Sucrose synthase as well as coding gene, mutant and application thereof

Abstract

The invention belongs to the technical field of genetic engineering and functional enzyme preparations, particularly relates to sucrose synthase and a coding gene and a mutant thereof, and further discloses application of the sucrose synthase. According to the invention, sucrose synthase is obtained from candidate methylobacterium and is further mutated, so that a sucrose synthase mutant of which the enzyme activity, the conversion rate and the stability are remarkably improved is obtained, and the activity and the efficiency of a stevioside synthesis process are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and functional enzyme preparation technology, specifically relating to a sucrose synthase and its encoding gene, mutant, and further disclosing its applications. Background Technology

[0002] In recent years, excessive intake of high-calorie sugars has led to serious obesity, diabetes, hypertension, and cardiovascular diseases worldwide. Therefore, functional sugars have attracted widespread attention in the food industry. Stevia glycosides, in particular, are highly praised for their high sweetness, low calories, and high safety. Among them, stevia and rebaudioside A (Reb A), which are relatively abundant, are widely used as sweeteners in beverages and food products.

[0003] Steviol glycosides are 250-300 times sweeter than sucrose, but their bitter aftertaste significantly impacts their palatability as sweeteners. However, rebaudioside D (RebD) / rebaudioside M (RebM), present in lower amounts among steviol glycosides, exhibit higher sweetness than RebA and steviol, with reduced bitterness and a faster onset of sweetness, thus providing a better palatability. They are considered promising next-generation sweeteners, especially RebM, which has higher commercial value than RebD. However, RebM is present in only 0.4%-0.5% of dried stevia leaves, about one-tenth the amount of RebA. This makes traditional leaf extraction methods unsuitable for RebM extraction. Furthermore, the cumbersome and complex extraction methods make large-scale production from stevia leaves difficult to achieve and meet market demand. Therefore, the production of rebaudioside M using bio-enzymatic methods is a key breakthrough direction for stevia manufacturers both domestically and internationally, and sucrose synthase is the main enzyme preparation product used in this process.

[0004] Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) widely found in plants, and its use in bacteria has been increasingly discovered in recent years. This enzyme catalyzes the reversible transfer of glucose residues between fructose and uridine diphosphate (UDP) (sucrose + UDP = UDP glucose + fructose). Using UDPGlc obtained from sucrose synthase as a glycosyl donor, UDP glycosyltransferase catalyzes the synthesis of major glycosides such as steviol glycosides and rebaudioside D. However, the synthesis of rebaudioside D or M currently requires expensive disodium uridine diphosphate, which is not only costly but also suffers from low activity due to the fact that the commonly used sucrose synthase gene is mainly derived from rice. Although modified, it still suffers from low activity, thus affecting the amount of disodium uridine diphosphate used and increasing enzyme dosage, leading to higher product costs and severely limiting industrial application. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a sucrose synthase and its mutant, thereby solving the problem of unsatisfactory enzyme activity in the prior art catalyzing the synthesis of steviol glycosides by sucrose synthase;

[0006] The second technical problem to be solved by the present invention is to provide the application of the above-mentioned sucrose synthase and its mutants in the process of catalytic synthesis of steviol glycosides.

[0007] To address the aforementioned technical problems, this invention obtains sucrose synthase from candidate methylocaldum szegediense and further mutates it to obtain a sucrose synthase mutant with significantly improved enzyme activity, conversion rate, and stability, effectively improving the activity and efficiency of the steviol glycoside synthesis process.

[0008] In a first aspect, the present invention provides a sucrose synthase comprising the amino acid sequence shown in SEQ ID NO: 1;

[0009] Preferably, the amino acid sequence of the sucrose synthase is shown in SEQ ID NO: 1.

[0010] The sucrose synthase provided by this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0011] In some specific embodiments, the sucrose synthase can be obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing the expression of the recombinant genetically engineered bacterium to obtain the sucrose synthase.

[0012] In a second aspect, the present invention provides a gene encoding the sucrose synthase, comprising the nucleotide sequence shown in SEQ ID NO: 2;

[0013] Preferably, the nucleotide sequence of the gene encoding the sucrose synthase is shown in SEQ ID NO: 2.

[0014] Specifically, the genes or nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0015] Thirdly, the present invention provides a sucrose synthase mutant, which is obtained by mutating the 578th amino acid of the sucrose synthase described in the first aspect, and its catalytic activity and stability are further improved compared with the above-mentioned sucrose synthase.

[0016] Specifically, the sucrose synthase mutant includes the amino acid sequence shown in SEQ ID NO: 3;

[0017] Preferably, the amino acid sequence of the sucrose synthase mutant is shown in SEQ ID NO: 3.

[0018] The mutant of sucrose synthase provided by the present invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0019] In some embodiments, the above-mentioned sucrose synthase is obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing expression of the recombinant genetically engineered bacterium to obtain a mutant of sucrose synthase.

[0020] Fourthly, the present invention also provides a gene encoding the sucrose synthase mutant, comprising the nucleotide sequence shown in SEQ ID NO: 4;

[0021] Preferably, the nucleotide sequence of the gene encoding the sucrose synthase mutant is shown in SEQ ID NO: 4.

[0022] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0023] Fifthly, the present invention also provides a recombinant vector carrying the gene encoding sucrose synthase or the gene encoding a sucrose synthase mutant.

[0024] Specifically, the recombinant vector includes a cloning vector and an expression vector, wherein the cloning vector is used to replicate the relevant sequence and the expression vector is used to express the relevant gene.

[0025] In some embodiments, the recombinant vectors include pET-MSSUS and / or pET-ΔMSSUS, which are obtained by replacing the sequence between the XbaI and BamHI restriction sites of pET-28a(+) with the nucleic acid molecule encoding the above-mentioned sucrose synthase or the nucleic acid molecule of the mutant of the above-mentioned sucrose synthase, while keeping the remaining sequences unchanged.

[0026] In a sixth aspect, the present invention also provides a recombinant host that expresses the recombinant vector.

[0027] In some embodiments, the recombinant host induces the production of the above-mentioned sucrose synthase or a mutant of the above-mentioned sucrose synthase.

[0028] In some embodiments, the method for constructing the recombinant host can employ methods known in the art, such as constructing the recombinant vector using the encoding gene of the sucrose synthase or a sucrose synthase mutant, transforming the recombinant vector into host cells, and inducing the expression of sucrose synthase or a mutant of the aforementioned sucrose synthase.

[0029] In some embodiments, the recombinant vector is any of the recombinant vectors described above, and the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably Escherichia coli BL21(DE3).

[0030] As a specific factual approach, the above-mentioned method for constructing a recombinant host includes, for example, the following steps:

[0031] (1) MSSUS gene amplification of sucrose synthase;

[0032] (2) Obtaining the mutant ΔMSSUS of the sucrose synthase gene;

[0033] (3) Construction of recombinant expression plasmids pET-MSSUS and pET-ΔMSSUS;

[0034] (4) Transform the recombinant expression plasmids pET-MSSUS and pET-ΔMSSUS into host cells;

[0035] (5) Positive clones were obtained by screening with resistant culture medium.

[0036] There are no special requirements for the culture method and conditions of the recombinant host, as long as the cells can grow normally and express sucrase synthase or its mutant.

[0037] Specifically, the recombinant host described in this invention includes, but is not limited to, recombinant cell lines or genetically engineered bacteria.

[0038] In some embodiments, the recombinant cells may be recombinant bacteria W and recombinant bacteria ΔW, and the culture medium used when the recombinant genetically engineered bacteria express sucrose synthase or its mutants may be a culture medium in the art that enables the recombinant genetically engineered bacteria to grow and produce the sucrose synthase or its mutants of the present invention, preferably LB medium.

[0039] There are no special requirements for the culture method and culture conditions of the genetically engineered bacteria, as long as the genetically engineered bacteria can grow normally and express sucrase synthase or its mutant.

[0040] In a seventh aspect, the present invention provides a method for preparing sucrose synthase or a sucrose synthase mutant, the method comprising:

[0041] The gene encoding sucrose synthase or the gene encoding a sucrose synthase mutant is ligated into an expression vector to construct a recombinant vector; the recombinant vector is then transformed into a host cell for culture and expression, and the desired sucrose synthase or sucrose synthase mutant is isolated from the resulting culture; or...

[0042] The recombinant vector is transferred into a host cell for culture and expression, and the desired sucrose synthase or sucrose synthase mutant is isolated from the resulting culture; or,

[0043] The recombinant host was induced and cultured, and the desired sucrose synthase or sucrose synthase mutant was isolated from the obtained culture.

[0044] The methods for inducing the culture of recombinant hosts and for isolating sucrose synthase or its mutants from the cultures are conventional methods in this field.

[0045] Eighthly, the present invention provides a sucrose synthase or a sucrose synthase mutant prepared based on the method described above.

[0046] In a ninth aspect, the present invention provides the application of the sucrase, the gene encoding the sucrase, the sucrase mutant, the gene encoding the sucrase mutant, the recombinant vector, the recombinant host, or the sucrase synthase or the sucrase mutant in the field of steviol glycoside synthesis.

[0047] In a tenth aspect, the present invention provides a method for preparing steviol glycosides, comprising the step of carrying out a catalytic reaction in a catalytic reaction system using steviol glycosides and sucrose as substrates, using the sucrose synthase, the sucrose synthase mutant, or the sucrose ribozyme or the sucrose ribozyme mutant as an enzyme catalyst;

[0048] Preferably, the steviol glycosides include rebaudioside A and / or rebaudioside D.

[0049] Preferably, in the method for preparing the steviol glycosides, the catalytic reaction system comprises the following components: 10-100 g / L rebaudioside A and / or rebaudioside D, 1-10 g / L MgCl2, 180-280 g / L sucrose, 0.1-10 g / L uridine diphosphate disodium salt, 1-20 g / L UDP-glycosyltransferase, and 0.3-0.8 g / L sucrose synthase and / or sucrose synthase mutant;

[0050] Preferably, the initial pH of the catalytic reaction system is 6-8; and / or,

[0051] Preferably, the temperature of the catalytic reaction is 30-80°C; and / or,

[0052] Preferably, the catalytic reaction takes 10-72 hours.

[0053] In some embodiments, the temperature in the above catalytic reaction is 30-80°C, for example 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, or any value or range between these values, preferably 50°C.

[0054] In some embodiments, the initial pH of the catalytic reaction system described above is 6-8, which can be adjusted using phosphate buffer (e.g., 50 mM PBS), for example, to pH 7.0.

[0055] In some embodiments, the method for preparing the steviol glycosides may use the sucrose synthase or its mutant in whole-cell form of engineered bacteria, or in the form of unpurified crude enzyme, or in the form of partially or completely purified enzyme; the sucrose synthase or its mutant of the present invention may also be prepared into an immobilized enzyme or a catalyst in immobilized cell form using immobilization techniques known in the art.

[0056] This invention obtains sucrose synthase from candidate methylocaldum szegediense, which can catalyze the synthesis of rebaudioside M using rebaudioside A and / or rebaudioside D as substrates, and exhibits ideal enzyme activity and catalytic efficiency.

[0057] This invention is based on the mutation of the wild-type sucrose synthase, and a sucrose synthase mutant with significantly improved enzyme activity, conversion rate and stability was obtained. It can efficiently catalyze the synthesis of steviol glycosides, and its enzyme activity can reach 72.5 U / mg, which is 1.3 times that of the original sucrose synthase, effectively improving the activity and efficiency of the steviol glycoside synthesis process.

[0058] The method for enzyme-catalyzed synthesis of steviol glycosides described in this invention, based on screened sucrose synthase and sucrose synthase mutants as catalysts, can effectively improve the conversion rate of the target product in a catalytic reaction system containing rebaudioside A and / or rebaudioside D and sucrose as substrates, resulting in significant improvements in industrial efficiency and industrial costs. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0060] In the following embodiments of the present invention, the methylocaldum szegediense used can be obtained from the NCBI and is available to the public from Wanhua Chemical Group Co., Ltd.

[0061] In the following embodiments of the present invention, pET-28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., with product catalog number B540183.

[0062] Example 1: Preparation of sucrose synthase

[0063] In this embodiment, the preparation of the sucrose synthase includes the steps of constructing a recombinant bacterium containing sucrose synthase and the step of culturing the recombinant bacterium to prepare the sucrose ribozyme.

[0064] The sucrose synthase gene from Methylocaldum zegediense was obtained by gene mining and screening in the NCBI database. Its nucleotide sequence is shown in SEQ ID NO: 2 (denoted as MSSUS). The DNA shown in SEQ ID NO: 2 was synthesized and used as a template for PCR amplification with the following primers to obtain a fragment containing the desired gene.

[0065] Forward primer: 5'-aacaattcccctctagaATGAGCGAAAGAGCG-3' (SEQ ID NO: 5);

[0066] Reverse primer: 5'-agctcgaattcggatccTCAGGCCATAGCCTGGG-3' (SEQ ID NO: 6).

[0067] pET-28a(+) was digested with XbaI and BamHI to obtain the vector fragment; the fragment containing the MSSUS gene was digested with XbaI and BamHI to obtain the gene fragment; the vector fragment and the gene fragment were ligated to obtain the recombinant expression plasmid pET-MSSUS, and the plasmid was sent for sequencing to confirm the correctness of the results.

[0068] The obtained recombinant expression plasmid pET-MSSUS was chemically transformed into E. coli BL21(DE3), and screened on LB solid medium containing 25 μg / mL kanamycin to obtain recombinant strain W expressing the glycosyltransferase MSSUS. It was verified that the recombinant strain W could express sucrose synthase encoded by SEQ ID NO: 2.

[0069] The recombinant strain W was inoculated into LB medium and cultured on a shaker at 37°C and 200 rpm for 8 hours to obtain a seed culture. The obtained seed culture was then inoculated into TB medium at a 10% inoculation rate (volume percentage) for fermentation, with initial OD controlled. 600 The concentration was 0.3, the aeration rate was 1 VVM, the stirring speed was 500 rpm, and the fermentation was carried out at 37℃ for 6 h. Then the temperature was lowered to 18℃, and IPTG with a final concentration of 0.1 mmol / L was added to induce fermentation for another 24 h to obtain the fermentation broth.

[0070] The LB medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being water, and pH 7.0.

[0071] The TB culture medium comprises: 15 g / L tryptone, 20 g / L yeast extract, 10 g / L sodium chloride, 2 g / L glucose, 2 g / L lactose, with the remainder being water, and pH 7.0.

[0072] The fermentation broth was collected and centrifuged at 5°C and 4000 rpm for 10 min using a disc centrifuge. The collected cells were then crushed using a high-pressure homogenizer and cyclically crushed three times at 800 bar and 5°C. The supernatant was then collected by centrifugation at 4°C and 4000 rpm and freeze-dried in a vacuum freeze dryer for 30 h to obtain sucrose synthase powder.

[0073] The amino acid sequence of the sucrose synthase has been verified as shown in SEQ ID NO: 1.

[0074] SEQ ID NO: 1:

[0075] MSERARSDDLIEQLRGFLDACPSVGHRVLHQIKKLERSFLQRSELCDAFADVCAAEDVPDGLRQSPLGKVIRLTQEAAVNDAWVYLAVRVRIASWRYVRIALEGMAVEEVTVRDFLRFKECLALGQHDFDEWLLEIDLGPFSREFPKLLEARSIGRGVEFLNKHLSLQLFDELGEGGERILNFLRIHSFRGQTLMLNEQIKSVSDLRRALRRADEVLAGHEPTATWEDVAAPLRALGFEVGWGREVARIRDTMSLLRDLLEAPDPRGLETFLARLPMVFSLAIISPHGYFGQANVLGRPDTGGQVVYILDQVRALETEMRSRLFEQGLDIEPQIVVLTRLIPQAEGTTCDQRLEPISGTRNARILRVPFRNASGEIVTHWISRFEVWPYLERYTLDSERELLAELGGRPDLIVGNYSDGNLVATLLSQRLGVTQCNIAHALEKTKYRHADLFWQENEAQYHFSCQFTADLIAMNAADFIITSTYQEIAGTRESVGQYESHTAFTMPKLFRVVNGIDVYDPKFNIVSPGADAAAYFPYTAVERRLPHLHTEIEQLVFGVDERVDARGVLTERDKPLLFTMARLDRIKNIVGLVEWFSACEALRKEANLLVISGHVDPERSSDTEELEQIRCMHALFNRYDLDRQVRWLGLRLPKDLAGEFYRYVADGRGAFVQPALFEAFGLTVIEAMASGLPCFATCFGGPSEIIEDGVSGFHIDPNHGDAAAERIARFFERTRQDPEYWNRISEGALKRVAERYTWQHYAERMMTLSRVYGFWRHVTDLERRETQRYLQALYSLQFRRLAQAMA。

[0076] SEQ ID NO:2:

[0077]

[0078] Example 2: Preparation of sucrose synthase mutant

[0079] In this embodiment, the method for preparing the sucrose synthase T578N mutant includes the steps of constructing a recombinant bacterium containing the mutant and culturing the recombinant bacterium to prepare the sucrose ribozyme mutant.

[0080] The sucrose synthase gene from *Methylocaldum szegediense*, screened in Example 1, was amplified by PCR using SEQ ID NO: 2 as a template and the following primers, yielding the gene fragment shown in SEQ ID NO: 4, denoted as ΔMSSUS.

[0081] Forward primer: 5'-CAAGCCGCTGTTGTTTAACATGGCGCGGCTGG-3' (SEQ ID NO: 7);

[0082] Reverse primer 6: 5'-TACGGTCCAGCCGCGCCATGTTAAACAACAGC-3' (SEQ ID NO: 8).

[0083] pET-28a(+) was digested with XbaI and BamHI to obtain the vector fragment; the fragment containing the ΔMSSUS gene was digested with XbaI and BamHI to obtain the gene fragment; the vector fragment and the gene fragment were ligated to obtain the recombinant expression plasmid pET-ΔMSSUS. The plasmid was sequenced, confirming the correctness of the results.

[0084] The recombinant expression plasmid pET-ΔMSSUS was transformed into *E. coli* BL21(DE3) by electroporation, and the culture was screened on LB agar containing 25 μg / mL kanamycin to obtain the recombinant bacterium ΔW expressing the glycosyltransferase MSSUS. It was verified that the recombinant bacterium ΔW could express the sucrose synthase encoded by SEQ ID NO: 4.

[0085] The T578N mutant enzyme powder was prepared using recombinant genetically engineered bacteria ΔW according to the cultivation method described in Example 1 above. It was confirmed that the amino acid sequence of the sucrose synthase T578N mutant is shown in SEQ ID NO: 3, and it is a T578N mutant with the amino acid sequence shown in SEQ ID NO: 1.

[0086] SEQ ID NO: 3:

[0087] MSERARSDDLIEQLRGFLDACPSVGHRVLHQIKKLERSFLQRSELCDAFADVCAAEDVPDGLRQSPLGKVIRLTQEAAVNDAWVYLAVRVRIASWRYVRIALEGMAVEEVTVRDFLRFKECLALGQHDFDEWLLEIDLGPFSREFPKLLEARSIGRGVEFLNKHLSLQLFDELGEGGERILNFLRIHSFRGQTLMLNEQIKSVSDLRRALRRADEVLAGHEPTATWEDVAAPLRALGFEVGWGREVARIRDTMSLLRDLLEAPDPRGLETFLARLPMVFSLAIISPHGYFGQANVLGRPDTGGQVVYILDQVRALETEMRSRLFEQGLDIEPQIVVLTRLIPQAEGTTCDQRLEPISGTRNARILRVPFRNASGEIVTHWISRFEVWPYLERYTLDSERELLAELGGRPDLIVGNYSDGNLVATLLSQRLGVTQCNIAHALEKTKYRHADLFWQENEAQYHFSCQFTADLIAMNAADFIITSTYQEIAGTRESVGQYESHTAFTMPKLFRVVNGIDVYDPKFNIVSPGADAAAYFPYTAVERRLPHLHTEIEQLVFGVDERVDARGVLTERDKPLLFNMARLDRIKNIVGLVEWFSACEALRKEANLLVISGHVDPERSSDTEELEQIRCMHALFNRYDLDRQVRWLGLRLPKDLAGEFYRYVADGRGAFVQPALFEAFGLTVIEAMASGLPCFATCFGGPSEIIEDGVSGFHIDPNHGDAAAERIARFFERTRQDPEYWNRISEGALKRVAERYTWQHYAERMMTLSRVYGFWRHVTDLERRETQRYLQALYSLQFRRLAQAMA*。

[0088] SEQ ID NO:4:

[0089]

[0090] Example 3 Enzyme Activity Assay

[0091] The recombinant bacteria W obtained in Example 1 and the recombinant bacteria ΔW screened in Example 2 were respectively cultured and expanded according to the culture method described in Example 1.

[0092] Fermentation broth was collected and centrifuged at 8000 rpm for 10 min. Cells were collected and subjected to routine treatments such as disruption and freeze-drying to prepare lyophilized powders of sucrose synthase (also known as the original enzyme) and sucrose synthase mutant (also known as mutant enzyme), which were stored at -80℃.

[0093] In this embodiment, enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the generation of 1 μmol UDPG per minute or the amount of enzyme required to consume 1 μmol UDP per minute at a sucrose concentration of 100 g / L and a UDP concentration of 50 g / L.

[0094] In this embodiment, the enzyme activity testing method for the sucrase or sucrase synthase mutant uses HPLC for process monitoring, and the specific method is as follows:

[0095] (1) Take 1 mL of the reaction solution and centrifuge it at 8000 rpm for 1 min;

[0096] (2) After centrifugation, carefully aspirate 100 μL of the supernatant into a 2 mL centrifuge tube and add 900 μL of ultrapure water;

[0097] (3) Use a 1mL syringe to draw 600-800μL of the diluted fermentation broth supernatant, filter it through a 0.22μm filter membrane and add it to the corresponding liquid phase bottle;

[0098] (4) Place the liquid chromatography bottle on the sample rack, establish the method, and perform the analysis.

[0099] In this embodiment, the specific procedure for HPLC analysis is as follows: Chromatograph: Agilent 1260; Detector: Evaporative Light Scattering Detector (Alltech Chrom, ELSD6000); Injection: Agilent autosampler; Injection volume: 10 μL; Column: Prevail Carbohydrate Ecolumn-W (5 μm, 4.6 × 250 mm, Agela Technologies, China); Column temperature: 40℃; Mobile phase: 70% acetonitrile; Flow rate: 1 mL / min.

[0100] According to the above definition and method, the original enzyme and mutant enzyme obtained above were tested for enzyme activity. The original enzyme and mutant enzyme were measured to be 55.7 U / g and 72.5 U / g, respectively. It can be seen that the enzyme activity of the mutant enzyme is 1.3 times that of the original enzyme.

[0101] Example 4 Thermal stability test

[0102] The lyophilized powders of the original enzyme and the mutant enzyme obtained in Example 3 were taken and incubated at 50°C for different times. The residual enzyme activity was measured and the half-life was calculated according to the reaction conditions in Example 3. The half-lives of the two enzymes were recorded as shown in Table 1 below.

[0103] Table 1 Half-life of the original enzyme and mutant enzyme

[0104] enzymes t 1 / 2 (50 °C) (min) t 1 / 2 (50°C) (fold) primitive enzymes 112 1 mutant enzyme 560 5

[0105] The results show that the mutant enzyme prepared in this invention has good thermal stability, especially the half-life at 50℃, which is increased from 112 min to 560 min, a five-fold increase, and the thermal stability is greatly improved.

[0106] Example 5: Application of the mutant in the production of steviol glycosides

[0107] In a 1L Erlenmeyer flask, 500mL of 80g / L rebaudioside A, 6g / L MgCl2, 0.1g / L UDP-glycosyltransferase, 10g / L UDP-glycosyltransferase, and 260g / L sucrose were added, along with 0.5g / L sucrose synthase primitive enzyme and sucrose synthase mutant enzyme (prepared in Example 3). The mixture was then reacted in a 50℃ water bath for 12 hours. The product amount was then determined by HPLC according to the method described in Example 3.

[0108] Calculations show that the conversion rates of the original sucrase enzyme and the mutant sucrase enzyme described in this invention to produce steviol glycosides are 84% and 95.2%, respectively. The conversion rate is calculated as: (Amount of added rebaudioside A - Residual rebaudioside A) / Amount of added rebaudioside A * 100%.

[0109] It is evident that the transformation performance of the mutant enzyme is 1.13 times that of the original enzyme, and the synthesis efficiency is significantly improved.

[0110] Example 6

[0111] In a 1L Erlenmeyer flask, 500mL of 10g / L rebaudioside A, 10g / L MgCl2, 1g / L UDP, 280g / L sucrose, and 1g / L UDP-glycosyltransferase were added respectively. 0.3g / L of sucrose synthase primitive enzyme and sucrose synthase mutant enzyme (prepared in Example 3) were added and mixed. The mixture was reacted in a 30℃ water bath for 72 hours, and the product amount was measured.

[0112] Calculations show that the conversion rates of the original sucrose ribozyme and the mutant sucrose synthase described in this invention to produce steviol glycosides are 79.2% and 91.2%, respectively.

[0113] Example 7

[0114] In a 1L Erlenmeyer flask, 500mL of 100g / L rebaudioside D, 1g / L MgCl2, 10g / L UDP, 180g / L sucrose, and 20g / L UDP-glycosyltransferase were added respectively. 0.8g / L of sucrose synthase primitive enzyme and sucrose synthase mutant enzyme (prepared in Example 3) were added and mixed. The mixture was reacted in an 80℃ water bath. After 24 hours of reaction, the amount of product was measured.

[0115] Calculations show that the conversion rates of the original sucrose ribozyme and the mutant sucrose synthase described in this invention to produce steviol glycosides are 83.9% and 94.3%, respectively.

[0116] As can be seen, the wild-type sucrose synthase and the sucrose synthase mutant obtained by mutation screened in this invention are stable in terms of enzyme activity, conversion rate and stability, and can efficiently catalyze the synthesis of steviol glycosides, which can effectively improve the conversion rate of the target product.

[0117] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A sucrose synthase, characterized in that, The sucrose synthase comprises an amino acid sequence as shown in SEQ ID NO:

1. Preferably, the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO:

1.

2. A gene encoding the sucrose synthase of claim 1, characterized in that, The nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO:

2. Preferably, the nucleotide sequence of the gene encoding the sucrose synthase is as shown in SEQ ID NO:

2.

3. A sucrose synthase mutant, characterized in that, The sucrose synthase mutant is obtained by mutating the amino acid at position 578 of the sucrose synthase according to claim 1. Preferably, the sucrose synthase mutant comprises an amino acid sequence as shown in SEQ ID NO:

3. More preferably, the amino acid sequence of the sucrose synthase mutant is as shown in SEQ ID NO:

3.

4. A gene encoding the sucrose synthase mutant of claim 3, wherein the gene comprises the nucleic acid sequence of SEQ ID NO:

1. The nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO:

4. Preferably, the nucleotide sequence of the gene encoding the sucrose synthase mutant is as shown in SEQ ID NO:

4.

5. A recombinant vector, characterized in that, The recombinant vector carries the gene encoding the sucrose synthase according to claim 2 or the gene encoding the sucrose synthase mutant according to claim 4. Preferably, the recombinant vector comprises recombinant expression plasmids pET-MSSUS and / or pET-ΔMSSUS.

6. A recombinant host, wherein, The recombinant host expresses the recombinant vector according to claim 5. Preferably, the recombinant host comprises a recombinant host cell or a genetically engineered bacterium.

7. A method for preparing sucrose synthase or sucrose synthase mutants, characterized in that, The method comprises: connecting the gene encoding the sucrose synthase according to claim 2 or the gene encoding the sucrose synthase mutant according to claim 4 to an expression vector to construct a recombinant vector; transforming the recombinant vector into a host for culture and expression, and isolating the desired sucrose synthase or sucrose synthase mutant from the obtained culture; or, transforming the recombinant vector according to claim 5 into a host for culture and expression, and isolating the desired sucrose synthase or sucrose synthase mutant from the obtained culture; or, inducing the culture of the recombinant host according to claim 6, and isolating the desired sucrose synthase or sucrose synthase mutant from the obtained culture.

8. A sucrose synthase or sucrose synthase mutant prepared by the method according to claim 7.

9. Use of the sucrose synthase according to claim 1, the gene encoding the sucrose synthase according to claim 2, the sucrose synthase mutant according to claim 3, the gene encoding the sucrose synthase mutant according to claim 4, the recombinant vector according to claim 5, the recombinant host according to claim 6, or the sucrose synthase or sucrose synthase mutant according to claim 8 in the field of steviol glycoside synthesis.

10. A method of preparing steviol glycosides, characterized by, The method comprises the step of catalyzing the reaction by using the sucrose synthase according to claim 1, the sucrose synthase mutant according to claim 3, or the sucrose synthase or sucrose synthase mutant according to claim 8 as the enzyme catalyst in a catalytic reaction system containing steviol glycoside and sucrose as substrates. Preferably, the steviol glycoside comprises rebaudioside A and / or rebaudioside D. Preferably, the catalytic reaction system comprises the following components: 10-100 g / L rebaudioside A and / or rebaudioside D, 1-10 g / L MgCl2, 180-280 g / L sucrose, 0.1-10 g / L uridine diphosphate disodium salt, 1-20 g / L UDP-glycosyltransferase, and 0.3-0.8 g / L sucrose synthase and / or sucrose synthase mutant; Preferably, the initial pH of the catalytic reaction system is 6-8; and / or, Preferably, the temperature of the catalytic reaction is 30-80°C; and / or, Preferably, the time of the catalytic reaction is 10-72 h.