A glycosyltransferase derived from Camelina sativa and its application

Abstract

The present invention discloses a glycosyltransferase derived from Camelina sativa and its application. The amino acid sequence of the UDP-glycosyltransferase disclosed in the present invention is shown in SEQ ID NO:1, and the amino acid sequence of its mutant is shown in SEQ ID NO:3. The UDP-glycosyltransferase and its mutant provided by the present invention can efficiently catalyze the conversion of tyrosol to salidroside, and its mutant has good substrate tolerance, which is of great significance for the production and application of salidroside.

Description

Technical Field

[0001] The invention belongs to the technical field of functional enzyme screening, and relates to a UDP-glycosyltransferase derived from Camelina sativa L. and a mutant thereof, nucleic acid molecules encoding the same, a recombinant vector and a recombinant cell containing the nucleic acid molecules, a preparation method of the UDP-glycosyltransferase and the mutant thereof, and application of the UDP-glycosyltransferase and the mutant thereof in preparing salidroside. Background Art

[0002] Salidroside is the main active ingredient of the plant Rhodiola rosea, a phenylethanol derivative, with significant antioxidant, anti-inflammatory, anti-fatigue, anti-hypoxia, anti-aging, anti-radiation and many other beneficial effects. It has been widely added and applied in the production of various medicines, health products, cosmetics, etc. The traditional method of obtaining it is to extract it from plants, but the original content of salidroside in plants is low, resulting in high separation and extraction costs; it can also be synthesized by chemical methods, but the process requires the use of precious metal catalysts and multi-step reactions such as group protection and deprotection, which is not conducive to large-scale industrial production.

[0003] In recent years, researchers have tried to clarify the natural synthesis pathway of salidroside, hoping to produce it industrially by genetically engineering microorganisms. It is now clear that salidroside can be produced by glycosylation of tyrosol on its 8-OH group. The glycosylation reaction is catalyzed by uridine diphosphate glucosyltransferase (UDP–glycosyltransferase, UGT). This method has high atom economy, but the activity of UGT has always been the rate-limiting step of the reaction. At present, researchers have obtained enzyme genes such as UGT73B6, UGT74R1, UGT72B1, UGT73C5, UGT73C6, and UGT85A1 from Rhodiola rosea, Arabidopsis, and Bacillus subtilis, and cloned and heterologously expressed them, but there are still problems such as insufficient enzyme activity, low substrate conversion rate, and poor substrate tolerance, which greatly limit the production and application of salidroside. Therefore, it is very important to obtain glycosyltransferases with high catalytic activity to create an efficient biosynthetic pathway for the synthesis of salidroside. Summary of the invention

[0004] The object of the present invention is to provide a UDP-glycosyltransferase derived from Camelina sativa L. and a mutant thereof, which is intended to solve the problem of limited enzyme activity and substrate tolerance in the existing glycosyltransferase-catalyzed synthesis of salidroside.

[0005] Specifically, the present invention obtains a UDP-glycosyltransferase from Camelina sativa, whose amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence after codon optimization is shown in SEQ ID NO:2. By further mutating it, a UDP-glycosyltransferase mutant with significantly improved enzyme activity and substrate tolerance is obtained, whose amino acid sequence is shown in SEQ ID NO:3 and the nucleotide sequence is shown in SEQ ID NO:4.

[0006] In a first aspect, the present invention provides a UDP-glycosyltransferase, whose amino acid sequence is shown in SEQ ID NO:1.

[0007] The above UDP-glycosyltransferase provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and this active polypeptide can be a product of natural purification, a product of chemical synthesis, or a product produced using recombinant techniques from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0008] In some embodiments, the above UDP-glycosyltransferase is obtained by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (such as E. coli BL21(DE3)) to construct a recombinant genetic engineering strain, then culturing the strain, and adding an inducer to induce expression to obtain the UDP-glycosyltransferase.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above UDP-glycosyltransferase, whose nucleotide sequence is shown in SEQ ID NO:2, and this nucleotide sequence is a sequence after codon optimization.

[0010] The above nucleic acid molecule provided by the present invention can generally be obtained by using a PCR instrument for amplification or by artificial synthesis.

[0011] In a third aspect, the present invention provides a mutant of the above UDP-glycosyltransferase, whose amino acid sequence is shown in SEQ ID NO:3, and its catalytic activity and substrate tolerance are further improved compared with the above UDP-glycosyltransferase. Compared with the amino acid sequence of the UDP-glycosyltransferase shown in SEQ ID NO:1, the following mutations exist in this mutant: Arg at position 64 is mutated to His, Leu at position 193 is mutated to Val, Gly at position 335 is mutated to Ser, and Ile at position 461 is mutated to Val.

[0012] The mutant of the above UDP-glycosyltransferase provided by the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression, such as obtained by expression using recombinant techniques from a prokaryotic host (Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0013] In some embodiments, the above UDP-glycosyltransferase mutant is constructed by transforming a recombinant vector containing its coding gene into an Escherichia coli expression host (such as E. coli BL21(DE3)), culturing the strain, and adding an inducer to induce expression to obtain the mutant.

[0014] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the above mutant, and its nucleotide sequence is as shown in SEQ ID NO:4.

[0015] The nucleic acid molecule of the mutant provided by the present invention can generally be obtained by using a PCR instrument for amplification or by artificial synthesis.

[0016] In a fifth aspect, the present invention provides a recombinant vector, which contains any one of the above nucleic acid molecules. Specifically, the recombinant vector includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes.

[0017] In some embodiments, the above recombinant vector is pET-ugt71D1 or pET-ugt-32, which are obtained by replacing the sequence between the HindIII and EcoRI cleavage sites of pET-28a(+) with the nucleic acid molecule encoding the above UDP-glycosyltransferase or the nucleic acid molecule of the above mutant, and the remaining sequences remain unchanged.

[0018] In a sixth aspect, the present invention provides a recombinant cell, which contains any one of the above recombinant vectors.

[0019] In some embodiments, the recombinant cell expresses the above UDP-glycosyltransferase or mutant after induction.

[0020] In some embodiments, the construction method of the recombinant cell includes the following:

[0021] Transform the recombinant vector into an expression host cell, culture it and add an inducer to induce expression to obtain the above UDP-glycosyltransferase or mutant.

[0022] Furthermore, the recombinant vector is any one of the above recombinant vectors, and the expression host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., and preferably the Escherichia coli expression host E. coli BL21(DE3).

[0023] In some embodiments, the recombinant cells are recombinant bacterium U and recombinant bacterium 32, and the recombinant cells may be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacteria express UDP-glycosyltransferase or its mutant may be a culture medium that can enable the recombinant genetically engineered bacteria to grow and express the UDP-glycosyltransferase or its mutant of the present invention in the art, such as LB medium.

[0024] There are no special requirements for the culture method and culture conditions. It is only necessary to ensure the normal growth of the recombinant genetically engineered strain and induce the expression of UDP-glycosyltransferase and its mutant at a temperature of, for example, 18°C.

[0025] More specifically, the method for constructing the above-mentioned recombinant cells comprises the following steps:

[0026] (1) Amplification of the UDP-glycosyltransferase gene;

[0027] (2) Obtaining the gene of the UDP-glycosyltransferase mutant;

[0028] (3) Construction of the recombinant expression plasmids pET-ugt71D1 and pET-ugt-32;

[0029] (4) Transformation of the recombinant expression plasmids pET-ugt71D1 and pET-ugt-32 into the host cell;

[0030] (5) Screening positive clone strains, recombinant bacterium U and recombinant bacterium 32, on the plate resistant medium;

[0031] In a seventh aspect, the present invention provides a method for preparing UDP-glycosyltransferase or its mutant, comprising the following steps:

[0032] 1) Culturing any of the above-mentioned recombinant cells and adding an inducer to induce the expression of UDP-glycosyltransferase or its mutant to obtain a culture;

[0033] 2) Isolating the above-mentioned UDP-glycosyltransferase or its mutant from the culture;

[0034] Among them, the method for culturing and inducing the recombinant cells and the method for isolating UDP-glycosyltransferase and its mutant from the culture are all conventional methods in the art.

[0035] In an eighth aspect, the present invention provides the application of the above-mentioned UDP-glycosyltransferase, any of the above-mentioned nucleic acid molecules, the above-mentioned mutant, the above-mentioned recombinant vector, the above-mentioned recombinant cells and / or the UDP-glycosyltransferase and its mutant prepared by the above-mentioned method in the preparation of salidroside.

[0036] In a ninth aspect, the present invention provides a method for preparing salidroside, comprising the following steps: using the above UDP-glycosyltransferase, the above mutant, the above recombinant cell and / or the UDP-glycosyltransferase or its mutant prepared by the above method as a catalyst to catalyze the substrate tyrosol to obtain salidroside.

[0037] In some embodiments, in the above preparation method, the reaction conditions for the catalysis are: the reaction temperature is 25 - 50 °C, such as 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, or any value or range between any two of these values, and preferably 30 °C as the reaction temperature; the reaction pH is 6 - 8, which can be adjusted with a phosphate buffer, for example, using 50 mM PBS (pH 7.5) to adjust the pH of the reaction; the reaction time is 0.1 - 5 h, such as 0.1 h, 1 h, 2 h, 3 h, 4 h, 5 h, or any value or range between any two of these values, and preferably 3 h.

[0038] Tyrosol is used as the substrate for the enzymatic catalysis reaction of the present invention. In some embodiments, its final concentration is 2 - 50 mM, such as 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or any value or range between any two of these values, and preferably 30 mM.

[0039] In some embodiments, the catalytic reaction further contains magnesium ions (Mg 2+ ), and the concentration of the magnesium ions is 0.1 - 1 mM, such as 0.1 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, or any value or range between any two of these values, and preferably 1 mM.

[0040] The above recombinant cell or its freeze-dried powder can be used as a catalyst for whole-cell catalysis to produce salidroside. If the catalyst is freeze-dried powder, the dosage is 5 - 100 mg / g tyrosol. If the catalyst is the culture solution of recombinant cells, the dosage can be 50 - 200 mg of the bacterial solution (OD 600 is 2) / g tyrosol. It should be understood that the UDP-glycosyltransferase or its mutant provided by the present invention can be used in the form of whole cells of engineered bacteria, or in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzymes, and the UDP-glycosyltransferase or its mutant of the present invention can also be made into a catalyst in the form of an immobilized enzyme or an immobilized cell using immobilization techniques known in the art.

[0041] The present invention first provides a UDP - glycosyltransferase derived from Camelina sativa, and further performs mutation treatment on it to obtain a UDP - glycosyltransferase mutant. When this mutant catalyzes the conversion of tyrosol to salidroside, the enzyme activity can reach 50.9 U / mL, which is 2.36 times that of the original enzyme. When the concentration of tyrosol increases to 50 mM, the catalytic conversion rate of the mutant enzyme is 9.7%, which is 3 times that of the original enzyme, facilitating the further increase of the yield of salidroside. The UDP - glycosyltransferase and its mutant provided by the present invention can efficiently catalyze the conversion of tyrosol to salidroside, and the mutant has good substrate tolerance, which is of great significance for the production and application of salidroside. Brief Description of the Drawings

[0042] Figure 1 It is the electrophoresis diagram of colony PCR verification of recombinant bacterium 32.

[0043] Figure 2 It is the HPLC chromatogram. Among them, (a) is the HPLC chromatogram of tyrosol standard, (b) is the HPLC chromatogram of salidroside standard, and (c) is the HPLC chromatogram of the reaction solution for mutant enzyme activity determination. Detailed Embodiments

[0044] The following further illustrates the present invention in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art or the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels unless otherwise specified.

[0045] pET - 28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., and the product catalog number is B540183.

[0046] Example 1: Obtaining the Gene Sequence of UDP - glycosyltransferase

[0047] 1. Log in to the NCBI official website. The Gene ID of UDP - glycosyltransferase (UDP - glycosyltransferase 71D1) derived from Camelina sativa L. is 104789205, and the amino acid sequence of the encoded enzyme is shown in SEQ ID NO: 1. After alignment, the homology of the gene sequence of this enzyme with the gene sequence of UGT71D1 (Gene ID: 817523) derived from Arabidopsis thaliana is 87%.

[0048] MRNAELIFIPTPTIGHLVPFLEFARRLIEQDARIRITILLMKLQGQSHMDSYVKSIASSLPFVRFIDVPELEEKPTLGTTQSVEAYVYDIIERNIPLVRNIVMDILTSLALDGVNVNGIVADLFCLPMIDVAKDASLPFHVFLTTNSGFLAMMKYLADRHCKDTSLFVKDSGEMLSIPGFVNPVPAKVLPSALFLEDGYDAYVKLAILFTKAKGVLVNSSFDIEPCSVSHFHHEQNYPSVYAVGPIFNRKAHPHPDQDLARRDELMKWLDDQPEASVVFLCFGSMGKLRGPLVKEIAHGLELCQYRFLWSLRTEEVTNVELLLPEGFLDRVAGRGMICGWSPQVEILAHKAVGGFVSHCGWNSIVESLWFGVPIVTWPMYAEQQLNAFLMVKELNLAVDMRLDYRAHSDELVSANEIETAVRCVMNKDNNVVRKRVMDISKMAREATMNGGSSYLAIEKFIQDVIGTKP(SEQ ID NO:1)

[0049] 2. To achieve the heterologous expression of the enzyme in Escherichia coli, the gene sequence of the enzyme was codon-optimized. The optimized sequence is shown in SEQ ID NO:2 and was artificially gene-synthesized.

[0050]

[0051] 3. Using the DNA shown in SEQ ID NO:2 obtained in step 2 as a template, with upstream primer 1 (5'- aagctt atgaggaatgctgaactaatatttattcccactc-3', SEQ ID NO:5) and downstream primer 2 (5'- gaattc ttatggtttggtgccaataacatcttgaat-3', SEQ ID NO:6) for PCR amplification to obtain a DNA fragment containing the UDP-glycosyltransferase gene.

[0052] Example 2: Obtaining the gene sequence of UDP-glycosyltransferase mutants using error-prone PCR technology

[0053] Error-prone PCR can change and increase the natural error rate of the polymerase on the basis of standard PCR. Compared with the basic PCR reaction (1.5 mM), the error-prone PCR reaction usually contains a higher concentration of magnesium chloride (7 mM) to stabilize non-complementary pairs. In this example, error-prone PCR technology was used to obtain mutants of the UDP-glycosyltransferase gene.

[0054] Using the DNA shown in SEQ ID NO:2 as a template, with upstream primer 1 and downstream primer 2 as primers, error-prone PCR was carried out according to the following reaction system, and a total of 90 mutants of the UDP-glycosyltransferase gene were obtained.

[0055] Error-prone PCR reaction system: 5 μl of 10× amplification buffer, 4 μl of each of the 4 dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μL of Taq DNA polymerase, Mg 2+ 7 mmol / L, add pure water to 50 μl.

[0056] Example 3: Cloning of the UDP-glycosyltransferase gene (ugt71D1) and construction of an expression strain

[0057] 1. Double-digest the DNA fragment containing the UDP-glycosyltransferase gene obtained in Example 1 with HindIII and EcoRI to obtain a gene fragment; double-digest pET-28a(+) with HindIII and EcoRI to obtain a vector fragment; ligate the gene fragment and the vector fragment to obtain a recombinant expression plasmid, which was named pET-ugt71D1, and send this plasmid for sequencing, and the result was consistent with the expectation.

[0058] 2. The recombinant expression plasmid pET-ugt71D1 obtained in step 1 was transformed into E. coli DH5α competent cells by heat shock, spread on LB solid medium containing 50 μg / mL kanamycin, and the corresponding monoclonal strains were cultured. After amplification and plasmid extraction, the pET-ugt71D1 plasmid was obtained. The obtained plasmid was transformed into E. coli BL21(DE3) by chemical transformation, and spread on LB solid medium containing 50 μg / mL kanamycin for screening to obtain the recombinant bacterium U expressing UDP-glycosyltransferase.

[0059] Example 4: Cloning of UDP-glycosyltransferase gene mutants and construction of expression strains

[0060] According to the method of Example 3, 90 mutants of the UDP-glycosyltransferase gene obtained in Example 2 were used to construct recombinant plasmids pET-ugt-1 to pET-ugt-90 respectively, and recombinant bacteria 1 - recombinant bacteria 90 expressing UDP-glycosyltransferase mutants were obtained.

[0061] Example 5: Obtaining UDP-glycosyltransferase mutants with high catalytic efficiency by high-throughput screening

[0062] The recombinant bacteria constructed in Example 4 were cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin respectively, and IPTG with a final concentration of 0.5 mM was added at 18 °C to induce protein expression. After 16 h, the OD 600 value of the bacterial solution was measured, and the bacterial solution was diluted with water to make the OD 600 value about 2. The diluted bacterial solution was added into 96-well plates for reaction, and the enzyme-coupled method was used to indirectly measure the generated UDP to monitor the catalytic ability of each mutant to the substrate tyrosol. The reaction of NADH generating NAD+ would cause a change in the light absorption at 340 nm.

[0063] The principle of the enzyme-coupled method is as follows:

[0064]

[0065]

[0066]

[0067] The specific reaction system was 250 μL, containing 5 mM tyrosol, 5 mM UDP-glucose, 1 mM Mg 2+, 5 mM NADH, 5 mM PEP (phosphoenolpyruvate), 1 mM PK (phosphokinase) and 1 mM LDH (lactate dehydrogenase), 70 mg of bacterial solution (equivalent to 100 mg of bacterial solution / g tyrosol), and the buffer was 50 mM PBS (pH 7.5). The prepared reaction system was incubated in an oscillating mixer at 30 °C and 120 rpm for 1 h. After the reaction, the reaction was terminated by heating at 95 °C for 5 min, and 100 μl of the supernatant was taken by centrifugation. After mixing with 200 μl of water, the absorbance was measured at a wavelength of 340 nm.

[0068] The strain corresponding to the reaction solution with the lowest absorbance (recombinant strain 32) was subjected to plasmid extraction. After extraction, plasmid sequencing was performed, and the gene sequence encoding the UDP-glycosyltransferase mutant was obtained as shown in SEQ ID NO: 4, and the amino acid sequence of the UDP-glycosyltransferase mutant encoded by it was as shown in SEQ ID NO: 3. Compared with the amino acid sequence of the UDP-glycosyltransferase shown in SEQ ID NO: 1, the following mutations exist in this UDP-glycosyltransferase mutant: Arg at position 64 was mutated to His, Leu at position 193 was mutated to Val, Gly at position 335 was mutated to Ser, and Ile at position 461 was mutated to Val.

[0069] MRNAELIFIPTPTIGHLVPFLEFARRLIEQDARIRITILLMKLQGQSHMDSYVKSIASSLPFVHFIDVPELEEKPTLGTTQSVEAYVYDIIERNIPLVRNIVMDILTSLALDGVNVNGIVADLFCLPMIDVAKDASLPFHVFLTTNSGFLAMMKYLADRHCKDTSLFVKDSGEMLSIPGFVNPVPAKVLPSAVFLEDGYDAYVKLAILFTKAKGVLVNSSFDIEPCSVSHFHHEQNYPSVYAVGPIFNRKAHPHPDQDLARRDELMKWLDDQPEASVVFLCFGSMGKLRGPLVKEIAHGLELCQYRFLWSLRTEEVTNVELLLPEGFLDRVAGRSMICGWSPQVEILAHKAVGGFVSHCGWNSIVESLWFGVPIVTWPMYAEQQLNAFLMVKELNLAVDMRLDYRAHSDELVSANEIETAVRCVMNKDNNVVRKRVMDISKMAREATMNGGSSYLAIEKFVQDVIGTKP (SEQ ID NO: 3)

[0070]

[0071] Colony PCR verification of recombinant bacterium 32 was carried out using upstream primer 1 and downstream primer 2. The electrophoresis pattern is as Figure 1 shown, where M represents DNA Marker, and lanes 1-8 are the colony PCR products of recombinant bacterium 32.

[0072] Example 6: Preparation of enzyme and determination of enzyme activity

[0073] Recombinant bacterium U and recombinant bacterium 32 were respectively subjected to enlarged culture. After the enlarged culture, the fermentation broth was centrifuged (8000 rpm, 10 min), cell disrupted, and freeze-dried to prepare freeze-dried powders of UDP-glycosyltransferase (original enzyme) and its mutant (mutant enzyme), which were stored at -80 °C.

[0074] Definition of enzyme activity unit (U): When the reaction system is 10 ml, containing 5 mM substrate tyrosol, 5 mM UDP-glucose, 35 mg freeze-dried powder (equivalent to 50 mg freeze-dried powder / g tyrosol), 1 mM Mg 2+ , and the buffer is 50 mM PBS (pH 7.5), at a temperature of 30 °C, the amount of enzyme required to catalyze the formation of 1 μmol of salidroside per minute or the amount of enzyme required to consume 1 μmol of tyrosol per minute is 1 enzyme activity unit.

[0075] The reaction equation is as follows:

[0076]

[0077] The enzyme activities of the original enzyme and the mutant enzyme were measured to be 21.5 U / mL and 50.9 U / mL respectively. Compared with the original enzyme, the enzyme activity of the mutant enzyme increased by 1.36 times. The contents of substrate tyrosol and product salidroside can be detected by HPLC.

[0078] The specific HPLC detection method is as follows: Take 1 mL of the reaction solution and centrifuge it at 8000 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm filter membrane. Select a Shimadzu liquid chromatograph and use a 4.6×250 mm C18 column; set the detection wavelength to 225 nm; mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is methanol, and the volume ratio is 20% A / 80% B, with a flow rate of 1 mL / min; the column temperature is 35 °C; the injection volume is 10 μL.

[0079] The HPLC chromatograms of the standard product and the reaction solution for mutant enzyme activity determination are as Figure 2 shown. The retention time of the tyrosol standard product is 12.8 min, the retention time of the salidroside standard product is 10.9 min, the retention time of tyrosol in the reaction solution for mutant enzyme activity determination is 12.8 min, and the retention time of salidroside is 10.9 min.

[0080] Example 7: Substrate Tolerance Test

[0081] Prepare a reaction system in an Erlenmeyer flask, containing 2 - 50 mM tyrosol and an equal amount of UDP - glucose, 1 mM Mg 2+ , 100 mg of bacterial solution (OD 600 is 2) / g tyrosol, the buffer is 50 mM PBS (pH 7.5), carry out the reaction in a water bath at 30 °C, end the reaction after 3 h, and obtain the conversion rates of the two enzymes under different substrate concentration conditions (conversion rate = (initial reaction concentration of tyrosol - residual tyrosol concentration measured in the liquid phase) / initial reaction concentration of tyrosol * 100%), as shown in Table 1.

[0082] Table 1

[0083]

[0084] The results show that the mutant enzyme has good tolerance to the substrate tyrosol. When the tyrosol concentration is between 2 - 20 mM, the conversion rate of the mutant enzyme can reach more than 22%. Compared with the original enzyme, its tolerance is greatly improved; when the tyrosol concentration rises to 50 mM, the conversion rate of the original enzyme is as low as 3.2%, while the conversion rate of the mutant enzyme is 9.7%.

[0085] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A mutant of UDP - glycosyltransferase, whose amino acid sequence is shown in SEQ ID NO:

3.

2. A nucleic acid molecule encoding the mutant according to claim 1, whose nucleotide sequence is shown in SEQ ID NO:

4.

3. A recombinant vector, which contains the nucleic acid molecule according to claim 2.

4. A recombinant cell, which contains the recombinant vector according to claim 3.

5. A method for preparing a mutant of UDP - glycosyltransferase, comprising the following steps: 1) Cultivate the recombinant cell as described in claim 4 and induce the expression of the mutant of UDP - glycosyltransferase; 2) Isolate the mutant as described in claim 1 from the culture obtained in step 1).

6. Use of the mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3 and / or the recombinant cell according to claim 4 in the preparation of salidroside.

7. A method for preparing salidroside, comprising the following steps: Use the mutant as described in claim 1 and / or the recombinant cell as described in claim 4 as a catalyst to catalyze the substrate tyrosol to obtain salidroside.

8. According to the method of claim 7, characterized in that: The catalytic reaction conditions are as follows: the reaction temperature is 20 - 50 °C, the reaction pH is 6 - 8, and the reaction time is 0.1 - 5 h; the concentration of the substrate tyrosol is 2 - 50 mM, and the Mg 2+ concentration is 0.1 - 1 mM.

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