Sucrose phosphorylase mutant and application thereof in synthesis of alpha-matrix chlorogenoside

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a sucrose phosphorylase mutant and application thereof in synthesis of alpha-mogroside. Streptococcus parasanguinis The present application is obtained by simultaneously mutating amino acid multiple sites of 13th, 206th and 243rd of wild-type sucrose phosphorylase derived from Parahaemolyticus (Parahaemolyticus), and constructing recombinant Escherichia coli by using the sucrose phosphorylase mutant, and then fermenting the recombinant sucrose phosphorylase to significantly improve the efficiency of producing alpha-mogroside by using sucrose and hydroquinone as raw materials. The conversion rate of producing alpha-mogroside by using sucrose and hydroquinone as raw materials can reach 90%, and the product concentration reaches 123 g / L, so the present application has strong industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a sucrose phosphorylase mutant and its application in the synthesis of α-arbutin. Background Technology

[0002] Arbutin, also known as arbutin or hydroquinone glucoside, is a natural active polyphenol compound that can be extracted from various plants. Arbutin has two isomers: α-arbutin (4-hydroxyphenyl-α-glucopyranoside) and β-arbutin (4-hydroxyphenyl-β-glucopyranoside). Studies have found that α-arbutin has higher activity in inhibiting human tyrosinase activity and less cytotoxicity, making it more valuable than β-arbutin and naturally occurring arbutin, and it has been widely used in various skin-whitening products.

[0003] Alpha-arbutin, a popular product in the skin whitening industry, is highly sought after by consumers, and its market demand is growing daily. Enzymatic methods and whole-cell conversion methods are the main production methods for alpha-arbutin. However, the inventors discovered that the catalytic efficiency of enzymes in existing technologies is relatively low, which limits the production of alpha-arbutin. Summary of the Invention

[0004] To overcome the above problems, this invention provides a sucrose phosphorylase mutant and its application in α-arbutin synthesis. This invention modifies the enzyme molecule using directed evolution theory, thereby successfully obtaining a sucrose phosphorylase mutant with high catalytic efficiency. Based on the above research results, this invention is thus completed.

[0005] Specifically, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a sucrose phosphorylase mutant obtained by mutating serine at position 13 to alanine, aspartic acid at position 206 to cysteine, and phenylalanine at position 243 to glutamic acid in the wild-type sucrose phosphorylase as shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention provides a nucleic acid molecule that encodes the sucrose phosphorylase mutant described in the first aspect.

[0008] In this invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0009] A third aspect of the present invention provides an expression cassette comprising the nucleic acid molecule described in the second aspect.

[0010] In this invention, the expression cassette includes a promoter capable of initiating transcription of the nucleic acid molecule and the nucleic acid molecule. Further, it may also include a termination sequence and an enhancer sequence, etc.

[0011] In a fourth aspect, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0012] The recombinant expression vector of the present invention is obtained by ligating a vector to the nucleic acid molecule. The vector can be constructed using any method known in the art or can be commercially available. For example, in one or more embodiments of the present invention, the vector is plasmid pET-28a, which can be expressed in *Escherichia coli*.

[0013] A fifth aspect of the present invention provides a recombinant bacterium comprising the nucleic acid molecule described in the second aspect or the recombinant expression vector described in the fourth aspect.

[0014] The recombinant bacteria can specifically be recombinant Escherichia coli.

[0015] A sixth aspect of the present invention provides the use of the sucrose phosphorylase mutant described in the first aspect or the recombinant bacteria described in the fifth aspect in the catalytic synthesis of α-arbutin.

[0016] A seventh aspect of the present invention provides a method for synthesizing α-arbutin, comprising:

[0017] Using the recombinant bacteria described in the fifth aspect above as a catalyst, wet bacterial cells obtained through induced culture or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, and sucrose and hydroquinone as substrates, α-arbutin is synthesized in a liquid environment.

[0018] The liquid environment is preferably water.

[0019] The amount of catalyst used is 5-25 g / L (preferably 20 g / L) based on the total weight of wet bacterial cells, the final concentration of sucrose is 0.1-5 M (preferably 1 M), and the final concentration of hydroquinone is 0.05-1 M (preferably 0.5 M).

[0020] In this invention, the wet bacterial cells can be prepared by the following method: the recombinant bacteria are inoculated into LB liquid medium containing kanamycin and cultured at 37 ℃ and 200 rpm for 12 h. Then, the bacteria are inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1-2% and cultured at 37 ℃ and 200 rpm until the bacterial OD600 reaches 0.6-0.8. IPTG is added to a final concentration of 0.1 mM and induced at 25 ℃ for 16 h. After induction culture, the bacteria are centrifuged at 4 ℃ and 8000 rpm for 20 min, the supernatant is discarded, and the precipitate is collected to obtain the wet bacterial cells.

[0021] Furthermore, the crude enzyme solution is obtained by extracting the wet bacterial cells through ultrasonic disruption.

[0022] The beneficial effects of one or more of the above technical solutions:

[0023] The above technical solution involves the treatment of Streptococcus parahaemolyticus (S. parahaemolyticus) Streptococcus parasanguinis Simultaneous mutations at multiple amino acid sites (13, 206, and 243) of wild-type sucrose phosphorylase yielded a sucrose phosphorylase mutant. Recombinant Escherichia coli was constructed and fermented to produce recombinant sucrose phosphorylase, significantly improving its efficiency in producing α-arbutin from sucrose and hydroquinone. Experimental verification showed that the conversion rate of α-arbutin from sucrose and hydroquinone could reach 90%, with a product concentration of 123 g / L, thus demonstrating strong potential for industrial application. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 This is a flowchart of the reaction process for the synthesis of α-arbutin catalyzed by the sucrose phosphorylase mutant in Example 5 of the present invention. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. The DNA sequencing results of each sucrose phosphorylase mutant in the embodiments are completely consistent with the expected designed mutations.

[0029] The culture medium formulations used in the following examples are as follows: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0030] LB plates: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 18 g / L agar.

[0031] The concentration of the product α-arbutin was determined by high performance liquid chromatography (HPLC). The analytical method was as follows: a UV detector was used, and the mobile phase consisted of 95% water and 5% methanol, containing 0.1% acetic acid. v / v The chromatographic conditions were set as follows: detection wavelength 282 nm, injection volume 10 µL, column temperature 30 ℃, and flow rate 0.5 mL·min. -1 .

[0032] Sample preparation: Take 10 μL of the sample after the reaction is completed, dilute it 10 times with water, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0033] Example 1

[0034] Through library mining, a strain originating from *Streptococcus parahaemolyticus* was screened. Streptococcus parasanguinous The sucrose phosphorylase, NCBI accession number WP_350012079.1, was synthesized in its entirety by Nanjing GenScript Biotech Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

[0035] Primers F1 and R1 were designed based on the nucleotide sequence shown in SEQ ID NO.1, and the following were introduced into the primers respectively. EcoR I and Xhosa I. Restriction enzyme cleavage site.

[0036] F1: 5'-GAATTCATGCCATTCAAAATAAAACAATGCT-3' (SEQ ID NO. 3);

[0037] R1: 5'-CTCGAGTTAGTCTTTATTTTGACGGGTAATCACG-3' (SEQ ID NO. 4);

[0038] Using pET-28a plasmid as an expression vector, Escherichia coli was constructed E. coli BL21(DE3) / pET28a.

[0039] Construction of expression plasmid: Under the initiation of primers F1 and R1, using the target gene as a template, amplification was performed using high-fidelity DNA polymerase to obtain the sucrose phosphorylase gene sequence. After sequencing, the expression plasmid was then used... EcoR I and Xhosa The amplified fragment was processed with restriction endonuclease I (TaKaRa) and then ligated with the pET-28a vector fragment treated with the same restriction endonuclease using T4 DNA ligase (TaKaRa) to construct the plasmid pET28a-SPase.

[0040] Preparation of competent cells: Escherichia coli in glycerol cryovials E. coli BL21(DE3) was activated by streaking on antibiotic-free LB solid medium. Single colonies with good morphology were selected and placed in 5 mL of antibiotic-free LB liquid medium and incubated overnight at 37°C and 220 rpm. 1% of the bacterial culture was then transferred to 50 mL of LB liquid medium and incubated until the bacterial OD value reached 90%. 600 The concentration was between 0.4 and 0.6. The cells were collected by centrifugation at 8000 rpm at 4 °C. The cell pellet was resuspended in pre-cooled 0.1 mol / L CaCl2 aqueous solution and then placed on ice for 50 min. The cells were then centrifuged at 5000 rpm at 4 °C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in pre-cooled 0.1 mol / L CaCl2 aqueous solution containing 15% glycerol. 100 μL was dispensed into each centrifuge tube and stored at -80 °C.

[0041] Construction of recombinant Escherichia coli: First, bacteria stored at -80 ℃... E. coli BL21(DE3) competent cells were placed on ice for 10 min, then 5 µL of pET28a-SPase was added in a clean bench, and the cells were incubated on ice for 30 min, followed by a 42 °C water bath for 90 s, an ice bath for 2 min, and then 400 µL of LB medium was added. The cells were incubated at 37 °C and 200 rpm for 45 min. The cells were then plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37 °C for 8–12 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant E. coli containing the recombinant plasmid expression were screened for these cells. E. coli BL21 (DE3) / pET28a-SPase.

[0042] Example 2

[0043] Wet bacterial cells containing the sucrose phosphorylase gene: The recombinant Escherichia coli obtained in Example 1 were used respectively. E. coli BL21(DE3) / pET28a-SPase was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 °C and 200 rpm for 12 h, then inoculated with 1% ( v / vThe inoculum was injected into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and incubated at 37 °C and 200 rpm until the bacterial cell OD reached its maximum. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM, induce culture at 25 ℃ for 16 h, centrifuge at 4 ℃ and 8000 rpm for 20 min, discard the supernatant, and the precipitate is the wet bacterial cell containing sucrose phosphorylase.

[0044] Example 3

[0045] Constructed using Example 2 E. coli BL21(DE3) / pET28a-SPase is the starting strain.

[0046] The complex model was obtained by modifying the sucrose phosphorylase crystal structure using directed evolution theory and docking it with homology modeling. The amino acid residues surrounding the substrate sucrose and hydroquinone in this structure were analyzed. Site-directed saturation mutagenesis was performed at sites 13, 58, 206, and 375. Based on protein folding free energy calculations, stability-enhancing sites P2K, M223I, F243E, and N387Q were also selected for site-directed mutagenesis. Primer design is shown in Table 1. Since site-directed saturation mutagenesis was performed at sites 13, 58, 206, and 375, degenerate bases were introduced when designing primers for these mutation sites. N represents any base (any one of A, T, C, or G); K represents G or T; and M represents A or C.

[0047] Mutant PCR system (50 μL): 10×KOD Buffer 5 μL, dNTPs 5 μL, MgSO4 3 μL, primers 1.5 μL each, template 1 μL, KOD polymerase 1 μL, add ddH2O to 50 μL.

[0048] PCR conditions were as follows: 94 °C pre-denaturation for 2 min, followed by 30 cycles: 98 °C for 10 s, 68 °C for 3 min 30 s, and a final extension at 68 °C for 10 min. PCR results were verified by DNA agarose gel electrophoresis. After successful verification, the DNA was digested with DpnI enzyme at 37 °C for 1 hour, followed by inactivation at 65 °C for 1 min. The digested PCR products were then transformed into... E. coli In BL21 (DE3).

[0049] Table 1. Primer design for site-directed mutagenesis of sucrose phosphorylase

[0050]

[0051] Example 4

[0052] Screening of sucrose phosphorylase gene mutant libraries: Single clones were picked from the plates obtained in Example 3 and inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance. The cultures were incubated at 37 °C and 200 rpm for 12 h, then inoculated with 2% ( v / v Inoculate the culture medium into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and incubate at 37 °C and 200 rpm until the bacterial cell OD reaches zero. 600 When the concentration reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM, induce at 25 ℃ for 16 h, centrifuge at 4 ℃ and 8000 rpm for 15 min, and the precipitate is the wet bacterial cells containing the sucrose phosphorylase gene mutant library.

[0053] (1) Initial screening:

[0054] Preparation of reaction solution (1 mL): Sucrose with a final concentration of 1 M, hydroquinone with a final concentration of 0.4 M, and catalyst dosage of 10 g / L based on the total weight of wet bacterial cells. Purified water was used as the reaction medium. Reaction conditions: After reacting for 24 hours on a shaker at 30 ℃ and 200 rpm, 10 μL of the reaction sample was taken, diluted 10-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 2.

[0055] Table 2 Initial screening reaction results

[0056]

[0057] (2) Secondary screening

[0058] The positive strains obtained from the initial screening were subjected to secondary screening. A reaction solution (1 mL) was prepared using sucrose at a final concentration of 1 M, hydroquinone at a final concentration of 0.4 M, and a catalyst dosage of 10 g / L based on the total wet cell weight. Purified water was used as the reaction medium. The reaction conditions were: 24 hours at 30 ℃ and 200 rpm in a reactor. After the reaction, samples were taken, diluted 10-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 3.

[0059] Table 3 Results of the secondary screening reaction

[0060]

[0061] Example 5

[0062] Application of sucrose phosphorylase mutants in the catalytic synthesis of α-arbutin

[0063] The recombinant sucrose phosphorylase mutant with the highest activity obtained in Example 4 E. coliThe BL21 (DE3) / pET28a-SPase-S13A-D206C-F243E strain was used for scale-up reactions. The reaction system (20 mL) consisted of 1 M sucrose, 0.5 M hydroquinone, and a catalyst concentration of 20 g / L based on the total wet cell weight. Purified water was used as the reaction medium. The reaction process is as follows: Figure 1 As shown, the reaction conditions were: reaction at 30 ℃ and 200 rpm on a shaker for 24 hours, followed by a 10-fold dilution, filtration through a 0.22 μm filter, and HPLC analysis. The HPLC results showed that the α-arbutin concentration was 123 g / L, with a conversion rate of 90%.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sucrose phosphorylase mutant, characterized in that, It was obtained by mutating serine at position 13 to alanine, aspartic acid at position 206 to cysteine, and phenylalanine at position 243 to glutamic acid in wild-type sucrose phosphorylase, as shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the sucrose phosphorylase mutant of claim 1.

3. An expression box, characterized in that, The expression cassette comprises the nucleic acid molecule of claim 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.

5. A recombinant bacterium, characterized in that, The recombinant bacteria comprise the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 4.

6. The recombinant bacteria as described in claim 5, characterized in that, The recombinant bacteria is recombinant Escherichia coli.

7. The use of the sucrose phosphorylase mutant of claim 1 or the recombinant bacteria of any one of claims 5-6 in the catalytic synthesis of α-arbutin.

8. A method for synthesizing α-arbutin, characterized in that, include: Using the wet bacterial cells obtained by induced culture of the recombinant bacteria as described in any one of claims 5-6, or the crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a catalyst, and sucrose and hydroquinone as substrates, α-arbutin is synthesized in a liquid environment.

9. The method as described in claim 8, characterized in that, The liquid environment is water.

10. The method as described in claim 8, characterized in that, The amount of catalyst used is 5-25 g / L based on the total weight of wet bacterial cells, the final concentration of sucrose is 0.1-5 M, and the final concentration of hydroquinone is 0.05-1 M.

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