A recombinant sucrose phosphorylase or a functional fragment thereof, a preparation method and application thereof

Abstract

This invention relates to the field of enzyme engineering technology, and particularly to a recombinant sucrose phosphorylase or its functional fragment, its preparation method, and its applications. The recombinant sucrose phosphorylase has at least one amino acid sequence as described in SEQ ID NO:1. The recombinant sucrose phosphorylase exhibits high activity and high specificity, specifically catalyzing the formation of glycosidic bonds between polyphenols and glucosides, and can specifically catalyze the formation of glycosidic bonds between polyphenols and glucose at the 4' and 4'' positions.

Description

A recombinant sucrose phosphorylase or its functional fragment, its preparation method and application Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a recombinant sucrose phosphorylase or its functional fragment, its preparation method and application. Background Technology

[0002] Epigallocatechin gallate (EGCG) is the most abundant catechin found in green tea, accounting for approximately 50% of its total polyphenols. It possesses excellent antioxidant, anti-inflammatory, skin-whitening, and anti-cancer activities. However, EGCG is highly susceptible to oxidative degradation in neutral and alkaline environments, leading to a loss of its biological activity and resulting in poor bioavailability. Furthermore, due to its low solubility, EGCG presents challenges in developing human-acceptable formulations, making it currently unsuitable as an ideal active ingredient for medical, commercial food, or cosmetic applications.

[0003] Enzymatic glycosylation modification technology is an effective way to improve the water solubility and stability of EGCG. Previous studies have shown that glycoside hydrolases (such as sucrase phosphorylase and cyclodextrin glucosyltransferase) and glycosyltransferases (such as dextran sucrase) can catalyze the specific binding of glucosyl groups to EGCG, generating EGCG-glucosides (EGCG-Gs). Compared to EGCG, EGCG-Gs exhibit significantly improved water solubility and stability. On the other hand, the number and substitution position of glycosyl groups directly affect the solubility and stability of EGCG derivatives. For example, EGCG-2G has higher aqueous solution stability than EGCG-1G. Therefore, EGCG-2G shows broader application potential and is expected to serve as an active ingredient in medical, commercial food, or cosmetic applications.

[0004] However, significant obstacles remain in the enzymatic glycosylation modification technology of EGCG. Because EGCG has numerous glycosylation sites, and neither glycoside hydrolases nor glycosyltransferases are specific synthases for EGCG, EGCG-2G cannot be synthesized accurately and specifically using these enzymes, resulting in a low proportion of EGCG-2G in the enzymatic synthesis product. Therefore, there is an urgent need for an enzyme capable of highly specifically synthesizing EGCG-2G. Summary of the Invention

[0005] This invention relates to a recombinant sucrose phosphorylase or its functional fragment, its preparation method and application. The recombinant sucrose phosphorylase can synthesize EGCG-2G with high specificity using EGCG and sucrose as substrates, which greatly improves the synthesis efficiency of EGCG-2G.

[0006] On one hand, the present invention provides a recombinant sucrose phosphorylase or a functional fragment thereof having at least one amino acid sequence as shown in SEQ ID NO:1.

[0007] Those skilled in the art will understand that the complete amino acid sequence of the recombinant sucrose phosphorylase may be based on the sequence shown in SEQ ID NO:1, with one or more additional amino acid fragments (e.g., linker peptides, signal peptides, purification tags, or domain linkers) at its N-terminus, C-terminus, and / or the middle of the sequence; or, it may contain conserved substitutions for one or more non-essential amino acids in SEQ ID NO:1. Sequences containing such variations fall within the scope of protection of this invention, provided that these additions, deletions, or substitutions do not substantially affect the catalytic activity of the enzyme in generating EGCG-2G from sucrose and EGCG as substrates.

[0008] In some embodiments, the recombinant sucrose phosphorylase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence shown in SEQ ID NO:1.

[0009] On the other hand, the present invention provides a polynucleotide comprising a polynucleotide sequence encoding the above-mentioned recombinant sucrose phosphorylase.

[0010] In some embodiments, the polynucleotide comprises a polynucleotide sequence as shown in SEQ ID NO:2. Translation of the polynucleotide sequence shown in SEQ ID NO:2 within a host cell yields an enzyme protein having the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the polynucleotide comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the polynucleotide sequence shown in SEQ ID NO:2.

[0012] On the other hand, the present invention provides an expression vector comprising the aforementioned polynucleotides. In some embodiments, the recombinant polynucleotides of the expression vector are operatively linked to a control sequence. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0013] On the other hand, the present invention provides a host cell comprising the aforementioned polynucleotides or the aforementioned expression vector. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell. In some preferred embodiments, the host cell is a bacterial cell, such as Escherichia coli or Bacillus subtilis.

[0014] On the other hand, the present invention provides an intracellular extract of the aforementioned host cell. In some embodiments, the intracellular extract is selected from the group consisting of: cell debris, intracellular proteins, DNA, mRNA, mitochondria, cell nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, ribosomes, genomic DNA, tRNA, rRNA, non-coding RNA, small metabolic molecules, carbohydrates, lipids, ions, or any combination thereof.

[0015] On the other hand, the present invention provides a method for preparing the above-mentioned recombinant sucrose phosphorylase, comprising the following steps:

[0016] S1. Synthesize a recombinant DNA plasmid having the nucleotide sequence shown in SEQ ID NO:2;

[0017] S2. Transfect the DNA plasmid into competent cells, expand the culture, and then add an inducer to induce expression.

[0018] S3. The competent cells are broken, the intracellular extract is taken, and the recombinant sucrose phosphorylase is obtained by purification.

[0019] In some embodiments, the inducing agent is isopropyl-β-D-thiopyranoside.

[0020] In some embodiments, the working concentration of the inducer is 0.5 mmol / L.

[0021] On the other hand, the present invention provides the application of the above-mentioned recombinant sucrose phosphorylase in the synthesis of EGCG-2G.

[0022] On the other hand, the present invention provides a method for preparing EGCG-2G, comprising the step of reacting EGCG and sucrose as substrates under the catalysis of the above-mentioned recombinant sucrose phosphorylase.

[0023] In some embodiments, the concentration of EGCG is 2.5 g / L, the concentration of sucrose is 500 g / L, and the concentration of recombinant sucrose phosphorylase is 30 U / mL.

[0024] In this invention, the term EGCG-2G refers to (-)-epigallocatechin gallate-4',4''-O-α-D-dipyranoside.

[0025] The present invention has the following advantages and effects:

[0026] 1. The recombinant sucrose phosphorylase of the present invention is characterized by high activity and high specificity. It is specifically catalyzed for the formation of α-glycosidic bonds between polyphenols and glucosides, and can specifically catalyze the formation of glycosidic bonds between the 4' and 4'' positions of polyphenols and glucose. Therefore, the recombinant sucrose phosphorylase of the present invention can specifically catalyze the transglycosylation reaction of EGCG to obtain EGCG-2G with high purity.

[0027] 2. The recombinant sucrose phosphorylase of the present invention has the maximum conversion rate at a concentration of 30 U / mL, proving that the recombinant sucrose phosphorylase of the present invention can achieve a highly efficient catalytic reaction at low concentrations. Attached Figure Description

[0028] Figure 1 shows the conversion rate of EGCG glycosylation reaction by three different sucrose phosphorylases in Test Example 1 of this invention.

[0029] Figure 2 is a combined HPLC result of EGCG standard, reaction solution and EGCG-2G standard in test example 3 of the present invention. Detailed Implementation

[0030] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of the appended claims.

[0031] Unless otherwise specified, all experimental reagents and materials used in this invention are commercially available.

[0032] Example 1 Preparation of recombinant sucrose phosphorylase

[0033] 1. Construction of recombinant expression vectors

[0034] The recombinant expression vector DNA was synthesized according to the polynucleotide sequence shown in SEQ ID NO:2.

[0035] 2. Transfection and Induced Expression

[0036] 2.1 Transfection

[0037] Take 50 μL of competent BL21 cells, add 10 ng of recombinant expression vector DNA, incubate on ice for 30 min, then heat shock at 42℃ for 45 s, and immediately transfer to ice to cool for 2 min; then add 500 μL of LB medium, and culture at 37℃ with shaking for 1 h; finally, take an appropriate amount of bacterial culture and spread it on LB solid plates containing 50 μg / mL kanamycin, and incubate upside down at 37℃ for 12-16 h until single colonies appear.

[0038] 2.2 Induced Expression

[0039] BL21 strain containing recombinant expression vector DNA was selected and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37℃ and 220 rpm for 16 h. After culturing, 2% of the culture was inoculated into TB liquid medium containing 50 μg / mL kanamycin, and cultured at 37℃ and 220 rpm until the bacterial density reached OD500. 600 After reaching a concentration of ≥0.6, isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and induced for 24 h at 25 °C and 220 rpm.

[0040] 2.3 Extraction of recombinant sucrose phosphorylase

[0041] After induction, the bacterial culture was collected and centrifuged at 10,000 rpm for 15 min at 4°C. The cells were then washed twice with 50 mM K₂HPO₄ / KH₂PO₄ buffer (pH 6.5) and collected. The cells were resuspended in 50 mM phosphate buffer (pH 6.5) and sonicated to disrupt the cells (400 W, 2 s working time, 4 s interval, total 60 min). After centrifugation (10,000 rpm, 15 min, 4°C), the supernatant was collected to obtain recombinant sucrose phosphorylase SP. LM Crude enzyme solution.

[0042] 2.4 Enzyme activity assay of recombinant sucrose phosphorylase

[0043] Take 1 mL of 5% sucrose solution and 900 μL of 50 mM phosphate buffer (pH 6.5), add 100 μL of the above crude enzyme solution, react at 35 °C for 10 min, then immediately add 3 mL of DNS reagent and boil in a water bath for 15 min. Measure the absorbance at 540 nm and calculate the fructose content in the reaction solution. The amount of enzyme required to hydrolyze sucrose to produce 1 μmol of fructose per minute is defined as one unit (U) of enzyme activity for sucrase phosphorylation.

[0044] Comparative Example 1: Preparation of Sucrose Phosphorylase

[0045] The only difference from the preparation process in Example 1 is that the polynucleotide sequence of the recombinant expression vector DNA is as shown in SEQ ID NO:3. The final product contains sucrose phosphorylase SP. LR Crude enzyme solution. SP LR Derived from Lactobacillus reuteri, SP LR It can be obtained by purifying intracellular proteins of Lactobacillus reuteri. This invention aims to obtain a higher content of SP. LR However, when engineered bacteria were used for expression, the SPs prepared by the two methods mentioned above were... LR There was no significant difference in activity.

[0046] Test Example 1: EGCG transglycosylation reaction

[0047] Take 5 g / L of EGCG and 100 g / L of sucrose, and use 15 U / mL SP respectively. LM SP LR And commercially available sucrose phosphorylase (purchased from Nanjing Dulai Biotechnology) SP 商品化 As a catalyst, the reaction was carried out at pH 6.5, 25℃ and 220 rpm for 18 h. The product EGCG-Gs was collected and the EGCG glycosylation conversion rate was calculated.

[0048] The results are shown in Figure 1, SP LM The conversion rate of the catalytic EGCG glycosylation reaction was 76.89%, SP LR The conversion rate of the catalytic EGCG glycosylation reaction was 57.23%, SP 商品化 The conversion rate of the catalytic EGCG glycosylation reaction was 64.76%, proving that the SP of this invention... LM It has a higher conversion rate.

[0049] Additionally, take 2.5 g / L of EGCG and 500 g / L of sucrose, and use 30 U / mL SP. LM As a catalyst, the reaction was carried out at pH 6.5, 25℃, and 220 rpm for 18 h. The product EGCG-Gs was collected, and the EGCG glycosylation conversion rate was calculated. The results showed that SP LM The conversion rate of the catalytic EGCG glycosylation reaction was 97.46%.

[0050] Test Example 2: Polyphenol transglycosylation reaction

[0051] Take 15 g / L of tea polyphenols and 500 g / L of sucrose, and use 30 U / mL SP. LM As a catalyst, the reaction was carried out at pH 6.5, 25℃ and 220 rpm for 18 h. The product, tea polyphenols, was inoculated with glucosides, and the glycosylation conversion rate of tea polyphenols was calculated.

[0052] The results showed that SP LM The conversion rate of the catalytic tea polyphenol glycosylation reaction was 73.4%, indicating that the SP of this invention... LM It exhibits excellent catalytic performance and high conversion efficiency in polyphenol glycosylation reactions.

[0053] Test Example 3: Identification of Product Structure

[0054] Take SP from test case 1 LM The catalytically obtained EGCG-Gs were separated into reaction products using a Sephadex LH-20 column (25 mm × 400 mm). After loading, sugars (sucrose, fructose, and glucose) were removed by elution with distilled water, followed by elution with 70% (v / v) ethanol. The eluent was then concentrated using a rotary evaporator at 40 °C.

[0055] Using Morphling WD-C18 ( Purification was performed using a 10 μm column (Nanjing HeXi Biotechnology CO.,LTD). The chromatographic conditions were as follows:

[0056] The column temperature was 30°C, the detection wavelength was 280 nm, the flow rate was 30 mL / min, and the injection volume was 1 mL. The mobile phase consisted of methanol (A) and 0.25% (v / v) aqueous acetic acid (B). The gradient elution program was as follows: 0-15 min: 85% B phase, 15-20 min: 85%-75% B phase, 20-60 min: 75% B phase, with a total run time of 60 min.

[0057] The HPLC results are shown in Figure 2. Figure 2 shows that the characteristic peak of EGCG was not observed in the chromatogram of the reaction solution, but the maximum characteristic peak appeared at a retention time of 42 min, which is consistent with the retention time of the characteristic peak of EGCG-2G.

[0058] The largest fraction was identified by NMR, and the results are as follows:

[0059] 1H NMR (400 MHz, MeOD) δ 6.92 (s, 2H), 6.56 (s, 2H), 6.01 – 5.93 (m,2H), 5.59 – 5.51 (m, 1H), 5.13 (d, J = 3.7 Hz, 1H), 5.01 (s, 1H), 4.99 (d, J= 3.8 Hz, 1H), 4.17 (dddd, J = 9.8, 7.3, 5.0, 2.4 Hz, 2H), 3.87 – 3.78 (m,4H), 3.72 (dddd, J = 11.8, 9.1, 5.1 Hz, 2H), 3.55 (ddd, J = 9.5, 3.9, 2.7 Hz,2H), 3.46 – 3.36 (m, 2H), 3.00 (dd, J = 17.4, 4.6 Hz, 1H), 2.86 (dd, J =17.5, 2.5 Hz, 1H).

[0060] 13 C NMR (100 MHz, MeOD) δ 166.9, 158.0, 157.9, 157.0, 151.9, 151.5,139.4, 137.1, 135.1, 127.8, 110.3, 107.0, 105.6, 104.9, 99.3, 96.7, 95.9,78.3, 75.2, 75.1, 75.0, 75.0, 73.5, 73.3, 71.1, 70.9, 70.4, 62.3, 62.2, 26.8.

[0061] The above results prove that SP in test case 1 LM The EGCG-Gs obtained by catalysis are EGCG-2G, and EGCG-2G has extremely high purity, proving that SP LM The synthesis of EGCG-2G is highly specific.

[0062] In this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0063] Although specific embodiments of the invention have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of the invention. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A recombinant sucrose phosphorylase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A polynucleotide encoding the recombinant sucrose phosphorylase as described in claim 1.

3. An expression vector comprising the polynucleotide as described in claim 2.

4. A host cell comprising the polynucleotide of claim 2 or the expression vector of claim 3.

5. An intracellular extract of a host cell as described in claim 4, characterized in that, Including the recombinant sucrose phosphorylase as described in claim 1.

6. A method for preparing recombinant sucrose phosphorylase as described in claim 1, characterized in that, Includes the following steps: S1. Synthesize a recombinant DNA plasmid having the nucleotide sequence shown in SEQ ID NO:2; S2. Transfect the DNA plasmid into competent cells BL21, expand the culture, and then add an inducer to induce expression, wherein the inducer is isopropyl-β-D-thiogalactopyranoside; S3. Disrupt the competent cells BL21, take the intracellular extract, and purify it to obtain the recombinant sucrose phosphorylase.

7. The preparation method according to claim 6, characterized in that, The working concentration of the inducer is 0.5 mmol / L.

8. The application of the recombinant sucrose phosphorylase as described in claim 1 in the synthesis of EGCG-2G, wherein the EGCG-2G is (-)-epigallocatechin gallate-4',4''-O-α-D-dipyranoside.

9. A method for preparing EGCG-2G, characterized in that, The reaction includes a step of using EGCG and sucrose as substrates and reacting under the catalysis of the recombinant sucrose phosphorylase as described in claim 1; wherein the EGCG-2G is (-)-epigallocatechin gallate-4',4''-O-α-D-dipyranoside.

Images