Amino acid mutant of glycosyltransferase and its application

Abstract

The present invention discloses an amino acid mutant of a glycosyltransferase and its application. The amino acid mutant is obtained by replacing the 141st proline of the amino acid sequence of the glycosyltransferase PhUGT derived from the Panicum hallii GT 1 family with glycine; the amino acid sequence of the amino acid mutant is shown in SEQ ID NO.3. The present invention implements directional modification of a glucosyltransferase with excellent characteristics or a certain ability to convert xylosyl groups through genetic engineering technology, and obtains a method for efficiently converting apigenin into apigenin 5-O-xyloside through a single-point mutation method. The novel amino acid mutant obtained by the present invention has strong specificity and high directional conversion efficiency, and a non-natural flavonoid glycoside compound apigenin 5-O-xyloside and a preparation process thereof are created.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and bioengineering, and specifically relates to an amino acid mutant of a glycosyltransferase and an application thereof. Background Art

[0002] Glycosyltransferase (GT, EC 2.4.xy) is a general term for a class of enzymes that can specifically catalyze the connection of activated sugars to different receptor molecules to produce glycosylated products. Its catalytic substrates are relatively broad, including sugars, proteins, lipids, flavonoids, terpenes, steroids and other compounds. Uridine diphosphate glycosyltransferase (UDP-glycosyltransferase, UGT), that is, glycosyltransferases that use sugar molecules activated by uridine diphosphate (UDP) as glycosyl donors, is the largest superfamily of glycosyltransferases in plants. Many glycosyltransferases related to the glycosylation of plant hormones and secondary metabolites belong to this superfamily. According to the glycosylation site of the receptor molecule, glycosyltransferases can be divided into O-, N-, C-, and S-glycotransferases. Among them, natural flavonoid glycosides are mainly synthesized by O-glycosyltransferases.

[0003] There are various types of flavonoid glycoside compounds. The same flavonoid aglycone can be connected to different types and numbers of glycosyl donors at different substitution positions. In the process of biosynthesis, they can be catalyzed by different types of glycosyltransferases and sugar donors. Common sugar donors include UDP-glucose, UDP-glucuronic acid, UDP-xylose, UDP-rhamnose, UDP-arabinose, UDP-galactose, UDP-galacturonic acid and UDP-mannose. Among them, UDP-glucose is the most widely used sugar donor in the study of flavonoid glycosylation, but different glycosyl moieties help to construct the diversity of flavonoid-O-glycoside structures, and it is expected that flavonoid compounds with stronger biological activity can be mined. For example, puerarin 6″-O-xyloside has better anti-tumor activity than puerarin glucoside, and quercetin-3-O-β-D-xyloside has more significant antioxidant effect than quercetin-3-O-glucoside.

[0004] Apigenin is a natural flavonoid compound found in many plants, such as Thymelaeaceae, Verbenaceae, Selaginaceae and other plants. Apigenin has a variety of pharmacological activities, such as anti-tumor, cardiovascular and cerebrovascular protection, antiviral, antibacterial, etc. Compared with other flavonoids (quercetin, kaempferol flavonoids), it has the characteristics of low toxicity and non-mutagenicity. However, apigenin has low water solubility and poor oral bioavailability, which limits its application in animal pharmacology research and clinical practice. Glycosylation modification is an effective means to improve the bioavailability of apigenin. However, common glycosyltransferases have low catalytic efficiency and poor conversion ability, and most of them can only catalyze UDP-glucose. In order to further improve the glycosylation efficiency of apigenin and expand the types of apigenin glycosides, the general research and development direction is to further explore and screen new glycosyltransferases, but it is difficult to achieve the expected results. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above-mentioned and / or existing problems in the prior art that the catalytic efficiency of common glycosyltransferases is low and most of them can only be used for UDP-glucose catalysis, the present invention is proposed.

[0007] One of the purposes of the present invention is to provide an amino acid mutant of a glycosyltransferase, to further explore the glycosyltransferase through genetic engineering technology combined with bioinformatics, to carry out directed modification of glucosyltransferases with excellent properties or with certain xylosylation capabilities, and to obtain a method for efficiently converting apigenin into apigenin 5-O-xyloside through a single-point mutation method.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: an amino acid mutant of a glycosyltransferase, wherein the amino acid mutant is derived from millet Panicum hallii The glycosyltransferase PhUGT of the GT 1 family was obtained by replacing the proline at position 141 with glycine in the amino acid sequence;

[0009] The amino acid sequence of the amino acid mutant is shown in SEQ ID NO.3.

[0010] As a preferred embodiment of the amino acid mutant of the glycosyltransferase of the present invention, the amino acid sequence of the glycosyltransferase PhUGT is shown in SEQ ID NO.1.

[0011] Another object of the present invention is to provide a gene encoding the amino acid mutant as described above, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0012] Another object of the present invention is to provide a recombinant plasmid comprising the gene as described above.

[0013] Another object of the present invention is to provide a host cell, wherein the host cell contains the recombinant plasmid as described above.

[0014] As a preferred embodiment of the host cell of the present invention, the host cell comprises one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae and Bacillus subtilis.

[0015] Another object of the present invention is to provide a recombinant Escherichia coli expressing the amino acid mutants described above.

[0016] Another object of the present invention is to provide the use of the above-mentioned amino acid mutant in the in vitro conversion of apigenin to apigenin 5-O-xyloside, using the amino acid mutant, apigenin as a substrate, and the target product is apigenin-5-O-xyloside; the reaction temperature is 25~55°C, pH 8.0~12.0, the apigenin substrate concentration is 0.5 mM, and the reaction time is 30 min; the in vitro conversion rate is 36%.

[0017] Another object of the present invention is to provide the use of the recombinant Escherichia coli as described above in the conversion of apigenin into apigenin 5-O-xyloside in vivo, using the recombinant Escherichia coli, taking apigenin as a substrate, and the target product is apigenin 5-O-xyloside;

[0018] Among them, the IPTG concentration was 0.5 mM, the induction temperature was 16~42℃, the fermentation temperature was 20~45℃, the cellobiose concentration was 0~25 g / L, the apigenin substrate concentration was 1 g / L, and the reaction time was 72 h; the in vivo conversion rate reached more than 80%.

[0019] Another object of the present invention is to provide the use of glycosyltransferase PhUGT in in vitro conversion of apigenin, wherein the glycosyltransferase PhUGT is derived from millet. Panicum hallii GT 1 family, the amino acid sequence of which is shown in SEQ ID NO.1;

[0020] The glycosyltransferase PhUGT is used, apigenin is used as a substrate, and the target product is apigenin-5-O-glucoside; the reaction temperature is 25-55°C, the pH is 5.5-12.0, the apigenin substrate concentration is 0.5 mM, and the reaction time is 30 min; the in vitro conversion rate is more than 50%; it is also found that the glycosyltransferase PhUGT cannot use UDP-xylose as a sugar donor in vitro to catalyze the conversion of apigenin into apigenin 5-O-xyloside.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention further explores glycosyltransferases through genetic engineering technology combined with bioinformatics methods, implements directed modification of glucosyltransferases with excellent characteristics or certain xylosylation capabilities, and obtains a novel glycosyltransferase mutant PhUGT-P141G that can efficiently convert apigenin into apigenin 5-O-xyloside through a single-point mutation method; at the same time, a method for efficiently enzymatically converting apigenin into apigenin 5-O-xyloside is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 is the SDS-PAGE image of the glycosyltransferase PhUGT of the present invention;

[0025] Figure 2 The enzymatic properties of the glycosyltransferase PhUGT of the present invention (a. optimum pH; b. optimum temperature; c. pH stability; d. temperature stability);

[0026] Figure 3 It is a liquid phase analysis diagram of the glycosyltransferase PhUGT and its mutant PhUGT-P14G of the present invention;

[0027] Figure 4 The enzymatic properties of the glycosyltransferase mutant PhUGT-P141G of the present invention (a. optimum pH; b. optimum temperature);

[0028] Figure 5 The diagram for optimizing the expression conditions of the glycosyltransferase mutant PhUGT-P14G of the present invention (a. optimal IPTG concentration; b. induction temperature; c. fermentation temperature; d. cellobiose concentration);

[0029] Figure 6 This is a reaction history diagram of the conversion of glycosyltransferase mutant PhUGT-P14G and the conversion of apigenin over time. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0034] The DNA polymerase (Prime STAR HS DNA Polymerase), deoxyribonucleic acid (dNTP), ligase (T4 DNA ligase), phosphorylase (T4 Polynucleotide Kinase) and related buffers used in the examples were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (TaKaRa).

[0035] Example 1

[0036] (1) Cloning and plasmid construction of glycosyltransferase PhUGT

[0037] Glycosyltransferase PhUGT (GenBank accession no. XP_025803933.1, amino acid sequence as SEQ ID NO.1, gene sequence as SEQ ID NO.2), optimized according to Escherichia coli codons, was synthesized by Bioengineering (Shanghai) in pET28a. Bam H I and XOt I cloning site to obtain the recombinant plasmid pET-28a-PhUGT.

[0038] (2) Expression and purification of glycosyltransferase PhUGT

[0039] Take the recombinant plasmid and add 1 μL of the recombinant plasmid pET28a-PhUGT to the competent cells of Escherichia coli BL21 (DE3), mix well and place on ice for 10 min. Incubate in a 42°C water bath for 2 min and place on ice for 5 min. Add 1 mL of SOC medium and culture at 37°C for 1 h. Then spread on an LB plate containing kanamycin (the final concentration of kanamycin is 50 mg / L) to screen out transformants. Pick several single colonies and culture them in LB liquid culture medium (add kanamycin to a final concentration of 50 mg / L) for 12 h. Then take 1 mL of the bacterial solution for sequencing to determine the Escherichia coli BL21 (DE3) strain containing the pET28a-PHUGT recombinant plasmid.

[0040] The recombinant Escherichia coli BL21 (DE3) was streaked on LB plates (the final concentration of kanamycin was 50 mg / L) for activation, cultured at 37°C for 12 h, and inoculated into 50 mL LB liquid culture medium until a single colony grew out. The culture was then shaken at 37°C and 180 rpm for 3-4 h until the OD 600 = 0.6~0.8, add IPTG with a final concentration of 0.01 mM for induction, and express at 20℃ for 12 h. At the same time, Escherichia coli BL21 (DE3) containing empty plasmid pET28a was used as a negative control (Control). 1 mL of bacterial solution was aspirated and centrifuged at 12000r / min for 3min to collect the bacteria, discard the supernatant, wash the bacteria twice with 1 mL PBS buffer, resuspend the bacteria with 500 µL PBS, and then perform ultrasonic lysis. The working conditions are: ultrasonic 3 s, interval 4 s, a total of 50 times. Centrifuge at 12000 r / min for 10 min, aspirate 15 µL of supernatant for later use, and filter the remaining crude enzyme solution through a 0.22 μm filter membrane, and then purify it with a GST affinity column to obtain the purified glycosyltransferase PhUGT. For the transformant expressing whole cells containing the PhUGT gene, 30 µL of the crude enzyme solution and the purified glycosyltransferase PhUGT were added with 6 µL of 5×SDS loading buffer, mixed, boiled for 5 min, and 10 µL of each was taken for SDS-PAGE electrophoresis to identify the expressed protein, such as Figure 1 shown.

[0041] (3) Determination of PhUGT activity

[0042] The crude enzyme was reacted with 0.5 mM apigenin as substrate at an appropriate temperature and pH for 30 min, and the reaction was terminated with 400 μL methanol. The sample was analyzed by HPLC at a wavelength of 340 nm. The results showed that PhUGT could catalyze the glycosylation reaction at the 5-OH position of apigenin to produce apigenin 5-O glycoside.

[0043] The reaction system is 100 μL, and the proportions of each substance are as shown in Table 1:

[0044] Table 1 Glycosyltransferase activity assay system

[0045]

[0046] The enzyme activity unit (U) is defined as the amount of enzyme required to synthesize 1 μmol of the product apigenin-5-O-glucoside per minute under the most suitable temperature and pH conditions for the enzyme.

[0047] (4) Study on the enzymatic properties of recombinant enzyme PhUGT

[0048] ① Determination of the optimal reaction pH of recombinant enzyme PhUGT

[0049] The recombinant enzyme PhUGT was measured in a KH2PO4-Na2HPO4 buffer solution at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and a glycine-NaOH system at pH 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 12.0 in a 40°C water bath according to the standard enzyme activity assay method. The highest enzyme activity value measured was taken as 100% relative to calculate the relative enzyme activity of the recombinant enzyme PhUGT at each pH to determine the effect of the reaction pH on the activity of the recombinant enzyme PhUGT. The reaction pH of 10.0 when the enzyme activity reached the highest was the optimal reaction pH of PhUGT ( Figure 2 a).

[0050] ② Determination of the optimal reaction temperature of recombinant enzyme PhUGT

[0051] The recombinant enzyme PhUGT was tested for its activity at pH 10.0 in a water bath at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C according to the standard enzyme activity assay method. The highest enzyme activity measured was 100%, and the relative enzyme activity of the recombinant enzyme PhUGT at each reaction temperature was calculated to determine the effect of reaction temperature on the activity of the recombinant enzyme PhUGT. The reaction temperature of 45°C when the enzyme activity reached the highest was the optimal reaction temperature of the recombinant enzyme PhUGT ( Figure 2 b).

[0052] ③ Study on pH stability of recombinant enzyme PhUGT

[0053] In the absence of substrate, the purified and concentrated recombinant enzyme PhUGT was diluted to an appropriate sample concentration using glycine-NaOH buffers at pH 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, and 12.0, respectively. After each sample was incubated at 4°C for 12 h, the residual enzyme activity of the enzyme was determined by HPLC. The enzyme activity of the recombinant enzyme PhUGT that was not incubated with pH buffer was set to 100%, and the relative residual enzyme activity of the samples treated with buffers of different pH values ​​was calculated to determine the pH stability trend of the recombinant enzyme PhUGT. The recombinant enzyme was placed in glycine-NaOH buffer at pH 9.0-10.5 for 12 h, and its relative enzyme activity was maintained at 60% or above ( Figure 2 c).

[0054] ④ Study on the temperature stability of recombinant enzyme PhUGT

[0055] The recombinant enzyme PhUGT was incubated at 25℃, 30℃, and 35℃ for 0, 15, 30, 45, 60, 75, and 90 min, respectively, and the residual enzyme activity of the samples taken out at each time point under the three groups of temperatures was measured. The recombinant protein activity without incubation treatment was set to 100%. The relative residual enzyme activity (%) of the recombinant enzyme PhUGT after different incubation times at the three groups of temperatures was calculated to determine the temperature stability trend of the recombinant enzyme PhUGT. After being placed at 25 and 30℃ for 90 min, the enzyme activity remained about 60%. After being placed at 35℃ for half an hour, the enzyme activity of PHUGT decreased by about 60% ( Figure 2 d).

[0056] Example 2 Construction of amino acid mutants of glycosyltransferase PhUGT

[0057] By performing saturation mutagenesis on point P141, the mutation site was finally determined to be PhUGT-P141G.

[0058] Design of mutation primers: Using bioinformatics analysis and homology modeling, we changed some amino acids in the glycosyltransferase PhUGT molecule and designed mutation points. We used inverse PCR to obtain the mutation sequence, and designed two forward and reverse oligonucleotide sequences for each mutation site. The mutation site selected the preferred codon of Escherichia coli, and the mutation primers are shown in Table 2.

[0059] Table 2 Site-directed mutagenesis primers

[0060]

[0061] Using the recombinant plasmid pET-28a in Example 1 -PhUGT was used as a template, and P141 was further mutated by reverse PCR to obtain the recombinant plasmid pET-28a-PhUGT-P141G carrying the mutant gene SEQ ID NO.4.

[0062]

[0063] Table 4 Reaction conditions of inverse PCR

[0064]

[0065] The 5' end phosphorylation reaction of the PCR fragment was prepared as shown in the following table and reacted at 37°C for 1 h.

[0066] Table 5 Preparation of phosphorylation reaction solution

[0067]

[0068] After phosphorylation at 37°C, 1 μL T4 ligase was added and ligation was carried out at 16°C for 12 h. Plasmid DNA was transformed, and mutants were selected and identified by sequencing.

[0069] The amino acid mutant of glycosyltransferase PhUGT is obtained by replacing the 141st proline of the amino acid sequence of glycosyltransferase PhUGT with glycine, and its amino acid sequence is shown in SEQ ID NO.3.

[0070] Example 3 Analysis of enzymatic properties of glycosyltransferase PhUGT mutants

[0071] (1) Determination of the optimal reaction pH of the mutant enzyme PhUGT-P141G

[0072] The activity of the mutant enzyme PhUGT-P141G was measured in a glycine-NaOH buffer system of 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, and 12.0 in a 35°C water bath according to the standard enzyme activity assay method. The highest enzyme activity value measured was taken as 100% relative to calculate the relative enzyme activity of the mutant enzyme PhUGT-P141G at each pH to determine the effect of the reaction pH on the activity of the mutant enzyme PhUGT-P141G. The reaction pH of 9.0, when the enzyme activity reached the highest, was the optimal reaction pH of PhUGT-P141G ( Figure 4 a).

[0073] (2) Determination of the optimal reaction temperature of the mutant enzyme PhUGT-P141G

[0074] The activity of the mutant enzyme PhUGT-P141G was measured at pH 9.0 in a water bath at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C according to the standard enzyme activity assay method. The highest enzyme activity measured was 100%, and the relative enzyme activity of the mutant enzyme PhUGT-P141G at each reaction temperature was calculated to determine the effect of reaction temperature on the activity of the mutant enzyme PhUGT-P141G. The reaction temperature of 30°C when the enzyme activity reached the highest was the optimal reaction temperature of the recombinant enzyme PhUGT ( Figure 4 b).

[0075] Example 4 Performance measurement of recombinant enzyme PhUGT in vitro conversion of apigenin

[0076] The conversion capacity of recombinant enzyme PhUGT to apigenin was determined. The concentration of apigenin was 0.5 mM. PhUGT was used for conversion at 45°C and pH 10.0 for 0.5 h. The results were detected by HPLC. The results showed that after 0.5 h of reaction, the conversion rate of PhUGT to apigenin-5-O-glucoside was greater than 50%. However, it was found that the recombinant enzyme PhUGT could not catalyze the conversion of apigenin to apigenin-5-O-xyloside using UDP-xylose as the sugar donor.

[0077] Example 5 Determination of the performance of mutant PhUGT-P141G in in vitro conversion of apigenin

[0078] The conversion ability of mutant PhUGT-P141G to apigenin was determined. The concentration of apigenin was 0.5 mM. The mutant PhUGT-P141G was converted at 30 ℃ and pH 9.0 for 0.5 h. The results were detected by HPLC. The results showed that after site-directed mutagenesis, the sugar donor preference of glycosyltransferase was successfully changed. Mutant PhUGT-P141G no longer catalyzed the glycosylation reaction with UDP-glucose as the sugar donor, but could only catalyze the xylosylation reaction. After 0.5 h of reaction, the conversion rate of PhUGT-P141G to apigenin-5-O-xyloside was 36%.

[0079] Example 6 Effect of recombinant enzyme PhUGT and its mutants on the transformation process of apigenin

[0080] To determine the effect of the sugar donor preference of the recombinant enzyme PhUGT and its mutants, the recombinant plasmids pACYCDuet-Cep-UgpA and pCDFDuet-Peuxs1-Ecugd (the recombinant plasmid pACYCDuet-Cep-UgpA contains Saccharophagus degradans The amino acid sequence of the cellobiose phospholyase Cep is shown in SEQ ID NO.5, which is derived from Bifidobacterium bifidumThe amino acid sequence of glucose-1-phosphate uridyltransferase UgpA is shown in SEQ ID NO.6; the recombinant plasmid pCDFDuet-Peuxs1-Ecugd contains Paenibacillus elgii The nucleotide sequence of UDP-glucuronic acid decarboxylase Peuxs1 is shown in SEQ ID NO.7, which is derived from Escherichia coli The nucleotide sequence of UDP-glucose dehydrogenase Ecugd is shown in SEQ ID NO.8) and PhUGT and its mutant plasmids were respectively transferred into Escherichia coli BL21 (DE3) competent cells, mixed and placed on ice for 10 min, incubated in a 42°C water bath for 2 min and placed on ice for 5 min, 1 mL of SOC medium was added thereto, and the cells were cultured at 37°C for 1 h, and then spread on LB plates containing kanamycin, spectinomycin, and chloramphenicol (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L) to screen out transformants, and single colonies were picked and cultured in LB liquid culture medium supplemented with kanamycin, spectinomycin, and chloramphenicol resistance for 12 h, and then inoculated into 5 mL of fresh LB liquid culture medium, and cultured at 37°C and 180 rpm for 1-2 h until OD 600 = 0.6~0.8, IPTG was added at a final concentration of 0.01 mM for induction, and the expression was carried out at 20℃ for 12 h. 10 g / L cellobiose and 500 mg / L apigenin were added, and the culture was carried out at 30℃ and 180 rpm for 48 h. Then, samples were taken for HPLC detection. The results showed that after the single-point mutation, the conversion rate of apigenin to apigenin-5-O-xyloside by PhUGT-P141G increased by 35%, as shown in Figure 2. Figure 3 .

[0081] The recombinant strain PhUGT-P141G mutant was inoculated into 5 mL of LB medium containing the corresponding resistance, and the activated bacterial solution was transferred to 5 mL of fresh LB medium at a 1% inoculum. Grow at 37°C until OD 600 Reach 0.6~0.8. Different concentrations of IPTG were added to the fermentation broth, and cellobiose and apigenin were added for fermentation after 12 h of induction expression. The effects of induction temperature (16~42 ℃), IPTG (0~2.0 mM), fermentation temperature (20~45℃), and cellobiose concentration (0~25 g / L) on the production of apigenin 5-O-xyloside were determined. The results are shown in Figure 4 When the induction temperature was 25℃, IPTG was 0.5 mM, fermentation temperature was 25℃, and cellobiose concentration was 15 g / L, the conversion efficiency of apigenin by the recombinant bacteria was the highest.

[0082] Determination of the reaction curve of PHUGT-P141G over time: The recombinant strain PhUGT-P141G mutant was inoculated into 5 mL of LB medium containing the corresponding resistance, and the activated bacterial solution was transferred to 50 mL of LB medium. 600 When the pH value reached 0.6-0.8, 0.5 mM IPTG was added, and after induction at 25 °C for 12 h, 20 g / L cellobiose and 1 g / L apigenin were added. The culture was shaken at 30 °C, and samples were taken at regular intervals to measure the OD 600 , until 144 h, HPLC analysis was performed, and the results are shown in Figure 6 The density of the recombinant bacteria OD600 reached 10 at 72h, but the density of the strain did not change significantly during the fermentation process. After 72h of fermentation, the conversion rate of apigenin by the recombinant bacteria reached more than 80%. With the extension of fermentation time, the yield of apigenin 5-O-xyloside in the fermentation broth did not increase significantly.

[0083] The present invention Panicum hallii A glycosyltransferase gene was cloned from the genome. Compared with the prior art, the glycosyltransferase PhUGT provided by the present invention has excellent catalytic activity, and the enzyme activity is highest under the conditions of 45°C and pH 10.0. The enzyme has high enzyme activity in a slightly alkaline environment and good pH stability. When placed in a glycine-NaOH buffer solution at 4°C and pH 9.0-10.5 for 12 hours, its relative enzyme activity can be maintained at 60% or above. The enzyme has potential application value.

[0084] The present invention mutates PhUGT P141 to G, successfully changing its sugar donor preference. The mutant can specifically convert apigenin into apigenin 5-O-xyloside, and the conversion rate of apigenin 5-O-xyloside is increased by 35% compared with the original enzyme. This provides a theoretical basis for the analysis of the key sites of sugar donor preference of GT1 family glycosyltransferases.

[0085] The present invention establishes a method for converting apigenin into apigenin-5-O-xyloside by bioenzymatic conversion, and the conversion efficiency is high. After 72 hours of shaking fermentation, the conversion rate of apigenin into apigenin-5-O-xyloside by the recombinant bacteria is greater than 80%.

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0087] SEQ ID NO.1: PhUGT

[0088] MASAAPTIVLVPLCVPGHLPSLFEAGKRLLRTSGGAMSLTVLFMQMTMEANLMSDVADLIRREAGSGLDIRFHHLPAVELPTDSHGTENFIMHFIQLHAPHVKATLSGLASPVAAVVVDYFCTTLFDAIRELALPVYVYVPCSAAMLAIILRLPALDEEVAGDLGDMEGAVEVPGMPPVPAALLPTPLLKKGPNYAWMVYHGRRIMEAAGIIVYTVAELEPPVLAAIADGRFPTVYPIGPALSSVKAPREQPHRCVVWLDAQPPASVVLLCFGSMGGSFPAPQVSEIADALERSGHRFLWVLRGPVPAGARSPYPSDANVDELLPEGFLERTKDRGLVWPTWAPQKAIIAHAAVGGFVTHCGWNSVLESMWHGVPLAPWPQHADQHLNAFQLVSMAGVAVAMEVDRKRGNFVEAAELERAVRSLMGGESEEGRKAREKAAEAKALFRRAVEEGGSSDVAMKKLAREMLEHRGCEAEASAT

[0089] ASLVR

[0090] SEQ ID NO.2:PhUGT

[0091]

[0092] SEQ ID NO.3:PhUGT-P141G

[0093] MASAAPTIVLVPLCVPGHLPSLFEAGKRLLRTSGGAMSLTVLFMQMTMEANLMSDVADLIRREAGSGLDIRFHHLPAVELPTDSHGTENFIMHFIQLHAPHVKATLSGLASPVAAVVVDYFCTTLFDAIRELALPVYVYVGCSAAMLAIILRLPALDEEVAGDLGDMEGAVEVPGMPPVPAALLPTPLLKKGPNYAWMVYHGRRIMEAAGIIVYTVAELEPPVLAAIADGRFPTVYPIGPALSSVKAPREQPHRCVVWLDAQPPASVVLLCFGSMGGSFPAPQVSEIADALERSGHRFLWVLRGPVPAGARSPYPSDANVDELLPEGFLERTKDRGLVWPTWAPQKAIIAHAAVGGFVTHCGWNSVLESMWHGVPLAPWPQHADQHLNAFQLVSMAGVAVAMEVDRKRGNFVEAAELERAVRSLMGGESEEGRKAREKAAEAKALFRRAVEEGGSSDVAMKKLAREMLEHRGCEAEASAT

[0094] ASLVR

[0095] SEQ ID NO.4:PhUGT-P141G

[0096]

[0097] SEQ ID NO.5:Cep

[0098] MKFGHFDDKAREYVITDPKTPYPWINYLGNEDFFSLVSNTGGGYSFYKDAKFRRLTRYRYNNVPVDNGGKYFYINDSGDVWSPGWKPVKAELDAYSCAHGLSYTRITGERNGIQAEVLSFIPLGTWAEIQKVSLKNTSGATKKFKLFSFAEWCLWNAEDDMTNFQRNFSTGEVEVEDSVIYHKTEFKERRNHYAFYSVNAPIQGFDTDRDKWKGLYNDFDKPDAVFEGEPRNSEAHGWSPIASHYLEVELAPGESKDLIFVLGYIEVAPENKWESKGVINKSPAKELIARFDSVEKVDAELTKLADYWANLLSTYSVESGDEKLDRMVNIWNQYQCMVTFNMSRSASFFESGIGRGMGFRDSNQDLIGFVHQVPERARERIIDIASTQFEDGSAYHQYQPLTKRGNNAIGGNFNDDPLWLILSTTDYIKETGDFSILEEQVPYDNDASKATSHFEHLKRSFYHTVNNLGPHGLPLIGRADWNDCLNLNCFSEDPNESFQTTGNKTGRTAESLMIAGLFVLYGNEFVKLCREIGQDGEAAEAQAHIDQMVEAVKKHGWDGEWFLRAYDYYGKKVGSKENEEGKIFIESQGFCGMAGIGLEDGLVEKSMDSVKEWLDCDYGIVLQQPAFTKYYIEYGEISTYPAGYKENAGIFCHNNPWIMITETLLGRGDKAFEYYRKIAPAYLEEISDLHKVEPYAYCQMIAGKDAYLPGEGKNSWLTGTASWNFAAITQYILGVKPDYSGLAINPCIPSSWDGFKVTRKYRGATYNIIVTNPTHVSKGVKSLTLNGNAIDGYIVPPQQAGTVCNVEVTLG

[0099] SEQ ID NO.6:UgpA

[0100] MFAEDLKRTEKMTVDDVFEQSAQKMREQGMSEIAISQFRHAYHVWASEKESAWIREDAVEPLHGVRSFHDVYKTIDHDKAVHAFAKTAFLKLNGGLGTSMGLQCAKSLLPVRRHKARQMRFLDIILGQVLTARTRLNVPLPVTFMNSFRTSDDTMKALRHQRKFKQTDIPLEIIQHQEPKIDAATGAPASWPANPDLEWCPPGHGDLFSTLWESGLLDTLLEHGFEYLFISNSDNLGARPSRTLAQYFEDTGAPFMVEVANRTYADRKGGHIVRDTATGRLILREMSQVHPDDKDAAQDIAKHPYFNTNNIWVRIDVLRVMLAEHDGVLPLPVIINNKTVDPTDPQSPAVVQLETAMGAAIGLFEGAICVQVDRMRFLPVKTTNDLFIMRSDRFHLTDSYEMEDGNYIFPNVDLDPRYYKNIEDFNERFPYNVPSLAAANSVSIKGDWTFGRDVIMFADARLEDRNEPSYVPNGEYVGPMGIEPGDWV

[0101] SEQ ID NO.7:Peuxs1

[0102] ATGAAACGCATTGTGATTACTGGGGCCGCAGGCTTTCTGGGCTCACATCTGACCCGTTCACTGCTGGAACAGAATCATCATGTGATTGGTATTGATGTGCTGTCAACCGGCCTGATTAGCAATCTGCATCAGGAACTGACCCATCCGAATTTTGAATTTCTGTTAATGGATATTAGCGCCGATGTGAGCGCCGAACTGGAAAAAATTGGTCATGTGGATGAAATTTATCATTTAGCAAGTCCGGCCTCTCCGAAATTTTATCAGAATGATCCGATTGGCACCATGCGCGTTAATACCTTAGGCACCCAGAATATGCTGGAACTGGCCAAAGCAACCAAAGCCAAAATGCTGTATACCTCTACCAGCGAAGCCTATGGCGATCCGGAAGTGCATCCGCAGACCGAAGAATATCGCGGCAATGTTAATACCTGGGGCCCGCGTGCCTGTTATGATGAAGCCAAACGTTTAGGTGAAGTGTATTGTTATGAATATCATAAACGCTATGGTGTTGAAGTTAAAGTTGCACGCATTTTTAATACCTATAGTGCAGGTTTACGTAATGATGATGGTCGCGTTATTTCTAATTTTGTGAGTCAGGCCATTCGCGGCGAGGACCTGACCGTGTATGGCGATGGGACTCAGACCCGCTCTTTTTGTTATGTGGATGATAATATTCGTGGCTTACAGATGATGATGGAAAGCGATCTGGCAACCGGCGAAATTATTAATATTGGTAATCCGAAAGAATATCAGATTCTGGAAGTTGCACAGCTGATTAAAAAACTGGCCAATACCGATAGTAATATTACCTTTCATCAGCTGCCGGTGGATGATCCGAAAGTTCGTCGCCCGAATATTGAAAAAGCCAAACGCTTACTGAATTGGGAACCGATTATTGAATTAGAAGAAGGCTTACTGCGCACCATTGATGCCTATCGTGCCAAACATGAAGCAGATATGAAACAGAATGTTGTGTAA

[0103] SEQ ID NO.8:Ecugd

[0104]

Claims

1. An amino acid mutant of a glycosyltransferase, characterized in that: The amino acid mutant is derived from millet Panicum hallii The glycosyltransferase PhUGT of the GT 1 family was obtained by replacing the proline at position 141 with glycine in the amino acid sequence; The amino acid sequence of the amino acid mutant is shown in SEQ ID NO.

3.

2. A gene encoding the amino acid mutant according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the gene according to claim 2.

4. A host cell, characterized in that: The host cell contains the recombinant plasmid according to claim 3, wherein the host cell is one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Bacillus subtilis.

5. A recombinant Escherichia coli expressing the amino acid mutant according to claim 1.

6. Use of the amino acid mutant according to claim 1 in the in vitro conversion of apigenin into apigenin 5-O-xyloside, characterized in that: The amino acid mutant is used, apigenin is used as a substrate, and the target product is apigenin-5-O-xyloside; the reaction temperature is 25-55°C, the pH is 8.0-12.0, the apigenin substrate concentration is 0.5 mM, the reaction time is 30 min, and the in vitro conversion rate is 36%.

7. Use of the recombinant Escherichia coli as claimed in claim 5 in converting apigenin into apigenin 5-O-xyloside in vivo, characterized in that: The recombinant Escherichia coli is used, apigenin is used as a substrate, and the target product is apigenin 5-O-xyloside; Among them, the IPTG concentration was 0.5 mM, the induction temperature was 16~42℃, the fermentation temperature was 20~45℃, the cellobiose concentration was 0~25 g / L, the apigenin substrate concentration was 1 g / L, and the reaction time was 72 h; the in vivo conversion rate reached more than 80%.

8. Use of glycosyltransferase PhUGT in the in vitro conversion of apigenin to apigenin-5-O-glucoside, characterized in that: The glycosyltransferase PhUGT is derived from millet Panicum hallii GT 1 family, the amino acid sequence of which is shown in SEQ ID NO.1; The glycosyltransferase PhUGT is used, apigenin is used as a substrate, and the target product is apigenin-5-O-glucoside; the reaction temperature is 25-55°C, the pH is 5.5-12.0, the apigenin substrate concentration is 0.5 mM, and the reaction time is 30 min; the in vitro conversion rate is more than 50%.

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