Method for efficiently preparing trehalose and application thereof

Abstract

This invention discloses a highly efficient method for preparing trehalose and its application, relating to the field of enzyme engineering. The invention obtains mutants with significantly increased enzyme activity by site-directed mutagenesis (D142T, F183A, R216Y) of trehalose synthase derived from *Streptomyces cerevisiae*. The triple mutant (D142T / F183A / R216Y) exhibits a specific enzyme activity of 72.1 U / mg, a 105% increase compared to the wild type; after incubation at 40℃ for 1 hour, the enzyme activity retention rate exceeds 90%. Using recombinant *E. coli* for shake-flask fermentation with 300 g / L maltose as a substrate, the trehalose yield reaches 269.8 g / L, with a conversion rate of 89.9%. The mutants and preparation method provided by this invention have advantages such as high enzyme activity, strong thermal stability, and high conversion rate, making them suitable for the industrial production of trehalose.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and fermentation engineering technology, specifically to a highly efficient method for preparing trehalose and its application. Background Technology

[0002] Trehalose is a non-reducing disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond, and is widely found in bacteria, fungi, yeast, and insects. It possesses unique biological functions, such as protecting the stability of biological macromolecular structures, providing stress resistance (to drying, low temperatures, hyperosmotic pressure, etc.), and inhibiting starch retrogradation and protein denaturation. Therefore, it has wide applications in the food, pharmaceutical, cosmetic, and agricultural fields.

[0003] Currently, the main methods for producing trehalose include direct extraction, fermentation, chemical synthesis, and enzymatic conversion. Among these, enzymatic conversion is considered the most promising industrial production method due to its mild reaction conditions, high specificity, and lack of pollution. Trehalose synthase (TrES) can catalyze the production of trehalose from maltose, representing an economical and efficient synthetic route. However, natural trehalose synthases suffer from low enzyme activity, poor thermal stability, and insufficient conversion rates, limiting their industrial application.

[0004] To address the aforementioned issues, existing technologies (such as CN111378631B and CN111500566B) have used directed evolution to mutate trehalose synthase from *Streptomyces coelicolor*, resulting in mutants with increased enzyme activity. Furthermore, CN108977480B improved trehalose yield by optimizing the fermentation medium composition (adding fatty acids, lactose, etc.). CN120060405A optimized the fermentation process using a composite enzyme catalysis technique. However, the catalytic efficiency and stability of existing mutants still need further improvement, and systematic mutation studies targeting key amino acid sites are lacking.

[0005] Therefore, this invention, based on trehalose synthase derived from Streptomyces azurite, has obtained a series of novel mutants (including single-point, double-point, and triple-point mutants) through rational design and site-directed mutagenesis. These mutants significantly improve enzyme activity, thermal stability, and trehalose conversion rate, and optimize the fermentation process, providing a new strategy for the efficient industrial production of trehalose. Summary of the Invention

[0006] To achieve the above-mentioned objective, the present invention first provides a trehalose synthase mutant, wherein the amino acid sequence of the trehalose synthase mutant is the sequence shown in SEQ ID NO: 9 obtained by mutation of at least one of D142T, F183A, and R216Y.

[0007] The present invention also provides that the mutant is a double mutant D142T / R216Y or a triple mutant D142T / F183A / R216Y.

[0008] The present invention also provides nucleic acid molecules of the above-mentioned mutants.

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid molecules, wherein the vector is pET-28a or pXMJ19.

[0010] The present invention also provides a host cell containing the above-mentioned recombinant expression vector, wherein the host cell is Escherichia coli or Corynebacterium glutamicum.

[0011] The present invention also provides an efficient method for preparing trehalose, which utilizes the above-mentioned host cells for fermentation culture and converts maltose as a substrate to produce trehalose.

[0012] In some embodiments, the following steps are included:

[0013] a) Host cells were inoculated into a fermentation medium to induce the expression of a trehalose synthase mutant;

[0014] b) Collect bacterial cells and carry out whole-cell catalytic reactions using maltose as a substrate;

[0015] c) Extract and purify trehalose.

[0016] In some embodiments, the fermentation medium contains 100-500 g / L of maltose, IPTG as the inducer, and an induction temperature of 16-28°C.

[0017] The present invention also provides the application of the above mutant as a catalyst for trehalose production in the food, pharmaceutical or cosmetic fields.

[0018] This invention also provides a method for improving trehalose conversion rate by using the above-mentioned mutant to catalyze maltose conversion. The conversion rate can reach over 75%.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The novel mutant (D142T / F183A / R216Y) obtained by rational design in this invention has an enzyme activity that is 1.8 times higher than that of the wild type and a trehalose conversion rate that is 75.2% higher;

[0021] (2) The mutant exhibits significantly enhanced thermostability, with enzyme activity retention exceeding 85% after incubation at 40℃ for 1 hour;

[0022] (3) The optimized fermentation process can achieve high-concentration substrate (300g / L maltose) conversion, with high production efficiency and low cost;

[0023] (4) The recombinant strain has good genetic stability and is suitable for large-scale industrial production. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Construction of Trehalose Synthase Mutant

[0026] 1. See CN111378631B using the genome of Streptomyces coelicolor GDM4.65 as a template, using ScT-F: 5'-tgggtcgcggatccgaattcATGATCGTCAACGAGCCCGT-3' (SEQ ID No. 1);

[0027] ScT-R: 5'-tcgagtgcggccgcaagcttTCAGGCGGCGTCCTTGCGCA-3' (SEQ ID No. 2), PCR amplification of the trehalose synthase gene (TreS, SEQ ID NO: 9), cloned into the pET-28a vector, to obtain the recombinant plasmid pET28a-TreS.

[0028] SEQ ID NO: 9:

[0029]

[0030] 2. Designed site-directed mutagenesis primers (Table 1), and introduced the mutation sites (D142T, F183A, R216Y) using overlap PCR technology.

[0031] Table 1. Site-directed mutagenesis primer sequences

[0032] mutation site Primer sequence (5'→3') D142T-F 5'-ccgacggcccctacggcacctactacgtgtgggccgacga-3'(SEQ ID NO: 3) D142T-R 5'-tcgtcggcccacacgtagttggtcgccgtaggggcctgccg-3' (SEQ ID NO: 4) F183A-F 5'-gtacttcttccaccgcttcgcatcccaccagccggatctc-3' (SEQ ID NO: 5) F183A-R 5'-gagatccggctggtgggatgcgaagcggtggaagaagtac-3'(SEQ ID NO: 6) R216Y-F 5'-acctgggaatcgacggcttctacctcgatgccgtgccgta-3'(SEQ ID NO: 7) R216Y-R 5'-tacggcacggcatcgaggtagaagccgtcgattcccaggt-3' (SEQ ID NO: 8)

[0033] 3. PCR reaction system: template pET28a-TreS 1 μL, primers 0.5 μL each, PrimeSTAR enzyme 25 μL, ddH2O added to 50 μL. Program: 95℃ / 5 min; 95℃ / 30 s, 55℃ / 30 s, 72℃ / 3 min, 30 cycles; 72℃ / 10 min.

[0034] 4. The vector pET28a was double-digested with EcoRI and HindIII in a water bath at 37°C for 1 hour. Then, the fragment was ligated into the vector via homologous recombination and transformed. E. coli After sequencing verification, mutant plasmids (pET28a-D142T, pET28a-F183A, pET28a-R216Y, pET28a-D142T / R216Y, pET28a-D142T / F183A / R216Y) were obtained for DH5α.

[0035] Example 2: Mutant enzyme activity assay

[0036] 1. Transform the above recombinant plasmid. E. coli BL21(DE3) was inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C until OD600=0.6. Then, 0.5 mM IPTG was added and the mixture was induced at 16°C for 12 h.

[0037] 2. Collect the bacterial cells, sonicate them, centrifuge to collect the supernatant, and purify the enzyme using a Ni-NTA column.

[0038] 3. Enzyme activation reaction system: 100 μL pure enzyme (1 mg / mL), 900 μL maltose solution (100 g / L, pH 7.0 sodium phosphate buffer), react at 35℃ for 1 h, and terminate the reaction by boiling in a water bath for 10 min.

[0039] 4. HPLC determination of trehalose production: NH₂ column, mobile phase acetonitrile:water = 75:25, flow rate 1.0 mL / min, RID detector. Enzyme activity definition: The amount of enzyme required to produce 1 μmol of trehalose per minute is 1 U.

[0040] 5. Results (Table 2): Data are expressed as mean ± standard deviation (n=3).

[0041] Table 2. Comparison of enzyme activity and thermal stability of mutants

[0042] Enzyme type Enzyme activity (U / mg) Relative enzyme activity (%) Residual enzyme activity at 40℃ (%) wild type 35.2 ± 1.5 100 60.2 ± 2.1 D142T 48.9 ± 2.1** 139** 75.5 ± 1.8** F183A 41.5 ± 1.8* 118* 70.3 ± 2.0* R216Y 50.3 ± 2.3** 143** 78.2 ± 1.9** D142T / R216Y 65.2 ± 2.5*** 185*** 85.6 ± 2.2*** Three mutants 72.1 ± 2.7*** 205*** 90.1 ± 2.5***

[0043] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 (relative to wild type).

[0044] Example 3: Production of Trehalose by Recombinant Bacterial Shake-Flavor Fermentation

[0045] 1. Seed culture: The recombinant bacteria were inoculated into LB medium and cultured at 37°C and 180 rpm for 12 h.

[0046] 2. Fermentation culture: Transfer to TB medium (15 g / L peptone, 25 g / L yeast extract, 10 mL / L glycerol, 300 g / L maltose) at a 5% inoculation rate, culture at 37℃ until OD600=1.0, add 0.5 mM IPTG, and induce at 16℃ for 12 h.

[0047] 3. Whole-cell transformation: Collect bacterial cells by centrifugation, resuspend in pH 7.0 sodium phosphate buffer to OD600=40, add 300g / L maltose solution, and react at 35℃ and 200rpm for 24h.

[0048] 4. HPLC determination of trehalose yield and conversion rate (Table 3).

[0049] Table 3. Results of shake-flask fermentation (n=3, mean ± standard deviation)

[0050] strain Trehalose yield (g / L) Conversion rate (%) wild type 175.4 ± 5.2 58.5 D142T 210.6 ± 6.1* 70.2* R216Y 225.9 ± 5.8** 75.3** D142T / R216Y 255.3 ± 6.5*** 85.1*** Three mutants 269.8 ± 7.2*** 89.9***

[0051] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 (relative to wild type).

[0052] Conclusion: The trehalose yield and conversion rate of the triple mutant (D142T / F183A / R216Y) were significantly higher than those of the wild type and other mutants (P<0.001), making it suitable for industrial production.

[0053] This invention obtains mutants with significantly enhanced enzyme activity by site-directed mutagenesis (D142T, F183A, R216Y) of trehalose synthase derived from *Streptomyces cerevisiae*. The triple mutant (D142T / F183A / R216Y) exhibits a specific enzyme activity of 72.1 U / mg, a 105% increase compared to the wild type; after incubation at 40°C for 1 hour, the enzyme activity retention rate exceeds 90%. Using recombinant *E. coli* for shake-flask fermentation with 300 g / L maltose as a substrate, the trehalose yield reaches 269.8 g / L, with a conversion rate of 89.9%. The mutants and preparation method provided by this invention have advantages such as high enzyme activity, strong thermal stability, and high conversion rate, making them suitable for the industrial production of trehalose.

[0054] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A trehalose synthase mutant, characterized in that, The amino acid sequence of the trehalose synthase mutant is the sequence shown in SEQ ID NO: 9, modified by the D142T / R216Y double mutant or the D142T / F183A / R216Y triple mutant.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. A recombinant expression vector containing the nucleic acid molecule of claim 2, wherein the vector is pET-28a or pXMJ19.

4. A host cell containing the recombinant expression vector of claim 3, wherein the host cell is Escherichia coli or Corynebacterium glutamicum.

5. A highly efficient method for preparing trehalose, characterized in that, The host cell described in claim 4 is used for fermentation culture, and trehalose is produced by conversion using maltose as a substrate.

6. The method according to claim 5, characterized in that, Includes the following steps: a) Host cells were inoculated into a fermentation medium to induce the expression of a trehalose synthase mutant; b) Collect bacterial cells and carry out whole-cell catalytic reactions using maltose as a substrate; c) Extract and purify trehalose.

7. The method according to claim 6, characterized in that, The fermentation medium contains 100-500 g / L of maltose, IPTG as the inducer, and the induction temperature is 16-28℃.

8. The application of the mutant of claim 1 as a catalyst for trehalose production in the food, pharmaceutical or cosmetic fields.

9. A method for improving trehalose conversion rate, characterized in that, Using the mutant described in claim 1 to catalyze the conversion of maltose, the conversion rate reaches over 75%.