Sucrose synthase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a sucrose synthase mutant and application thereof. The sucrose synthase mutant disclosed by the invention is obtained by performing the following mutation on a sucrose synthase amino acid sequence as shown in SEQ ID NO.1: valine at the 159th site is mutated into threonine. The sucrose synthase mutant is high in enzyme activity and can be applied to biocatalytic synthesis of neohesperidin. The invention also provides a method for preparing neohesperidin by using the sucrose synthase mutant, hesperetin-7-O-glucoside, sucrose and uridine diphosphate are taken as substrates, whole-cell crude enzyme liquid of rhamnosyltransferase, rhamnose synthase and sucrose synthase is added, neohesperidin is obtained through catalysis, and the conversion rate is relatively high.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a sucrose synthase mutant and an 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 grapefruits.

[0003] Neohesperidin will generate neohesperidin dihydrochalcone after high-pressure hydrogenation. Neohesperidin dihydrochalcone is a new sweetener with a sweetness 1500-1800 times that of sucrose. It is not only sweeter than commonly used sweeteners such as saccharin, sodium cyclamate, and aspartame, but also has good palatability and tastes similar to white sugar. At the same time, it is also a new bitterness shielding agent, which is used in bitter tablets or liquid preparations to correct the strong bitterness of chemical drugs and improve the palatability of tablets or liquid preparations. With the increase in market demand for neohesperidin dihydrochalcone, the demand for its raw material neohesperidin is also increasing.

[0004] At present, there are three main methods for preparing 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 main 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, such as using abundant hesperidin as raw material, and using glycoside hydrolases, glycosyltransferases, and UDP-rhamnose synthases to prepare neohesperidin through biosynthesis. Although the biotransformation method is currently the most economical, effective and relatively green production method, it cannot meet the requirements for high conversion rates and still needs to be continuously improved. 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 applied to the 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 solution of the present invention is as follows: 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.

[0007] The sucrose synthase is derived from Arabidopsis thaliana, and its amino acid sequence is shown in SEQ ID NO.1: .

[0008] The present invention provides application of the sucrose synthase mutant in preparing neohesperidin.

[0009] The present invention also provides a method for preparing the neohesperidin, comprising the following steps: (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 (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.

[0010] Catalytic conditions: pH 7.0-7.5, temperature 30-37°C, the addition amount of hesperidin-7-O-glucoside, sucrose, UDP and crude enzyme solution can be adjusted according to actual needs.

[0011] The rhamnosyltransferase is derived from orange, and its amino acid sequence is shown in SEQ ID NO: 2: MGMDTKHQDKPSILMLPWLAHGHIAPHLELAKKLSQKNFHIYFCSTPNNLAAFGRNVEKNFSSSIQLIELQLPNTFPELPSQNQTTKNLPPHLIYTLVGAFEDAKPAFCNILE TLKPTLVMYDLFQPWAAEAAYQYDIAAILFLPLSAVACSFLLHNIVNPSLKYPFFESDYQDRESKNINYFLHLTANGTLNKDRFLKAFELSCKFVFIKTSREIESKYLDYFPSL MGNEIIPVGPLIQEPTFKEDDTKIMDWLSQKEPRSVVYASFGSEYFPSKDEIHEIASGLLLSEVNFIWAFRLHPDEKMTIEEALPQGFAEEIERNNKGMIVQGWVPQAKILRHG SIGGFLSHCGWGSVVEGMVFGVPIIGVPMAYEQPSNAKVVVDNGMGMVVPRDKINQRLGGEEVARVIKHVVLQEEAKQIRRKANEISESMKKIGDAEMSVVVEKLLQLVKKSE.

[0012] The rhamnose synthase is derived from Camelina tinctoria, and its amino acid sequence is shown in SEQ ID NO: 3: .

[0013] In step (1), the method for preparing the whole-cell crude enzyme solution comprises the following steps: a. construct plasmid vectors carrying rhamnosyltransferase, rhamnose synthase and sucrose synthase mutant genes respectively; the plasmid vector is a pET vector; b. transforming the plasmid vector into a host cell; the host cell is a bacterial cell, preferably Escherichia coli; c. Select positive clones for shake flask fermentation. 600 =0.6-0.8, add IPTG to make the final concentrations 1.0mmol / L, 0.4mmol / L and 1.0mmol / L respectively, set the rotation speed to 120rpm, 200rpm, 200rpm respectively, and induce at 16℃ for 16-24h to obtain the bacterial solution containing the target plasmid; d. Centrifuge the bacterial solution at 8000 rpm and 4°C for 20 min, discard the supernatant, add 1×PBS to resuspend the precipitate, the amount of 1×PBS used is 1 / 100 of the volume of the bacterial solution used, and then use an ultrasonic disruptor to disrupt the solution. The disruption program is 70% power (total power is 560 W), disrupt for 5 s, stop for 3 s, and disrupt for 5 min, thereby obtaining the whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants, respectively.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 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.

[0015] 2. The present invention provides a method for preparing neohesperidin by a multi-enzyme one-pot process, with a conversion rate of more than 95%. The catalytic synthesis process is as follows: first, sucrose and UDP generate UDP-glucose under the action of sucrose synthase, then UDP-glucose obtains UDP-rhamnose under the action of rhamnose synthase, and finally hesperidin-7-O-glucoside and UDP-rhamnose generate neohesperidin under the action of rhamnosyltransferase.

[0016] 3. In the process of converting UDP-glucose into UDP-rhamnose, rhamnose synthase can only catalyze in the presence of NAD and NADPH. However, the present invention uses the crushed whole cells as the crude enzyme solution, so there is no need to add NAD and NADPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a catalytic synthesis pathway diagram of the neohesperidin; Figure 2 It is the HPLC chart of the 0h sample of the catalytic system; Figure 3 It is the HPLC chart of the 5h sample of the catalytic system; Figure 4 It is the HPLC chart of the 24h sample of the catalytic system; Figure 5 This is the standard curve of the neohesperidin standard product; Figure 6 This is the HPLC chart of 0.1 mg / mL neohesperidin standard; Figure 7 This is the HPLC chart of 0.2 mg / mL neohesperidin standard; Figure 8 This is the HPLC chart of 0.4 mg / mL neohesperidin standard; Fig. 9 This is the HPLC chart of 0.6 mg / mL neohesperidin standard; Fig.10 This is the HPLC chart of 0.8 mg / mL neohesperidin standard; Fig.11 HPLC chart of 0.2 mg / mL hesperetin-7-O-glucoside. DETAILED DESCRIPTION

[0018] The embodiments of the present invention are only used as further explanations of the present invention and cannot be used as limiting the content or scope of the present invention. For molecular biology experimental methods not specifically described in the present embodiments, reference can be made to the Molecular Cloning Experiment Guide.

[0019] Example 1 Determination of sucrose synthase mutant enzyme activity 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: 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).

[0020] Table 1 Site-directed mutagenesis primers

[0021] 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.

[0022] Table 2 Enzyme activity of sucrose synthase mutants

[0023] Example 2 Preparation of neohesperidin catalyzed by sucrose synthase mutant V159T The preparation method of neohesperidin comprises the following steps: (1) Prepare whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants respectively; a. construct plasmid vectors carrying rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutant genes respectively; b. Transform the plasmid vector into the host cell Escherichia coli; c. Select positive clones for shake flask fermentation. 600=0.6-0.8, add IPTG to make the final concentrations 1.0mmol / L, 0.4mmol / L and 1.0mmol / L respectively, set the rotation speed to 120rpm, 200rpm, 200rpm respectively, and induce at 16℃ for 24h to obtain the bacterial solution containing the target plasmid; d. Centrifuge the bacterial solution at 8000 rpm and 4°C for 20 min, discard the supernatant, add 1×PBS to resuspend the precipitate, the amount of 1×PBS used is 1 / 100 of the volume of the bacterial solution used, and then use an ultrasonic disruptor to disrupt the solution. The disruption program is 70% power (total power is 560 W), disrupt for 5 s, stop for 3 s, and disrupt for 5 min, thereby obtaining the whole-cell crude enzyme solutions of rhamnosyltransferase, rhamnose synthase, and sucrose synthase mutants, respectively.

[0024] (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; wherein, the catalytic system was 10 mL, and the system composition was shown in Table 3. Each 100 mL buffer solution included 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), and the volume was fixed to 100 mL with ultrapure water; the catalytic system pH was 7.2, the temperature was 30 °C, and the catalysis was carried out at 200 rpm for 24 h; Figure 1 The figure is a catalytic synthesis pathway diagram of the neohesperidin.

[0025] Table 3 Catalytic system composition

[0026] 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 hesperidin-7-O-glucoside and neohesperidin during the catalytic process are shown in Table 4. 28 g / L of hesperidin-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.

[0027] Table 4 Concentrations of hesperidin-7-O-glucoside and neohesperidin during the catalytic process

[0028] 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( Fig. 9 )、0.8mg / mL( Fig.10 ), a linear regression equation was established with concentration as the horizontal axis and peak area as the vertical axis.

[0029] Fig.11 HPLC chart of 0.2 mg / mL hesperetin-7-O-glucoside.

[0030] The sample was subjected to HPLC determination in the following manner: diluted 5 times with acetonitrile, centrifuged at 10000rpm for 10min, filtered and analyzed, the HPLC column was SHIMSEN Superb II C18, 5µm, 1.6mm×250mm, the detector was PDA detector, the detection wavelength was 282nm, the liquid phase detection procedure was: the volume ratio of acetonitrile to water was 45:55, the flow rate was 1.0mL / min, the column temperature was 40℃, and the analysis was 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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