Sucrose synthase mutants and their applications

Abstract

The present invention belongs to the technical field of enzyme engineering, and particularly relates to a sucrose synthase mutant and its application. The sucrose synthase mutant described in the present invention is obtained by the following mutations on the amino acid sequence of sucrose synthase shown in SEQ ID NO.1: valine at position 159 is mutated to threonine. The sucrose synthase mutant has high enzyme activity and can be applied to the biocatalytic synthesis of neohesperidin. The present invention also provides a method for preparing neohesperidin using the sucrose synthase mutant. Using hesperetin-7-O-glucoside, sucrose, and uridine diphosphate as substrates, and adding the whole-cell crude enzyme solution of rhamnosyltransferase, rhamnose synthase, and sucrose synthase, neohesperidin is catalytically obtained with a relatively high conversion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a sucrose synthase mutant and application thereof. Background Art

[0002] The molecular formula of neohesperidin is C 28 H 34 O 15 , with a molecular weight of 610.56, belongs to the dihydroflavonoids among flavonoids. It was first discovered in plants and is usually distributed in sour oranges, fructus aurantii, immature pomelo.

[0003] Neohesperidin undergoes high-pressure hydrogenation to produce neohesperidin dihydrochalcone, a novel sweetener 1,500-1,800 times sweeter than sucrose. Not only is it sweeter than commonly used sweeteners like saccharin, cyclamate, and aspartame, it also offers excellent palatability, with a taste similar to that of white sugar. It is also a novel bitterness-blocking agent, used in bitter tablets and liquid formulations to correct the strong bitterness of chemical drugs and improve their palatability. As market demand for neohesperidin dihydrochalcone increases, so too does demand for its raw material, neohesperidin.

[0004] At present, there are three main methods for the preparation of neohesperidin reported in research: 1. Plant extraction, which is mainly extracted from Rutaceae plants (such as sour orange, sweet orange, Citrus aurantium, Citrus aurantium) and Rubiaceae plants (such as Uncaria tomentosa), which is the most important method for preparing neohesperidin, but the content of neohesperidin in natural plants is low and the extraction rate is low; 2. Chemical synthesis, which uses readily available raw materials such as naringin for conversion, but the process involves acid and alkali catalysts; 3. Biocatalytic synthesis, which refers to the use of enzymes or enzyme-producing microorganisms as catalysts for the synthesis of neohesperidin. For example, using abundant hesperidin as raw material, neohesperidin is prepared by biosynthesis using glycoside hydrolases, glycosyltransferases, and UDP-rhamnose synthases. Although the biotransformation method is currently the most economical, efficient, and relatively environmentally friendly production method, it cannot meet the requirements for high conversion rates and still needs continuous improvement. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a sucrose synthase mutant with high enzyme activity, which can be used for biocatalytic synthesis of neohesperidin. The present invention also provides a method for preparing neohesperidin using the sucrose synthase mutant with high conversion rate.

[0006] The technical solutions of the present invention are as follows:

[0007] A sucrose synthase mutant is obtained by subjecting the sucrose synthase amino acid sequence shown in SEQ ID NO. 1 to the following mutation: valine at position 159 is mutated to threonine, and is named V159T.

[0008] The sucrose synthase is derived from Arabidopsis thaliana, and its amino acid sequence is shown in SEQ ID NO.1:

[0009] .

[0010] The present invention provides the use of the sucrose synthase mutant in the preparation of neohesperidin.

[0011] The present invention also provides a method for preparing the neohesperidin, comprising the following steps:

[0012] (1) Prepare whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants respectively;

[0013] (2) Hesperidin-7-O-glucoside, sucrose, and uridine diphosphate (UDP) were used as substrates, and whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase were added to catalyze the production of neohesperidin.

[0014] Catalytic conditions: pH 7.0-7.5, temperature 30-37°C. The amount of hesperetin-7-O-glucoside, sucrose, UDP, and crude enzyme solution added can be adjusted according to actual needs.

[0015] The rhamnosyltransferase is derived from orange, and its amino acid sequence is shown in SEQ ID NO: 2:

[0016] MGMDTKHQDKPSILMLPWLAHGHIAPHLELAKKLSQKNFHIYFCSTPNNLAAFGRNVEKNFSSSIQLIELQLPNTFPELPSQNQTTKNLPPHLIYTLVGAFEDAKPAFCNILE TLKPTLVMYDLFQPWAAEAAYQYDIAAILFLPLSAVACSFLLHNIVNPSLKYPFFESDYQDRESKNINYFLHLTANGTLNKDRFLKAFELSCKFVFIKTSREIESKYLDYFPSL MGNEIIPVGPLIQEPTFKEDDTKIMDWLSQKEPRSVVYASFGSEYFPSKDEIHEIASGLLLSEVNFIWAFRLHPDEKMTIEEALPQGFAEEIERNNKGMIVQGWVPQAKILRHG SIGGFLSHCGWGSVVEGMVFGVPIIGVPMAYEQPSNAKVVVDNGMGMVVPRDKINQRLGGEEVARVIKHVVLQEEAKQIRRKANEISESMKKIGDAEMSVVVEKLLQLVKKSE.

[0017] The rhamnose synthase is derived from Camelina tinctoria, and its amino acid sequence is shown in SEQ ID NO: 3:

[0018] .

[0019] In step (1), the method for preparing the whole-cell crude enzyme solution comprises the following steps:

[0020] a. constructing plasmid vectors carrying rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutant genes, respectively; the plasmid vectors are pET vectors;

[0021] b. transforming the plasmid vector into a host cell; the host cell is a bacterial cell, preferably Escherichia coli;

[0022] c. Select positive clones for shake flask fermentation and culture at OD 600=0.6-0.8, add IPTG to a final concentration of 1.0 mmol / L, 0.4 mmol / L, and 1.0 mmol / L, respectively. Set the rotation speed to 120 rpm, 200 rpm, and 200 rpm, respectively. Induce at 16°C for 16-24 h to obtain a bacterial solution containing the target plasmid.

[0023] d. Centrifuge the bacterial suspension at 8000 rpm and 4°C for 20 min, discard the supernatant, and resuspend the pellet in 1× PBS (1 / 100 of the total bacterial suspension volume). Then, disrupt the pellet using an ultrasonic disruptor at 70% power (560 W total power), with a cycle of 5 s, 3 s rest, and 5 min. This will yield crude whole-cell enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants, respectively.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The sucrose synthase mutant V159T provided by the present invention has a higher enzyme activity, which is 1.45 times that of the wild type.

[0026] 2. The present invention proposes a multi-enzyme one-pot method for preparing neohesperidin with a conversion rate of over 95%. The catalytic synthesis process is as follows: first, sucrose and UDP are reacted to form UDP-glucose under the action of sucrose synthase, then UDP-glucose is reacted to form UDP-rhamnose under the action of rhamnose synthase, and finally, hesperetin-7-O-glucoside and UDP-rhamnose are reacted to form neohesperidin under the action of rhamnosyltransferase.

[0027] 3. In the process of converting UDP-glucose into UDP-rhamnose, rhamnose synthase requires the presence of NAD and NADPH to catalyze. However, the present invention uses the crushed whole cells as the crude enzyme solution, so there is no need to add additional NAD and NADPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a catalytic synthesis pathway diagram of the neohesperidin;

[0029] Figure 2 HPLC chart of the 0h sample of the catalytic system;

[0030] Figure 3 HPLC chart of the 5h sample of the catalytic system;

[0031] Figure 4 HPLC chart of 24h sample of catalytic system;

[0032] Figure 5 This is the standard curve of neohesperidin standard product;

[0033] Figure 6This is the HPLC chart of 0.1 mg / mL neohesperidin standard;

[0034] Figure 7 This is the HPLC chart of 0.2 mg / mL neohesperidin standard;

[0035] Figure 8 This is the HPLC chart of 0.4 mg / mL neohesperidin standard;

[0036] Figure 9 This is the HPLC chart of 0.6 mg / mL neohesperidin standard;

[0037] Figure 10 This is the HPLC chart of 0.8 mg / mL neohesperidin standard;

[0038] Figure 11 HPLC chart of 0.2 mg / mL hesperetin-7-O-glucoside. DETAILED DESCRIPTION

[0039] The examples of the present invention are only used to further illustrate the content of the present invention and cannot be used to limit the content or scope of the present invention. For molecular biology experimental methods not specifically described in this example, please refer to the "Molecular Cloning Experiment Guide".

[0040] Example 1 Determination of sucrose synthase mutant enzyme activity

[0041] In this example, the sucrose synthase sequence shown in SEQ ID NO: 1 was subjected to site-directed mutagenesis (see Table 1 for site-directed mutagenesis primers) to obtain the following mutants:

[0042] The valine at position 159 was mutated to threonine (V159T), aspartic acid (V159D), glutamine (V159Q), and asparagine (V159N), respectively; the asparagine at position 777 was mutated to valine (N777V).

[0043] Table 1 Site-directed mutagenesis primers

[0044]

[0045] The enzyme activity was determined by staining method (reference: Molecular modification of sucrose synthase and its efficient preparation of UDP-glucose_Zhao Liting). The results are shown in Table 2. The enzyme activities of V159T, V159D, V159Q, V159N, and N777V were 1.45 times, 0.64 times, 0.64 times, 1.20 times, and 1.04 times that of the wild type, respectively.

[0046] Table 2 Enzyme activities of sucrose synthase mutants

[0047]

[0048] Example 2 Preparation of neohesperidin using sucrose synthase mutant V159T

[0049] The preparation method of the neohesperidin comprises the following steps:

[0050] (1) Prepare whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants respectively;

[0051] a. Construct plasmid vectors carrying rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutant genes respectively;

[0052] b. Transforming the plasmid vector into host cells Escherichia coli;

[0053] c. Select positive clones for shake flask fermentation and culture at OD 600 =0.6-0.8, add IPTG to a final concentration of 1.0 mmol / L, 0.4 mmol / L, and 1.0 mmol / L, respectively. Set the rotation speed to 120 rpm, 200 rpm, and 200 rpm, respectively. Induce at 16°C for 24 h to obtain a bacterial solution containing the target plasmid.

[0054] d. Centrifuge the bacterial suspension at 8000 rpm and 4°C for 20 min, discard the supernatant, and resuspend the pellet in 1× PBS (1 / 100 of the total bacterial suspension volume). Then, disrupt the pellet using an ultrasonic disruptor at 70% power (560 W total power), with a cycle of 5 s, 3 s rest, and 5 min. This will yield crude whole-cell enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants, respectively.

[0055] (2) Hesperidin-7-O-glucoside, sucrose, and UDP were used as substrates, and whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase were added to catalyze the production of neohesperidin. The catalytic system was 10 mL, and the system composition was shown in Table 3. Each 100 mL of buffer solution contained 4 g of trisodium citrate dihydrate, 1.67 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of EDTA, and 1 g of AEO-9 (surfactant). The volume was adjusted to 100 mL with ultrapure water. The catalytic system had a pH of 7.2, a temperature of 30 °C, and a catalytic reaction at 200 rpm for 24 h. Figure 1 The figure is a catalytic synthesis pathway diagram of the neohesperidin.

[0056] Table 3 Catalytic system composition

[0057]

[0058] Samples were taken at 0h, 5h and 24h during the catalytic process, and the samples were boiled at 100℃ for 10min to terminate the reaction. HPLC determination was performed, such as Figure 2、 Figure 3 and Figure 4 The changes in the concentrations of hesperetin-7-O-glucoside and neohesperidin during the catalytic process are shown in Table 4. 28 g / L of hesperetin-7-O-glucoside can be converted into 22.12 g / L of neohesperidin. The conversion rate of the catalytic system reaches more than 95% in 24 hours.

[0059] Table 4 Concentrations of hesperetin-7-O-glucoside and neohesperidin during the catalytic process

[0060]

[0061] When calculating the concentration of generated neohesperidin, a standard curve of neohesperidin standard was constructed, e.g. Figure 5 , the concentration points used were 0.1 mg / mL ( Figure 6 )、0.2mg / mL( Figure 7 )、0.4mg / mL( Figure 8 )、0.6mg / mL( Figure 9 )、0.8mg / mL( Figure 10 ), a linear regression equation was established with concentration as the horizontal axis and peak area as the vertical axis.

[0062] Figure 11 HPLC chart of 0.2 mg / mL hesperetin-7-O-glucoside.

[0063] The samples were subjected to HPLC analysis as follows: diluted 5-fold with acetonitrile, centrifuged at 10,000 rpm for 10 min, filtered, and analyzed. The HPLC column was a SHIMSEN Superb II C18, 5µm, 1.6mm×250mm, the detector was a PDA detector, the detection wavelength was 282nm, and the liquid phase detection procedure was: acetonitrile:water volume ratio of 45:55, flow rate of 1.0mL / min, column temperature of 40°C, and analysis for 10min.

Claims

1. A sucrose synthase mutant, characterized in that: The sucrose synthase amino acid sequence shown in SEQ ID NO.1 is obtained by subjecting the amino acid sequence to the following mutation: the valine at position 159 is mutated to threonine.

2. The sucrose synthase mutant according to claim 1, characterized in that: The sucrose synthase is derived from Arabidopsis thaliana.

3. Use of the sucrose synthase mutant according to claim 1 in the preparation of neohesperidin.

4. A method for preparing neohesperidin using the sucrose synthase mutant according to claim 1, characterized in that: The following steps are involved: (1) Prepare whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants respectively; (2) Hesperidin-7-O-glucoside, sucrose and uridine diphosphate were used as substrates, and whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase and sucrose synthase were added to catalyze the production of neohesperidin.

5. The method according to claim 4, characterized in that: The rhamnosyltransferase is derived from orange, and its amino acid sequence is shown in SEQ ID NO:

2.

6. The method according to claim 4, characterized in that: The rhamnose synthase is derived from Camelina tinctoria, and its amino acid sequence is shown in SEQ ID NO:

3.

7. The method according to claim 4, characterized in that: The catalytic conditions in step (2) are: pH 7.0-7.5, temperature 30-37°C.

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