Mutants of enzymes, mutants of glycosyltransferases and their use in the preparation of rhodioside

Abstract

The present application relates to the field of bioengineering, and particularly to a mutant of an enzyme, a mutant of a glycosyltransferase and application thereof in preparing rhodiolin. The present application provides a mutant of an enzyme, which has the following amino acid site mutations on the basis of a wild-type glycosyltransferase: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S and the 386th site is mutated from S to G, and the amino acid sequence of the wild-type glycosyltransferase is shown as SEQ ID NO: 1. The present application obtains a glycosyltransferase and a sucrose synthase with improved thermal stability through structural rational design and deep learning modification, and can glycosylate tyramine to generate rhodiolin in the form of regenerating ADP-glucose, and the price of ADP is only about 20% of UDP, which has a cost advantage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to mutants of enzymes, mutants of glycosyltransferases and their applications in preparing salidroside. BACKGROUND

[0002] Rhodiola, a perennial herb on the plateau, its dried roots and rhizomes can be used as medicine, which has the functions of benefiting qi and promoting blood circulation, and can also relieve angina and asthma. The main effective components are salidroside and tyrosol. Rhodiola is known as one of the four famous Tibetan medicines together with Cordyceps sinensis, saffron and Saussurea laniceps. In recent years, it has been used in pharmaceuticals, health products and skin care products. There are more than 90 species of Rhodiola, such as Rhodiola crenulata, Rhodiola kirilowii, Rhodiola sachalinensis and Rhodiola rosea. Due to overexploitation and destruction of the growing environment, the wild resources are becoming increasingly depleted. Although Rhodiola kirilowii has been artificially cultivated in Gannan region, the long growth period and high cost have not formed large-scale planting, which cannot meet the market demand.

[0003] Thanks to the gene mining of glycosyltransferases for synthesizing salidroside in plants, the Institute of Process Engineering and Tianjin University have carried out research on the fermentation of salidroside using Escherichia coli and Saccharomyces cerevisiae as the host cells. However, the conversion of tyrosol to salidroside is not complete due to the low activity of glycosyltransferases and insufficient supply of UDP-glucose in cells, which limits the industrial application. SUMMARY

[0004] Therefore, the present application provides mutants of enzymes, mutants of glycosyltransferases and their applications in preparing salidroside. The present application obtains glycosyltransferases and sucrose synthases with improved thermal stability through structural rational design and deep learning modification, and can glycosylate tyrosol to generate salidroside in the form of regenerating ADP-glucose, which has a cost advantage as the price of ADP is only about 20% of that of UDP.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides mutants of enzymes, which have the following amino acid site mutations based on wild-type glycosyltransferases: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S, and the 386th site is mutated from S to G. The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO: 1.

[0007] In some embodiments of the present application, the amino acid sequence of the mutant is shown in SEQ ID NO: 5.

[0008] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the wild-type glycosyltransferase described above is shown as SEQ ID NO: 2.

[0009] The present application also provides a mutant of the glycosyltransferase described above, which has the following amino acid site mutations based on the mutant described above: the 21st site is mutated from A to I, the 83rd site is mutated from M to N, and the 407th site is mutated from Q to P.

[0010] In some embodiments of the present application, the amino acid sequence of the mutant of the glycosyltransferase described above is shown as SEQ ID NO: 7.

[0011] The present application also provides an enzyme composition comprising: the mutant of the enzyme described above and / or the mutant of the glycosyltransferase described above, and a sucrose synthase.

[0012] In some embodiments of the present application, the amino acid sequence of the sucrose synthase in the enzyme composition described above is shown as SEQ ID NO: 3.

[0013] The present application also provides a nucleic acid molecule encoding the mutant of the enzyme described above, the mutant of the glycosyltransferase described above, and / or the enzyme composition described above.

[0014] In some embodiments of the present application, the nucleic acid molecule described above comprises:

[0015] the nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme described above is shown as SEQ ID NO: 6; and / or

[0016] the nucleotide sequence of the nucleic acid molecule encoding the mutant of the glycosyltransferase described above is shown as SEQ ID NO: 8; and / or

[0017] the nucleotide sequence of the nucleic acid molecule encoding the sucrose synthase described above is shown as SEQ ID NO: 4.

[0018] The present application also provides a recombinant vector comprising: the nucleic acid molecule described above and an acceptable genetic element.

[0019] The present application also provides a host transformed and / or transfected with the recombinant vector described above.

[0020] The present application also provides a product comprising: the mutant of the enzyme described above, the mutant of the glycosyltransferase described above, the enzyme composition described above, the nucleic acid molecule described above, the recombinant vector described above, and / or the host described above and an acceptable adjuvant or excipient.

[0021] The present application also provides the use of the mutant of the enzyme described above, the mutant of the glycosyltransferase described above, the enzyme composition described above, the nucleic acid molecule described above, the recombinant vector described above, the host described above, and / or the product described above in the preparation of rhodiolin.

[0022] The present application also provides a preparation method of rhodioloside, wherein a raw material is converted into rhodioloside by any of the following methods:

[0023] (a) a mutant of the above enzyme; or

[0024] (b) a mutant of the above glycosyltransferase; or

[0025] (c) the above enzyme composition; or

[0026] (d) the above nucleic acid molecule; or

[0027] (e) the above recombinant vector; or

[0028] (f) the above host; or

[0029] (g) the above product.

[0030] The raw material includes sucrose, tyrosol and ADP.

[0031] In some embodiments of the present application, in the above preparation method, the temperature during the conversion is 30-60°C.

[0032] In some embodiments of the present application, in the above preparation method, when a mutant of the above enzyme is used, the temperature during the conversion is 30°C.

[0033] In some embodiments of the present application, in the above preparation method, when a mutant of the above glycosyltransferase is used, the temperature during the conversion is 60°C.

[0034] In some embodiments of the present application, in the above preparation method, the enzyme activity of the mutant of the enzyme is 13.3 U / mL; the enzyme activity of the mutant of the glycosyltransferase is 80.3 U / mL; and the enzyme activity of the sucrose synthase is 20.6-83.8 U / mL.

[0035] In some embodiments of the present application, in the above preparation method, the final concentration of the sucrose is 204.5-409 mM; the final concentration of the tyrosol is 202.7-405.3 mM; and the final concentration of the ADP is 0.2 mM.

[0036] The beneficial effects of the present application include:

[0037] (1) The UDP glycosyltransferase after modification can use cheap ADP as a substrate to replace expensive UDP;

[0038] (2) The ADP glycosyltransferase after secondary modification can react at a high temperature of 60°C, which greatly shortens the reaction time compared with 30°C, and can complete the reaction at a high substrate concentration of more than 400 mM. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0040] Figure 1 Electrophoresis results of AtUGT85A1 and AcSUS proteins expressed in shake flasks;

[0041] Figure 2 The alignment results of the predicted AtUGT85A1 structure with the SrUGT76G1-UDP-RA composite crystal structure are shown, with UDP displayed as a ball-and-stick model.

[0042] Figure 3 The SrUGT76G1 residues that interact with UDP are shown (the rectangular boxes are labeled with the corresponding residues of AtUGT85A1).

[0043] Figure 4 The predicted structure of AtUGT85A1 shows residues Ser307, Ile308, Val333, Trp363, and Ser386 that may interact with UDP.

[0044] Figure 5 Local sequence alignment of AtUGT85A1 residues that may interact with UDP is shown;

[0045] Figure 6 Showing the Pythia energy heatmap of global single-point mutation in AtUGT85A1;

[0046] Figure 7 Showing the high-performance liquid chromatography (HPLC) chromatogram of pure sedin;

[0047] Figure 8 The pure quality spectrum of rhodioloside is shown. Detailed Implementation

[0048] This invention discloses mutants of enzymes, mutants of glycosyltransferases, and their application in the preparation of rhodioloside.

[0049] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0050] The use of the terms "including," "containing," or "comprising" and variations thereof, are intended to be open-ended, and include the presence of sub- elements or additional elements. Variations of "or" mean any or.

[0051] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0052] The use of any and all examples or exemplary language herein is intended to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0053] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are merely intended to convey general information as to the scope of the application. Consistent with the statement above, unless otherwise specified, all ranges include endpoints. Certain numerical ranges and parameters are presented herein with numerical values representing the lower and upper bounds of the range. All such ranges are open-ended, and always include the minimum and maximum values of the ranges. All ranges and numerical values are "approximations", meaning features to not have to be literally reciprocal, ratio, linear, or other such strict mathematical property, as such terms are commonly used in the art. It should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between and including the minimum value of 1 and the maximum value of 10, e.g., 1 to 6.1, 3.5 to 4.5, etc.

[0054] MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSITVLSVKQLVEFAWGLAGSGKEFLWVIRPDLVAGEEAMVPPDFLMETKDRSMLASWCPQEKVLSHPAIGGFLTHCGWNSILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO: 1);

[0055]

[0056] Amino acid sequence of sucrose synthase AcSUS from Acidithiobacillus caldus: MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQQGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME (SEQ ID NO:3);

[0057]

[0058] Amino acid sequence of engineered ADP glycosyltransferase AtUGT85A1 -I308Q / V333R / W363S / S386G (AGT): MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO: 5);

[0059]

[0060] Amino acid sequence of engineered ADP glycosyltransferase AGT-A21I / M83N / Q407P: MGSQIIHNSQKPHVVCVPYPIQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDNDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQPMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD (SEQ ID NO: 7);

[0061]

[0062] In Comparative Example 1 and Examples 1 to 5 of this invention, all raw materials and reagents used can be purchased from the market.

[0063] The present invention will be further illustrated below with reference to the embodiments:

[0064] Comparative Example 1: Synthesis of Rhodioloside by UDP Glycosyltransferase and Sucrose synthase

[0065] The UDP glycosyltransferase AtUGT85A1 from Arabidopsis thaliana and the sucrose synthase AcSUS from Acidithiobacillus caldus were selected. Codon optimization was performed on the host Escherichia coli using the Invitrogen GeneArt online tool GeneOptimizer. The optimized nucleotide sequences were then synthesized into the whole gene and constructed into the pET-22b vector.

[0066] The obtained plasmids pET22b-atUGT85A1 and pET22b-acSUS were transformed into BL21(DE3) competent cells and cultured to 2000 mL LB liquid medium (Amp). Expression was induced at 30℃ under 0.1 mM IPTG conditions. Cells were collected by low-temperature centrifugation, and resuspended in lysis buffer (100 mM K2HPO4∙3H2O, 10 mM KH2PO4, 200 mM NaCl, pH 7.6) at a ratio of 1 g: 4 mL of wet cells. The cells were then sonicated to obtain crude enzyme solution. Protein expression was detected by electrophoresis. Figure 1 AtUGT85A1 and AcSUS are both primarily expressed in the supernatant.

[0067] Weigh 35g of sucrose (204.5mM, reaction volume 500mL), 14g of tyrosol (202.7mM), and 0.04g of UDP (0.2mM) and dissolve them in 400mL of pure water. Adjust the pH to 6.0 and bring the volume to 450mL. After preheating at 30℃, add 30mL of AtUGT85A1 crude enzyme solution (12.5U / mL) and 20mL of AcSUS crude enzyme solution (20.6U / mL) to start the reaction.

[0068] The reaction process involved diluting 100 μL of the solution with 900 μL of methanol, then taking another 100 μL of the solution and diluting it with 900 μL of methanol, resulting in a total dilution of 100 times. The resulting product was then analyzed by high-performance liquid chromatography (HPLC) for tyrosol and rhodioloside. As shown in Table 1, the conversion rate was 86.85% after 48 hours of reaction, but the reaction was time-consuming.

[0069] Table 1

[0070]

[0071] Example 1 Glycoside selectivity engineering of UDP glycosyltransferases

[0072] The AlphaFold-predicted structure of AtUGT85A1 was globally aligned with the reference model of the Stevia-derived glycosyltransferase SrUGT76G1-UDP-RA complex (PDB: 6INI) Figure 2 ), with a backbone RMSD of 1.006 Å indicating a similar structure, especially in the region that binds UDP. LigPlot was used to display residues that interact with UDP Figure 3 ), with Val309 and Trp338 of SrUGT76G1 constraining the uracil ring of UDP and Ser283 and Ser361 interacting with the diphosphate of UDP likely involved in glycosyl transfer. Sequence and structure alignment Figure 4 and Figure 5 ) showed residues Ser307 (Ser283), Ile308 (Thr284), Val333 (Val309), Trp363 (Trp338), and Ser386 (Ser361) of AtUGT85A1 that likely interact with UDP, with the corresponding SrUGT76G1 residues in parentheses.

[0073] To accommodate the bulkier adenine ring, Val333 and Trp363 were chosen as the sites for engineering in AtUGT85A1, while Ser307, Ile308, and Ser386 were fine-tuned to accommodate the shift of the diphosphate. The primers in Table 2 were designed to construct the plasmids of the mutants using the QuickChange Site-Directed Mutagenesis Kit (Agilent).

[0074] Table 2

[0075]

[0076] The resulting plasmids were transformed into BL21 (DE3) competent cells, and the cultures were expanded to 200 mL of LB liquid medium (Amp) and induced to express at 30 °C with 0.1 mM IPTG. The bacterial cells were collected by low-temperature centrifugation, resuspended in 9 mL of lysis buffer per 1 g of wet bacterial cells, and then sonicated to obtain the corresponding crude enzyme solution.

[0077] 1 mL of the crude enzyme solution was taken into a final volume of 10 mL of reaction solution (10 mM tyrosol, 5 mM ADP-glucose, pH 6.0), and the reaction was carried out at 30 °C and 300 rpm overnight. The reaction was terminated by adding 5 mL of acetonitrile, and the supernatant was centrifuged and injected into a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system for detection of salidroside. As shown in Table 3, V333R was the optimal single-point mutant.

[0078] Table 3

[0079]

[0080] In Table 3, * The product concentration of the wild type is defined as: "-" less than 1.5 times; "+" 1.5-2.5 times; "++" 2.5-3.5 times; "+++" more than 3.5 times.

[0081] On the basis of AtUGT85A1-V333R mutant, other mutation sites are superimposed, and the expression, culture, and broken reaction are constructed, cultured, and broken in the same manner as described above. The results are shown in Table 4, and AtUGT85A1-I308Q / V333R / W363S / S386G is the optimal mutation.

[0082] Table 4

[0083]

[0084] In Table 3, * The product concentration of the AtUGT85A1-V333R mutant is defined as: "+" less than 1.5 times; "++" 1.5-2 times; "+++" more than 2 times.

[0085] Example 2 Sugar transferase mutant, sucrose synthase synthesizes salidroside

[0086] AtUGT85A1-I308Q / V333R / W363S / S386G mutant BL21 (DE3) glycerol bacteria are expanded to 2000 mL of LB liquid medium (Amp), and expression is induced at 30°C under the condition of 0.1 mM IPTG. The bacterial cells are collected by low-temperature centrifugation, resuspended with a crushing solution (100 mM K2HPO4·3H2O, 10 mM KH2PO4, 200 mM NaCl, pH 7.6) at a ratio of 1 g of wet bacterial cells to 4 mL, and then ultrasonically broken to obtain a crude enzyme solution.

[0087] Sucrose 70 g (204.5 mM, reaction volume 1000 mL), tyrosol 28 g (202.7 mM) are dissolved in 800 mL of pure water, adjusted to pH 6.0 and constant volume to 900 mL, divided into two portions, respectively, ADP 0.04 g (0.2 mM), UDP 0.04 g (0.2 mM) are dissolved, 30°C preheating, then adding AtUGT85A1-I308Q / V333R / W363S / S386G crude enzyme solution (13.3 U / mL) 30 mL, AcSUS crude enzyme solution (20.6 U / mL) 20 mL, starting reaction.

[0088] Reaction process sampling 100 μL + 900 μL methanol dilution, and then 100 μL + 900 μL methanol is taken from it, a total of 100-fold dilution, and then high performance liquid chromatography (HPLC) is used to detect tyrosol and salidroside. As shown in Table 5, the conversion rate of the ADP group after 46h of reaction was 87.37%, which was comparable to the effect of synthesizing salidroside by AtUGT85A1 enzyme, AcSUS enzyme and UDG-glucose in Comparative Example 1, while the conversion rate of the UDP group after 46h of reaction was only 38.01%, indicating that the modified AtUGT85A1-I308Q / V333R / W363S / S386G was an ADP-glucose selective glycosyltransferase, named "AGT".

[0089] Table 5

[0090]

[0091] Example 3 Thermal stability modification of ADP glycosyltransferase AGT

[0092] The AlphaFold-predicted AtUGT85A1 structure was uploaded to the protein mutation prediction tool Pythia based on structural self-supervised learning, and a global single-point mutation energy heat map was generated. Figure 6 The top 20 single-point mutations with the lowest △△G values are shown in Table 6, and their structures were analyzed.

[0093] Table 6

[0094]

[0095] The M83, V310, Q407, L467, M472 and A21, G151 sites located on the surface and affecting the flexibility of Loop were selected, and the primers in Table 7 were designed to construct the plasmid of the mutant using the QuickChange site-directed mutagenesis kit (Agilent) with the nucleotide sequence of AGT (SEQ ID NO. 6) as the template.

[0096] Table 7

[0097]

[0098] In Table 7, * The product concentration of AGT is defined as follows: "-" is less than 1.5 times; "+" is 1.5-3 times; "++" is 3-6 times; "+++" is more than 6 times.

[0099] The obtained plasmid was transformed into BL21 (DE3) competent cells, and the culture was expanded to 200 mL of LB liquid medium (Amp) and induced to express at 30°C and 0.1 mM IPTG. The bacterial cells were collected by low-temperature centrifugation, resuspended by adding 9 mL of crushing liquid to 1 g of wet bacterial cells, and then ultrasonically crushed to obtain the corresponding crude enzyme solution.

[0100] Take 1 mL of crude enzyme solution to a final volume of 10 mL reaction solution (10 mM tyrosol, 5 mM ADP-glucose, pH 6.0), 60°C 300 rpm overnight reaction, add 5 mL acetonitrile to terminate the reaction, centrifuge the supernatant into liquid chromatography-tandem mass spectrometry (LC-MS / MS) for detection of salidroside. The results are shown in Table 7, Q407P is the optimal single-point mutation.

[0101] On the basis of AGT-Q407P mutant, superimposed other mutation sites, constructed, cultured, broken down and reacted in the same way as described above. The results are shown in Table 8, AGT-A21I / M83N / Q407P is the optimal mutation.

[0102] Table 8

[0103]

[0104] In Table 8, * Compared with the product concentration of AGT-Q407P mutant: “-” less than 1 times; “+” 1-2 times; “++” 2-3 times; “+++” more than 3 times.

[0105] Example 4 ADP glycosyltransferase, sucrose synthase synthesis salidroside

[0106] AGT-A21I / M83N / Q407P mutant BL21 (DE3) glycerol bacteria were expanded to 2000 mL of LB liquid medium (Amp), and induced to express at 30°C and 0.1 mM IPTG. The bacterial cells were collected by low-temperature centrifugation, resuspended with 4 mL of breaking solution (100 mM K2HPO4·3H2O, 10 mM KH2PO4, 200 mM NaCl, pH 7.6) according to 1 g of wet bacterial cells, and then ultrasonically broken to obtain crude enzyme solution.

[0107] Weigh sucrose 35 g (204.5 mM, reaction volume 500 mL), tyrosol 14 g (202.7 mM), ADP 0.04 g (0.2 mM) in 400 mL pure water, adjust pH 6.0 and constant volume to 480 mL, 60°C preheating, then add AGT-A21I / M83N / Q407P crude enzyme solution (80.3 U / mL) 10 mL, AcSUS crude enzyme solution (83.8 U / mL) 10 mL, start reaction.

[0108] The reaction process was sampled 100 μL + 900 μL methanol dilution, and then 100 μL + 900 μL methanol was taken from it, a total of 100-fold dilution, and then high performance liquid chromatography (HPLC) was used to detect tyrosol and salidroside. As shown in Table 9, the conversion rate was 88.72% after 22 hours of reaction, compared with the AGT enzyme and AcSUS enzyme reaction in Example 2, the reaction time was greatly shortened, and the enzyme amount was lower.

[0109] Table 9

[0110]

[0111] Example 5 ADP glycosyltransferase, sucrose synthase synthesis of high concentration of salidroside

[0112] Sucrose 70 g (409.0 mM, reaction volume 500 mL), tyrosol 28 g (405.3 mM), ADP 0.04 g (0.2 mM) were dissolved in 400 mL pure water, the pH was adjusted to 6.0 and the volume was adjusted to 480 mL, and then 60°C preheating AGT-A21I / M83N / Q407P crude enzyme solution (80.3 U / mL) 15 mL, AcSUS crude enzyme solution (83.8 U / mL) 10 mL were added, and the reaction was started.

[0113] The reaction process was sampled 100 μL + 900 μL methanol dilution, and then 100 μL + 900 μL methanol was taken from it, a total of 100-fold dilution, and then high performance liquid chromatography (HPLC) was used to detect tyrosol and salidroside. As shown in Table 9, the conversion rate was 88.72% after 22 hours of reaction, compared with the AGT enzyme and AcSUS enzyme reaction in Example 2, the reaction time was greatly shortened, and the enzyme amount was lower.

[0114] The reaction solution was removed from the protein by ceramic membrane, and then purified by C18 preparation liquid phase with mobile phase 30% methanol. The target peak was collected, rotary evaporated and vacuum dried at constant temperature to obtain the crude product. The crude product was dissolved in 5 times the volume of methanol / ethyl formate (1:1, v / v) at 50°C, then cooled to 4°C to crystallize, and vacuum dried at constant temperature to obtain the pure product. The purity was 99.98% (HPLC) and the mass spectrum (MS) was characterized as salidroside. Figure 7 Figure 8

[0115] Table 10

[0116]

[0117] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should be considered as the protection scope of the present application.​​

Claims

1. A mutant of an enzyme, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

5.

2. A mutant of a glycosyltransferase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

7.

3. An enzyme composition, characterized in that, include: The mutant of the enzyme as described in claim 1 and / or the mutant of the glycosyltransferase as described in claim 2, and sucrose synthase.

4. Nucleic acid molecules encoding mutants of the enzyme as described in claim 1, mutants of the glycosyltransferase as described in claim 2, and / or enzyme compositions as described in claim 3.

5. The nucleic acid molecule as described in claim 4, characterized in that, include: The nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme is shown in SEQ ID NO:6; and / or The nucleotide sequence of the nucleic acid molecule encoding the mutant glycosyltransferase is shown in SEQ ID NO:8; and / or The nucleotide sequence of the nucleic acid molecule encoding the sucrose synthase is shown in SEQ ID NO:

4.

6. A recombinant vector, characterized in that, include: The nucleic acid molecule as described in claim 4 or 5, and the acceptable gene element.

7. The host, characterized in that, Transformation and / or transfection with the recombinant vector as described in claim 6.

8. The product, characterized in that, include: The mutant of the enzyme as claimed in claim 1, the mutant of the glycosyltransferase as claimed in claim 2, the enzyme composition as claimed in claim 3, the nucleic acid molecule as claimed in claim 4 or 5, the recombinant vector as claimed in claim 6, and / or the host as claimed in claim 7, as well as acceptable adjuvants or excipients.

9. The use of the mutant of the enzyme as described in claim 1, the mutant of the glycosyltransferase as described in claim 2, the enzyme composition as described in claim 3, the nucleic acid molecule as described in claim 4 or 5, the recombinant vector as described in claim 6, the host as described in claim 7, and / or the product as described in claim 8 in the preparation of rhodioloside.

10. A method for preparing rhodioloside, characterized in that, Rhodioloside was obtained by converting the raw material through any of the following methods; (a) A mutant of the enzyme as described in claim 1; or (b) A mutant of the glycosyltransferase as described in claim 2; or (c) The enzyme composition as described in claim 3; or (d) The nucleic acid molecule as described in claim 4 or 5; or (e) the recombinant vector as described in claim 6; or (f) The host as described in claim 7; or (g) The product as described in claim 8; The raw materials include: sucrose, tyrosol, and ADP.

11. The preparation method according to claim 10, characterized in that, The conversion temperature is 30~60℃.

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