A bacterial-derived glycosyltransferase mutant

Abstract

The application provides a bacterial-derived glycosyltransferase mutant, and belongs to the technical field of enzyme engineering. Paenibacillus durus The wild-type glycosyltransferase is subjected to protein engineering. The specific enzyme activity of the optimal mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G reaches 58.5+ / -0.2 U / mg (increased by 189.6% compared with the wild type), the regioselectivity for salidroside is 99.9%, and the melting temperature (T m ) is 62.0 DEG C. The mutant still retains about 60% residual activity after incubation at 55 DEG C for 6 h, and the thermal stability and catalytic efficiency are significantly improved. The mutant provides an efficient biological catalyst for the industrial green production of salidroside.

Description

Technical Field

[0001] This invention relates to a bacterial glycosyltransferase mutant, belonging to the field of enzyme engineering technology. Background Technology

[0002] Rhodioloside (2-(4-hydroxyphenyl)ethyl-beta-D-glucopyranoside) is the main active phenylethanol glycoside found in the medicinal plant Rhodiola rosea, possessing a wide range of pharmacological effects including anti-fatigue, anti-hypoxia, neuroprotection, and cardiovascular protection. Currently, the industrial supply of rhodioloside mainly relies on physical extraction from wild Rhodiola rosea plants, but this method is severely limited by the slow plant growth cycle and the depletion of ecological resources. While chemical synthesis is feasible, it suffers from poor atom economy, cumbersome multi-step protection / deprotection procedures, and environmental pollution.

[0003] In recent years, the synthesis of rhodioloside via the glycosylation of tyrosol catalyzed by UDP-glucosyltransferases (UGTs) has become a promising green production pathway due to its mild reaction conditions and high specificity. Plant-derived UGTs (such as...) At UGT85A1 exhibits excellent regioselectivity for the primary hydroxyl group of tyrosol, but it suffers from drawbacks such as difficulty in heterologous expression in microbial hosts and extremely low solubility, making it difficult to meet the needs of large-scale industrial production. Microbial-derived UGTs typically have excellent soluble expression levels and high basic catalytic activity, but their regioselectivity is generally poor, easily leading to the mixed formation of rhodioloside and its regiomeric byproduct (icariin D2), greatly increasing downstream purification costs.

[0004] In 2025, Bingfang He et al. engineered the UDP-glycosyltransferase UGT derived from Bacillus licheniformis using protein engineering. BL 1. This method achieves a regioselectivity of 99.2% for rhodioloside, but its specific enzyme activity is only 0.4 U / mg, limiting its industrial application. Therefore, there is an urgent need to develop a novel glycosylation biocatalyst that combines high catalytic activity, excellent regioselectivity, robust structural stability, and superior soluble expression. Summary of the Invention

[0005] The first objective of this invention is to provide a bacterial glycosyltransferase mutant, based on the amino acid sequence shown in SEQ ID NO. 1, by performing at least one mutation at positions 11, 13, 45, 67, 81, 116, 130, 144, 146, 242, 323, 324, 325, and 388.

[0006] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO. 1, and involves any of the following mutations: (1) Replace the isoleucine at position 116 with leucine to obtain mutant I116L; (2) Replace the alanine at position 146 with phenylalanine to obtain mutant A146F; (3) Replace the methionine at position 324 with phenylalanine to obtain the mutant M324F; (4) Replace isoleucine at position 116 with leucine and alanine at position 146 with phenylalanine to obtain mutant I116L / A146F. (5) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine to obtain mutant I116L / A146F / M324F. (6) Replace isoleucine at position 11 with phenylalanine, isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine to obtain mutant I116L / A146F / M324F / I11F. (7) Replace the alanine at position 13 with leucine, the isoleucine at position 116 with leucine, the alanine at position 146 with phenylalanine, and the methionine at position 324 with phenylalanine to obtain the mutant I116L / A146F / M324F / A13L. (8) Replace isoleucine at position 116 with leucine, isoleucine at position 67 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine to obtain mutant I116L / A146F / M324F / I67L. (9) Replace the 81st alanine with tyrosine, the 116th isoleucine with leucine, the 146th alanine with phenylalanine, and the 324th methionine with phenylalanine to obtain the mutant I116L / A146F / M324F / A81Y. (10) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, valine at position 144 with asparagine, and methionine at position 324 with phenylalanine to obtain mutant I116L / A146F / M324F / V144N. (11) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, and methionine at position 324 with phenylalanine to obtain mutant I116L / A146F / M324F / Q323L. (12) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain mutant I116L / A146F / M324F / H325S. (13) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine to obtain the mutant I116L / A146F / M324F / I11F / A13L; (14) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N; (15) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, and methionine at position 324 with phenylalanine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L; (16) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S; (17) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y; (18) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L; (19) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / D45L; (20) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / E388D; (21) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, glutamic acid at position 130 with proline, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / E130P; (22) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, proline at position 242 with glycine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / P242G; (23) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid to obtain the mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / D45L / E388D; (24) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, and proline at position 242 with glycine. The mutant I116L / A146F / M324F / I11F / A13L / V144N / Q323L / H325S / A81Y / I67L / D45L / E388D / P242G was obtained by replacing glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid. (25) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, glutamic acid at position 130 with proline, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, and proline at position 242 with... The mutants were obtained by replacing glycine with leucine at position 323, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid.

[0007] A second objective of this invention is to provide a gene encoding the glycosyltransferase mutant.

[0008] A third objective of this invention is to provide an expression vector for expressing the mutant or carrying the gene, using pET28a(+) as the vector.

[0009] A fourth object of the present invention is to provide recombinant microbial cells carrying the gene or the expression vector.

[0010] In one embodiment, the recombinant microbial cells use bacteria or fungi as the host.

[0011] A fifth objective of this invention is to provide a recombinant *E. coli* strain expressing the glycosyltransferase mutant, using pET28a(+) as a vector, and... E. coli BL21(DE3) is the host.

[0012] A sixth object of the present invention is to provide a method for producing the glycosyltransferase mutant, wherein microbial cells expressing the glycosyltransferase mutant are inoculated in 2×YT medium and cultured at 35-37°C until OD. 600 When the concentration is 0.6~0.8, add the inducing agent IPTG and induce at 20℃ for 12~16 h.

[0013] In one embodiment, the method involves inoculating the recombinant Escherichia coli into a 2×YT expression medium containing kanamycin and culturing it at 37°C with shaking at 200 r / min until OD500 is reached. 600 When the concentration is 0.6-0.8, add IPTG to 0.4 mM and induce at 20℃ for 12-16 h to express the glycosyltransferase mutant enzyme.

[0014] In one embodiment, bacterial cells after induced expression are collected, the bacterial cells are lysed and the supernatant is collected, the supernatant is filtered through a 0.22 μm aqueous filter membrane, and the glycosyltransferase mutant is obtained by separation using a His Trap HP column.

[0015] A seventh object of the present invention is to provide the use of the mutant, or the gene, or the expression vector, or the recombinant microbial cell in the catalytic synthesis of rhodioloside and other active glycosides in the food, pharmaceutical or chemical fields.

[0016] In one embodiment, the mutant or the fermentation product of the recombinant Escherichia coli is added to a system containing tyrosol for reaction.

[0017] In one embodiment, the system further contains UDP-glucose, magnesium salt, and buffer solution.

[0018] In one embodiment, the reaction conditions are: temperature 40°C to 55°C, pH 7.5 to 9.0.

[0019] Beneficial effects: This invention constructs a variety of UDP-glycosyltransferase mutants through protein engineering. Among them, the specific enzyme activity of mutant I116L / A146F / M324F was 44.8±0.2 U / mg, which was 122.2% higher than that of wild type, and the regioselectivity for rhodioloside was 81.7%; the specific enzyme activity of mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S was 46.1±0.5 U / mg, and the regioselectivity for rhodioloside was 99.9%; the specific enzyme activity of mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G was 58.5±0.2 U / mg. U / mg, with a regioselectivity of 99.9% for rhodioloside, and a melting temperature (T m The specific activity was 62.0℃, which is 11.2℃ higher than that of the wild type. Compared with the wild type enzyme, this mutant enzyme has significantly improved specific activity, regioselectivity and thermostability, which is conducive to realizing the industrial-scale production of rhodioloside biosynthesis. Attached Figure Description

[0020] Figure 1 SDS-PAGE electrophoresis images of wild-type and mutant enzymes. M: Standard protein marker; 1-3 are, in order, wild-type lysate supernatant, wild-type lysate, and wild-type purified enzyme.

[0021] Figure 2 Bar charts showing the specific activity and regioselectivity of the pure enzymes for wild-type and mutant enzymes under the following conditions: 40℃, pH 8.0 (wild-type); 42℃, pH 8.0 (I116L / A146F / M324F); 42℃, pH 8.5 (I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S); 47℃, pH 8.5 (I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G). WT represents wild-type.

[0022] Figure 3 The optimal pH curves for wild-type and mutant enzymes measured at 40°C are shown.

[0023] Figure 4 The graph shows the optimal temperature curves for wild-type and mutant enzymes at pH 8.0, with WT representing wild-type.

[0024] Figure 5The graphs show the thermostability of the wild-type and mutant enzymes at 40℃ and 55℃, respectively. Detailed Implementation

[0025] 1. Culture medium LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0026] 2×YT expression medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L.

[0027] 2. Methods for determining the activity, selectivity, and enzymatic properties of glycosyltransferases (1) Reaction system and conditions: The total enzymatic reaction volume was 500 μL, consisting of: 22.5 mM tyrosol, 25 mM UDP-glucose, 17.5 mM MgCl2, 75 mM Bicine buffer (pH 8.0), and an appropriate concentration of purified enzyme solution. The reaction was initiated at 40 °C for 3 min, then terminated by heating in a 95 °C metal bath for 10 min. The reaction solution was centrifuged at 13000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane for subsequent assays. The enzyme activity assay conditions for each mutant were adjusted according to its optimal temperature and pH, adjusting the reaction temperature and buffer pH accordingly.

[0028] (2) High-performance liquid chromatography (HPLC) detection method: The concentrations of rhodioloside and icariin D2 were quantitatively determined using high-performance liquid chromatography (HPLC). Mobile phase: Phase A was 0.1% formic acid aqueous solution, and Phase B was methanol. Elution conditions: A:B = 80:20 (isocratic elution). Flow rate: 0.7 mL / min. Column temperature: 25℃. Detection wavelength: 280 nm.

[0029] (3) Calculation of evaluation indicators: ① Enzyme activity definition: Under the above standard reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol of rhodioloside per minute is defined as 1 enzyme activity unit (U).

[0030] ② Specific enzyme activity calculation: Specific enzyme activity (U / mg) = Total enzyme activity (U) / Total amount of protein added to the reaction system (mg). Relative enzyme activity calculation: Using the specific enzyme activity of wild type as 100%, calculate the percentage of enzyme activity of mutant relative to wild type.

[0031] ③ Definition of regional selectivity: Regional selectivity (%) = [Rhodioloside production / (Rhodioloside production + Icariin D2 production)] × 100%.

[0032] (4) Determination of the optimal pH: In a standard reaction system, keeping other conditions constant, only the pH gradient of the Bicine buffer was adjusted, and the relative enzyme activity under different pH conditions was measured to determine the optimal pH of the enzyme.

[0033] (5) Determination of the optimal temperature: Under optimal pH conditions, an enzymatic reaction was carried out at a temperature gradient ranging from 30°C to 55°C, and the relative enzyme activity at each temperature was measured to determine the optimal reaction temperature.

[0034] (6) Temperature stability test: Purified enzyme solutions of a certain concentration were incubated at 40℃ (wild type or mutant), 45℃ (wild type or mutant), or 55℃ (mutant), respectively. Samples were taken at different time points and cooled on ice. The residual enzyme activity was then measured under optimal reaction conditions, with the enzyme activity without incubation taken as 100%, to evaluate the thermal inactivation characteristics of the enzyme.

[0035] Melting temperature (T) was determined using the protein thermal displacement analysis (DSF) method. m The purified enzyme solution was mixed with a fluorescent dye, and the change in fluorescence intensity with increasing temperature was monitored using a qPCR instrument. The temperature at which the protein undergoes thermal denaturation, T, was determined by analyzing the derivative peak of the fluorescence curve. m The value is used to evaluate the structural thermal stability of enzymes.

[0036] The half-life is calculated based on the first-order inactivation reaction equation of the enzyme catalytic reaction, as shown in formulas ① and ②: First-order deactivation reaction equation: ln( A / A 0 ) = - K D t ① Half-life: t 1 / 2 = ln2 / K D ② in A To maintain heat at 45℃ t Residual enzyme activity after time A 0 represents the initial enzyme activity. K D t is the inactivation constant of the enzyme at 45℃. 1 / 2 This is the half-life of the enzyme at 45°C.

[0037] Example 1: Recombinant Escherichia coli BL21 / pET28- Pd Construction and purification of MGT 1. Recombinant plasmid pET28- PdMGT Construction choose Paenibacillus durus The derived glycosyltransferase is wild-type, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence encoding the glycosyltransferase is shown in SEQ ID NO.2. The nucleotide sequence SEQ ID NO.2 was synthesized in its entirety by Suzhou Anshengda Biotechnology Co., Ltd., and inserted into the pET28 vector. Bam HI and Hind In the III restriction site, the recombinant plasmid pET28- was obtained. Pd MGT. The recombinant plasmid obtained from the construction was transferred into... E. coli BL21(DE3) competent cells were incubated on ice for 30 min, then heat-shocked at 42℃ for 90 s. 1 mL of antibiotic-free LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) was added, and the cells were incubated at 37℃ in a shaker for 1 h. The culture was then centrifuged at 5000 rpm for 2 min, and most of the supernatant was removed using a pipette, retaining 100-200 μL for resuspending the cells. The resuspended cells were spread on antibiotic-resistant solid medium containing kanamycin and incubated overnight at 37℃. Positive transformants were then collected.

[0038] 2. Expression of recombinant glycosyltransferase The correctly identified recombinant E. coli BL21 / pET28- Pd MGT single colonies were inoculated into 5 mL of LB liquid medium with a kanamycin concentration of 50 μg / mL and activated by shaking at 37°C and 200 r / min for 6-8 h.

[0039] The above cultures were inoculated at a rate of 2% (v / v) into 50 mL of 2×YT expression medium containing 50 μg / mL kanamycin. The cultures were incubated at 37℃ with shaking at 200 r / min until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.4 mM, and expression was induced at 20℃ for 12-16 h. The fermentation broth was centrifuged at 8000 rpm for 10 min to collect the cells. The cells were then analyzed at OD600... 600 The precipitate was ultrasonically disrupted under conditions of 5, followed by centrifugation at 13000 rpm for 10 min. The precipitate was resuspended in an equal volume of deionized water. The supernatant and precipitate were analyzed and identified using SDS-PAGE, respectively. Figure 1 As shown.

[0040] 3. Purification of recombinant glycosyltransferase The collected recombinant bacterial cells were suspended in 10 mL of binding buffer (50 mmol / L Na₂HPO₄, 50 mmol / L NaH₂PO₄, 500 mmol / L NaCl, 20 mmol / L imidazole) and sonicated. The disrupted bacterial suspension was centrifuged at 13000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter. A 1 mL His Trap HP affinity chromatography column was equilibrated with 10 column volumes of binding buffer. After loading the sample, non-specifically adsorbed proteins were washed away with 15 column volumes of binding buffer, followed by a linear gradient elution with 27 column volumes of buffer containing 20-500 mmol / L imidazole. The purified target enzyme solution was collected and analyzed by SDS-PAGE for identification. Figure 1 As shown. At 40℃, the specific activity and regioselectivity of the wild-type pure enzyme were detected. The obtained wild-type enzyme was... Pd The specific enzyme activity of the purified MGT enzyme was 20.2 ± 0.3 U / mg, and the regioselectivity for rhodioloside was 81.2%. Figure 2 ).

[0041] Example 2: Glycosyltransferase Pd Construction of MGT mutants 1. Glycosyltransferase Pd Construction of MGT single mutation site Depending on the purpose of the modification, this invention employs two different primer design strategies for whole-plasmid PCR amplification. The PCR system and conditions are shown in Table 1. The PCR products are then... Dpn After digestion of the parental template by enzyme I, the cells were transformed into competent cells. E. coli JM109 was used to obtain a clone carrying the coding mutant.

[0042] (1) For sites related to activity and selectivity: using primers containing NNK degenerate codons (as shown in Table 1), with pET28- Pd MGT was used as a template for site-directed saturation mutagenesis. Sequencing screening yielded mutants with single mutation sites at I116, A146, M324, I11, A13, V144, Q323, I67, A81, and H325. Taking the construction of mutant I116L as an example, pET28- Pd Using MGT as a template, saturation mutation PCR (Table 2) and subsequent screening were performed using I116X-F and I116X-R primers to obtain the recombinant plasmid pET28- Pd MGT-I116L.

[0043] (2) For sites related to thermal stability: using primers containing specific target amino acid mutant codons (as shown in Table 1), with pET28- Pd MGT was used as a template for targeted construction. Single mutants D45L, E388D, P242G, and E130P were obtained. Taking the construction of mutant D45L as an example, pET28- Pd Using MGT as a template and D45L-F and D45L-R, which contain specific mutant codons, as primers, full-plasmid PCR was performed, and the resulting recombinant plasmid was named pET28- Pd MGT-D45L.

[0044] Transform the recombinant plasmids of the single mutants that were correctly sequenced. E. col From strain i BL21(DE3), recombinant strains expressing each single mutant were obtained. Taking the construction of a recombinant strain expressing mutant I116L as an example, the recombinant strain was named BL21 / pET28- Pd MGT-I116L.

[0045] Table 1 Primers

[0046] Table 2. PCR amplification reaction system for whole plasmids

[0047] The PCR amplification reaction conditions were as follows (30 cycles): 98℃, pre-denaturation, 1 min; 98℃, denaturation, 30 s; Annealing at 55℃ for 30 seconds; 72℃, extended, 1 min 10 s; 72℃, extended, 5 min.

[0048] 2. Glycosyltransferase Pd Construction of combined mutants of MGT The optimal single mutant plasmid obtained by the above saturation mutation screening (such as pET28-) Pd Using MGT-I116L as a template, and primers containing specific mutant codons as shown in Table 3, site-directed mutagenesis was performed according to the full plasmid PCR procedure described above to construct a recombinant mutant. Taking the construction of the recombinant mutant I116L / A146F as an example, pET28- Pd Using MGT-I116L as a template, full plasmid PCR was performed using primers (A146F-F and A146F-R) containing a specific mutation in A146F. The PCR products were then processed... DpnAfter digestion and transformation, the recombinant plasmid obtained is pET28- Pd MGT-I116L / A146F. Transform the correctly sequenced recombinant plasmid. E. coli The BL21(DE3) strain was used to obtain a recombinant strain named BL21 / pET28- Pd MGT-I116L / A146F.

[0049] (Note: The construction of other higher-level combined mutants is carried out using this direct iterative method, which uses the recombinant plasmid obtained in the previous round as a template and introduces new mutations sequentially using specific mutation primers targeting the target site.) Table 3 Primers

[0050] 3. Glycosyltransferase Pd Characterization of enzyme activity, regioselectivity, and thermostability of MGT mutants Wild-type [type of organism] was obtained according to the expression, purification, and enzymatic property determination methods described in the foregoing embodiments. Pd Pure enzyme solutions of MGT and its mutants were prepared, and reactions and detections were performed using tyrosol as a substrate. Wild type Pd The basic assay data for MGT (WT) are as follows: specific enzyme activity 20.2 ± 0.3 U / mg (detection conditions: 40℃, pH 8.0), regioselectivity for rhodioloside 81.2%, and melting temperature (T). m The activity was 50.8℃, and the residual activity was 15% after incubation at 40℃ for 1 h. The half-life at 45℃ was 6.3 min.

[0051] The results of modifications targeting key active sites are shown in Table 4. Through iteration at these sites, the final mutant I116L / A146F / M324F exhibited an activity of 44.8 ± 0.2 U / mg, representing a 122.2% increase compared to the wild type, with a regioselectivity of 81.7% and a melting temperature (T0). m The activity was 51.8℃, and the residual activity was 41% after incubation at 40℃ for 1 h. The half-life was 14.6 min at 45℃.

[0052] Table 4 Results of Activity Modification

[0053] Based on the activity modification, modifications were made to key selectivity sites, and the results are shown in Table 5. Through iterative modifications at these sites, the specific enzyme activity of the combined mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S was 46.1 ± 0.5 U / mg, an increase of 128.2% compared to the wild type. The regioselectivity was 99.9%, an increase of 19.8% compared to the wild type. The melting temperature (T) was also improved. m The activity was 51.6℃, and the residual activity was 35% after incubation at 40℃ for 1 h. The half-life at 45℃ was 13.5 min.

[0054] Table 5 Results of Selective Modification

[0055] Based on the modifications to activity and selectivity, modifications were made to improve thermal stability, and the results are shown in Table 6. The final combined mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G had a specific enzyme activity of 58.5 ± 0.2 U / mg, a regioselectivity of 99.9%, and a melting temperature (T0). m The residual activity was 98% after incubation at 40℃ for 1 h, and the half-life was 802 min at 45℃.

[0056] Table 6 Results of Stability Modification

[0057] Example 3: Determination of Optimal pH The total reaction volume of the assay system was 500 μL, containing 22.5 mM tyrosol, 25 mM UDP-glucose (UDPG), 17.5 mM MgCl2, 75 mM Bicine buffer with different pH gradients, and pure enzyme solution with a final concentration of 10 μg / mL. The pH gradient of the different Bicine buffers was set in the range of 7.5-9.0, and the Bicine buffers and each substrate stock solution were adjusted to the corresponding reaction pH by adding appropriate amounts of HCl or NaOH. Wild-type and mutant proteins were added to the reaction systems containing different pH values, reacted at 40 °C for 3 min, and then heated in a 95 °C metal bath for 10 min to inactivate the protein, followed by enzyme activity measurement.

[0058] like Figure 3As shown, with the highest enzyme activity of the mutant and wild-type enzymes each recorded as 100%, the optimal pH for both the wild-type and mutant I116L / A146F / M324F is 8.0, while the optimal pH for the combined mutants I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S and I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G is increased to 8.5, indicating that their catalytic adaptability under slightly alkaline conditions has been improved.

[0059] Example 4: Determination of Optimal Temperature The total reaction volume of the assay system was 500 μL, containing 22.5 mM tyrosol, 25 mM UDPG, 17.5 mM MgCl2, 75 mM Bicine buffer (pH 8.0), and purified enzyme solution with a final concentration of 10 μg / mL. The reaction systems containing wild-type or mutant enzymes were incubated at different temperature gradients for 3 min each, followed by heating at 95 °C for 10 min to inactivate the protein, and the corresponding enzyme activities were measured.

[0060] like Figure 4 As shown, with the highest enzyme activity of the mutant and wild-type enzymes respectively defined as 100%, the optimal reaction temperature for the wild-type enzyme is 40℃, while the optimal reaction temperature for mutants I116L / A146F / M324F and I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S is increased to 42℃, and the optimal reaction temperature for mutants I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G is significantly increased to 47℃.

[0061] Example 5: Thermal stability determination Wild-type or mutant purified enzyme solutions at a concentration of 1 mg / mL were incubated at 40°C (wild-type or mutant) and 55°C (mutant) for different times. After incubation for a certain period, samples were taken, and the incubated purified enzyme solution was added to a 500 μL system (containing 22.5 mM tyrosol, 25 mM UDPG, 17.5 mM MgCl2, and 75 mM Bicine buffer, pH 8.0) to a final concentration of 10 μg / mL. After a standard reaction at 40°C for 3 min, the protein was inactivated by heating at 95°C for 10 min, and the residual enzyme activity was measured.

[0062] like Figure 5 As shown, the highest enzyme activity of the unincubated mutant and wild-type enzymes is defined as 100%. After incubation at 40℃ for 1 h, the residual activity of the wild-type enzyme decreased to only 15%. In contrast, the mutant I116L / A146F / M324F / I11F / A13L / I67L / A81Y / V144N / Q323L / H325S / D45L / E388D / E130P / P242G retained 98% of its residual enzyme activity after incubation at 40℃ for 1 h, and 89% after incubation at 6 h; after incubation at 55℃ for 6 h, the residual activity was still 60%. These data indicate that the mutant has achieved a significant breakthrough in thermal stability compared to the wild type, meeting the requirements of industrial high-temperature catalysis to a certain extent.

[0063] Comparative Example 1: Glycosyltransferase Pd Construction and relative enzyme activity of other MGT mutants For specific implementation methods, refer to Examples 1 and 2, the difference being that the primers described in Table 7 were used to construct mutant strains at other sites, such as pET28- Pd MGT-P148A and pET28- Pd MGT-F88A and other mutant strains were analyzed, and their specific enzyme activities were measured. The results showed that at 40℃, the relative enzyme activities of mutants P148A and F88A were 10.8% and 102.5%, respectively. The specific enzyme activities of these two single-point mutants showed a significant decrease or a change within the error range compared to the wild type. The specific enzyme activities of these two single-point mutants differed significantly from those of the aforementioned dominant mutants I116L and A146F; therefore, no further studies were conducted on them.

[0064] Table 7 Primers

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glycosyltransferase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 1, at least one mutation is performed at position 11, position 13, position 45, position 67, position 81, position 116, position 130, position 144, position 146, position 242, position 323, position 324, position 325, and position 388.

2. The mutant according to claim 1, characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 1, perform any of the following mutations: (1) Replace isoleucine at position 116 with leucine; (2) Replace the alanine at position 146 with phenylalanine; (3) Replace the methionine at position 324 with phenylalanine; (4) Replace isoleucine at position 116 with leucine and alanine at position 146 with phenylalanine; (5) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine. (6) Replace isoleucine at position 11 with phenylalanine, isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine. (7) Replace the alanine at position 13 with leucine, the isoleucine at position 116 with leucine, the alanine at position 146 with phenylalanine, and the methionine at position 324 with phenylalanine. (8) Replace isoleucine at position 116 with leucine, isoleucine at position 67 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine. (9) Replace the 81st alanine with tyrosine, the 116th isoleucine with leucine, the 146th alanine with phenylalanine, and the 324th methionine with phenylalanine. (10) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, valine at position 144 with asparagine, and methionine at position 324 with phenylalanine. (11) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, and methionine at position 324 with phenylalanine. (12) Replace isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (13) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine. (14) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, and methionine at position 324 with phenylalanine. (15) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, and methionine at position 324 with phenylalanine. (16) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (17) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (18) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (19) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (20) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid. (21) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, glutamic acid at position 130 with proline, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (22) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, proline at position 242 with glycine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, and histidine at position 325 with serine. (23) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid. (24) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, proline at position 242 with glycine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid. (25) Replace isoleucine at position 11 with phenylalanine, alanine at position 13 with leucine, aspartic acid at position 45 with leucine, isoleucine at position 67 with leucine, alanine at position 81 with tyrosine, isoleucine at position 116 with leucine, glutamic acid at position 130 with proline, valine at position 144 with asparagine, alanine at position 146 with phenylalanine, proline at position 242 with glycine, glutamine at position 323 with leucine, methionine at position 324 with phenylalanine, histidine at position 325 with serine, and glutamic acid at position 388 with aspartic acid.

3. The gene encoding the mutant of claim 1 or 2.

4. Expressing the mutant of claim 1 or 2 or an expression vector carrying the gene of claim 3.

5. Recombinant microbial cells carrying the gene of claim 3 or the expression vector of claim 4.

6. The recombinant microbial cell as described in claim 5, characterized in that, It uses bacteria or fungi as hosts.

7. A recombinant *E. coli* expressing the glycosyltransferase mutant of claim 1 or 2, characterized in that, Using pET28a(+) as a vector, E. coli BL21(DE3) is the host.

8. A method for producing the mutant of claim 1 or 2, characterized in that, After fermenting the recombinant Escherichia coli of claim 7 in a culture medium for a period of time, IPTG was added to induce expression.

9. A method for producing rhodioloside, characterized in that, The fermentation product of the mutant of claim 1 or 2 or the recombinant Escherichia coli of claim 7 is added to a system containing tyrosol for reaction.

10. The method according to claim 9, characterized in that, The reaction conditions are: temperature 40℃~55℃, pH 7.5-9.0.

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