Method for improving transglycosylation activity of cyclodextrin glycosyl transferase on polyphenol compound

Abstract

The invention discloses a method for improving the transglycosylation activity of cyclodextrin glycosyl transferase to polyphenol compounds, and belongs to gene engineering or enzyme engineering. According to the method, based on two principles of hydrophobicity enhancement and steric hindrance reduction, a key residue influencing substrate specificity at a receptor subsite is subjected to rational mutation, a cyclodextrin glycosyl transferase mutant is constructed, and the binding effect of polyphenol at the receptor subsite is improved, so that the glycosylation activity of the cyclodextrin glycosyl transferase on the polyphenol is improved. According to the method disclosed by the invention, the efficiency of synthesizing the alpha-glucosyl polyphenol by the cyclodextrin glycosyl transferase is improved through site-directed mutagenesis, the synthesis of the alpha-glucosyl polyphenol with higher yield under a lower substrate concentration is facilitated, and the commercial application of the alpha-glucosyl polyphenol product is expanded.

Description

Technical Field

[0001] The present invention relates to a method for improving the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenolic compounds, and belongs to the field of genetic engineering or enzyme engineering. Background Art

[0002] Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) belongs to glycoside hydrolase family 13 (GH13), and has four activities: cyclization, disproportionation, hydrolysis and coupling. It has been widely concerned because of its remarkable transglycosylation activity. In addition, CGTase has been reported to have good substrate promiscuity and can use a series of compounds such as small molecule sugars (fructose, maltose), polyphenols (daidzein, quercetin, resveratrol), terpenoids (stevioside, ginsenoside) and vitamins (ascorbic acid) as acceptors to catalyze glycosylation reactions. In summary, the two characteristics of CGTase endow it with the potential for large-scale production of glycosylated products.

[0003] In recent decades, plant-derived natural products have received extensive attention, and polyphenols are one of the most widely studied bioactive compounds in this category. Epidemiological studies have shown that long-term intake of polyphenols has multiple benefits for preventing diabetes, obesity, aging and neurodegenerative diseases. However, the low solubility and poor stability of polyphenols pose challenges to the in vivo therapeutic delivery of such compounds. α-Glucosyl polyphenol products are glycosylated modifications based on polyphenols, which can significantly improve solubility and stability. Therefore, various α-glucosyl polyphenol products have been widely used in the fields of food and cosmetics. For example, α-glucosyl hesperidin has been approved as a food additive. Based on this, cyclodextrin glycosyltransferase has become an effective tool for the production of polyphenol glycosylated products and has been used in commercial production. However, in the actual production process, there is a problem of low glycosylation efficiency of natural polyphenol substrates. Even when using a higher substrate concentration, only a small increase in the yield can be achieved, resulting in an increase in the production cost of α-glucosyl polyphenol products.

[0004] The team has previously identified two major factors restricting the glycosylation efficiency of natural substrates, including low substrate accessibility due to the low solubility of natural substrates and poor substrate adaptability due to poor reaction specificity of the enzyme. To address the problem of low substrate solubility, cyclodextrin complexation was previously used to improve the solubility of polyphenols. At the same time, cyclodextrin can serve as a donor for CGTase, effectively improving the glycosylation yield of polyphenols without introducing additional solubilizing media. To address the problem of poor substrate specificity of CGTase, engineering modification of the acceptor subsite is an effective means to improve the catalytic performance of the enzyme. For example, the combined mutant K228R / M230L of the Bacillus stearothermophilus NO2CGTase +2 acceptor subsite increased the glycosylation efficiency of ascorbic acid by 2.69 times. The present invention uses molecular docking to analyze the binding mode of polyphenol substrates at the acceptor subsite, obtaining mutants with significantly improved polyphenol glycosylation activity and achieving a substantial increase in the yield of α-glucosyl polyphenols. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a method for improving the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenol substrates. Based on the analysis of the differences in the action modes of polyphenol and oligosaccharide substrates at the acceptor subsite of cyclodextrin glycosyltransferase, the key amino acids affecting the polyphenol glycosylation activity at the +1 and +3 subsites are determined, and site-directed mutagenesis is carried out according to the two principles of enhanced hydrophobicity and reduced steric hindrance to create a catalytic environment more suitable for the stable binding of polyphenol substrates. The transglycosylation activity of the mutant towards polyphenol substrates is significantly improved, thereby increasing the substrate utilization rate and the yield of α-glucosyl polyphenol products.

[0006] The first technical solution provided by the present invention is a cyclodextrin glycosyltransferase mutant with improved transglycosylation activity towards polyphenolic compounds. By rationally mutating the key amino acids affecting the reaction specificity of polyphenolic compounds in cyclodextrin glycosyltransferase, a cyclodextrin glycosyltransferase mutant is prepared to improve the transglycosylation activity towards polyphenol substrates, thereby increasing the yield of α-glucosyl polyphenol products. The key amino acids are tyrosine Y at the +1 subsite residue and glutamate E at the +3 subsite residue.

[0007] The mutant is based on cyclodextrin glycosyltransferase as the parent, and on the basis of the amino acid sequence of the parent, the tyrosine Y at the sixth position in the IYXNL Y DLAX sequence is mutated to phenylalanine F to obtain the amino acid sequence; or the glutamate E at the tenth position in the FGEWFLXXX E sequence in the amino acid sequence of the parent is mutated to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; where X is not conserved in cyclodextrin glycosyltransferases from different sources.

[0008] In some embodiments, the parental amino acid sequence is an amino acid sequence derived from any one of cyclodextrin glycosyltransferases from Alkalophilic Bacillus sp. 1011, Bacillus ohbensis, Bacillus cereus, Niallia circulans, Brevibacillus brevis, Cytobacillus firmus, Paenibacillus macerans.

[0009] In some embodiments, the parental amino acid sequence is any one of the following with the accession numbers on NCBI: AAA22308.1 (Alkalophilic Bacillus sp. 1011), P27036.2 (Bacillus ohbensis), AGT45478.1 (Bacillus cereus), AAG31622.1 (Niallia circulans), O30565.1 (Brevibacillus brevis), AGR66230.1 (Cytobacillus firmus), P31835.1 (Paenibacillus macerans).

[0010] In some embodiments, the amino acid sequence of the mutant is any one of the following amino acid sequences selected from:

[0011] (1) The amino acid sequence obtained by mutating the tyrosine (Y) at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Alkalophilic Bacillus sp. 1011 to phenylalanine (F) and / or mutating the glutamate (E) at position 264 to alanine (A), valine (V), glycine (G), aspartic acid (D), threonine (T) or cysteine (C);

[0012] (2) The amino acid sequence obtained by mutating the tyrosine (Y) at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Bacillus ohbensis to phenylalanine (F) and / or mutating the glutamate (E) at position 261 to alanine (A), valine (V), glycine (G), aspartic acid (D), threonine (T) or cysteine (C);

[0013] (3) The amino acid sequence obtained by mutating the tyrosine (Y) at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Bacillus cereus to phenylalanine (F) and / or mutating the glutamate (E) at position 261 to alanine (A), valine (V), glycine (G), aspartic acid (D), threonine (T) or cysteine (C);

[0014] (4) An amino acid sequence obtained by mutating the tyrosine Y at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Niallia circulans to phenylalanine F and / or mutating the glutamate E at position 264 to alanine A, valine V, glycine G, aspartic acid D, threonine T, or cysteine C;

[0015] (5) An amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Brevibacillus brevis to phenylalanine F and / or mutating the glutamate E at position 260 to alanine A, valine V, glycine G, aspartic acid D, threonine T, or cysteine C;

[0016] (6) An amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Cytobacillus firmus to phenylalanine F and / or mutating the glutamate E at position 261 to alanine A, valine V, glycine G, aspartic acid D, threonine T, or cysteine C;

[0017] (7) An amino acid sequence obtained by mutating the tyrosine Y at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Paenibacillus macerans to phenylalanine F and / or mutating the glutamate E at position 265 to alanine A, valine V, glycine G, aspartic acid D, threonine T, or cysteine C.

[0018] In certain embodiments, the parental amino acid sequence is the cyclodextrin glycosyltransferase sequence derived from Alkalophilic Bacillus sp. 1011, and the amino acid sequence is as shown in SEQ ID NO.2.

[0019] In certain embodiments, the mutant is any one of the following mutations made to the parental amino acid sequence shown in SEQ ID NO.2:

[0020] (a) Mutating tyrosine Y at position 195 to phenylalanine F;

[0021] (b) Mutating glutamate E at position 264 to alanine A;

[0022] (c) Mutating glutamate E at position 264 to valine V;

[0023] (d) Mutating glutamate E at position 264 to glycine G;

[0024] (e) Mutate the glutamic acid E at position 264 to aspartic acid D;

[0025] (f) Mutate the glutamic acid E at position 264 to threonine T;

[0026] (g) Mutate the glutamic acid E at position 264 to cysteine C;

[0027] (h) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to alanine A;

[0028] (i) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to valine V.

[0029] The second technical solution provided by the present invention is a gene encoding the cyclodextrin glycosyltransferase mutant described in the first technical solution.

[0030] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0031] In some embodiments, the recombinant vector uses a pET series plasmid as the expression vector.

[0032] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0033] In some embodiments, the recombinant cell uses bacteria or fungi as the expression host.

[0034] The fifth technical solution provided by the present invention is a method for improving the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenolic compounds. The method rationally mutates the key amino acids in cyclodextrin glycosyltransferase that affect the reaction specificity of polyphenolic substrates, improves the transglycosylation activity towards polyphenolic substrates, and thus increases the yield of α-glucosyl polyphenol products.

[0035] In some embodiments, the method is to mutate the parental cyclodextrin glycosyltransferase, and the mutations include: on the basis of the amino acid sequence of the parental cyclodextrin glycosyltransferase, mutate the Y (tyrosine) at the sixth position in the IYXNL Y DLAX sequence to phenylalanine; or mutate the E (glutamic acid) at the last position in the sequence containing FGEWFLXXX E in the parental amino acid sequence to alanine, valine, glycine, aspartic acid, threonine or cysteine.

[0036] In some embodiments, the method is to perform any one of the following mutations on the cyclodextrin glycosyltransferase parent with the amino acid sequence shown in SEQ ID NO. 2:

[0037] (a) Mutate tyrosine Y at position 195 to phenylalanine F;

[0038] (b) Mutate glutamate E at position 264 to alanine A;

[0039] (c) Mutate glutamate E at position 264 to valine V;

[0040] (d) Mutate glutamate E at position 264 to glycine G;

[0041] (e) Mutate glutamate E at position 264 to aspartic acid D;

[0042] (f) Mutate glutamate E at position 264 to threonine T;

[0043] (g) Mutate glutamate E at position 264 to cysteine C;

[0044] (h) Mutate tyrosine Y at position 195 to phenylalanine F, and mutate glutamate E at position 264 to alanine A;

[0045] (i) Mutate tyrosine Y at position 195 to phenylalanine F, and mutate glutamate E at position 264 to valine V.

[0046] The sixth technical solution provided by the present invention is a method for synthesizing α-glucosyl polyphenol. The method is to use the cyclodextrin glycosyltransferase mutant described in the first technical solution as a catalyst, and use cyclodextrin and polyphenol as a donor and a receptor respectively to catalyze a glycosylation reaction. After the reaction is completed, the reaction mixture is treated with glucoamylase derived from Aspergillus niger to obtain α-glucosyl polyphenol.

[0047] The polyphenols include daidzein and rutin.

[0048] The seventh technical solution provided by the present invention is the application of the cyclodextrin glycosyltransferase mutant or the gene vector or the recombinant cell or the method for improving the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenolic compounds in the preparation of α-glucosyl polyphenol products.

[0049] The technical effects of the present invention are as follows:

[0050] By analyzing the interaction modes between the acceptor subsites of cyclodextrin glycosyltransferase and polyphenols or oligosaccharides, the key amino acids with different interaction forces with the two substrates were determined. Based on this, two strategies of enhancing hydrophobicity and reducing steric hindrance were adopted for site-directed mutagenesis to obtain cyclodextrin glycosyltransferase mutants, which is beneficial to the stable binding of polyphenol substrates. The present invention significantly improves the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenol substrates and the substrate utilization rate, thereby increasing the yield of α-glucosyl polyphenol products, reducing production costs, and contributing to the expansion of the commercial application of α-glucosyl polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the structure of alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase with polyphenol (left, taking daidzein as an example) and oligosaccharide (right, taking maltotriose as an example) (residues D229, E257, and D328 are the catalytic triad).

[0052] Figure 2 Characterization of the relative transglycosylation activity of wild-type and mutant forms of alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase towards daidzein.

[0053] Figure 3 Comparison of the relative yields of α-glucosyl daidzein of wild-type and mutant forms of cyclodextrin glycosyltransferase. DETAILED DESCRIPTION OF THE INVENTION

[0054] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0055] Testing method:

[0056] 1. Construction of glycosylation system: Weigh a certain molar concentration of β-cyclodextrin / γ-cyclodextrin and a certain molar concentration of daidzein / rutin into a 20 mM, pH 6.0 Na2HPO4-NaH2PO4 buffer solution, pre-mix them in the dark at 60 °C for 12 h, add wild-type and mutant enzyme solutions in a ratio of 0.1 mL (4 U) per 1 mL of the total volume of the system, react at 60 °C and 500 rpm for 12 h, inactivate the enzyme in a boiling water bath for 15 min, and filter using a 0.22 μm water membrane. Use glucoamylase to digest for 4 h to convert all the mixed glycosylation products into α-glucosyl daidzein / rutin.

[0057] 2. Detection of transglycosylation activity:

[0058] (1) α-Glucosyl daidzein: The HPLC determination conditions are as follows: Use an XBridge C18 column, 5 μm, 4.6×250 mm; column temperature: 30 °C; detector: UV detector; injection volume: 10 μL; flow rate: 0.8 mL / min. Mobile phase A: pure methanol, mobile phase B: ultrapure water containing 0.1% formic acid. The gradient elution program of mobile phase A and mobile phase B is as follows: 0 - 5 min, 5% A; 5 - 20 min, 5 - 20% A; 20 - 30 min, 50 - 90% A; and 30 - 32 min, 90 - 20% A.

[0059] (2) α-Glucosyl rutin: The HPLC determination conditions are as follows: Use an XBridge C18 column, 5 μm, 4.6×250 mm; column temperature: 30 °C; detector: UV detector; injection volume: 10 μL; flow rate: 0.8 mL / min. Mobile phase A: pure acetonitrile, mobile phase B: ultrapure water containing 0.1% formic acid. The gradient elution program of mobile phase A and mobile phase B is as follows: 0 - 12 min, 13 - 20% A; 12 - 15 min, 20 - 50% A; 15 - 16 min, 50 - 80% A; 16 - 18 min, 80% A; and 18 - 24 min, 80 - 13% A.

[0060] Raw materials used in the examples:

[0061] 1. The plasmid pETM-11 was provided by Suzhou Genewiz Biotechnology Co., Ltd. and the target gene was introduced.

[0062] 2. Escherichia coli BL21(DE3) and Escherichia coli DH5α were sourced from Nanjing Novoprotein Scientific Co., Ltd.

[0063] 3. LB medium, including peptone and yeast extract, was all sourced from Oxoid (UK) Ltd.

[0064] Example 1

[0065] A rational mutation of key amino acids of cyclodextrin glycosyltransferase and an analysis of the effect of the mutation on the improvement of the transglycosylation activity of polyphenols, including the following steps:

[0066] 1. Selection of key amino acid residues at the acceptor subsite

[0067] Compare the roles of residues within the binding range of polyphenols (taking daidzein as an example) and oligosaccharides (taking maltotriose as an example) at the acceptor subsite of the cyclodextrin glycosyltransferase of Alkalophilic Bacillus sp. 1011 within the range ( Figure 1)。Both daidzein and maltotriose have similar hydrogen bond interactions with residues K232, H233, and F259, but there are differences in their interactions with residues E264 and Y195. Residue Y195 is located near the +1 receptor subsite and has a hydrogen bond interaction with maltotriose, but this interaction is not found in daidzein. Residue E264 is located in part of the +3 subsite, creating a large steric hindrance to the planar and rigid daidzein substrate and thus hindering its binding. For maltotriose with a relatively flexible chain structure, its conformation can be freely adjusted to adapt to the steric hindrance and is thus less affected, and there is a hydrogen bond interaction that is beneficial for positioning. In summary, tyrosine at position 195 and glutamate at position 264 in Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase synergistically regulate substrate specificity, and these two sites were selected for rational mutagenesis.

[0068] 2. Design mutant primers

[0069] Mutate tyrosine at position 195 of the above-selected Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase to phenylalanine to maintain a similar conformation while enhancing hydrophobicity; mutate glutamate at position 264 to alanine, valine, glycine, aspartic acid, threonine, or cysteine to reduce steric hindrance. The primer design is shown in Table 1 below:

[0070] Table 1 Primer sequences of mutants

[0071]

[0072] Note: The underlined bases are the mutated bases

[0073] The combination of the above two sets of primers is the primer for constructing the combinatorial mutants Y195F / E264A and Y195F / E264V.

[0074] 3. Construction of cyclodextrin glycosyltransferase

[0075] Artificially synthesize the gene fragment of Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase with the nucleotide sequence shown in SEQ ID NO.1, construct the recombinant plasmid pETM-11 / CGTase using the plasmid pETM-11 with 5’XbaI and 3’XhoI restriction enzyme sites as the expression vector, and express it using the E. coli BL21(DE3) expression strain.

[0076] 4. Construction of cyclodextrin glycosyltransferase mutants

[0077] Using pETM-11 / CGTase as a template, the target amino acids were mutated by whole plasmid PCR. The PCR program was set as follows: pre-denaturation at 98°C for 3 min, followed by 30 cycles (denaturation at 98°C: 20 s; annealing at 55°C: 30 s; extension at 68°C: 9 min), and continued extension at 68°C for 10 min.

[0078] The PCR product after the reaction was digested with DpnⅠ enzyme to remove the template, and the recombinant plasmid was introduced into Escherichia coli DH5α competent cells by heat shock method for cloning. The competent cells were cultured overnight on LB solid medium (containing 50 μg / mL kanamycin), single colonies were picked and cultured in LB liquid medium (containing 50 μg / mL kanamycin), plasmids were extracted, and the plasmids with correct sequencing were transformed into the expression host Escherichia coli BL21(DE3) competent cells.

[0079] 5. Expression and purification of wild-type and mutant cyclodextrin glycosyltransferase

[0080] (1) The recombinant Escherichia coli containing wild-type and mutant genes obtained were respectively inoculated into LB medium containing kanamycin (50 μg / mL), cultured at 37°C and 200 rpm for 8 - 12 h to obtain seed solutions. The obtained seed solutions were added to a larger volume of LB medium containing kanamycin (100 μg / mL) for expansion. When the OD 600 nm reached 0.4 - 0.6, after ice-bathing for 30 min, isopropyl-β-thiogalactoside (IPTG, 0.4 mM) was added, and enzyme production was induced at 20°C and 160 rpm for 20 h to respectively prepare fermentation broths;

[0081] (2) The obtained fermentation broths were centrifuged at 4°C and 8000×g for 30 min to collect cell precipitates. The cell precipitates were resuspended according to the ratio of 1 g of cells dissolved in 10 mL of 25 mM Tris-HCl (250 mM NaCl, pH 7.5), and then ultrasonically disrupted in an ice bath for 20 min (30% power, ultrasonic for 2 s, pause for 3 s). The disrupted cell solution was centrifuged at 4°C and 8000×g for 30 min, and the obtained supernatant was the crude enzyme solution;

[0082] (3) The crude enzyme solution obtained in step (2) was purified by nickel affinity chromatography and eluted successively with 20 mM Tris-HCl (250 mM NaCl, pH 7.5) and 20 mM Tris-HCl (250 mM NaCl, pH 7.5) containing 50 mM imidazole. The eluate containing cyclodextrin glycosyltransferase activity was collected, and purified enzymes of cyclodextrin glycosyltransferase wild type and single-point mutants Y195F, E264A, E264V, E264G, E264D, E264T, E264C, as well as combined mutants Y195F / E264A and Y195F / E264V were obtained respectively.

[0083] 6. Determination of the improvement effect of cyclodextrin glycosyltransferase mutants on the transglycosylation activity towards polyphenolic compounds

[0084] Using the purified wild type of Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase and its mutants as catalysts respectively, under the condition that the concentrations of β-cyclodextrin and daidzein were 8:2 mM, α-glucosyl daidzein was obtained according to the reaction steps in the test method, and the transglycosylation activities of the wild type and its mutants towards daidzein were determined. One activity unit (U) was defined as the amount of enzyme required to catalyze the formation of 1 μmol of α-glucosyl daidzein per minute.

[0085] The relative transglycosylation activities of daidzein of the wild type of Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase and its mutants are as Figure 2 shown. Defining the transglycosylation activity of the wild type enzyme as 100%, compared with the wild type enzyme, the cyclodextrin glycosyltransferase mutants achieved a significant effect of improving the transglycosylation activity towards polyphenolic substrates. The transglycosylation activities of single-point mutants Y195F, E264A, E264V, E264G, E264D, E264T, E264C and combined mutants Y195F / E264A, Y195F / E264V increased by 59.2%, 43.4%, 49.8%, 38.3%, 41.3%, 38.1%, 26.5%, 51.5% and 74.1% respectively.

[0086] Example 2:

[0087] Analysis of the conservation of key amino acids of cyclodextrin glycosyltransferase and the effect of improving the yield of α-glucosyl daidzein, including the following steps:

[0088] 1. Comparing the sequences of 29 cyclodextrin glycosyltransferases from different sources, it was found that IYXNL in the sequence YThe amino acids in the non-X part of DLAX are conserved, and the Y (tyrosine) at the sixth position corresponds to the tyrosine at the 195th position in the cyclodextrin glycosyltransferase sequence from Alkalophilic Bacillus sp. 1011.

[0089] The sequences of the above 28 cyclodextrin glycosyltransferases from different sources have the following accession numbers on NCBI: AAA22308.1 (Alkalophilic Bacillus sp.1011), P05618.1 (Bacillus sp.1011), P27036.2 (Bacillus ohbensis), AGT45478.1 (Bacillus cereus), AAG31622.1 (Nialliacirculans), P43379.1 (Niallia circulans), CAA48401.1 (Niallia circulans), P30920.1 (Niallia circulans), O30565.1 (Brevibacillus brevis), AGR66230.1 (Cytobacillusfirmus), P31835.1 (Paenibacillus macerans), AAC04359.1 (Paenibacillus macerans), AGG09664.1 (Paenibacillus sp.xw-6-66), ACO70966.1 (Paenibacillus sp.ZY-8), ACB71089.1 (Paenibacillus sp.JB-13), CAO05752.1 (Paenibacillus pabuli), ACA01964.1 (Paenibacillus graminis), AAR32682.1 (Bacillus sp.I-5), P09121.2 (Bacillus sp.38-2), P30921.1 (Bacillus sp.17-1), AND01239.1 (Bacillus sp.SD5), P31746.1 (Bacillus sp.1-1), P31747.1 (Bacillus sp.6.3.3), P14014.1 (Bacilluslicheniformis), P17692.1 (Bacillus sp.B1018), BAE87038.1 (Bacillus sp.G-825-6), USU43408.1 (Evansella caseinilytica), BAB91217.2 (Evansella clarkii).

[0090] 2. The sequences of 20 cyclodextrin glycosyltransferases from different sources were compared, and it was found that in the sequences, FGEWFLXXX EThe amino acids in the non-X part are conserved. The E (glutamic acid) at the tenth position corresponds to the glutamic acid at the 264th position in the cyclodextrin glycosyltransferase sequence from Alkalophilic Bacillus sp. 1011.

[0091] The sequences of the above 20 different sources of cyclodextrin glycosyltransferase have the following accession numbers on NCBI: AAA22308.1 (Alkalophilic Bacillus sp. 1011), P05618.1 (Bacillus sp. 1011), P27036.2 (Bacillus ohbensis), AGT45478.1 (Bacillus cereus), AAG31622.1 (Nialliacirculans), P43379.1 (Niallia circulans), O30565.1 (Brevibacillus brevis), AGR66230.1 (Cytobacillus firmus), P31835.1 (Paenibacillus macerans), AGG09664.1 (Paenibacillus sp. xw-6-66), ACB71089.1 (Paenibacillus sp. JB-13), AAR32682.1 (Bacillus sp. I-5), P09121.2 (Bacillus sp. 38-2), P30921.1 (Bacillus sp. 17-1), AND01239.1 (Bacillus sp. SD5), P31746.1 (Bacillus sp. 1-1), P17692.1 (Bacillussp. B1018), P31797.1 (Geobacillus stearothermophilus), AAB00845.1 (Thermoanaerobacterium thermosulfurigenes), CAH61550.1 (Anaerobrancagottschalkii).

[0092] 3. The following lists the specific amino acid fragment IYXNL corresponding to the sequences of 7 different sources of cyclodextrin glycosyltransferase Y DLAX and FGEWFLXXX E 。

[0093] Table 2 Display of specific amino acid fragments in cyclodextrin glycosyltransferase from different sources

[0094]

[0095]

[0096] By comparing specific amino acid fragments in cyclodextrin glycosyltransferase sequences from different sources, it can be found that the sites corresponding to tyrosine at position 195 and glutamate at position 264 of the cyclodextrin glycosyltransferase from Alkalophilic Bacillus sp. 1011 used in the present invention are conserved in cyclodextrin glycosyltransferases from a variety of different sources, indicating that the improvement effect of the selected site mutations on the transglycosylation activity of polyphenolic compounds is universal among different cyclodextrin glycosyltransferases.

[0097] 4. Determination of the effect of cyclodextrin glycosyltransferase mutants on improving the yield of α-glucosyl daidzein:

[0098] The wild-type (WT) of cyclodextrin glycosyltransferase from Alkalophilic Bacillus sp. 1011 and its mutants were used for effect verification. Under the condition of donor:receptor = 6:1, the addition concentrations of β-cyclodextrin and daidzein were 9:1.5 mM; under the condition of donor:receptor = 10:1, the addition concentrations of β-cyclodextrin and daidzein were 15:1.5 mM. α-Glucosyl daidzein was obtained according to the reaction steps in the test method.

[0099] The content of α-glucosyl daidzein was determined by HPLC, and the ability of the wild-type of cyclodextrin glycosyltransferase and its mutants to produce α-glucosyl daidzein at different substrate concentrations was calculated. The conversion rate (%) of α-glucosyl daidzein = (the number of moles of α-glucosyl daidzein generated / the initial number of moles of daidzein) × 100%.

[0100] The yields of α-glucosyl daidzein catalyzed by different mutants were measured respectively, and the results are shown in Table 3.

[0101] Table 3 Yields of α-glucosyl daidzein of the wild-type of cyclodextrin glycosyltransferase and its mutants

[0102]

[0103] According to Table 3, the relative ratio of the yields of α-glucosyl daidzein catalyzed by the wild-type and its mutants is as Figure 3As shown, mutants Y195F, E264A, E264V, E264G, E264D, E264T, E264C, Y195F / E264A, and Y195F / E264V can achieve a similar or higher yield of α-glucosyl daidzein compared to the wild type at a higher substrate concentration at a lower substrate concentration. In particular, the combined mutants Y195F / E264A (33.19%) and Y195F / E264V (33.56%) had a 5.01% and 5.38% increase in yield compared to the wild type (28.18%), respectively. In addition, at a higher substrate concentration, the yields of the mutants were significantly higher than that of the wild type. The combined mutants Y195F / E264A and Y195F / E264V were 28.21% and 31.94% higher than the wild type, respectively, reaching 2.0 and 2.1 times that of the wild type. Similarly, the effect of increasing the yield of α-glucosyl daidzein also exists in cyclodextrin glycosyltransferases conserved at two sites.

[0104] Example 3:

[0105] An analysis of the effect of cyclodextrin glycosyltransferase mutants on improving the yield of α-glucosyl rutin includes the following steps:

[0106] Using the wild type of Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase and its mutants as catalysts, the molar concentrations of γ-cyclodextrin and rutin in the system were 20:2.5 mM, and α-glucosyl rutin was obtained according to the reaction steps in the test method.

[0107] The content of α-glucosyl rutin was determined by HPLC, and the ability of the wild type of cyclodextrin glycosyltransferase and its mutants to produce α-glucosyl rutin was calculated. The conversion rate of α-glucosyl rutin (%) = (moles of α-glucosyl rutin produced / initial moles of rutin) × 100%.

[0108] The yields of α-glucosyl rutin produced by the wild type of Alkalophilic Bacillus sp. 1011 cyclodextrin glycosyltransferase and the combined mutants Y195F / E264A and Y195F / E264V are shown in Table 4.

[0109] Table 4 Yields of α-glucosyl rutin of the wild type of cyclodextrin glycosyltransferase and combined mutants

[0110]

[0111] According to Table 4, the yields of α-glucosyl rutin catalyzed by cyclodextrin glycosyltransferase mutants Y195F / E264A and Y195F / E264V are both increased by about 15% compared with the wild type. A higher glycosylation yield is achieved within a shorter catalytic time (30 min), significantly improving the reaction efficiency. This indicates that the improvement effect of the transglycosylation activity of cyclodextrin glycosyltransferase mutants is applicable to a wide range of polyphenolic compounds represented by daidzein and rutin. The cyclodextrin glycosyltransferase mutants designed in the present invention have universality for improving the glycosylation yield of polyphenolic substrates.

[0112] Comparative Example 1

[0113] Replace the cyclodextrin glycosyltransferase mutant in Example 2 with mutant E264Y (negative control, increased steric hindrance). The concentrations of β-cyclodextrin and daidzein are 15 mM and 1.5 mM, respectively. Other conditions or parameters are the same as those in Example 3. The conversion rate of α-glucosyl daidzein obtained by glucose amylase digestion is 22.42%.

[0114] Comparative Example 2

[0115] Replace the cyclodextrin glycosyltransferase mutant in Example 2 with mutant E264K (negative control, increased steric hindrance). The concentrations of β-cyclodextrin and daidzein are 15 mM and 1.5 mM, respectively. Other conditions or parameters are the same as those in Example 3. The conversion rate of α-glucosyl daidzein obtained by glucose amylase digestion is 27.72%.

[0116] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A cyclodextrin glycosyltransferase mutant, characterized in that, The amino acid sequence of the mutant is any one of the following amino acid sequences selected from: (1) The amino acid sequence obtained by mutating the tyrosine Y at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Alkalophilic Bacillus sp.1011 to phenylalanine F and / or mutating the glutamate E at position 264 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (2) The amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Bacillus ohbensis to phenylalanine F and / or mutating the glutamate E at position 261 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (3) The amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Bacillus cereus to phenylalanine F and / or mutating the glutamate E at position 261 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (4) The amino acid sequence obtained by mutating the tyrosine Y at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Niallia circulans to phenylalanine F and / or mutating the glutamate E at position 264 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (5) The amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Brevibacillus brevis to phenylalanine F and / or mutating the glutamate E at position 260 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (6) The amino acid sequence obtained by mutating the tyrosine Y at position 192 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Cytobacillus firmus to phenylalanine F and / or mutating the glutamate E at position 261 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C; (7) The amino acid sequence obtained by mutating the tyrosine Y at position 195 of the amino acid sequence of cyclodextrin glycosyltransferase derived from Paenibacillus macerans to phenylalanine F and / or mutating the glutamate E at position 265 to alanine A, valine V, glycine G, aspartic acid D, threonine T or cysteine C.

2. The mutant according to claim 1, wherein The mutant is any one of the following mutations made to the parent with the amino acid sequence shown in SEQ ID NO.2: (a) Mutating tyrosine Y at position 195 to phenylalanine F; (b) Mutating glutamate E at position 264 to alanine A; (c) Mutating glutamate E at position 264 to valine V; (d) Mutate the glutamic acid E at position 264 to glycine G; (e) Mutate the glutamic acid E at position 264 to aspartic acid D; (f) Mutate the glutamic acid E at position 264 to threonine T; (g) Mutate the glutamic acid E at position 264 to cysteine C; (h) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to alanine A; (i) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to valine V.

3. A gene encoding the cyclodextrin glycosyltransferase mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector uses a pET series plasmid as the expression vector; optionally, the recombinant vector uses plasmid pETM-11 as the expression vector.

6. A recombinant cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant vector according to claim 4 or 5.

7. The recombinant cell according to claim 6, wherein The recombinant cell uses bacteria or fungi as the expression host; optionally, the recombinant cell uses Escherichia coli as the expression host.

8. A method for improving the transglycosylation activity of cyclodextrin glycosyltransferase towards polyphenolic compounds, characterized in that, The method is to perform any one of the following mutations on the cyclodextrin glycosyltransferase parent with the amino acid sequence shown in SEQ ID NO.2: (a) Mutate the tyrosine Y at position 195 to phenylalanine F; (b) Mutate the glutamic acid E at position 264 to alanine A; (c) Mutate the glutamic acid E at position 264 to valine V; (d) Mutate the glutamic acid E at position 264 to glycine G; (e) Mutate the glutamic acid E at position 264 to aspartic acid D; (f) Mutate the glutamic acid E at position 264 to threonine T; (g) Mutate the glutamic acid E at position 264 to cysteine C; (h) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to alanine A; (i) Mutate the tyrosine Y at position 195 to phenylalanine F, and mutate the glutamic acid E at position 264 to valine V.

9. A method for synthesizing α-glucosyl polyphenols, characterized in that, The method is to use the cyclodextrin glycosyltransferase mutant according to claim 1 or 2 as a catalyst, respectively use cyclodextrin and polyphenol as donors and acceptors to catalyze the glycosylation reaction, and after the reaction, use glucoamylase derived from Aspergillus niger to treat the reaction mixture to obtain α-glucosyl polyphenol.

10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant vector according to claim 4 or 5, or the recombinant cell according to claim 6 or 7, or the method according to claim 8 in the preparation of α-glucosyl polyphenol products.

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