloning site, lacI gene, and KanR (kanamycin resistance gene), including ori (ori), T7lac promoter, pET-30a(+) sequence, fl ori (ori of replication), lacI gene, and KanR gene. pET-30a(+) provides the T7lac promoter for gene expression, the lacI gene for regulation, and the KanR gene for selection; ori and fl ori ensure plasmid replication in the host cell.

[0058] 6. Transformation and screening: The ligated plasmids are transformed into *E. coli* or other host cells. Positive clones containing the KanR gene are selected through antibiotic screening.

[0059] 7. Verification and follow-up operations:

[0060] Positive clones were validated to ensure that the CcDac gene was correctly inserted into the plasmid and that the plasmid structure was intact.

[0061] Sequencing was used to further confirm the correctness of the gene insertion.

[0062] The validated plasmid was used to express and purify the CcDac protein.

[0063] Example 1: Referring to Figure 1, the following details the construction and identification steps of the recombinant Escherichia coli expressing deacetylesterase according to the present invention:

[0064] 1. Plasmid construction

[0065] Escherichia coli BL21(DE3) and DH5α competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid pET-30a(+) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; all gene and primer synthesis and sequencing services were provided by Suzhou Genewise Biotech Co., Ltd. Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases NdeI and XhoI were purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0066] (1) Deacetylesterase gene acquisition

[0067] The deacetylesterase (CcDac) gene was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0068] Design amplification primers:

[0069] Upstream primer CcDac-pET-F:

[0070] (The bolded text indicates the NdeI restriction site)

[0071] Downstream primer CcDac-pET-F:

[0072] (The bolded text indicates the XhoI restriction site)

[0073] The PCR amplification procedure is as follows: [The procedure is described in the original text, but the translation is incomplete and cannot be translated.] Max Super-Fidelity DNA Polymerase was used to amplify the target fragment. The specific reaction system and amplification procedure are shown in Table 1.

[0074] Table 1. PCR reaction system and amplification procedure for Phanta DNA polymerase.

[0075] (2) Enzyme digestion and enzyme chain

[0076] The amplified DNA fragments or plasmids are digested with restriction endonucleases. Different restriction enzymes require different digestion times, and the appropriate digestion time must be selected according to the specific requirements of the restriction enzyme. This invention uses the Fastdigest series of restriction endonucleases, and the reaction system is shown in Table 2.

[0077] Table 2 Enzyme digestion reaction system

[0078] After purification following enzyme digestion, the T4 DNA ligase, purified DNA fragment, and vector plasmid fragment were mixed and incubated in a metal bath at 22°C for 2.5 hours to complete the ligation reaction. The ligation reaction system is shown in Table 3.

[0079] Table 3 Enzyme ligation reaction system

[0080] (3) Verification of transformation

[0081] The above enzyme-linked product was transformed (using conventional chemical transformation) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 μg / ml kanamycin) were sent for sequencing. After successful sequencing, the positive plasmid pET30a(+)-CcDac was returned.

[0082] 2. Construction of deacetylesterase expression strains

[0083] The constructed plasmid was transformed (using conventional chemical transformation) into competent Escherichia coli BL21(DE3) cells and cultured overnight. Validation primers (upstream primer test-pET-F: 5'-CATCGGTGATGTCGGCGATATAG-3', downstream primer test-pET-R: 5'-CCGGATATAGTTCCTCCTTTCAGCA-3') were designed to verify single colonies grown in a screening plate (50 μg / ml kanamycin) using colony PCR. This constructed an inducible recombinant E. coli strain, E. coli W30E, expressing deacetylesterase.

[0084] Example 2: The induction and expression process of recombinant Escherichia coli W30E of the present invention is as follows:

[0085] 1. Induced expression

[0086] Single colonies of engineered E. coli W30E were inoculated into LB medium (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) and cultured overnight at 37°C and 220 rpm. A 1% inoculum was then transferred to 200 ml of TB medium (TB medium formula: 12 g / L peptone, 24 g / L yeast extract, 0.4% glycerol, 0.017 M potassium dihydrogen phosphate, 0.072 M dipotassium hydrogen phosphate) and cultured at 37°C and 220 rpm until the OD600 reached 0.8. IPTG was added to a final concentration of 0.1 mM, and the mixture was incubated at 16°C for 20 h to induce expression. The mixture was then centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was kept for later use.

[0087] 2. Protein electrophoresis

[0088] Electrophoresis buffer: Weigh 7.5g Tris, 36g glycine, and 2.5g SDS, dissolve in water, and bring the volume to 500ml. Store at 4℃. Dilute 5 times before use.

[0089] Staining solution: Weigh 0.2g of Coomassie Brilliant Blue R-250, measure 80ml of anhydrous ethanol and 20ml of glacial acetic acid, dissolve in water and bring the volume to 200ml. Store at 4℃. It can be recycled after use.

[0090] Decolorizing solution: Measure 450ml of anhydrous ethanol, 50ml of glacial acetic acid, and 550ml of distilled water, mix well, and store at room temperature.

[0091] Prepare 10% engineered and control bacterial suspensions, and homogenize them using high-pressure homogenization (900 bar). Take 16 μL of the supernatant (intracellular soluble protein) and the precipitate resuspended in 8M urea (inclusion bodies), mix with 4 μL of 5x protein loading buffer, and treat at 100℃ for 10 min. Spot the mixture into the wells of a protein gel and electrophoresis at 150V for 60 min. Place the gel in staining solution, microwave for 2 min, and then place it on a multi-functional destaining shaker for further staining for 15 min. Discard the staining solution, wash the protein gel once with water, and then place it in destaining solution on a multi-functional destaining shaker for destaining. Change the destaining solution periodically depending on the destaining effect. The total destaining time is approximately 2-4 hours. Observe the destaining gel directly on a gel imaging system. The protein electrophoresis results are shown in Figure 2. Compared with the control strain (BL21(DE3) strain that does not express deacetylesterase), a target protein band (35.9 kDa) is clearly present at the 35 kDa position.

[0092] 3. Enzyme activity assay

[0093] Following the enzyme activity assay method in the basic examples, 0.1% (0.1 g) of centrifuged precipitate (wet bacterial cells) was weighed into a shake flask, and 100 ml of PBS (0.15 M, pH 7.4) containing 2% B sugar was added. The reaction was carried out at 38°C and 220 rpm for 3 min. 250 μL of the reaction solution was mixed with 750 μL of methanol, and the amount of C sugar produced was detected by HPLC. The specific activity of the induced enzyme expression of recombinant E. coli W30E was 1443 U / g.

[0094] Example 3: Optimization of IPTG induction concentration after induction of recombinant E. coli W30E expression:

[0095] Following the induction expression method described in Example 2, IPTG concentrations were set to 0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM, and 0.5 mM, respectively. After induction, bacterial cells were collected by centrifugation, and the enzyme activity of the expressed bacterial cells induced by different IPTG concentrations was measured. Samples were analyzed by HPLC, as shown in Figure 3. At an IPTG induction concentration of 0.025 mM, the engineered strain E. coli W30E exhibited the highest deacetylesterase activity, at 1681.3 U / g.

[0096] Example 4: The process of high-density fermentation and expression induction of engineered strain E. coli W30E in a 100L tank is as follows:

[0097] Seed culture medium (LB): sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L.

[0098] Fermentation medium: potassium dihydrogen phosphate 7g / L, ammonium sulfate 1g / L, citric acid monohydrate 1.6g / L, magnesium sulfate heptahydrate 2g / L, yeast powder 2g / L, vitamin B1 1ml / L (B1 concentration prepared at 0.5g / L, filtered and sterilized), biotin 1ml / L (concentration prepared at 0.1g / L, filtered and sterilized), trace elements 1ml / L (filtered and sterilized), defoamer 0.2ml / L.

[0099] Trace elements: calcium chloride 10g / L, ferrous sulfate heptahydrate 6g / L, zinc sulfate heptahydrate 2g / L, manganese chloride tetrahydrate 2g / L, cobalt chloride hexahydrate 0.2g / L, copper sulfate pentahydrate 0.2g / L (filtered for sterilization).

[0100] Specifically, the steps include the following:

[0101] (1) Pick single colonies of engineered bacteria from a plate and inoculate them into 5ml LB test tubes. Incubate at 37℃ and 200rpm for 12h. Inoculate the plates with an OD of 3-4 into the fermenter.

[0102] (2) Inoculate 4% of the solution into a 100L fermenter and add 50μg / ml of kanamycin;

[0103] (3) Add carbon source to the bottom tank until the final concentration is 5g / L, fill the liquid system with 0.5, set the initial tank pressure to 0.03Mpa (upper limit 0.08Mpa), the rotation speed to 200rpm, the initial air volume to 0.8vvm, the pH to 6.9±0.1, and control the dissolved oxygen linkage speed at 30%±5%. When the sugar in the bottom tank is exhausted, use the minimum sugar replenishment process to replenish sugar: set the sugar replenishment speed, replenish for 8min, stop for 2min (verification is frequent in the early stage, and can be verified once every half hour in the later stage of fermentation). Mainly observe whether the dissolved oxygen curve can rise after stopping sugar for 2 minutes. If it rises, it means that the sugar replenishment is appropriate, and the sugar replenishment speed can be increased. If it cannot rise, the sugar replenishment flow rate is reduced accordingly until a) the maximum activity of the strain is reached (the rotation speed when the dissolved oxygen does not rise after increasing the replenishment speed, and the replenishment speed is reduced so that the dissolved oxygen can rise) or b) the maximum performance of the fermenter (the maximum rotation speed, aeration volume and pressure of the fermenter are reached. After the maximum performance of the fermenter is reached, the replenishment speed can be appropriately reduced to keep the dissolved oxygen from being too low).

[0104] (4) When the OD reaches 20-25 (approximately), add 0.025mM IPTG to induce expression;

[0105] (5) After adding the inducer IPTG, take a sample (100ml) every 3 hours, centrifuge and store the precipitate at -80℃. Stop fermentation when the enzyme activity reaches its maximum.

[0106] Following the enzyme activity assay method in the basic embodiment, 0.1% (0.1 g) of the above centrifuged precipitate (wet bacterial cells) was weighed into a shake flask, and 100 ml of PBS (0.15 M, pH 7.4) solution containing 2% B sugar was added. The mixture was reacted at 38°C and 220 rpm for 3 min. 250 μL of the reaction solution was then mixed with 750 μL of methanol, and the amount of C sugar generated was detected by HPLC. The specific activities of the induced enzyme in recombinant *E. coli* W30E at 3.3 h, 6.2 h, 9.2 h, 12.2 h, 13.2 h, 14.2 h, 15.2 h, and 16.2 h after the addition of the inducer were 1518 U / g, 3093 U / g, 4493 U / g, 5147 U / g, 4862 U / g, 5126 U / g, 5149 U / g, and 5421 U / g, respectively. This induction expression process significantly improved the enzyme activity of E. coli W30E deacetylesterase, and the enzyme activity reached its highest and stabilized (5126-5421 U / g) after 19.6-23.6 h of fermentation (12.2-16.2 h of induction).

[0107] Application Example 1: Preparation of sucralose catalyzed by deacetylesterase

[0108] Weigh 0.025% (0.1 g) of wet bacterial cells (e.g., E. coli W30E, precipitated after 16.2 h of induction during high-density fermentation in a 100L tank) into a shake flask, add 400 ml of PBS (0.15 M, pH 7.4) containing 2% β-glucose, and react at 37°C and 220 rpm for 30 min. After the reaction, take a sample, dilute it 4-fold with pure methanol, and filter it through a 0.22 μm organic filter membrane to obtain the sucralose liquid chromatography sample.

[0109] The sample solution, along with ammonium acetate, sucralose-6-ethyl ester, and sucralose standards, were subjected to HPLC analysis, as shown in Figure 7. Figure 5 of this invention shows the HPLC chromatograms of sucralose and sucralose-6-ethyl ester standards; Figure 6 shows the HPLC chromatogram of ammonium acetate standards. As can be seen from the HPLC chromatogram of the deacetylation enzymatic reaction solution in Figure 7, only peaks of ammonium acetate and PBS (retention time 1.405 min) and a peak of sucralose (3.129 min) were present in the sample, indicating virtually no reaction impurities and a complete reaction.

[0110] In this invention, the amino acid sequence of the esterase is SEQ ID NO.1 (Clostridium clariflavum), and the specific sequence is as follows:

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An esterase for preparing sucralose, characterized in that: The amino acid sequence of the esterase is shown in SEQ ID NO.

1.

2. The application of the esterase according to claim 1 in the preparation of sucralose, using the esterase with the amino acid sequence shown in SEQ ID NO.1 as a catalyst, sucralose-6-ethyl ester as a substrate, and PBS solution as a pH adjuster, to prepare sucralose by hydrolysis.

3. The application of the esterase according to claim 2 in the preparation of sucralose, characterized in that, Wet bacterial cells were added to a PBS solution containing sucralose-6-ethyl ester to produce a mixture containing sucralose.

4. The application of the esterase according to claim 2 in the preparation of sucralose, characterized in that, Weigh 0.1g of wet bacterial cells and add them to 300-600ml of PBS solution containing 1-5% B sugar. React at 30-40℃ for 10-60min.

5. A gene encoding the esterase according to claim 1.

6. A recombinant vector containing the gene according to claim 5.

7. A genetically engineered bacterium obtained by transformation of the recombinant vector according to claim 5.

8. The genetically engineered bacteria according to claim 7, characterized in that: The genetically engineered bacteria were obtained by transforming the positive plasmid pET30a into Escherichia coli BL21(DE3) competent cells.

9. The genetically engineered bacterium according to claim 7, characterized in that, Furthermore, IPTG was used as an inducer to induce expression through high-density fermentation in a fermentation medium.

10. The genetically engineered bacteria according to claim 9, wherein during the high-density fermentation induction expression process, IPTG is added to induce expression when the optical density (OD) of the bacterial cells is 20-25.

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