Sucrose synthase mutant, method for preparing same, product and use thereof

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to a sucrose synthase mutant, its preparation method, product, and application. The mutant is based on the amino acid sequence of the wild-type sucrose synthase shown in SEQ ID NO.1, with the 94th amino acid mutated from P to N, resulting in the amino acid sequence shown in SEQ ID NO.2; or the 660th amino acid mutated from Y to H, resulting in the amino acid sequence shown in SEQ ID NO.3. This mutant can be used for in vitro glycosylation reactions, plant genetic improvement, or industrial biocatalysis, solving the technical problems of poor stability and low catalytic efficiency of natural enzymes.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a sucrose synthase mutant, its preparation method, product and application. Background Technology

[0002] Sucrose synthase (SuSy; EC 2.4.1.13) is a key enzyme catalyzing the reversible reaction between sucrose and nucleoside diphosphate (NDP) to produce the corresponding NDP-glucose and fructose. The reaction direction is pH-regulated; generally, slightly acidic conditions (pH 5.5-7.5) favor sucrose cleavage, while slightly alkaline conditions (pH 7.5-9.5) favor sucrose synthesis. The uridine diphosphate (UDP) to glucose diphosphate (UDPG) catalyzed by SuSy is particularly important, as it is an indispensable activated sugar donor in organisms. Under the catalysis of glycosyltransferases (such as UDP-glucosyltransferase, UGT), UDPG directly participates in the synthesis of various macromolecules such as starch, glycogen, and glycosides, and can be easily converted into other important nucleoside sugars (such as UDP-glucuronic acid and UDP-galactose), widely participating in key physiological processes, including protein glycosylation. Of particular note is that, using UDPG as a sugar donor, UGT can catalyze the synthesis of a variety of high-value glycosides, such as ginsenoside Rh2, gastrodin, and rebaudioside D (RD) and rebaudioside M (RM) from steviol glycosides.

[0003] However, efficient production of UDPG using SuSy faces significant challenges. Both plant- and bacterial sucrose synthases exhibit strong affinity for UDP and ADP. Generally, plant-derived sucrose synthases (e.g., AtSUS from *Arabidopsis thaliana*) have a higher affinity for UDP than ADP, while bacterial ones show the opposite. Although high affinity usually accompanies high catalytic efficiency, kinetic analysis of both plant- and bacterial SuSys indicates that low Km substrates often induce strong substrate inhibition, resulting in lower practical maximum rates. Improving the catalytic activity and stability of SuSy for UDP is crucial for synthesizing more UDPG in a shorter time, extending enzyme lifetime, and thus significantly reducing the production cost of UDPG and its downstream high-value glycosides.

[0004] Therefore, developing sucrose synthase mutants with high catalytic activity, excellent stability, and easy expression in E. coli for UDP substrates is key to solving the problem of low-cost UDPG supply and thus realizing the economical and efficient biosynthesis of high-value glycosides. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a sucrose synthase mutant that significantly improves the enzyme's catalytic performance and stability, enabling it to demonstrate outstanding application value in industrial biocatalysis, in vitro glycosylation reactions, and plant genetic improvement. Compared to natural sucrose synthase, the mutant of this invention effectively solves key problems in existing technologies such as insufficient enzyme stability and low catalytic efficiency, providing a more efficient biocatalytic tool for related industries.

[0006] The technical solution of this invention is as follows:

[0007] On the one hand, the present invention provides a sucrose synthase mutant, which is based on the amino acid sequence shown in SEQ ID NO.1 and contains at least one substitution mutation selected from the following positions: the 94th amino acid is mutated from P to N, and the 660th amino acid is mutated from Y to H.

[0008] Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0009] In another aspect, the present invention provides a nucleic acid encoding an understated sucrose synthase mutant, the sequence of which is as shown in SEQ ID NO.7 or SEQ ID NO.8.

[0010] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0011] Specifically, the expression vector is selected from plasmids, bacteriophages, viruses, or artificial chromosomes.

[0012] Preferably, the expression vector is a plasmid.

[0013] Preferably, the plasmid is selected from the pET series, pQE series, pGEX series, or pMAL series;

[0014] The virus is selected from lentiviruses, adenoviruses, or baculoviruses;

[0015] The artificial chromosome is selected from BAC, YAC, or PAC.

[0016] Preferably, the plasmid is pET-30a.

[0017] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0018] Specifically, the host cell is a prokaryotic cell or a eukaryotic cell.

[0019] Preferably, the host cells include, but are not limited to, Escherichia coli, yeast, Bacillus, or Lactobacillus.

[0020] Preferably, the host cell is Escherichia coli.

[0021] Preferably, the host cell has the accession number CGMCC No. 34494.

[0022] In another aspect, the present invention provides a method for preparing the aforementioned sucrose synthase mutant, comprising the following steps:

[0023] S1. Express the nucleic acid encoding the mutant in the host cell;

[0024] S2. Culture the host cells and induce protein expression;

[0025] S3. Separate and purify to obtain the sucrose synthase mutant.

[0026] In another aspect, the present invention provides a cell culture or extract, characterized in that it is obtained by culturing the aforementioned host cells.

[0027] In another aspect, the present invention provides an enzyme preparation comprising the aforementioned sucrose synthase mutant, cell culture, or extract.

[0028] Specifically, the enzyme preparation also includes pharmaceutically or industrially acceptable excipients.

[0029] Preferably, the excipients include, but are not limited to, buffers, stabilizers, preservatives, or lyophilizers.

[0030] In another aspect, the present invention provides the application of the aforementioned sucrose synthase mutants, nucleic acids, expression vectors, host cells, cell cultures, extracts, or enzyme preparations in the efficient production of UDPG.

[0031] In another aspect, the present invention provides the application of the aforementioned sucrose synthase mutants, nucleic acids, expression vectors, host cells, cell cultures, extracts, or enzyme preparations in plant genetic improvement to enhance carbohydrate metabolism efficiency.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) The sucrose synthase mutant provided by the present invention has high catalytic activity and excellent stability against UDP substrates, and can synthesize more UDPG in a shorter time, prolonging the enzyme's lifespan and helping to reduce production costs.

[0034] (2) The sucrose synthase mutant provided by the present invention is easy to express in Escherichia coli, which facilitates large-scale production and is suitable for industrial application.

[0035] (3) The mutants of the present invention can be used in multiple fields such as in vitro glycosylation reaction, plant genetic improvement or industrial biocatalysis, and have broad application prospects. They can provide cheap and readily available substrate raw materials for the synthesis of active sugars.

[0036] Preservation information:

[0037] Biomaterial R3;

[0038] Classification and nomenclature: Escherichia coli ( Escherichia coli );

[0039] Accession number: CGMCC No. 34494;

[0040] Deposit date: May 9, 2025;

[0041] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0042] Abbreviation of depositary institution: CGMCC;

[0043] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0044] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0045] Basic Implementation

[0046] The detection methods for sucrose and fructose are as follows:

[0047] Chromatographic column: Innoval NH2 (4.6*250mm, 5µm);

[0048] ELSD detector;

[0049] Flow rate: 1 mL / min;

[0050] Column temperature: 40℃;

[0051] Drift tube temperature: 45℃;

[0052] Gain: 5;

[0053] Pressure: 320 kPa;

[0054] Injection volume: 10 μL;

[0055] Sample preparation solvent: MEOH;

[0056] Mobile phase: 70% ACN;

[0057] Isocratic elution for 10 minutes.

[0058] Example 1: Construction and Identification of Recombinant Escherichia coli

[0059] Escherichia coli BL21(DE3) (Catalog No.: B528414) and DH5α (Catalog No.: A338951) competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid pET-30a(+) (Catalog No.: B540185) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; all gene and primer synthesis and sequencing services were provided by Suzhou Genewise Biotech Co., Ltd.; 2x Phanta Flash Master Mix high-fidelity DNA polymerase (Catalog No.: P510-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases NdeI (Code No. 1621) and XhoI (Code No. 1635) were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0060] 1.1 Gene Acquisition

[0061] The AtSUS gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0062] The amplification primers are shown in Table 1:

[0063] Table 1 Primer sequences

[0064]

[0065] Note: Italic and bold font CATATG This is the NdeI restriction site. CTCGAG This is the XhoI restriction site.

[0066] The PCR amplification program is as follows: the target fragment is amplified using 2x Phanta Flash Master Mix high-fidelity DNA polymerase. The specific reaction system and amplification program are shown in Table 2.

[0067] Table 2. PCR reaction system and amplification procedure for Phanta DNA polymerase.

[0068]

[0069] The above PCR amplification products were purified and recovered using a DNA gel recovery and purification kit (Zymo Research; D4008) to obtain the purified AtSUS gene fragment.

[0070] 1.2 Enzyme digestion and ligation

[0071] The amplified DNA fragments or plasmids were digested with restriction endonucleases. In this study, QuickCut™ series restriction endonucleases were used. Digestion was performed at 37°C for 5 min, and the reaction system is shown in Table 3.

[0072] Table 3 Enzyme digestion reaction system

[0073]

[0074] After purification, the digested fragments were mixed with T4 DNA ligase (Novizan; C301-01) and the purified AtSUS gene fragment and pET30a(+) plasmid fragment, and incubated in a metal bath at 22 ℃ for 2.5 h for ligation. The ligation reaction system is shown in Table 4.

[0075] Table 4 Enzyme ligation reaction system

[0076]

[0077] 1.3 Conversion Validation

[0078] The above enzyme-linked product was transformed (using conventional chemical transformation) into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, the positive plasmid pET30a(+)-AtSUS was returned.

[0079] Amino acid sequence SEQ ID NO.1 AtSUS:

[0080] MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH*

[0081] Nucleotide sequence SEQ ID NO.6 AtSUS:

[0082]

[0083] 1.4 Construction of mutant expression plasmids

[0084] Based on the pET30a(+)-AtSUS plasmid, PCR amplification was performed using the amplification primers in the table. The PCR products were purified by gel excision and transformed (using conventional chemical transformation method) into E. coli DH5α competent cells and cultured overnight. Single colonies grown in the selection plate (50 µg / mL kanamycin) were sent to Genewiz for sequencing. After successful sequencing, plasmids containing AtSUS mutant positive plasmids pET30a(+)-AtSUS(P94N), pET30a(+)-AtSUS(Y660H), pET30a(+)-AtSUS(L689Q), and pET30a(+)-AtSUS(K752D) were returned.

[0085] Table 5 List of primers for mutants

[0086]

[0087] Amino acid sequence SEQ ID NO.2 AtSUS (P94N):

[0088] MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRNGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH*

[0089] Amino acid sequence SEQ ID NO.3 AtSUS (Y660H):

[0090] MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRHICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH*

[0091] Amino acid sequence SEQ ID NO.4 AtSUS (L689Q):

[0092] MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGQPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH*

[0093] Amino acid sequence SEQ ID NO.5 AtSUS (K752D):

[0094] MANAERMITRVHSQRERLNETLVSDRNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEDYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDHHHHHH*

[0095] Nucleotide sequence SEQ ID NO.7 AtSUS (P94N):

[0096]

[0097] Nucleotide sequence SEQ ID NO.8 AtSUS (Y660H):

[0098]

[0099] Nucleotide sequence SEQ ID NO.9 AtSUS (L689Q):

[0100]

[0101] Nucleotide sequence SEQ ID NO.10 AtSUS (K752D):

[0102]

[0103] 1.5 Construction of Recombinant Strains

[0104] The constructed plasmid was transformed (using conventional chemical transformation) into competent Escherichia coli BL21(DE3) cells and cultured overnight. Validation primers (upstream primer (SEQ ID NO.11) test-pET-F: 5'-CATCGGTGATGTCGGCGATATAG -3', downstream primer (SEQ ID NO.12) test-pET-R: 5'-CCGGATATAGTTCCTCCTTTCAGCA -3') were designed to validate single colonies grown in a screening plate (50 µg / mL kanamycin) using colony PCR, thus constructing the corresponding recombinant strain.

[0105] Example 2: Induction and expression of recombinant Escherichia coli and preparation of purified enzyme solution

[0106] 2.1 Induced Expression

[0107] Single colonies of the engineered bacteria were inoculated into 2YT medium containing 50 µg / mL kanamycin (2YT medium formulation: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) and cultured at 37℃ and 220 rpm for 6 h. The culture was then transferred at a 1% (v / v) inoculation rate to 150 mL of 2YT medium containing 50 µg / mL kanamycin and cultured at 37℃ and 220 rpm until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.3 mM, and the culture was induced at 16℃ for 20 h. The culture was then centrifuged at 4℃ and 6000 rpm for 15 min, the supernatant was discarded, and the precipitate was kept for later use.

[0108] 2.2 Preparation of crude enzyme solution

[0109] A 10% (m / v) AtSUS mutant bacterial culture was prepared using a 50 mM PB (dipotassium hydrogen phosphate, potassium dihydrogen phosphate, pH 6.5) solution. The bacterial cells were homogenized under high pressure (900 bar) to release the target protein. The culture was centrifuged at 4°C and 6000 rpm for 15 min. The supernatant enzyme solution was collected to obtain crude AtSUS mutant enzyme solution for later use.

[0110] 2.3 Protein purification

[0111] (1) Nickel gravity column pretreatment: HisSep Ni-NTA Agarose Resin was loaded into a suitable purification column by gravity, the column was rinsed with 2 column volumes of deionized water, and the column was equilibrated with 2 column volumes of Tris.

[0112] (2) Sample loading: Add the crude enzyme solution prepared above to the purification column. Pay attention to controlling the sample loading speed to ensure that the target protein is in full contact with Ni2+ in order to improve the purification yield.

[0113] (3) Washing: Washing a 2-cylinder volume of Wash Buffer;

[0114] (4) Elution: Elute with 30 mL of Elution Buffer and collect the eluent, which is the target protein solution;

[0115] (5) Column regeneration: one column volume of 1M imidazole washing, followed by two column volumes of deionized water rinsing.

[0116] (6) Storage: Finally, store the resin in deionized water at 4°C;

[0117] (7) Ultrafiltration: Add 50mM PB buffer to the ultrafiltration tube, centrifuge at 5000 rpm and 4℃ for 10 min, and discard the centrifuged liquid; add an appropriate amount of the eluted target protein solution, centrifuge at 5000 rpm and 4℃ to a volume of about 1 mL; add an equal volume of 50mM PB buffer, centrifuge at 5000 rpm and 4℃ to a volume of about 1 mL, and repeat this step once to remove the high concentration of imidazole and obtain the protein purification solution.

[0118] Example 3: Enzyme activity assay of recombinant Escherichia coli (AtSUS mutant)

[0119] AtSUS mutant (UDP→UDPG) enzyme activity assay:

[0120] Definition: One unit of enzyme activity (U) is the amount of enzyme required to generate 1 µmol of fructose in 1 minute at a reaction temperature of 37°C.

[0121] (1) Enzyme-catalyzed reaction

[0122] In a 200 μL reaction system for a 96-well plate, 100 mM sucrose, 10 mM UDP, and 50 mM pH 6.5 PB buffer were added sequentially, along with 0.1 mg / mL purified protein. The mixture was reacted at 37°C for 5 min. After adding an equal volume of methanol to the reaction solution, the mixture was centrifuged and filtered through a 0.22 μm filter membrane. The amount of fructose generated was then determined by liquid chromatography.

[0123] (2) Enzyme activity calculation

[0124] Specific activity (U / g) = Fructose production (μmol) ÷ Reaction time (min) ÷ Protein mass (g)

[0125] The detection results, obtained using the enzyme activity assay method described above, are shown in Table 6.

[0126] Table 6

[0127]

[0128] As shown in Table 6, the AtSUS (P94N) mutant glycosyltransferase induced by recombinant Escherichia coli showed a significant increase in specific activity with UDP as substrate, reaching 42.45 U / mg, which was 697.93% higher than that of AtSUS.

[0129] Example 4: Determination of the thermal stability of enzyme activity in recombinant Escherichia coli (AtSUS mutant)

[0130] After incubating the above mutants in a buffer system at 50°C for 1 hour and 3 hours, the enzyme activity was measured according to the method described in Example 3. The results are shown in Table 7.

[0131] Table 7

[0132]

[0133] As shown in Table 7, the AtSUS mutant AtSUS(P94N) induced by recombinant E. coli retained 89.30% and 72.01% of its enzyme activity after incubation at 50℃ for 1 h and 3 h, respectively. Under the same treatment conditions, the wild-type enzyme activity retained 16.73% and 3.95%, respectively. The recombinant E. coli AtSUS mutant AtSUS(P94N) was named R3 (CGMCC No. 34494).

[0134] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A sucrose synthase mutant, characterized in that, The amino acid sequence of the sucrose synthase mutant is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. A nucleic acid encoding the sucrose synthase mutant of claim 1, characterized in that, The sequence of the nucleic acid is as shown in SEQ ID NO.7 or SEQ ID NO.

8.

3. An expression carrier, characterized in that, Includes the nucleic acid described in claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is a plasmid.

5. The expression vector according to claim 4, characterized in that, The plasmids are selected from the pET series, pQE series, pGEX series, or pMAL series.

6. The expression vector according to claim 4, characterized in that, The plasmid is pET-30a.

7. A host cell, characterized in that, Includes the nucleic acid as described in claim 2 or the expression vector as described in any one of claims 3-6.

8. The host cell according to claim 7, characterized in that, The host cell is Escherichia coli.

9. The host cell according to claim 8, characterized in that, The host cell has the accession number CGMCC No. 34494.

10. A method for preparing the sucrose synthase mutant of claim 1, characterized in that, Includes the following steps: S1. Express the nucleic acid encoding the sucrose synthase mutant in the host cell; S2. Culture the host cells and induce protein expression; S3. Separate and purify to obtain the sucrose synthase mutant.

11. A cell culture, characterized in that, Obtained by culturing the host cells as described in any one of claims 7-9.

12. An enzyme preparation, characterized in that, Includes the sucrose synthase mutant of claim 1 or the cell culture of claim 11.

13. The use of the sucrose synthase mutant of claim 1, the nucleic acid of claim 2, the expression vector of any one of claims 3-6, the host cell of any one of claims 7-9, the cell culture of claim 11, or the enzyme preparation of claim 12 in the efficient production of UDPG.

Images