High-temperature-resistant glycosyl transferase mutant as well as preparation method and application thereof

Abstract

The invention relates to a high-temperature-resistant glycosyl transferase mutant and application thereof, the amino acid sequence of the glycosyl transferase mutant is that T363I mutation or C445L mutation occurs on the basis of the amino acid sequence (UGT91C1-F208M (M0)) as shown in SEQ ID NO: 1, and the Tm value is increased by 7.3-8.4 DEG C compared with the glycosyl transferase UGT91C1-F208M (M0) on the premise of keeping the catalytic activity relatively unchanged. Therefore, the mutation sites T363I and C445L provided by the invention can obviously improve the heat resistance of the glycosyl transferase M0, and are beneficial to promotion of wide application of the glycosyl transferase M0 in the field of industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a glycosyltransferase mutant and an application thereof. Background Art

[0002] Sugar has many positive effects on the human body. First of all, sugar is an essential nutrient for the human body. It is mainly composed of carbon, hydrogen, and oxygen elements and is a type of carbohydrate. During exercise or fatigue, sugar can be quickly absorbed, increase blood sugar, replenish energy, relieve fatigue, and maintain normal body activities. At the same time, moderate intake of sugar can promote the secretion of dopamine, increase the serotonin content in the human brain, increase appetite, and make people happy. However, excessive intake of sugar can also bring adverse effects, such as increased blood sugar levels, causing diabetes, obesity, cardiovascular and cerebrovascular diseases, etc. In recent years, the risk of dental caries, obesity, diabetes, hypertension, and cardiovascular diseases has continued to increase worldwide. Therefore, consumers prefer low-calorie or calorie-free sweeteners.

[0003] Stevioside is a class of natural sweeteners with high sweetness extracted from stevia leaves. The most ideal components, rebaudioside D (Reb D) and rebaudioside M (Reb M), are highly sweet and have no bitter aftertaste. However, due to the low level of β(1-2)-glycosylation in stevia, their contents in the leaves are relatively low. UGT91C1 is a uridine diphosphateglycosyltransferase (UGTs) derived from rice. It can add a second glucose group at C13 or C19 on the stevioside substrate by forming a β(1-2)-glycosidic bond, such as generating Reb D from rebaudioside A (Reb A) in the latter way, so it plays an important role in the synthesis of high-quality sweeteners. In 2017, the U.S. Food and Drug Administration (FDA) also certified Reb D as "GRAS". Therefore, Reb D is a potential natural sweetener with better quality than Reb A.

[0004] As the taste requirements for sweeteners increase, the demand for high-end products such as Reb D is growing. However, in stevia plants, the content of Reb D is extremely low, accounting for only about 0.5% of the dry leaf weight, and the cost of separation and extraction is high, which cannot meet market demand. In addition, the thermal stability of the UGT91C1 mutant UGT91C1-F208M (M0), the glycosyltransferase with the highest known catalytic activity, is difficult to meet the needs of industrial production, which further increases the amount of glycosyltransferase used in industrial production, resulting in certain restrictions on industrial production. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a thermostable glycosyltransferase mutant and application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a thermostable glycosyltransferase mutant, wherein the amino acid sequence of the glycosyltransferase mutant is a T363I mutation or a C445L mutation occurring on the basis of the amino acid sequence shown in SEQ ID NO:1.

[0008] The amino acid sequence with T363I mutation is SEQ ID NO: 2;

[0009] The amino acid sequence with C445L mutation is SEQ ID NO: 3.

[0010] SEQ ID NO: 1:

[0011] MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEF LGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERMSLTLSRSSLVVGRSCVEFEPETVPL LSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAA VGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDLEHHH HHH

[0012] SEQ ID NO:2:

[0013] MDSGYSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERMSLTLSRSSLVVGRSCVEFEPETVLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRLWDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSIIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDLEHHHHHHHH

[0014] SEQ ID NO:3:

[0015] MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFL GTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERMSLTLSRSSLVVGRSCVEFEPETVPLLS TLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVG AFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMALHERYIDGFIQQLRSYKDLEHHHHHH

[0016] The present invention provides a thermostable glycosyltransferase mutant, which is a protein that undergoes an amino acid mutation as shown in SEQ ID NO: 1 (i.e., glycosyltransferase mutant UGT91C1-F208M (M0)) and has an enzyme activity that catalyzes rebaudioside A to rebaudioside D.

[0017] Preferably, the amino acid sequence of the glycosyltransferase mutant is based on the sequence shown in SEQ ID NO: 1, with the amino acid at position 363 changing from Thr to Ile.

[0018] Preferably, the amino acid sequence of the glycosyltransferase mutant is that the 445th amino acid of the glycosyltransferase shown in SEQ ID NO: 1 is changed from Cys to Leu.

[0019] The present invention significantly improves the thermal stability of glycosyltransferase UGT91C1-F208M (M0) by site-directed mutagenesis. Compared with glycosyltransferase M0, the optimal mutant T m The value was increased by 8.4℃, which can significantly reduce the amount of enzyme used and has good application prospects.

[0020] In a second aspect, the present invention provides a DNA molecule encoding the glycosyltransferase mutant according to the first aspect.

[0021] Preferably, the DNA molecule comprises a DNA sequence encoding the amino acid sequence SEQ ID NO:2 or SEQ ID NO:3.

[0022] In a third aspect, the present invention provides a recombinant expression plasmid, wherein the recombinant expression plasmid is connected to the DNA molecule described in the second aspect.

[0023] In a fourth aspect, the present invention provides a host cell, into which the recombinant expression plasmid described in the third aspect is transformed.

[0024] Preferably, the host cell is Escherichia coli.

[0025] Preferably, the Escherichia coli is an Escherichia coli C41 (DE3) strain.

[0026] In a fifth aspect, the present invention provides a method for preparing the glycosyltransferase mutant according to the first aspect, the method for preparing the glycosyltransferase mutant comprising:

[0027] The DNA molecule encoding the glycosyltransferase mutant described in the first aspect is inserted into an expression plasmid, the expression plasmid is transformed into a host cell, the host cell is fermented and cultured, and the glycosyltransferase mutant is separated and purified.

[0028] Preferably, the method for separating and purifying the glycosyltransferase mutant is to disrupt the host cells and extract the glycosyltransferase mutant protein.

[0029] Preferably, the method for disrupting the host cells may be a high-pressure homogenization disruption method, and the method for extracting the glycosyltransferase mutant protein may be a nickel column gradient elution method.

[0030] In a sixth aspect, the present invention provides the use of the glycosyltransferase mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant expression plasmid described in the third aspect, the host cell described in the fourth aspect, and the method for preparing the glycosyltransferase mutant described in the fifth aspect in the preparation of rebaudioside D.

[0031] In a seventh aspect, the present invention provides a method for preparing rebaudioside D, wherein the method utilizes the glycosyltransferase mutant described in the first aspect to catalyze a substrate to obtain the rebaudioside D.

[0032] Preferably, the substrate comprises rebaudioside A and a glycosyl donor.

[0033] Preferably, the glycosyl donor can be selected from guanosine diphosphate glucose (UDPG).

[0034] In an eighth aspect, the present invention provides the use of rebaudioside D prepared by the method for preparing rebaudioside D described in the seventh aspect in the field of food additives.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention significantly improves the thermal stability of glycosyltransferase UGT91C1-F208M (M0) by site-directed mutagenesis. Compared with glycosyltransferase M0, the optimal mutant T m The value was increased by 8.4℃, which can significantly reduce the amount of enzyme used and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a diagram showing the results of measuring the enzyme catalytic activity of the mutant protease prepared in Example 2;

[0038] Figure 2 is a graph showing the surface melting temperature characterization results of the mutant protease prepared in Example 2;

[0039] Figure 3 is the optimum temperature T of the mutant protease obtained in Example 2 opt Characterize the result line graph;

[0040] Figure 4 is the optimum temperature T of the mutant protease obtained in Example 2 opt Characterize the results in histogram;

[0041] Figure 5 is the half-inactivation temperature T of the mutant protease obtained in Example 2 50 Characterization result diagram;

[0042] Figure 6 is the half-life t of the mutant protease prepared in Example 2 at 40°C 1 / 2 Characterization result diagram;

[0043] Figure 7 is the half-life t of the mutant protease prepared in Example 2 at 45°C 1 / 2 Characterization results diagram. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0046] The Escherichia coli host strain C41 (DE3) used in the following examples is a commercial strain purchased from Beijing Qingke Biotechnology Co., Ltd.

[0047] The plasmid vector pET-32a used in the following examples is a commercial plasmid purchased from Beijing Qingke Biotechnology Co., Ltd.

[0048] Example 1 Acquisition of glycosyltransferase UGT91C1 gene and construction of mutants

[0049] The amino acid sequence of rice-derived glycosyltransferase UGT91C1 (accession number: XP_015629141.1) was downloaded from GenBank, and the sequence was optimized according to the codon preference of Escherichia coli. At the same time, the 208th amino acid was changed from Phe to Met to obtain the codon-optimized UGT91C1-F208M (M0) gene sequence, whose sequence is SEQ ID NO: 1.

[0050] The whole gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. and connected to the multiple cloning restriction site of the vector pET-32a to obtain the recombinant plasmid pET-32a-M0.

[0051] Using the recombinant plasmid pET-32a-M0 as a template, the following primer pairs were used for whole plasmid PCR and site-directed mutagenesis to construct the corresponding recombinant plasmids carrying mutants:

[0052] G151F-F:GATGATGCTGCTGTTTAGTGCCCAC; (SEQ ID NO:4)

[0053] G151F-R: GTGGGCACTAAACAGCAGCATCATC; (SEQ ID NO:5)

[0054] T230Y-F: TGAACCGGAATATGTGCCTCTGC; (SEQ ID NO: 6)

[0055] T230Y-R: GCAGAGGCACATATTCCGGTTCA; (SEQ ID NO:7)

[0056] T243V-F: GCGCGGTAAACCGATTGTGTTTCTGGGTCTGATGC; (SEQ ID NO: 8)

[0057] T243V-R: GCATCAGACCCAGAAACACAATCGGTTTACCGCGC; (SEQ ID NO:9)

[0058] V264L-F:GAAGATGCCACCCTGCGCTGGCTGG; (SEQ ID NO:10)

[0059] V264L-R:CCAGCCAGCGCAGGGTGGCATCTTC;(SEQ ID NO:11)

[0060] A272P-F:GGATGCACAGCCGCCGAAATCAGTTG;(SEQ ID NO:12)

[0061] A272P-R:CAACTGATTTCGGCGGCTGTGCATCC;(SEQ ID NO:13)

[0062] A323P-F:CAGATTTACTGCCGCCGGGTTTTGAAGAAC;(SEQ ID NO:14)

[0063] A323P-R:GTTCTTCAAAACCCGGCGGCAGTAAATCTG;(SEQ ID NO:15)

[0064] A349P-F:GTCTATTCTGGCACATCCGGCAGTTGGCGCATTTC;(SEQ ID NO:16)

[0065] A349P-R:GAAATGCGCCAACTGCCGGATGTGCCAGAATAGAC;(SEQ ID NO:17)

[0066] T363I-F:GTGGCTGGAATAGTATTATTGAAGGTC;(SEQ ID NO:18)

[0067] T363I-R:GACCTTCAATAATACTATTCCAGCCAC;(SEQ ID NO:19)

[0068] A399P-F:GTCTGCAAGTTCCGCGTAATGATGG;(SEQ ID NO:20)

[0069] A399P-R:CCATCATTACGCGGAACTTGCAGAC;(SEQ ID NO:21)

[0070] G411S-F:CTTTGATCGTGAAAGCGTTGCAGCAGC;(SEQ ID NO:22)

[0071] G411S-R:GCTGCTGCAACGCTTTCACGATCAAAG;(SEQ ID NO:23)

[0072] C445L-F: CAGATATGGCACTGCATGAACGTTATATTG; (SEQ ID NO: 24)

[0073] C445L-R: CAATATAACGTTCATGCAGTGCCATATCTG; (SEQ ID NO: 25)

[0074] G452E-F: GTTATATTGATGAATTTATTCAGCAGCTGC; (SEQ ID NO: 26)

[0075] G452E-R: GCAGCTGCTGAATAAATTCATCAATATAAC; (SEQ ID NO: 27)

[0076] The mutant plasmids obtained were sequenced and identified by Beijing Qingke Biotechnology Co., Ltd., and the plasmids pET-32a-M0-G151F, pET-32a-M0-T230Y, pET-32a-M0-T243V, pET-32a-M0-V264L, pET-32a-M0-A272P, pET-32a-M0-A232P, and pET-32a-M0-A3 49P, pET-32a-M0-T363I, pET-32a-M0-A399P, pET-32a-M0-G411S, pET-32a-M0-C445L, pET-32a-M0-G452E and template plasmid pET-32a-M0 were transformed into host bacteria C41 (DE3) competent cells, and then spread on the medium containing 50 μg / mL ampicillin sodium (Ampicillin sodium) LB solid plates (25 g / L LB Broth, 15 g / L Agar), inverted culture at 37 ° C, screening positive transformants, and then obtaining Escherichia coli strains containing the corresponding mutant plasmids, which were named: M0, M0-G151F, M0-T230Y, M0-T243V, M0-V264L, M0-A272P, M0-A232P, M0-A349P, M0-T363I, M0-A399P, M0-G411S, M0-C445L, and M0-G452E.

[0077] Example 2 Induced expression and extraction and purification of protease

[0078] The E. coli strain containing the mutant plasmid obtained in Example 1 was inoculated into LB medium (25 g / L LB Broth), and cultured with shaking at 170 rpm and 37° C. for 8-12 h to obtain a primary seed solution.

[0079] The first-level seed solution was inoculated into 1000 mL TB Media (12 g / L Tripton, 24 g / L Yeast Extract, 0.4% glycerol, 23.1 g / L KH2PO4, 164.32 g / L K2HPO4·3H2O) at a ratio of 10% (v / v), cultured at 170 rpm and 37°C, and the OD600 was cultured to 0.6-0.8 to obtain the second-level seed solution.

[0080] IPTG (final concentration 0.5 mM) was added to the secondary seed solution for induction, and the bacteria were placed at 170 rpm and 18 ° C for 16 hours to induce the expression of protease. The bacterial solution was collected and centrifuged at 4 ° C and 6000 rpm for 15 minutes, the supernatant was discarded, and the bacterial mud was resuspended in a disruption buffer (Tris 50 mM, NaCl 500 mM, imidazole 20 mM, glycerol 10% (v / v), β-mercaptoethanol 1 mM, 1% Tween 20, adjusted to pH 8) for disruption.

[0081] The bacterial solution was crushed using a high-pressure homogenizer, and 1% PMSF was added before crushing. The specific experimental conditions were 4°C, crushing pressure 1500 bar, and crushing 5 times. The cell crushing liquid was centrifuged at 4°C, 9000 rpm for 20 minutes, and the precipitate was discarded and the supernatant was collected.

[0082] The supernatant after centrifugation was filtered through a 0.45 μM filter head, and the filtrate was pumped to Ni in a 4°C chromatography cabinet. 2+ In the affinity chromatography column, the mutant protein with the His-tag binds to the Ni 2+ The affinity adsorbed material was collected on the column, and the flow-through was collected for subsequent verification.

[0083] After balancing the A and B pumps of the AKTAprimer plus system using Buffer A (Tris 50 mM, NaCl 500 mM, imidazole 20 mM, glycerol 10% (v / v), β-mercaptoethanol 1 mM, pH adjusted to 8) and Buffer B (Tris 50 mM, NaCl 500 mM, imidazole 250 mM, glycerol 10% (v / v), β-mercaptoethanol 1 mM, pH adjusted to 8), the nickel column bound to the mutant protein was connected and flushed with Buffer A until the baseline was stable. During this process, Buffer A can elute the unbound impurities in the nickel column. The content of Buffer B flowing through the nickel column was then set to 10%, 20%, and 30% in sequence. Different concentrations of imidazole were used to compete for the elution of impurities with weak binding. After the baseline was balanced again, Buffer B was adjusted to 100% to elute UGT91C1 and the peak liquid was collected.

[0084] In order to remove the influence of high concentration imidazole on subsequent experiments, Buffer B was replaced with Desalting Buffer (Tris 20 mM, glycerol 10%, β-mercaptoethanol 1 mM, adjusted to pH 8) using the molecular sieve principle of the desalting column.

[0085] First, use Desalting Buffer to balance the A and B pumps of the AKTAprimerplus system, connect the desalting column and balance the desalting column with Desalting Buffer, inject the protein sample into the desalting column after the baseline is balanced, and collect the peak liquid. The injection volume each time should not exceed 10% of the column volume to avoid premature outflow of the mutant protease.

[0086] The volume of the enzyme solution after desalting increases and its concentration decreases accordingly, so it is concentrated using an ultrafiltration tube with a molecular cutoff of 10 kDa.

[0087] The balanced ultrafiltration tube was centrifuged at 4°C and 5000 rpm for 10 min. The concentrated enzyme solution was measured for concentration by Nanodrop and then packaged and stored in a -80°C refrigerator.

[0088] According to the above method, 13 pure enzyme solutions of glycosyltransferase UGT91C1 mutants were obtained, and they were marked as M0, M0-G151F, M0-T230Y, M0-T243V, M0-V264L, M0-A272P, M0-A232P, M0-A349P, M0-T363I, M0-A399P, M0-G411S, M0-C445L, and M0-G452E, respectively.

[0089] Example 3 Enzyme activity determination of mutants

[0090] The three glycosyltransferase UGT91C1 mutant pure enzyme solutions obtained in Example 2 were respectively used to prepare reaction systems to determine the enzyme activity, wherein the M0 pure enzyme solution was used as a control, and the ambient temperature was 42° C. The 200 μL reaction system contained the following components:

[0091] The buffer is 50 mM phosphate, 5 mM MgCl2, 500 mM UDPG, 250 mM RebA, 0.8 mM protease, and the ambient pH is 7.0-8.0.

[0092] The reaction was carried out at 42°C and 170 rpm for 30 min. After the reaction, a sample was taken and heated at 95°C for 5 min to terminate the reaction. After cooling, an equal volume of methanol was added and centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μM filter membrane and used for high performance liquid chromatography detection to calculate the relative activity of the mutant protease.

[0093] The results of enzyme catalytic activity determination refer to the attached Figure 1Compared with M0, the relative activities of 3 of the 12 mutant proteases decreased significantly, while the relative activities of the remaining 9 mutant proteases remained above 80%.

[0094] Subsequently, V264L, A272P, A323P, A349P, T363I, A399P, G411S, C445L, and G452E with good relative activity were selected to investigate their surface melting temperatures T m , optimum temperature T opt , half inactivation temperature T 50 , half-life t 1 / 2 Thermostability parameters were measured to explore the effect of mutation on the thermal stability of protease.

[0095] It is easy for those skilled in the art to understand that, based on the amino acid sequence shown in SEQ ID NO: 1, it is easy to obtain a protein that still has the enzymatic activity of catalyzing the synthesis of rebaudioside D from rebaudioside A through conventional means such as substitution, deletion or addition of one or several amino acid sequences, and therefore these mutants also fall within the scope of the present invention.

[0096] Example 4 Surface melting temperature T of mutant proteases m Characterization

[0097] In characterizing enzyme stability, the surface melting temperature T m The value is often used as an important parameter for the thermal stability of the reaction enzyme and can be determined by circular dichroism (CD). In the thermal denaturation study of mutant proteases, the same enzyme concentration was controlled to be 0.7 μM, a 1 cm pool with a rotor was used, 222 nm was set as the detection wavelength, the temperature range was 20-95 ° C, the step was 1 ° C, and continuous acquisition was performed for 3600 s.

[0098] Data analysis, with temperature as the horizontal axis and θMRE as the vertical axis, the two-state model formula was used to fit the thermal denaturation results, and the fitted T m value.

[0099] Surface melting temperature T m The characterization results refer to the attached Figure 2 , compared with M0, the T m The value increased by 1-22℃.

[0100] Example 5 Optimum temperature T of mutant protease opt Characterization

[0101] Generally speaking, a lower reaction temperature will reduce the rate of intermolecular movement, thereby limiting the rate of enzymatic reaction; while an excessively high reaction temperature will cause irreversible changes in the structure of the enzyme, resulting in reduced enzyme activity or even complete loss of enzyme activity. After the purified mutant protein pure enzyme solution is appropriately diluted, its enzyme activity is measured at different temperatures of 15-65°C (5°C intervals). The highest enzyme activity is 100%, and the enzyme activity at other temperatures is the relative enzyme activity. Plot the relative enzyme activity as the ordinate and the temperature as the abscissa to determine its optimum temperature.

[0102] Optimum temperature T opt The characterization results refer to the attached Figure 3 , 4 , attached Figure 3 The enlarged diagram of the relative activity changes of each mutant protease in the 40-45℃ part. Compared with M0, the optimum temperature T opt Most of them reacted in the range of 40-45℃, among which C445L had the highest optimum temperature, reaching 45℃ and showing the highest relative enzyme activity. The optimum temperatures of other mutant proteases were T opt The difference between them and M0 is not much. And at their respective optimum temperatures, their catalytic activities are maintained at a high level.

[0103] Example 6 Half-inactivation temperature T of mutant protease 50 Characterization

[0104] The purified enzyme solution was incubated at 30-70°C for 10 min (5°C intervals), and immediately placed on ice for 10 min after incubation. The enzyme activity was determined using the above method. The enzyme activity under the unincubated condition was set as 100%, and the relative specific activity under each temperature condition was calculated. The temperature corresponding to the relative residual enzyme activity dropping to 50% was the half-inactivation temperature T. 50 . Plot the relative enzyme activity as the ordinate and the temperature as the abscissa to determine its half-inactivation temperature T 50 .

[0105] Half inactivation temperature T 50 The characterization results refer to the attached Figure 5 , where M0's T 50 The mutant proteases M0-T363I and M0-C445L have a T of about 42.5℃ compared to M0. 50 The increases were about 5°C and 7.5°C respectively, indicating that both still have certain catalytic activity under higher temperature conditions.

[0106] Example 7 Half-life of mutant proteases 1 / 2 Characterization

[0107] The purified enzyme solution was incubated at 40℃ and 45℃ for a certain period of time, and samples were taken at regular intervals. The samples were quickly placed on ice for 10 minutes and the enzyme activity was determined using the above method. The enzyme activity under the unincubated condition was set as 100%, and the relative enzyme activity under each time condition was calculated. The time corresponding to the relative residual enzyme activity dropping to 50% is the half-life t 1 / 2 The kd (kd is the inactivation constant, defined as ln (residual enzyme activity) / incubation time t) and half-life t were calculated by measuring the residual enzyme activity. 1 / 2 The calculation formula is t 1 / 2 =ln2 / kd.

[0108] Half-life at 40℃ and 45℃ 1 / 2 The characterization results refer to Figure 6 and Figure 7 Under the two incubation temperature conditions, the half-lives of the mutant proteases M0-T363I and M0-C445L were significantly longer than that of M0. Among them, the half-lives of M0-T363I were increased by 2.35 times and 3.38 times respectively compared with M0 under the two temperature gradient conditions, and the half-lives of M0-C445L were increased by 3.78 times and 4.63 times respectively compared with M0 under the two temperature gradient conditions.

[0109] In summary, the glycosyltransferase mutant provided by the present invention has stronger heat resistance and is more suitable for application in the field of food additives.

[0110] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0111] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0112] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A thermostable glycosyltransferase mutant, characterized in that: The amino acid sequence of the glycosyltransferase mutant is a T363I mutation or a C445L mutation occurring on the basis of the amino acid sequence shown in SEQ ID NO:

1.

2. A DNA molecule, characterized in that The DNA molecule encodes the glycosyltransferase mutant according to claim 1.

3. The DNA molecule according to claim 2, characterized in that The DNA molecule comprises a DNA sequence encoding an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid is connected to the DNA molecule according to claim 2 or 3.

5. A host cell, characterized in that The recombinant expression plasmid according to claim 4 is expressed in the host cell, and the host cell is Escherichia coli.

6. The method for preparing a glycosyltransferase mutant according to claim 1, characterized in that: The preparation method comprises: The DNA molecule encoding the glycosyltransferase mutant according to claim 1 is inserted into an expression plasmid, the expression plasmid is transformed into a host cell, the host cell is fermented and cultured, and the glycosyltransferase mutant is separated and purified.

7. Use of the glycosyltransferase mutant according to claim 1, the DNA molecule according to claim 2 or 3, the recombinant expression plasmid according to claim 4, the recombinant cell according to claim 5, and the preparation method of the glycosyltransferase mutant according to claim 6 or 7 in the preparation of rebaudioside D.

8. A method for preparing rebaudioside D, characterized in that: The preparation method comprises: The rebaudioside D is obtained by catalyzing the substrate using the glycosyltransferase mutant described in claim 1.

9. The method for preparing rebaudioside D according to claim 8, characterized in that: The substrate includes rebaudioside A and a glycosyl donor.

10. Use of rebaudioside D obtained by the method for preparing rebaudioside D according to claim 8 or 9 in the field of food additives.

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