A trehalose-6-phosphate phosphatase and mutants and uses thereof

Abstract

The application discloses a trehalose-6-phosphate phosphatase and a mutant and application thereof, and belongs to the technical field of biology. The amino acid sequence of the wild-type LcTre6PPase is shown as SEQ ID NO:1, and the encoding nucleotide sequence is shown as SEQ ID NO:2. The mutant is obtained by mutating the isoleucine at the 571th position of the wild-type enzyme into leucine (I571L), the amino acid sequence is shown as SEQ ID NO:3, and the corresponding encoding nucleotide sequence is shown as SEQ ID NO:4. Bidirectional enzyme activity analysis shows that, compared with the wild type, the I571L mutant is improved by about 1.2 times in the direction of catalyzing glucose-6-phosphate and beta-D-glucose-1-phosphate to synthesize trehalose-6-phosphate, and the activity in the direction of catalyzing trehalose-6-phosphate to phosphorolyze is reduced to 0.8 times of the wild type. The mutant shows good application potential in the efficient synthesis of trehalose-6-phosphate, and provides a new enzyme tool for preparing trehalose-6-phosphate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a trehalose-6-phosphate phosphorylase, its mutants, and their applications. Background Technology

[0002] Trehalose-6-phosphate (Tr6P) is a key intermediate in the sugar metabolism pathway. In plants, it not only participates in carbon source allocation but also plays a crucial role in regulating sugar metabolism homeostasis, promoting growth and development, and increasing crop yield. Recent studies have shown that in vitro spraying of trehalose-6-phosphate or its precursors can improve the yield of crops such as wheat (Membrane-permeable trehalose 6-phosphate precursor spray increases wheat yields in field trials, Nature biotechnology, 2025). Therefore, developing efficient methods for synthesizing trehalose-6-phosphate is of great significance for agricultural production research.

[0003] Currently, there are two main known biosynthetic pathways for Tre6P in nature. The first is widely found in plants and microorganisms, catalyzed by trehalose-6-phosphate synthase (TPS) using uridine diphosphate glucose (UDP-Glc) and glucose-6-phosphate (Glc6P) as substrates. The drawback of this pathway is the high cost of the substrate UDP-Glc and the complex metabolic processes involved, leading to high in vitro production costs. The second pathway, discovered in some microorganisms, involves the reversible catalysis of Glc6P and β-D-glucose-1-phosphate (βGlc1P) to synthesize Tre6P by trehalose-6-phosphate phosphorylase (Tre6PPase). This pathway directly utilizes structurally simple phosphorylated monosaccharides as substrates, eliminating the need for expensive cofactors, and provides a highly attractive alternative for low-cost, large-scale production of Tre6P.

[0004] However, as a reversible enzyme, Tre6PPase in its natural form typically catalyzes the phospholysis of Tre6P (the breakdown of Tre6P into Glc6P and βGlc1P), while its efficiency in the synthetic direction (catalyzing the conversion of Glc6P and βGlc1P into Tre6P) is relatively low. This catalytic imbalance towards phospholysis severely limits the application potential of wild-type Tre6PPase in the efficient synthesis of Tre6P. Therefore, discovering new Tre6PPases or molecularly modifying existing Tre6PPases to enhance their synthetic activity and weaken their phospholysis activity is crucial for achieving efficient enzymatic synthesis of Tre6P.

[0005] Although a few microbial Tre6PPases have been reported in the prior art (Bioscience, Biotechnology, and Biochemistry, 2017, 81, 8, 1512–1519; J. Agric. Food Chem. 2025, 73, 30, 19065–19075), wild-type trehalose-6-phosphate phosphorylase remains highly efficient in catalyzing phospholysis, limiting its application in Tre6P synthesis. Obtaining Tre6PPase mutants with superior catalytic performance through directed evolution or rational design using protein engineering is an effective way to address this issue; however, there are currently no reports on molecular modification of reversible reaction processes catalyzed by Tre6PPase. Summary of the Invention

[0006] To address the common catalytic reversibility issues of existing trehalose-6-phosphate phosphorylases (Tre6PPase), particularly their low catalytic efficiency and high phospholysis activity in the synthesis of trehalose-6-phosphate (Tre6P), which restricts their application in the efficient synthesis of Tre6P, this invention aims to provide a novel trehalose-6-phosphate phosphorylase (LcTre6PPase), its high-performance mutants, and their applications. Furthermore, it optimizes the catalytic reaction conditions to improve the synthesis efficiency of Tre6P, providing an effective enzymatic tool for the large-scale preparation of Tre6P.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant trehalose-6-phosphate phosphorylase LcTre6PPase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] Secondly, the present invention provides the encoding gene of the recombinant trehalose-6-phosphate phosphorylase LcTre6PPase, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0010] Thirdly, the present invention provides a trehalose-6-phosphate phosphorylase mutant, which is obtained by replacing isoleucine at position 571 with leucine (I571L) based on the amino acid sequence shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:3.

[0011] Fourthly, the present invention provides the encoding gene of the trehalose-6-phosphate phosphorylase mutant, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0012] Fifthly, the present invention provides a recombinant expression vector into which the coding gene of the recombinant trehalose-6-phosphate phosphorylase LcTre6PPase or the coding gene of the trehalose-6-phosphate phosphorylase mutant is inserted.

[0013] Based on the above technical solution, the recombinant expression vector further includes the pRSFDuet-1 plasmid.

[0014] In a sixth aspect, the present invention provides a recombinant engineered bacterium carrying the aforementioned recombinant expression vector.

[0015] Based on the above technical solution, the recombinant engineered bacteria further includes Escherichia coli BL 21(DE3).

[0016] In a seventh aspect, the present invention provides a method for preparing the recombinant trehalose-6-phosphate phosphorylase LcTre6PPase or the trehalose-6-phosphate phosphorylase mutant, wherein the recombinant engineered bacteria are inoculated into a culture medium and cultured until OD0.05. 600 When the concentration reaches 0.4-0.8, IPTG is added to induce protein expression for 10-20 hours. The bacterial cells are collected, lysed, and purified by nickel affinity chromatography to obtain recombinant trehalose-6-phosphate phosphorylase LcTre6PPase or trehalose-6-phosphate phosphorylase mutant.

[0017] Based on the above technical solution, the culture medium further includes LB liquid culture medium, with culture conditions of 15~38℃ and 150~250 rpm; the final concentration of IPTG is 0.1~0.3 mM.

[0018] Eighthly, the present invention provides the application of the recombinant trehalose-6-phosphate phosphorylase LcTre6PPase, the trehalose-6-phosphate phosphorylase mutant, the recombinant expression vector, or the recombinant engineered bacteria in catalyzing the synthesis of trehalose-6-phosphate from glucose-6-phosphate (Glc6P) and β-D-glucose-1-phosphate (βGlc1P).

[0019] Based on the above technical solution, the reaction is further carried out in a buffer system with a pH of 3.0 to 8.5, and the reaction temperature is 20 to 40°C.

[0020] Based on the above technical solution, the pH of the buffer system is further defined as 5.0~6.0.

[0021] Based on the above technical solution, the buffer system further includes citrate / sodium citrate buffer, MES buffer, Bis-Tris buffer and Tris-HCl buffer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention is the first to clone and fully characterize a novel trehalose-6-phosphate phosphorylase, LcTre6PPase, from a specific microbial source, thus expanding the resource library of this enzyme.

[0024] 2. This invention successfully obtained the I571L mutant through semi-rational design site-directed mutagenesis. The enzyme activity of this mutant in the direction of Tre6P synthesis is significantly increased by about 1.2 times compared with the wild type, while the phosphorylation activity is reduced to about 0.8 times that of the wild type, thus making it more inclined to synthesize Tre6P efficiently.

[0025] 3. This invention systematically studied the effect of reaction conditions on catalytic efficiency, and found that pH 6.0 is the optimal reaction condition in application, providing a key parameter for the efficient utilization of the enzyme and its mutants.

[0026] 4. This invention combines novel recombinant enzyme resources, high-performance mutants, and optimized reaction conditions to form a highly efficient Tre6P enzymatic synthesis scheme. It has advantages such as high catalytic efficiency in Tre6P synthesis and low efficiency in phosphorolysis, and has broad application prospects in industrial production. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1 The SDS-PAGE results of the protein solutions used in the purification of LcTre6PPase were presented to express this information.

[0029] Figure 2 The figure shows the effect of pH on the directional activity (a) and phospholysis activity (b) of LcTre6PPase catalyzing the synthesis of trehalose-6-phosphate.

[0030] Figure 3 The figure shows the effect of metal ions on the catalytic activity of LcTre6PPase in the synthesis of trehalose-6-phosphate.

[0031] Figure 4 The results of sequence analysis (a) and substrate complex analysis (b) of LcTre6PPase are shown.

[0032] Figure 5 The figure shows the screening results of the LcTre6PPase mutant's catalytic activity in the synthesis of trehalose-6-phosphate.

[0033] Figure 6 The screening results for the LcTre6PPase mutant's catalytic activity in the direction of trehalose-6-phosphate phosphate hydrolysis are shown in the figure. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, unless otherwise specified, the experimental methods used are all conventional molecular biology methods, and the reagents and materials used are commercially available.

[0035] Example 1: Cloning and Construction of Recombinant Expression Vector for LcTre6PPase Gene

[0036] This invention screened a potential trehalose-6-phosphate phosphorylase (Tre6PPase) from *Lactococcus lactis*, with the amino acid sequence shown in SEQ ID NO:1. However, experimental data to clarify its specific function are currently lacking. To achieve efficient expression of this gene in *E. coli* systems, the coding gene corresponding to this amino acid sequence was optimized based on *E. coli* codon bias, resulting in an optimized nucleotide sequence (as shown in SEQ ID NO:2). The optimized gene sequence was then commissioned to a professional company for whole-gene synthesis. The synthesized product was double-digested with BamHI and HindIII and ligated into the multiple cloning site of the pRSFDuet-1 vector, which was linearized with the same restriction enzymes, using conventional molecular cloning techniques. The ligation product was transformed into *E. coli* DH5α competent cells and screened on LB agar plates containing kanamycin (50 µg / mL). Single colonies were picked, cultured, and plasmids were extracted. Double digestion and DNA sequencing confirmed the correct insertion sequence, yielding the recombinant expression plasmid, named pRSFDuet1-LcTre6PPase.

[0037] Example 2: Culture of recombinant Escherichia coli, expression of LcTre6PPase, and cell collection

[0038] The correctly constructed recombinant plasmid pRSFDuet1-LcTre6PPase from Example 1 was heat-shocked and transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB agar plates containing 50 µg / mL kanamycin and incubated overnight at 37°C with inverted incubation. The following day, single colonies were picked and inoculated into 5 mL of LB liquid medium containing the same antibiotic and cultured at 37°C with shaking at 220 rpm until OD500. 600 The concentration was approximately 0.6. Subsequently, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.2 mM, and expression was induced for 16 hours at 16°C and 220 rpm. After induction, the bacterial culture was centrifuged at 4°C and 4500 rpm for 15 minutes to collect the bacterial cells, and the supernatant was discarded. The obtained wet bacterial cells were washed once with pre-cooled phosphate-buffered saline (PBS, 0.1 M, pH 7.4), centrifuged again, and the supernatant was discarded. The obtained bacterial precipitate could be used immediately for subsequent protein purification or stored at -20°C or below for later use.

[0039] Example 3: Isolation and purification of recombinant LcTre6PPase

[0040] The wet bacterial precipitate obtained in Example 2 was resuspended in pre-cooled lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 5% glycerol, pH 8.0) and intermittently sonicated using an ultrasonic cell disruptor under ice bath conditions. The disrupted bacterial solution was centrifuged at 4°C and 12,000 rpm for 30 minutes, and the supernatant was collected as crude enzyme extract.

[0041] The crude enzyme extract was loaded onto Ni, which had been equilibrated with lysis buffer. 2+ -NTA affinity chromatography columns are first thoroughly washed with wash buffer (50 mM Tris-HCl, 300 mM NaCl, 5% glycerol, pH 8.0) containing 25 mM imidazole to remove non-specifically bound contaminating proteins. Subsequently, elution is performed using elution buffer (50 mM Tris-HCl, 300 mM NaCl, 5% glycerol, pH 8.0, 350 mM imidazole), and the eluted peaks are collected.

[0042] Each purified fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the results showed a single main band at approximately 85 kDa. Figure 1 The molecular weight is consistent with the theoretical molecular weight of LcTre6PPase, indicating that high-purity recombinant protein was successfully obtained.

[0043] The affinity chromatography eluent was concentrated using an ultrafiltration centrifuge tube and replaced with gel filtration chromatography buffer (50 mM Tris-HCl, 300 mM NaCl, 5% glycerol, 1 mM DTT, pH 8.0). The concentrated sample was further purified using an ÄKTA pure protein purification system via a pre-equilibrated Superdex™ 200 Increase gel filtration chromatography column. The elution peaks corresponding to the target protein monomers were collected, concentrated again, and the protein concentration was adjusted to 5-10 mg / mL. The samples were then aliquoted and stored at -80°C for subsequent enzymatic characterization analysis.

[0044] Example 4: Effect of pH on the catalytic activity of LcTre6PPase in the synthesis and phospholysis of trehalose-6-phosphate.

[0045] Trehalose-6-phosphate phosphorylase reversibly catalyzes the following reactions: the synthesis of trehalose-6-phosphate from glucose-6-phosphate (Glc6P) and β-D-glucose-1-phosphate (βGlc1P) (synthetic direction), or the phospholysis of trehalose-6-phosphate to generate the two monosaccharides mentioned above (phospholysis direction). To clarify the effect of pH on the activity of LcTre6PPase described in this invention in two opposite catalytic directions, this embodiment systematically evaluated the changes in enzyme activity under different pH buffer systems.

[0046] Reaction systems with different pH values ​​were constructed using 50 mM citrate / sodium citrate buffer (pH 3.00–5.00), MES buffer (pH 5.01–6.00), Bis-Tris buffer (pH 6.01–7.00), and Tris-HCl buffer (pH 7.01–8.50). At each pH value, the catalytic activity of LcTre6PPase in the synthetic and phospholytic directions was measured.

[0047] The synthetic reaction system contained LcTre6PPase, 3 mM Glc6P and 3 mM βGlc1P, buffer solution corresponding to pH, enzyme and substrate molar ratio of 1:10000, and reaction at 30℃ for 5 minutes.

[0048] The phospholysis reaction system contained LcTre6PPase, 3 mM trehalose-6-phosphate and 3 mM sodium phosphate, a buffer solution corresponding to the pH, and an enzyme to substrate molar ratio of 1:10000. The reaction was carried out at 30°C for 5 minutes.

[0049] The amount of product generated or substrate consumed was detected by high performance liquid chromatography-mass spectrometry (refer to Example 9). The highest enzyme activity measured under all test conditions was defined as 100%, and the relative activity under each condition was calculated accordingly.

[0050] The relative activity results of LcTre6PPase in the synthetic direction are as follows: Figure 2 As shown in a, the catalytic activity of LcTre6PPase in the synthetic direction is highly pH-dependent. The enzyme activity remains at its highest level in the pH range of 5.00 to 6.00, and the relative activity reaches its peak at MES buffer (pH 6.00). When the pH is below 5.00 or above 6.00, the enzyme activity decreases sharply, and the relative activity is less than 10% at pH 3.00 and 8.50.

[0051] The relative activity results of LcTre6PPase in the phospholysis direction are as follows: Figure 2 As shown in b, in the opposite direction of synthesis, the catalytic activity of LcTre6PPase in the phospholysis direction remains stable over a wide pH range (5.00–8.00), with the relative activity remaining above 80%.

[0052] Example 5 Effect of metal ions on the catalytic activity of LcTre6PPase on trehalose-6-phosphate

[0053] To evaluate the influence of common metal ions on the catalytic function of LcTre6PPase, this example systematically detected the influence of Mg in the synthetic reaction direction. 2+ Mn 2+ Ca 2+ Co 2+ Ni 2+ Zn 2+ Cu 2+ and Fe 3+ The effects of eight metal ions at concentrations of 1 mM and 10 mM on enzyme activity were investigated. A reaction system without metal ions but with 1 mM EDTA added was used as a blank control, and its enzyme activity was defined as 100%. The relative activities of each experimental group were calculated.

[0054] The reaction system consisted of 3 mM substrate (3 mM Glc6P and 3 mM βGlc1P), 50 mM Bis-Tris reaction system, with an enzyme to substrate molar ratio of 1:1000, and the reaction was carried out at 30℃ for 5 minutes. The results were detected by high performance liquid chromatography-mass spectrometry.

[0055] The results are as follows Figure 3 As shown, under low concentration metal ion conditions (1 mM), the catalytic activity of LcTre6PPase is generally higher than that under high concentration metal ion conditions (10 mM), where Fe... 3+The inhibitory effect on enzyme activity was the most significant. This indicates that high concentrations of metal ions may inhibit enzyme activity by disrupting protein structural stability, a typical phenomenon of metal ions affecting enzyme activity. Further comparison of the effects of eight metal ions on LcTre6PPase revealed that, compared to the blank control group (EDTA treatment), Mg²⁺ had the most significant inhibitory effect. + The enzyme activity is minimally affected under both low and high concentration conditions, and is almost unaffected; while other metal ions inhibit enzyme activity to varying degrees.

[0056] Example 6: Structural and substrate interaction analysis of trehalose-6-phosphorylphosphatase

[0057] After gaining a thorough understanding of the basic enzymatic properties of LcTre6PPase, this embodiment identifies potential functional sites through sequence conservation and structural analysis to facilitate mutant design and screening. First, 24 enzyme sequences belonging to the GH65 family, to which LcTre6PPase belongs, were retrieved from the UniProt database. Using LcTre6PPase as a reference sequence, multiple sequence alignment was performed using ClustalW2, and the alignment results were displayed using ESPript 3.0. The alignment results showed that amino acid residue 480 was highly conserved phylogenetically, suggesting it may be the active site. Simultaneously, amino acid residues 570–573 in the LcTre6PPase subfamily exhibited strict conservation, while significant differences were observed in other GH65 family enzymes, indicating that these residues may determine the specific function of LcTre6PPase. Figure 4 a). Furthermore, based on the LcTre6PPase structure obtained from AlphaFold3 calculations, the AutoDock vina was used to predict the complex structure of LcTre6PPase with the substrate Tre6P, and this structure was compared and visualized with the LcTre6PPase-β-G1P complex using PyMOL software. Figure 4 b).

[0058] The analysis results showed that Lys589 and Trp342, the binding sites of the enzyme with trehalose-6-phosphate, are also potential binding sites for β-G1P. Based on the mechanism of trehalose-6-phosphate synthesis from β-G1P and G6P under the catalysis of LcTre6PPase, it is speculated that the sugar ring of Tre6P binding with Lys589 and Trp342 corresponds to the β-G1P part, while the other sugar ring, i.e., the part with the phosphate group, corresponds to the G6P part.

[0059] To improve the catalytic efficiency of trehalose-6-phosphate synthesis and suppress the reverse phospholysis reaction, this embodiment optimized the binding site of the G6P portion of trehalose-6-phosphate (Tre6P) while keeping the β-G1P binding site unchanged. Finally, four sites, R570, I571, D572, and R573, were identified as candidate mutation sites for subsequent saturation mutagenesis and screening experiments.

[0060] In summary, this embodiment, through a combination of sequence conservation analysis and substrate complex structure comparison, identified key sites affecting the specificity and catalytic efficiency of LcTre6PPase, providing a scientific basis and guidance for mutant design and subsequent screening experiments.

[0061] Example 7 Construction of Trehalose-6-phosphorylphosphatase mutant

[0062] To construct saturated mutants of the four key sites of LcTre6PPase, this embodiment uses the 22c-trick method to perform single-site saturation mutations on sites R570, I571, D572, and R573.

[0063] In primer design, three sets of degenerate primers were designed for each site, with the corresponding codons for the mutation site being NDT, VHG, and TGG, respectively (where N represents A / T / G / C, D represents A / G / T, V represents A / G / C, and H represents A / C / T). These three sets of primers were mixed in a molar ratio of 12:9:1 for PCR amplification. This mixing ratio ensures that in the final synthesized mutant gene library, all 20 natural amino acids are encoded by specific codons, thus reducing codon bias while achieving comprehensive coverage of all possible amino acid substitutions.

[0064] Table 1 Primer sequences for each mutation site

[0065]

[0066] In addition to primer design, conventional site-directed mutagenesis (whole plasmid PCR) was employed. Amplification was performed using a recombinant plasmid containing the gene shown in SEQ ID NO:2 as a template, and the aforementioned mixed primers were used. After the PCR product was digested with DpnI to remove the template plasmid, it was transformed into *E. coli* Dh5α competent cells and plated on kanamycin-resistant plates for overnight culture. 50-80 single clones were randomly selected from each plate for sequencing verification. Following actual sequencing using the above strategy and integration of valid data, a total of 50 different mutants were constructed.

[0067] Example 8: Two-way enzymatic property analysis of trehalose-6-phosphorylphosphatase mutant

[0068] To efficiently screen mutants with excellent catalytic performance, this embodiment first optimized the screening strategy: using lyophilized bacterial powder prepared from recombinant bacterial cells after induced expression as the enzyme source for activity determination. This method avoids cumbersome protein purification steps while effectively maintaining the stability of enzyme activity, ensuring the reliability and reproducibility of large-scale screening data.

[0069] Catalytic activity analysis in the synthesis direction: The catalytic activity of 50 single-point saturated mutants constructed in the synthesis of trehalose-6-phosphate was systematically evaluated.

[0070] The synthesis reaction system contained 3 mM Glc6P and 3 mM βGlc1P, MES buffer, 0.2 mg lyophilized whole cells, and reacted at 30°C for 5 minutes.

[0071] The amount of product generated was detected by high performance liquid chromatography-mass spectrometry. The highest enzyme activity measured under all test conditions was defined as 100%, and the relative activity of each mutant was calculated accordingly.

[0072] Activity assay results as follows Figure 5 As shown, the results indicate that: the high-performance mutant, I571L (amino acid sequence as shown in SEQ ID NO:3, nucleotide sequence of the encoding gene as shown in SEQ ID NO:4), exhibited the most significant activity enhancement, with a relative activity of approximately 120% of the wild type. Combined with protein structure model analysis, although site 571 does not directly participate in substrate binding, the spatial orientation of the hydrophobic side chain group changes after Ile is mutated to Leu, which may facilitate the entry and stabilization of the substrate glucose-6-phosphate (G6P) at the catalytic center, thereby improving synthesis efficiency. Neutral and low-activity mutants, such as D572K, R573K, and R573V, showed slightly decreased activity, maintaining between 75% and 90% of the wild-type activity; the activity of most other mutants was significantly reduced. Key residue identification: Notably, any amino acid substitution at site R570 resulted in a sharp loss of enzyme activity (the highest-activity mutant, R570I, had a relative activity of only 18%). Structural analysis revealed that the R570 residue side chain is very close to the binding site of the substrate T6P, and it is very likely that it will form a key electrostatic or hydrogen bond interaction with G6P during catalysis. Therefore, the conservation of this site is crucial for maintaining the enzyme's catalytic function.

[0073] Catalytic activity analysis in the direction of phosphorolysis: Thirteen representative mutants with good activity in the synthesis direction were selected to evaluate their catalytic activity in the direction of phosphorolysis.

[0074] The phosphate hydrolysis reaction system contained 3 mM trehalose-6-phosphate and 3 mM sodium phosphate, MES buffer, 0.2 mg lyophilized whole cells, and reacted at 30°C for 5 minutes.

[0075] The amount of product generated was detected by high performance liquid chromatography-mass spectrometry, and the conversion rate of the catalytic reaction of each mutant was calculated accordingly.

[0076] The results are as follows Figure 6 As shown, the results indicate that although the activities of each mutant in the direction of phosphorylation are relatively similar, they are all reduced, especially the I571L mutant, whose activity in the direction of phosphorylation is 0.8 times that of the wild-type enzyme.

[0077] Example 9: Detection of target product using high performance liquid chromatography-mass spectrometry

[0078] Tre6P was quantitatively analyzed using a high-performance liquid chromatography-mass spectrometry (LC-MS) system equipped with an electrospray ionization (ESI) source. For the detection and quantification of the target product, a Hi-Plex H column (300 mm × 7.7 mm) was used, with a flow rate of 0.6 mL / min, a mobile phase of 0.1% formic acid, and an injection volume of 2 μL.

[0079] The retention time of Tre6P standard (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number T339557) was determined to be 6.700 min by LC-MS. The supernatant after the reaction was analyzed, and the results showed that a new chromatographic peak consistent with the standard was generated at a retention time of 6.700 min, indicating that Tre6P was generated in the reaction solution. A 30 mM stock solution of Tre6P standard was prepared and serially diluted to 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM standard solutions. The target product was quantified and the yield was analyzed by standard curve.

[0080] In summary, this embodiment, through systematic measurements of the synthesis and phospholysis directions, clearly demonstrated a significant increase (1.2-fold) in the catalytic activity of the mutant I571L in the synthesis direction, while confirming a decrease (0.8-fold) in the phospholysis direction. The LcTre6PPase mutant obtained in this invention provides a key technology for its industrial application in the synthesis of trehalose-6-phosphate.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant trehalose-6-phosphate phosphatase, characterized in that, The mutant was obtained by replacing isoleucine at position 571 with leucine based on the amino acid sequence shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:

3.

2. The encoding gene of the trehalose-6-phosphate phosphorylase mutant according to claim 1.

3. A recombinant expression vector into which the encoding gene of claim 2 is inserted.

4. A recombinant engineered bacterium carrying the recombinant expression vector of claim 3.

5. The method for preparing the trehalose-6-phosphate phosphorylase mutant according to claim 1, characterized in that, The recombinant engineering bacteria of claim 4 are inoculated into culture medium and cultured to OD 600 0.4~0.8, IPTG is added to induce protein expression for 10~20h, the bacterial bodies are collected, and after being broken, the trehalose-6-phosphate phosphatase mutant is obtained through nickel column affinity chromatography purification.

6. The preparation method according to claim 5, characterized in that, The culture medium includes LB liquid medium, and the culture conditions are 15~38℃ and 150~250 rpm; the final concentration of IPTG is 0.1~0.3 mM.

7. The application of the trehalose-6-phosphate phosphorylase mutant of claim 1, the recombinant expression vector of claim 3, or the recombinant engineered bacteria of claim 4 in the catalytic synthesis of trehalose-6-phosphate from glucose-6-phosphate and β-D-glucose-1-phosphate.

8. The application according to claim 7, characterized in that, The synthesis reaction was carried out in a buffer system with a pH of 3.0 to 8.5 at a temperature of 20 to 40°C.

9. The application according to claim 8, characterized in that, The pH of the buffer system is 5.0~6.0.

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