Sucrose synthase mutant and its application in synthesis of udp-g

Abstract

This invention belongs to the field of enzyme engineering technology, and relates to a sucrose synthase mutant and its application in the catalytic synthesis of uridine diphosphate glucose (UDPG). The sucrose synthase mutant of this invention is derived from *Nitrosomonas cerevisiae* (European *Nitrosomonas*). Nitrosomonas europaea Wild-type sucrose synthase (ATCC 19718) was obtained through single-point or combined mutations. The in vitro enzyme activity, UDP conversion rate, and thermostability of the sucrose synthase mutant of this invention are significantly superior to the wild type. Fusion of a soluble tag at the N-terminus of the mutant significantly enhances its soluble expression and catalytic efficiency. Under conditions of 100 mM UDP and 100 mg / L enzyme loading, the mutant of this invention achieves a UDPG yield of 72.1 mM within 60 min, with a maximum UDP conversion rate of 73.7%, an improvement of approximately 20 percentage points compared to the wild type.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a highly soluble, highly active, and highly stable sucrose synthase mutant, and its application in the catalytic synthesis of uridine diphosphate glucose (UDPG). Background Technology

[0002] Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase that catalyzes the reaction of sucrose with nucleoside diphosphate (NDP) to produce equal proportions of nucleoside diphosphate glucose (NDP-Glc) and fructose. (See [link to article]). Picture 1 This enzyme is often used in combination with glycosyltransferases in in vitro glycosylation synthesis to construct an in situ regeneration system to maintain the supply of uridine diphosphate glucose (UDPG), thereby improving glycosylation efficiency.

[0003] Based on their origin, sucrose synthases are mainly divided into two categories: plant-derived and bacterial-derived. Plant-derived sucrose synthases generally exhibit high specificity for uridine diphosphate (UDP), but they commonly suffer from poor thermostability, low optimal reaction temperatures (typically 30-40℃), low soluble expression levels in common prokaryotic expression systems, and complex purification processes, which limit their large-scale preparation and application. Bacterial-derived sucrose synthases generally have better thermostability, with optimal reaction temperatures between 50-65℃, which is beneficial for improving reaction rates and reducing microbial contamination. They are also generally easier to express efficiently and solublely in systems such as *E. coli*, making them more suitable for industrial production. However, bacterial-derived sucrose synthases have a relatively broad substrate spectrum, acting on various nucleoside diphosphates such as UDP, ADP, and GDP. Currently reported bacterial-derived sucrose synthases still have limitations in catalytic efficiency for UDP and insufficient soluble expression under specific expression conditions, indicating room for further optimization of their catalytic performance.

[0004] Source Nitrosomonas europaea ATCC 19718 sucrose synthase ( NeSuSy has the potential to be used as an industrial biocatalyst. The literature (DIRICKS M, DE BRUYN F, VAN DAELE P, et al. Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes [J]. Applied Microbiology and Biotechnology, 2015, 99(20): 8465-8474.) reports that this enzyme has certain stability under high temperature conditions (60℃) and can catalyze reactions using uridine diphosphate (UDP) as a substrate. However, Ne The natural form of SuSy still faces some limitations in practical applications: its catalytic efficiency needs improvement, and its long-term operational stability (such as thermal stability) requires further optimization. Furthermore, its soluble expression level can be improved when recombinantly expressed in common prokaryotic expression systems (such as E. coli). Therefore, through... Ne SuSy was molecularly modified to obtain enzyme mutants with higher catalytic activity, better stability, and easy and efficient soluble expression, which is of great significance for expanding its application in industrial biocatalysis. Summary of the Invention

[0005] To improve the catalytic efficiency and long-term operational stability of sucrose synthase, this invention provides a sucrose synthase mutant and its application in the synthesis of UDPG. The sucrose synthase mutant of this invention is based on sucrose synthase... Ne Based on SuSy, single-point and combined mutations were performed on the predicted amino acids to obtain high-performance sucrose synthase mutants.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a sucrose synthase mutant obtained by mutating the wild-type sucrose synthase shown in SEQ ID NO:1 through at least one of the following mutations: N29G, S69P, V144S, N184D, N194S, T209D, M241L, R477K, N527E, G597A, K599D, L633I, M635S, R636Q, L637M, D638N, K639R, A642N, R683C, N693H, R695K, R695V, A706D, L720K, L766H, and L766V. The wild-type sucrose synthase is derived from *Nitrosomonas cerevisiae* (…). Nitrosomonas europaeaATCC 19718), whose amino acid sequence is shown in GenBank: CAD85125.1, and the specific sequence is shown in SEQ ID NO:1.

[0008] The N29G mentioned above indicates that the 29th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from asparagine (N) to glycine (G).

[0009] S69P indicates that the 69th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from serine (S) to proline (P).

[0010] V144S indicates that the 144th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from valine (V) to serine (S).

[0011] N184D indicates that the 184th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from asparagine (N) to aspartic acid (D).

[0012] N194S indicates that the 194th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from asparagine (N) to serine (S).

[0013] T209D indicates that the 209th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from threonine (T) to aspartic acid (D).

[0014] M241L indicates that the 241st amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from methionine (M) to leucine (L).

[0015] R477K indicates that the 477th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from arginine (R) to lysine (K).

[0016] N527E indicates that the 527th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from asparagine (N) to glutamic acid (E).

[0017] G597A indicates that the 597th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from glycine (G) to alanine (A).

[0018] K599D indicates that the 599th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from lysine (K) to aspartic acid (D).

[0019] L633I indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by a mutation of leucine (L) to isoleucine (I) at position 633.

[0020] M635S indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by a mutation of methionine (M) to serine (S) at amino acid position 635.

[0021] R636Q indicates that the 636th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from arginine (R) to glutamine (Q).

[0022] L637M indicates that the 637th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from leucine (L) to methionine (M).

[0023] D638N indicates that the 638th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from aspartic acid (D) to asparagine (N).

[0024] K639R indicates that the 639th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from lysine (K) to arginine (R).

[0025] A642N indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by a mutation of alanine (A) to asparagine (N) at amino acid position 642.

[0026] R683C indicates that the 683rd amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from arginine (R) to cysteine ​​(C).

[0027] N693H indicates that the 693rd amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from asparagine (N) to histidine (H).

[0028] R695K indicates that the 695th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from arginine (R) to lysine (K).

[0029] R695V indicates that the 695th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from arginine (R) to valine (V).

[0030] A706D indicates that the 706th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from alanine (A) to aspartic acid (D).

[0031] L720K indicates that the 720th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from leucine (L) to lysine (K).

[0032] L766H indicates that the 766th amino acid of the wild-type sucrose synthase shown in SEQ ID NO:1 is mutated from leucine (L) to histidine (H).

[0033] L766V indicates that the 766th amino acid of the wild-type sucrose synthase, as shown in SEQ ID NO:1, is mutated from leucine (L) to valine (V).

[0034] In one specific embodiment, the mutant comprises a combination of two, three, or four of the aforementioned site mutations, such as A642N-K639R, A642N-T209D, K639R-T209D, L637M-A642N, L637M-T209D, L637M-K639R, K639R-L637M-T209D, A642N-K639R-T209D, A642N-L637 M-T209D, L637M-K639R-A642N, A642N-K639R-T209D-L637M, A642N-K639R-V144S, A642N-K639R -R477K, A642N-T209D-V144S, A642N-T209D-R477K, A642N-T209D-L766V, A642N-K639R-L766V.

[0035] A642N-K639R indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by changing amino acid 642 from alanine (A) to asparagine (N) and amino acid 639 from lysine (K) to arginine (R).

[0036] A642N-T209D indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by changing amino acid 642 from alanine (A) to asparagine (N) and amino acid 209 from threonine (T) to aspartic acid (D).

[0037] K639R-T209D indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by changing the amino acid at position 639 from lysine (K) to arginine (R) and the amino acid at position 209 from threonine (T) to aspartic acid (D).

[0038] L637M-A642N indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by mutating amino acid leucine (L) to methionine (M) at position 637 and amino acid 642 by mutating amino acid alanine (A) to asparagine (N).

[0039] L637M-T209D indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by changing amino acid 637 (L) to methionine (M) and amino acid 209 (T) to aspartic acid (D).

[0040] L637M-K639R indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by changing the amino acid at position 637 from leucine (L) to methionine (M) and the amino acid at position 639 from lysine (K) to arginine (R).

[0041] K639R-L637M-T209D indicates that the amino acid sequence of wild-type sucrose synthase, as shown in SEQ ID NO:1, is modified by the following mutations: amino acid position 639 is changed from lysine (K) to arginine (R), amino acid position 637 is changed from leucine (L) to methionine (M), and amino acid position 209 is changed from threonine (T) to aspartic acid (D).

[0042] A642N-K639R-T209D indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 639 is mutated from lysine (K) to arginine (R), and amino acid position 209 is mutated from threonine (T) to aspartic acid (D).

[0043] A642N-L637M-T209D indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 637 is mutated from leucine (L) to methionine (M), and amino acid position 209 is mutated from threonine (T) to aspartic acid (D).

[0044] L637M-K639R-A642N indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by the mutation of amino acid 637 from leucine (L) to methionine (M), amino acid 639 from lysine (K) to arginine (R), and amino acid 642 from alanine (A) to asparagine (N).

[0045] A642N-K639R-T209D-L637M indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 639 is mutated from lysine (K) to arginine (R), amino acid position 209 is mutated from threonine (T) to aspartic acid (D), and amino acid position 637 is mutated from leucine (L) to methionine (M).

[0046] A642N-K639R-V144S indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by the following mutations: amino acid position 642 is modified by alanine (A) to asparagine (N), amino acid position 639 is modified by lysine (K) to arginine (R), and amino acid position 144 is modified by valine (V) to serine (S).

[0047] A642N-K639R-R477K indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 639 is mutated from lysine (K) to arginine (R), and amino acid position 477 is mutated from arginine (R) to lysine (K).

[0048] A642N-T209D-V144S indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by the mutation of amino acid position 642 from alanine (A) to asparagine (N), amino acid position 209 from threonine (T) to aspartic acid (D), and amino acid position 144 from valine (V) to serine (S).

[0049] A642N-T209D-R477K indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 209 is mutated from threonine (T) to aspartic acid (D), and amino acid position 477 is mutated from arginine (R) to lysine (K).

[0050] A642N-T209D-L766V indicates that the amino acid sequence of wild-type sucrose synthase as shown in SEQ ID NO:1 is modified by the following mutations: amino acid position 642 is modified by alanine (A) to asparagine (N), amino acid position 209 is modified by threonine (T) to aspartic acid (D), and amino acid position 766 is modified by leucine (L) to valine (V).

[0051] A642N-K639R-L766V indicates that in the wild-type sucrose synthase shown in SEQ ID NO:1, amino acid position 642 is mutated from alanine (A) to asparagine (N), amino acid position 639 is mutated from lysine (K) to arginine (R), and amino acid position 766 is mutated from leucine (L) to valine (V).

[0052] In a further embodiment, the sucrose synthase mutant is obtained by single-point mutation of the wild-type sucrose synthase shown in SEQ ID NO:1 as follows: V144S, L633I, L637M, K639R, A642N, L766H, L766V, T209D, M241L, and R477K; or ... The wild-type sucrose synthase shown in NO:1 was obtained through the following combination of mutations: A642N-T209D, A642N-K639R, K639R-T209D, L637M-K639R, A642N-T209D-R477K, A642N-K639R-R477K, A642N-T209D-V144S, K639R-L637M-T209D, A642N-K639R-T209D, L637M-K639R-A642N, A642N-K639R-T209D-L637M, A642N-T209D-L766V, or A642N-K639R-L766V.

[0053] In a further embodiment, the sucrose synthase mutant is a combination of the following mutants: A642N-T209D, A642N-K639R, and A642N-T209D-R477K. These three mutants significantly enhance the catalytic activity of UDP. Among them, A642N-T209D exhibits the highest specific enzyme activity and catalytic efficiency.

[0054] Furthermore, the present invention provides a sucrose synthase mutant with a soluble tag, that is, a soluble tag is fused to the N-terminus of the sucrose synthase mutant to enhance the soluble expression and enzyme activity of the mutant. The soluble tag is selected from one of GKGKG, P17-m, SKIK, T7B9, and N11.

[0055] In a second aspect, the present invention provides a nucleic acid encoding the above-described sucrose synthase mutant.

[0056] A third aspect of the present invention provides a biomaterial, said biomaterial being at least one of the following:

[0057] (a) An expression cassette containing the said nucleic acid;

[0058] (b) A recombinant expression vector containing the said nucleic acid;

[0059] (c) A recombinant expression vector containing the expression cassette described in (a);

[0060] (d) Recombinant microorganisms containing the said nucleic acid;

[0061] (e) Recombinant microorganisms containing the expression cassette described in (a);

[0062] (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

[0063] Furthermore, the starting vector for the recombinant expression vectors mentioned in (b) and (c) is an *E. coli* expression plasmid (i.e., a plasmid that can be expressed in *E. coli*), such as the pET series vectors, and more specifically pET-28a (+); the host microorganisms corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) are selected from prokaryotes, such as *E. coli*, and can be specifically adopted as follows: Escherichia coli BL21(DE3).

[0064] A fourth aspect of the present invention provides the application of the sucrose synthase mutant, the nucleic acid, and the biomaterial in the catalytic synthesis of UDPG.

[0065] A fifth aspect of the present invention provides a method for efficiently synthesizing UDPG: using a crude enzyme solution of the sucrose synthase mutant or the biological material as a catalyst, and UDPG and sucrose as substrates, the reaction is carried out under conditions of pH 5.5-8.0 and 40-70°C to generate UDPG and fructose. In a further embodiment, the reaction conditions are pH 6.5-7.0, 60°C, and a reaction time of 10 min. Even further, the pH is 6.5 or 7.

[0066] The beneficial effects of this invention are:

[0067] 1. This invention provides a sucrose synthase mutant, which improves sucrose synthase activity through site-directed mutagenesis. Ne The enzyme activity of SuSy was improved, and this mutant was used to achieve efficient catalytic synthesis of natural glycosides. The synthesis method is simple to operate, short in time, high in catalytic efficiency and high in yield, and has good application prospects.

[0068] 2. The sucrose synthase multi-point mutant A642N-T209D constructed in this invention is the mutant with the most significant improvement in enzyme catalytic performance, with a specific enzyme activity of 25.139 U / mg. The specific enzyme activity of the mutant is 3.09 times that of the original specific enzyme activity, and the catalytic efficiency (… k cat / K m The efficiency was increased to 6.44 times that of the original catalytic efficiency, and the thermal stability was significantly enhanced, with the half-life at 60℃ extended from 110.7 min to 228.2 min.

[0069] 3. The mutant exhibits high catalytic efficiency, with a UDP conversion rate of 73.7%, nearly 20 percentage points higher than the wild type, demonstrating strong potential for industrial application.

[0070] At an enzyme concentration of 100 mg / L, the sucrose synthase multipoint mutant A642N-T209D achieved a high conversion rate of 74.74% under 50 mM UDP conditions; when the UDP concentration was increased to 100 mM, the UDPG conversion rate still reached 72.77%, indicating that it still has high catalytic performance under high substrate loading.

[0071] 4. Applying short peptide soluble tags to Ne The SuSy mutant significantly increases soluble expression in E. coli, and the T7B9 tag increases relative enzyme activity by 180%.

[0072] 5. In the application of UDPG synthesis, the mutant A642N-T209D achieved a UDPG yield of 72.1 mM within 60 minutes under the conditions of 100 mM UDP and 100 mg / L enzyme loading, with a maximum UDP conversion rate of 73.73%, which significantly improved the production efficiency compared with the wild type. Attached Figure Description

[0073] Picture 1 Schematic diagram of the reaction catalyzed by sucrose synthase to generate UDPG from NDP;

[0074] Picture 2 HPLC technology detection Ne SuSy catalyzes the generation of UDPG from UDP;

[0075] Picture 3 : Ne The expression and purification of SuSy recombinant protein;

[0076] Picture 4 Standard curve for fructose content determination using the DNS method;

[0077] Picture 5 : Ne The enzyme activity of wild-type and mutant SuSy;

[0078] Picture 6 : Ne The optimal pH for SuSy wild-type and mutants;

[0079] Picture 7 : Ne The optimal reaction temperature for SuSy wild-type and mutants;

[0080] Picture 8 : Ne Thermal stability of SuSy wild-type and mutant;

[0081] Picture 9 Different enzyme concentrations NeConversion rates of UDPG synthesis between wild-type and mutant SuSy (A: 25 mg / L purified enzyme; B: 100 mg / L purified enzyme).

[0082] Picture 10 : Integrating different soluble labels Ne SDS-PAGE images of SuSy-A642N-T209D protein before and after induction of expression. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0084] The culture medium formula used in the examples is as follows:

[0085] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, solvent: water, pH 7.0;

[0086] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, solvent: water, pH 7.0.

[0087] The reagent formulations used for cell disruption and protein purification in this example are as follows:

[0088] Buffer A: 500 mM NaCl, 50 mM Tris-HCl (pH 7.4);

[0089] Buffer B: 100 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5% glycerol.

[0090] In this example, high-performance liquid chromatography (HPLC, HITACHI) was used to detect the concentration of UDPG, thereby calculating the enzyme activity of SuSy. The HPLC conditions for UDPG detection were as follows: after terminating the reaction and diluting the sample, impurities were removed by filtration through a 0.22 μm syringe filter. The concentration of UDPG in the sample was then analyzed by HPLC. The chromatographic column used was a Spursil C18 column (5 μm, 250 × 4.6 mm), the detection wavelength was 260 nm, mobile phase A contained 8 mM tetrabutylammonium bisulfate and 17 mM KH₂PO₄ (the pH of mobile phase A was adjusted to 6.5 using KOH), and mobile phase B was prepared by adding 30% methanol to 70% mobile phase A (pH 6.5). The flow rate was 1 mL / min, and the injection volume was 10 μL. Before use, the mobile phase was filtered through a 0.22 μm filter membrane and then ultrasonically agitated to remove air bubbles. The HPLC procedure for UDPG detection is shown in Table 1, and the analytical results of UDPG detection are presented in Table 1. Picture 2 middle.

[0091] Table 1. HPLC detection procedure for UDPG

[0092]

[0093] Example 1: Recombinant plasmid pET28a- Ne SuSy's Construction

[0094] wild Ne The amino acid sequence of SuSy is shown in SEQ ID NO:1. The amino acid sequence encoded by SuSy, synthesized by Sangon Biotech (Shanghai) Co., Ltd. Ne The gene fragment of SuSy (as shown in SEQ ID NO:2) was cloned between the NheⅠ and HindⅢ restriction sites of pET-28a, and the resulting recombinant plasmid was named pET28a- Ne SuSy.

[0095] Example 2: Construction of recombinant plasmids

[0096] (1) Construction of single mutant recombinant plasmids:

[0097] pET28a- constructed using Example 1 NeUsing SuSy as the starting plasmid, site-directed mutagenesis was performed on a total of 26 sites: N29G, S69P, V144S, N184D, N194S, T209D, M241L, R477K, N527E, G597A, K599D, L633I, M635S, R636Q, L637M, D638N, K639R, A642N, R683C, N693H, R695K, R695V, A706D, L720K, L766H, or L766V. Among them, V144S, G597A, K599D, L633I, M635S, R636Q, L637M, D638N, K639R, A642N, R695K, L766H, and L766V are single mutation sites identified in the previous screening based on enzyme activity; N29G, S69P, N184D, N194S, T209D, M241L, R477K, N527E, A706D, R683C, N693H, R695V, and L720K are single mutation sites identified in the previous screening based on stability.

[0098] The site-directed mutagenesis specifically uses pET28a- Ne Using SuSy as the DNA template, PCR amplification was performed using primers as shown in Table 2. The mutant PCR system (50 μL) consisted of: 25 μL of 2×TransStart FastPfu Fly PCR SuperMix premix, 1 μL each of the upstream and downstream mutant primers, 4 μL of DNA template (starting plasmid), and Nuclease-free water to a final volume of 50 μL. PCR conditions were: 94℃ pre-denaturation for 3 min, followed by 25 cycles: 94℃ for 20 s, 60-70℃ for 20 s, 72℃ for 2 min 30 s, final extension at 72℃ for 10 min, and final incubation at 4℃. The PCR amplification products were detected by agarose gel electrophoresis, and then the PCR products were digested with DMT enzyme at 37℃ for 2 h. The digested products were then excised and recovered from the gel.

[0099] E. coli DH5α competent cells stored at -80℃ were incubated at 0℃ for 10 min on ice. Then, 5 µL of the recovered mutant product was added to a clean bench, followed by an incubation at 0℃ for 30 min on ice, a heat shock at 42℃ for 45 s, and an incubation at 0℃ on ice for 2 min. 600 µL of antibiotic-free LB broth was added to the clean bench, and the cells were incubated at 37℃ and 200 rpm for 1 h on a shaker. Afterward, the cells were centrifuged at 5000 rpm for 5 min at room temperature, and the precipitated cells were spread onto LB agar plates containing 50 μg / mL kanamycin resistance. The plates were incubated upside down at 37℃ overnight. Single colonies were picked from the plates and inoculated into LB broth containing 50 mg / L kanamycin. After incubation at 37℃ and 200 rpm for 16 h, the plasmid was extracted and named pET28a-. Ne SuSy-N was submitted for testing and identification, where N represents the mutation site. For example, the plasmid obtained by site-directed mutagenesis of N29G is pET28a-. Ne SuSy-N29G.

[0100] Table 2. Primer design for site-directed mutagenesis of sucrose synthase

[0101]

[0102]

[0103] (2) Construction of recombinant plasmids for combined mutants:

[0104] Following the method described in "Construction of Single-Mutant Recombinant Plasmids" above, combined mutations were performed based on single-point mutations. The 17 combined mutation methods are as follows:

[0105] A642N-K639R mutant:

[0106] With recombinant plasmid pET28a- Ne Using SuSy-A642N as the DNA template, the K639R was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-K639R was ultimately submitted for testing and evaluation.

[0107] A642N-T209D mutant:

[0108] With recombinant plasmid pET28a- Ne Using SuSy-A642N as the DNA template, the T209D was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-T209D was ultimately submitted for testing and evaluation.

[0109] K639R-T209D mutant:

[0110] With recombinant plasmid pET28a- Ne Using SuSy-K639R as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-K639R-T209D was ultimately submitted for testing and evaluation.

[0111] L637M-A642N mutant:

[0112] With recombinant plasmid pET28a- Ne Using SuSy-L637M as a template, the A642N plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-L637M-A642N was ultimately submitted for testing and evaluation.

[0113] L637M-T209D mutant:

[0114] With recombinant plasmid pET28a- Ne Using SuSy-L637M as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-L637M-T209D was ultimately submitted for testing and evaluation.

[0115] L637M-K639R mutant:

[0116] With recombinant plasmid pET28a- Ne Using SuSy-L637M as a template, the K639R plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-L637M-K639R was ultimately submitted for testing and evaluation.

[0117] K639R-L637M-T209D mutant:

[0118] With recombinant plasmid pET28a- Ne Using SuSy-L637M-K639R as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-K639R-L637M-T209D was ultimately submitted for testing and evaluation.

[0119] A642N-K639R-T209D mutant:

[0120] With recombinant plasmid pET28a- Ne Using SuSy-A642N-K639R as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-K639R-T209D was ultimately submitted for testing and evaluation.

[0121] A642N-L637M-T209D mutant:

[0122] With recombinant plasmid pET28a- Ne Using SuSy-L637M-A642N as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-L637M-T209D was ultimately submitted for testing and evaluation.

[0123] L637M-K639R-A642N mutant:

[0124] With recombinant plasmid pET28a- Ne Using SuSy-L637M-A642N as a template, the K639R was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-L637M-K639R-A642N was ultimately submitted for testing and evaluation.

[0125] A642N-K639R-T209D-L637M mutant:

[0126] With recombinant plasmid pET28a- Ne Using SuSy-L637M-K639R-A642N as a template, the T209D plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-K639R-T209D-L637M was ultimately submitted for testing and evaluation.

[0127] A642N-K639R-V144S mutant:

[0128] With recombinant plasmid pET28a- Ne Using SuSy-A642N-K639R as a template, the V144S plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- NeThe SuSy-A642N-K639R-V144S was ultimately submitted for testing and evaluation.

[0129] A642N-K639R-R477K mutant:

[0130] With recombinant plasmid pET28a- Ne Using SuSy-A642N-K639R as a template, the R477K was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-A642N-K639R-R477K was ultimately submitted for testing and evaluation.

[0131] A642N-T209D-V144S mutant:

[0132] With recombinant plasmid pET28a- Ne Using SuSy-A642N-T209D as a template, the V144S plasmid was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-T209D-V144S was ultimately submitted for testing and evaluation.

[0133] A642N-T209D-R477K mutant:

[0134] With recombinant plasmid pET28a- Ne Using SuSy-A642N-T209D as a template, the R477K was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne SuSy-A642N-T209D-R477K was ultimately submitted for testing and evaluation.

[0135] A642N-T209D-L766V mutant:

[0136] With recombinant plasmid pET28a- Ne Using SuSy-A642N-T209D as a template, the L766V was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-A642N-T209D-L766V was ultimately submitted for testing and evaluation.

[0137] A642N-K639R-L766V mutant:

[0138] With recombinant plasmid pET28a- NeUsing SuSy-A642N-K639R as a template, the L766V was mutated using the mutagenesis method described in "Construction of Single Mutant Recombinant Plasmids". The resulting plasmid was named pET28a- Ne The SuSy-A642N-K639R-L766V was ultimately submitted for testing and evaluation.

[0139] Example 3: E. coli BL21(DE3) / pET28a- Ne Construction of SuSy or its mutant recombinant strains

[0140] Construction of recombinant Escherichia coli: Escherichia coli BL21(DE3) competent cells stored at -80℃ were incubated at 0℃ for 10 min, and then 5 µL of pET28a- obtained in Example 1 was added in a clean bench. Ne The mutant plasmid obtained from SuSy or Example 2 was subjected to a 30-minute ice bath at 0°C, followed by a 45-second heat shock in a 42°C water bath, and then a 2-minute ice bath at 0°C. 600 µL of antibiotic-free LB liquid medium was added to the culture medium, and the culture was carried out at 37°C and 200 rpm for 1 hour using a shaker. A portion of the bacterial cells was then plated onto LB solid medium plates containing 50 μg / mL kanamycin resistance and incubated overnight at 37°C. Single clones were randomly selected to obtain recombinant cells containing the recombinant expression plasmid. E. coli BL21(DE3) / pET28a- Ne SuSy or its mutant expression strains were stored for future use.

[0141] Example 4: Induced expression of recombinant proteins

[0142] The recombinant strain obtained in Example 3 was activated by inoculating LB solid medium plates. Single colonies were then picked and inoculated into Erlenmeyer flasks containing LB liquid medium with 50 μg / mL kanamycin resistance, and cultured overnight at 37 °C and 220 rpm. Then, a 1% (v / v) inoculum was added to fresh LB liquid medium containing 50 μg / mL kanamycin resistance, and cultured at 37 °C and 200 rpm until the bacterial OD reached the target cell count. 600 The concentration was increased to 0.6-0.8, and then isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM. The mixture was then induced and cultured at 16 ℃ and 150 rpm for 16-20 h.

[0143] Example 5: Preparation of crude enzyme solution

[0144] Collect the bacterial culture obtained from the induction culture in Example 4, and centrifuge at 12000 rpm for 15 min at 4°C. Discard the supernatant and collect the precipitate to obtain the wet bacterial cells of the sucrose synthase gene mutant library. Resuspend the wet bacterial cells in Buffer A containing 5 mM imidazole, add lysozyme to a final concentration of 1 mg / mL and PMSF to a final concentration of 1 mM, and then incubate on ice for 30 min. Sonicate the lysate for 30 min on an ice-water mixture (amplifier Ф6, power 35%, 3 s for disruption, 6 s for pause). Centrifuge the lysate at 12000 rpm for 30 min at 4°C. Discard the precipitate; the supernatant of the lysate is the crude enzyme solution. Add a cryoprotectant and store at 4°C for later use.

[0145] Example 6: Purification of recombinant protein

[0146] The crude enzyme solution obtained in Example 5 was filtered through a 0.22 μm filter membrane and then incubated with Ni-NTA magnetic beads at 4°C for 1 hour to promote the adhesion of the tag to Ni. 2+ After incubation, the crude enzyme solution was collected as the flow-through sample. It was eluted sequentially with Buffer A containing 5 mM imidazole and Buffer B containing 50 mM, 200 mM, and 300 mM imidazole, respectively. The elution fractions at different imidazole concentrations were collected to obtain the purified protein. SDS-PAGE was used to verify protein purity. SDS-PAGE results showed that, with increasing imidazole concentration, the recombinant protein eluted best at 200 mM imidazole, producing a single band (approximately 90 kDa). Picture 3 As shown (only wild-type sucrose synthase is shown in the figure).

[0147] Example 7: Catalytic effect of crude enzyme solution of sucrose synthase mutant

[0148] The enzyme activity of the mutants obtained in Example 5 was tested, and the test methods and results are as follows:

[0149] 1. Comparison of relative enzyme activities of sucrose synthase and its single-point mutants

[0150] The relative fructose content generated from the cleavage of sucrose by crude enzyme was determined using the DNS method. 10 μL of crude enzyme solution was added to 90 μL of reaction solution containing 200 mM sucrose, 5 mM UDP, and Tris-HCl buffer (50 mM, pH 6.5). The reaction was carried out at 60 °C for 10 min, and then terminated by heating at 100 °C for 5 min. A control experiment was conducted without the addition of crude enzyme solution to eliminate the influence of heat treatment on sucrose decomposition. One volume of DNS (dinitrosalicylic acid) colorimetric reagent was added to the inactivated reaction solution. After boiling in a water bath for 15 min, the solution was removed, cooled to room temperature, diluted 5 times with pure water, and the absorbance of the sample was measured at 540 nm. The control was as follows. Picture 4 The DNS standard curve is shown, and the relative fructose content is calculated. It should be noted that in the sucrose synthase-catalyzed reaction, fructose production and UDPG production are in a 1:1 ratio. Therefore, the relative fructose content can indirectly reflect the relative UDPG content, and thus the relative enzyme activity of sucrose synthase in UDPG production. Using the relative enzyme activity of wild-type sucrose synthase (WT) as a 100% baseline, the relative enzyme activities of single-point mutants are shown in Tables 3 and 4.

[0151] Table 3. Relative enzyme activity of crude enzyme solution of single-mutant sucrose synthase selected based on enzyme activity screening

[0152]

[0153] Table 4. Relative enzyme activity of crude sucrose synthase solution based on stability screening

[0154]

[0155] As can be seen from the test results in Table 2, among the single-mutant sucrose synthases screened based on enzyme activity, the relative enzyme activities of some single-point mutants were superior to those of the wild type. Specifically, the relative enzyme activities of V144S (1.48 times), L633I (1.25 times), L637M (1.70 times), K639R (1.60 times), A642N (1.52 times), L766H (1.37 times), and L766V (1.40 times) were significantly higher than those of the wild type.

[0156] As can be seen from the test results in Table 3, among the single-mutant sucrose synthases screened based on stability, the relative enzyme activities of some single-point mutants are better than those of wild type. Among them, the relative enzyme activities of T209D (1.39 times), M241L (1.15 times), and R477K (1.36 times) are significantly higher than those of wild type.

[0157] 2. Comparison of relative enzyme activities of sucrose synthase and its multi-point combination mutants

[0158] The relative enzyme activities of multi-point combination mutants were detected using the method described in "Comparison of Relative Enzyme Activities of Sucrose Synthase and Its Single-Point Mutants", as shown in Table 5.

[0159] Table 5. Relative enzyme activity of crude enzyme solutions obtained from multi-site mutant strains

[0160]

[0161] As shown in Table 5, the multi-point mutant combinations exhibit higher relative enzyme activities than the wild type. The mutant combinations with the highest to lowest relative enzyme activities are A642N-T209D (3.23 times), A642N-K639R (2.7 times), A642N-T209D-R477K (2.36 times), A642N-K639R-R477K (2.27 times), A642N-T209D-V144S (2.14 times), and A642N-K639R-V144S (2.1 times). The relative enzyme activities of these mutant combinations are all more than twice that of the wild type. Other combinations, such as K639R-T209D... The following chips also showed varying degrees of improvement: L637M-K639R (1.68x), K639R-L637M-T209D (1.28x), A642N-K639R-T209D (1.65x), L637M-K639R-A642N (1.20x), A642N-K639R-T209D-L637M (1.13x), A642N-T209D-L766V (1.96x), and A642N-K639R-L766V (1.54x).

[0162] Example 8: Catalytic effect of purified enzyme from sucrose synthase mutant

[0163] 1. Comparison of specific enzyme activities of sucrose synthase and some of its mutants

[0164] Three mutants—A642N-T209D, A642N-K639R, and A642N-T209D-R477K—with significantly enhanced UDP catalytic activity were selected from the combined mutants. The enzyme proteins were purified, and their specific enzyme activity was determined. Sucrose synthase activity (U) was defined as the amount of enzyme required to convert 1 μM fructose within 1 min, which is considered one enzyme activity unit. Specific enzyme activity (U / mg) was defined as the enzyme activity units contained per mg of protein.

[0165] The reaction system contained 50 mM Tris-HCl buffer (pH 6.5), 200 mM sucrose, and 5 mM UDP or ADP / CDP / GDP, as well as 100 mg / L purified enzyme solution. The reaction was carried out at 60℃ for 10 min, and the reaction was terminated by treating the sample at 100℃ for 5 min. The catalytic activity towards UDP was then assessed, with the reaction without added enzyme serving as a blank control. Results are as follows: Picture 5 As shown, the mutant A642N-T209D had the highest specific enzyme activity, at 25.139 U / mg, which is 3.09 times that of WT.

[0166] 2. Enzyme kinetic parameters of sucrose synthase and some of its mutants

[0167] Three mutants, A642N-T209D, A642N-K639R, and A642N-T209D-R477K, which showed a significant increase in UDP catalytic activity from the combined mutations, were selected. The enzyme protein was purified and its enzyme kinetic parameters were measured.

[0168] Enzyme activity was detected under reaction conditions of 60℃ and pH 6.5, with a UDP concentration gradient ranging from 0.1 to 10 mM. The experimental data were fitted using the Michaelis-Menten equation with GraphPad Prism software to calculate the enzyme activity. K m Enzyme activity and k cat Enzymatic kinetic parameters were determined. The results are shown in Table 6, for the three mutants. K m All three mutants showed lower affinity than the wild type, indicating a significant increase in their affinity for UDP; k cat and k cat / K m All were higher than the wild type, with A642N-T209D being the highest. k cat It is the highest, 3.01 times that of the wild type, with a catalytic efficiency ( k cat / K m It is also the best, being 6.44 times that of the wild type.

[0169] Table 6. Ne kinetic parameters of SuSy mutant against UDP

[0170]

[0171] Example 9: Determination of the optimal pH for purified sucrose synthase mutant enzyme

[0172] The relative fructose content of sucrose cleavage catalyzed by purified enzymes of A642N-T209D, A642N-K639R, and A642N-T209D-R477K mutants was determined using the DNS method. 10 μL of purified enzyme was added to 90 μL of reaction solution containing 200 mM sucrose, 5 mM UDP, 50 mM Tris-HCl buffer, and 100 mg / L of purified wild-type sucrose synthase (WT), A642N-T209D, A642N-K639R, or A642N-T209D-R477K mutant enzyme solution. A pH gradient of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9 was established. The reaction was carried out at 60 °C for 10 min, followed by heating at 100 °C for 5 min to terminate the reaction. A reaction system without enzyme solution was used as a blank control to eliminate the effect of heat treatment on sucrose decomposition. Add an equal volume of DNS colorimetric solution to the inactivated reaction solution, heat in a boiling water bath for 15 min, cool to room temperature, dilute 5 times with pure water, and measure the absorbance of the sample at a wavelength of 540 nm. Compare with control values ​​as follows: Picture 4 The DNS standard curve is shown, and the relative enzyme activity under different pH conditions is calculated with the relative enzyme activity of wild sucrose synthase as 100% as the baseline, and the pH-relative enzyme activity relationship curve is plotted.

[0173] like Picture 6 As shown, the optimal pH for the synthesis of UDPG catalyzed by wild-type, A642N-T209D, and A642N-T209D-R477K remained around 6.5, while that of A642N-K639R increased to around 7.

[0174] Example 10: Optimal reaction temperature of purified sucrose synthase mutant enzyme

[0175] The relative fructose content of sucrose cleavage catalyzed by purified enzymes of A642N-T209D, A642N-K639R, and A642N-T209D-R477K mutants was determined using the DNS method. 10 μL of purified enzyme solution was added to 90 μL of reaction solution containing 200 mM sucrose, 5 mM UDP, and 50 mM Tris-HCl buffer (pH 6.5), as well as 100 mg / L of purified wild-type sucrose synthase (WT), A642N-T209D, A642N-K639R, or A642N-T209D-R477K mutant enzyme solution. The reaction was carried out at temperature gradients of 30, 40, 50, 60, 70, and 80 °C for 10 min, and then terminated by heating at 100 °C for 5 min. A reaction system without purified enzyme was used as a blank control to eliminate the effect of heat treatment on sucrose decomposition. Add an equal volume of DNS colorimetric reagent to the inactivated reaction solution, heat in a boiling water bath for 15 min, cool to room temperature, dilute 5 times with pure water, and measure the absorbance of the sample at a wavelength of 540 nm. (Compare with control examples.) Picture 4 The DNS standard curve is shown, and the relative enzyme activity of the pure wild sucrose synthase enzyme is used as a 100% baseline to calculate the relative enzyme activity of the sucrose synthase mutant under different temperature conditions, and the temperature-relative enzyme activity relationship curve is plotted.

[0176] like Picture 7 As shown, the optimal reaction temperature for both wild-type and mutant pure enzymes is 60℃, with no significant change. However, the relative enzyme activity of the mutant is higher than that of the wild-type at 30-60℃, while the relative enzyme activity of A642N-T209D and A642N-T209D-R477K is higher than that of the wild-type at 60-75℃.

[0177] Example 11: Comparison of the thermostability of purified enzymes from sucrose synthase mutants

[0178] Take appropriate amounts of purified wild-type WT, A642N-T209D, A642N-K639R, and A642N-T209D-R477K mutant enzyme solutions and incubate them at 60℃ for 0 min, 30 min, 60 min, 90 min, 120 min, 180 min, and 240 min, respectively. Take 10 μL of the pure enzyme solution incubated for different time lengths and add it to 90 μL of reaction solution. The reaction system contains 50 mM Tris-HCl buffer (pH 6.5), 200 mM sucrose, 5 mM UDP, and 100 mg / L of purified wild-type sucrose synthase (WT), A642N-T209D, A642N-K639R, or A642N-T209D-R477K mutant enzyme solution. Incubate the reaction at 60℃ for 10 minutes, and then terminate the reaction by heat treatment at 100℃ for 5 minutes. All reactions were performed in triplicate, with a no-enzyme system serving as a blank control. The initial unincubated enzyme activity was taken as 100%, and the residual relative enzyme activity was calculated at different incubation times to reflect changes in thermostability. The inactivation rate constant Kd was obtained through nonlinear fitting, and then calculated according to the formula T... 1 / 2 = ln2 / Kd Calculate the heat-inactivation half-life of the enzyme.

[0179] like Picture 8 As shown, compared to wild-type sucrose synthase (WT), the thermostability of each mutant was significantly improved. After incubation at 60℃ for 240 min, the remaining relative enzyme activity of the wild-type was only 21.8%, while the remaining relative enzyme activities of mutants A642N-K639R, A642N-T209D, and A642N-T209D-R477K reached 29.4%, 43.0%, and 40.6%, respectively. Among them, mutant A642N-T209D showed the most significant improvement, with the heat inactivation half-life at 60℃ increasing from 110.7 min in the wild-type to 228.2 min, a 2.06-fold increase.

[0180] Example 12: Comparison of conversion rates of sucrose synthase mutant purified enzymes to substrate UDP

[0181] (1) Comparison of conversion rates at different UDP substrate concentrations

[0182] Using purified mutant A642N-T209D as a catalyst, and in 50 mM Tris-HCl buffer (pH 6.5), with 200 mM sucrose as the glycosyl donor and an enzyme concentration of 100 mg / L, UDP substrate concentration gradients of 50 mM, 100 mM, 150 mM, and 200 mM were established to construct a 1 mL reaction system. The catalytic reaction was carried out at 60 °C for 10 min. After terminating the reaction by heat treatment at 100 °C for 5 min, the amount of UDPG produced was determined by HPLC, and the UDP conversion rate at different UDP substrate concentrations was calculated. The results are shown in Table 7.

[0183] Table 7. Conversion efficiency of mutant A642N-T209D for different concentrations of substrate UDP

[0184]

[0185] Experimental results showed that at 60℃ and a UDP concentration of 50 mM, the mutant achieved a high conversion rate of 74.74%, with a UDPG yield of 37.3 mM. When the UDP concentration was increased to 100 mM, the mutant's conversion rate reached 72.77%, and the UDPG yield correspondingly increased to 72.8 mM. These results indicate that the mutant maintains high catalytic efficiency even under high substrate loading.

[0186] (2) Comparison of conversion rates under different enzyme dosages

[0187] Using purified wild-type sucrose synthase (WT) and the mutant A642N-T209D as catalysts, a 1 mL reaction system was constructed in 50 mM Tris-HCl buffer (pH 6.5) with 200 mM sucrose as the glycosyl donor and a fixed UDP substrate concentration of 100 mM. Two enzyme concentration gradients of 25 mg / L and 100 mg / L were established, and the reaction was carried out at 60 °C for 240 min. After terminating the reaction by heat treatment at 100 °C for 5 min, the amount of UDPG produced was determined by HPLC, and the UDP conversion rate at different UDP substrate concentrations was calculated.

[0188] The results are as follows Picture 9As shown in the figure. The results indicate that, within the same reaction time (120 min), the mutant A642N-T209D exhibited significantly superior catalytic performance compared to WT at different enzyme concentrations. At an enzyme concentration of 100 mg / L, the highest conversion rate was reached approximately 60 min, with the mutant achieving a UDP conversion rate of 72.1% and a UDPG yield of 72.1 mM, significantly higher than WT (53.4% ​​conversion rate, 53.4 mM yield). At a lower enzyme concentration of 25 mg / L, the highest conversion rate was reached approximately 180 min, with the mutant still maintaining a 72.4% conversion rate, significantly higher than WT (53.7% conversion rate, 53.7 mM yield).

[0189] This embodiment demonstrates that, compared with the wild type, the mutant A642N-T209D can achieve higher UDP conversion rate and UDPG yield over a wider range of substrate concentrations and enzyme dosages. In particular, it maintains high conversion efficiency even at lower enzyme dosages, proving that its catalytic performance is significantly improved and it is more suitable for efficient and economical UDPG enzymatic synthesis processes.

[0190] Example 13: Verification of Soluble Tag Fusion and Expression Effect

[0191] To improve Ne Soluble expression of the SuSy mutant A642N-T209D was performed. Five short peptide soluble tags—GKGKG, N11, P17-m, SKIK, and T7B9—were selected to replace the His tag. Information on the short peptides is shown in Table 8. These peptides were fused to the N-terminus of the mutant A642N-T209D via homologous recombination. The resulting plasmids were cloned into the pET-28a(+) expression vector using XbaI and NheI restriction sites (the NheI site was removed in the final clone). Ne SuSy-A642N-T209D, all plasmids were sequenced and verified to be correct.

[0192] Table 8. Soluble tags and their protein and DNA sequences

[0193]

[0194] Transform the recombinant plasmid into E. coli BL21 (DE3), after induction of expression, bacterial cells were collected and sonicated for disruption, and then expressed as OD21. 600 The supernatant from cell lysis at a concentration of 6 was used as crude enzyme. Soluble expression was analyzed by SDS-PAGE, and the results are as follows: Picture 10 As shown in Table 9, the relative enzyme activity was determined using the crude enzyme activity of the His-tagged mutant as a 100% baseline.

[0195] Table 9. Table with different labels Ne The relative enzyme activity of the crude enzyme solution obtained from strain SuSy-A642N-T209D

[0196]

[0197] The results showed that, compared with the control protein carrying only the His tag, fusion expression of a soluble short peptide tag altered the expression characteristics of the target protein and increased its enzymatic activity. (See [link to relevant documentation]). Picture 10 See Table 9.

[0198] Specifically, in terms of total expression, such as Picture 10 As shown, the fusion of GKGKG, P17-m, T7B9 and N11 tags all led to a decrease in the total amount of the target protein, with the N11 tag showing the smallest decrease, while the SKIK tag had no significant effect on the total protein amount.

[0199] Regarding soluble expression, such as Picture 10 As shown, although the expression levels of each tag fusion protein in the lysate supernatant were basically the same as those of the control, there were differences in the proportion of soluble protein: the proportion of soluble protein in the His tag control, SKIK and N11 fusion proteins was relatively low; while the proportion of soluble protein in GKGKG, P17-m and T7B9 fusion proteins was significantly increased, and the content was basically equivalent to the total protein content.

[0200] Regarding enzyme activity, as shown in Table 9, with the activity of the control protein carrying only the His tag as 100%, the relative enzyme activities of each fusion protein are as follows: T7B9 tag 180%, GKGKG tag 162%, P17-m tag 160%, SKIK tag 146%, and N11 tag 115%.

[0201] In summary, T7B9 is the optimal soluble tag, while GKGKG, P17-m, and SKIK can be considered as candidate tags, which can significantly improve the soluble expression and relative enzyme activity of mutants in E. coli.

[0202] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sucrose synthase mutant, characterized in that: The sucrose synthase mutant is obtained by mutating the wild-type sucrose synthase shown in SEQ ID NO:1 through at least one of the following mutations: (1) The 29th amino acid was mutated from asparagine to glycine. (2) The 69th amino acid was mutated from serine to proline. (3) The 144th amino acid was mutated from valine to serine. (4) The 184th amino acid was mutated from asparagine to aspartic acid. (5) The 194th amino acid is mutated from asparagine to serine. (6) The 209th amino acid is mutated from threonine to aspartic acid. (7) The 241st amino acid was mutated from methionine to leucine. (8) The 477th amino acid was mutated from arginine to lysine. (9) The 527th amino acid was mutated from asparagine to glutamic acid. (10) The 597th amino acid is mutated from glycine to alanine. (11) The 599th amino acid is mutated from lysine to aspartic acid. (12) The 633rd amino acid is mutated from leucine to isoleucine. (13) The 635th amino acid is mutated from methionine to serine. (14) The 636th amino acid is mutated from arginine to glutamine. (15) The 637th amino acid was mutated from leucine to methionine. (16) The amino acid at position 638 is mutated from aspartic acid to asparagine. (17) The amino acid at position 639 is mutated from lysine to arginine. (18) The amino acid at position 642 is mutated from alanine to asparagine. (19) The 683rd amino acid was mutated from arginine to cysteine. (20) The 693rd amino acid is mutated from asparagine to histidine. (21) The 695th amino acid was mutated from arginine to lysine. (22) The 695th amino acid is mutated from arginine to valine. (23) The 706th amino acid is mutated from alanine to aspartic acid. (24) The 720th amino acid is mutated from leucine to lysine. (25) The 766th amino acid is mutated from leucine to histidine. (26) The 766th amino acid was mutated from leucine to valine.

2. The sucrose synthase mutant according to claim 1, characterized in that: The sucrose synthase mutant was obtained by subjecting the wild-type sucrose synthase shown in SEQ ID NO:1 to the following single-point mutation: (1) The 144th amino acid was mutated from valine to serine. (2) The 209th amino acid is mutated from threonine to aspartic acid. (3) The 241st amino acid was mutated from methionine to leucine. (4) The 477th amino acid was mutated from arginine to lysine. (5) The amino acid at position 633 is mutated from leucine to isoleucine. (6) The 637th amino acid is mutated from leucine to methionine. (7) The 639th amino acid was mutated from lysine to arginine. (8) The amino acid at position 642 is mutated from alanine to asparagine. (9) The 766th amino acid is mutated from leucine to histidine. Or (10) the 766th amino acid is mutated from leucine to valine; Alternatively, the sucrose synthase mutant is obtained by mutating the wild-type sucrose synthase shown in SEQ ID NO:1 through a combination of the following mutations: (1) Amino acid at position 642 was mutated from alanine to asparagine, and amino acid at position 209 was mutated from threonine to aspartic acid. (2) Amino acid at position 642 was mutated from alanine to asparagine, and amino acid at position 639 was mutated from lysine to arginine. (3) The amino acid at position 639 was mutated from lysine to arginine, and the amino acid at position 209 was mutated from threonine to aspartic acid. (4) The amino acid at position 637 is mutated from leucine to methionine, and the amino acid at position 639 is mutated from lysine to arginine. (5) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 209 was mutated from threonine to aspartic acid, and amino acid at position 477 was mutated from arginine to lysine. (6) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 639 was mutated from lysine to arginine, and amino acid at position 477 was mutated from arginine to lysine. (7) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 209 was mutated from threonine to aspartic acid, and amino acid at position 144 was mutated from valine to serine. (8) The amino acid at position 639 was mutated from lysine to arginine, the amino acid at position 637 was mutated from leucine to methionine, and the amino acid at position 209 was mutated from threonine to aspartic acid. (9) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 639 was mutated from lysine to arginine, and amino acid at position 209 was mutated from threonine to aspartic acid. (10) Amino acid at position 637 was mutated from leucine to methionine, amino acid at position 639 was mutated from lysine to arginine, and amino acid at position 642 was mutated from alanine to asparagine. (11) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 639 was mutated from lysine to arginine, amino acid at position 209 was mutated from threonine to aspartic acid, and amino acid at position 637 was mutated from leucine to methionine. (12) Amino acid at position 642 was mutated from alanine to asparagine, amino acid at position 209 was mutated from threonine to aspartic acid, and amino acid at position 766 was mutated from leucine to valine. Or (13) the amino acid at position 642 is mutated from alanine to asparagine, the amino acid at position 639 is mutated from lysine to arginine, and the amino acid at position 766 is mutated from leucine to valine.

3. A sucrose synthase mutant according to claim 1, characterized in that: The sucrose synthase mutant was obtained by combining the following mutations to the wild-type sucrose synthase shown in SEQ ID NO:1: (1) Amino acid at position 642 was mutated from alanine to asparagine, and amino acid at position 209 was mutated from threonine to aspartic acid. (2) Amino acid at position 642 was mutated from alanine to asparagine, and amino acid at position 639 was mutated from lysine to arginine. Or (3) the amino acid at position 642 is mutated from alanine to asparagine, the amino acid at position 209 is mutated from threonine to aspartic acid, and the amino acid at position 477 is mutated from arginine to lysine.

4. A sucrose synthase mutant according to claim 1, characterized in that: The N-terminus of the sucrose synthase mutant is fused with a soluble tag.

5. A sucrose synthase mutant according to claim 4, characterized in that: The soluble label is selected from one of GKGKG, P17-m, SKIK, T7B9, and N11.

6. The nucleic acid encoding the sucrose synthase mutant according to any one of claims 1-5.

7. A biomaterial, characterized in that: The biomaterial is at least one of the following: (a) An expression cassette containing the nucleic acid of claim 6; (b) A recombinant expression vector containing the nucleic acid of claim 6; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant microorganisms containing the nucleic acid of claim 6; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

8. A biomaterial according to claim 7, characterized in that: The starting vector of the recombinant expression vector is an Escherichia coli expression plasmid, and the host microorganism corresponding to the recombinant microorganism is selected from Escherichia coli.

9. The use of the sucrose synthase mutant according to any one of claims 1-5, the nucleic acid according to claim 6, or the biomaterial according to claim 7 or 8 in the catalytic synthesis of uridine diphosphate glucose.

10. A method for catalytic synthesis of uridine diphosphate glucose, characterized in that, Includes the following steps: Using the crude enzyme solution of the sucrose synthase mutant according to any one of claims 1-5 or the biological material according to claim 7 as a catalyst, and nucleoside diphosphate and sucrose as substrates, the reaction is carried out under conditions of pH 5.5-8.0 and 40-70℃ to produce uridine diphosphate glucose and fructose.

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