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

Abstract

The application relates to the technical field of genetic engineering, in particular to a sucrose phosphorylase mutant and application thereof in synthesis of alpha-maronia. Streptococcus parasanguinis The application is characterized in that the histidine at the 152th position of the wild-type sucrose phosphorylase from Streptococcus sanguis is mutated into threonine, and the isoleucine at the 336th position is mutated into proline, so that the sucrose phosphorylase mutant is obtained, the catalytic activity and stability of the sucrose phosphorylase are significantly improved. When sucrose and hydroquinone are used as substrates, the yield of alpha-maronia is increased by more than 40% compared with the wild type, and the catalytic reaction time can be greatly shortened.

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 bearberry extract, is a natural glycoside compound belonging to the hydroquinone glycoside class. It is composed of glucose and hydroquinone linked by a glycosidic bond and originates from plants such as bearberry, wheat, and pear, primarily found in the peels and leaves of these plants. Arbutin can inhibit tyrosinase activity, blocking melanin synthesis and thus reducing skin pigmentation, achieving a skin-whitening effect. As a skin-brightening agent, it is widely used in various cosmetics and is a key ingredient in many whitening cosmetics. Furthermore, arbutin possesses antioxidant, antibacterial, and anti-inflammatory biological activities, making it suitable for applications in the pharmaceutical field. Because glucose and hydroquinone can form both α- and β-glycosidic bonds, arbutin is also classified into α-arbutin and β-arbutin. Compared to β-arbutin, α-arbutin offers advantages in both whitening efficacy and safety. α-Arbutin has a 10-fold stronger inhibitory effect on tyrosinase than β-arbutin, and it has no inhibitory effect on normal human cells, making it safer and more effective.

[0003] Arbutin is mainly obtained through plant extraction, chemical synthesis, and biological methods. Plant extraction of arbutin is cumbersome and has a low extraction rate. Chemical methods suffer from low catalytic efficiency, poor stereoselectivity of the product, harsh reaction conditions, and the generation of numerous byproducts. Biological methods utilize enzyme catalysis to synthesize α-arbutin from sucrose and hydroquinone. These methods produce fewer byproducts, a single, easily separated and extracted product, and cause less environmental pollution, making them the most commonly used method for producing α-arbutin.

[0004] α-Arbutin is a glycosylated derivative of hydroquinone and can be synthesized via glycosylation by sucrose phosphorylase. However, wild-type sucrose phosphorylase has drawbacks such as low expression levels and poor transglycosylation activity, resulting in low α-arbutin yield. Therefore, in order to catalyze the synthesis of α-arbutin more efficiently, there is an urgent need for sucrose phosphorylase with enhanced transglycosylation activity. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a sucrose phosphorylase mutant and its application in the synthesis of α-arbutin.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a sucrose phosphorylase mutant in which the histidine at position 152 of the wild-type sucrose phosphorylase, as shown in SEQ ID NO: 2, is mutated to threonine and / or the isoleucine at position 336 is mutated to proline.

[0008] A second aspect of the invention provides a gene encoding a sucrose phosphorylase mutant as described in the first aspect.

[0009] A third aspect of the invention provides an expression cassette comprising the gene described in the second aspect.

[0010] A fourth aspect of the present invention provides a recombinant expression vector comprising the gene described in the second aspect.

[0011] A fifth aspect of the present invention provides a recombinant bacterium comprising the genes described in the second aspect.

[0012] A sixth aspect of the present invention provides a transgenic cell line comprising the genes described in the second aspect.

[0013] A seventh aspect of the present invention provides the use of the sucrose phosphorylase mutant described in the first aspect, the encoding gene described in the second aspect, or the recombinant bacteria described in the fifth aspect in the catalytic synthesis of α-arbutin.

[0014] An eighth aspect of the present invention provides a method for synthesizing α-arbutin, comprising:

[0015] α-Arbutin was obtained by using wet bacterial cells obtained through induced culture of genetically engineered bacteria with sucrose phosphorylase mutant, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, or immobilized enzyme as catalysts, and sucrose and hydroquinone as substrates.

[0016] The genetically engineered bacteria are constructed by introducing the sucrose phosphorylase mutant described in the first aspect into a host bacterium.

[0017] In one or more embodiments, the host bacterium is a bacterium; preferably, the bacterium is Escherichia coli.

[0018] In one or more embodiments, the amount of catalyst used is 10-25 g / L based on the total weight of wet cells or immobilized enzymes, the final concentration of substrate sucrose is 0.8-1.2 mol / L, and the final concentration of hydroquinone is 0.3-0.5 mol / L.

[0019] In one or more embodiments, the reaction temperature is 28~35 °C; the reaction time is 18~36 h.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention utilizes the method of processing bacteria derived from Streptococcus sanguinis (S. serovar sero Streptococcus parasanguinis A sucrose phosphorylase mutant was obtained by mutating histidine at position 152 to threonine and isoleucine at position 336 to proline, significantly improving the catalytic activity and stability of the sucrose phosphorylase. When synthesizing α-arbutin using sucrose and hydroquinone as substrates, the yield was increased by more than 40% compared to the wild type, and the catalytic reaction time was significantly shortened. Attached Figure Description

[0022] 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.

[0023] Figure 1 The reaction process curves for the synthesis of α-arbutin catalyzed by recombinant sucrose phosphorylase mutant and parent material. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation 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.

[0025] 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.

[0026] α-Arbutin is a glycosylated derivative of hydroquinone and can be synthesized via glycosylation using sucrose phosphorylase. However, wild-type sucrose phosphorylase has drawbacks such as low expression levels, poor thermostability, and low transglycosylation activity, resulting in low α-arbutin yield. Therefore, to catalyze the synthesis of α-arbutin more efficiently, there is an urgent need for sucrose phosphorylases with enhanced transglycosylation activity.

[0027] To overcome the above problems, the present invention provides a sucrose phosphorylase mutant and its application in the synthesis of α-arbutin.

[0028] This invention obtains a sucrose phosphorylase mutant by mutating histidine at position 152 to threonine and isoleucine at position 336 to proline in the wild-type sucrose phosphorylase derived from *Streptococcus parasanguini*. This significantly improves the catalytic activity and stability of the sucrose phosphorylase. When synthesizing α-arbutin using sucrose and hydroquinone as substrates, the yield is increased by more than 40% compared to the wild type, and the catalytic reaction time is significantly shortened.

[0029] To enable those skilled in the art to better 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.

[0030] Example 1

[0031] (1) Construction of recombinant expression vector and engineered bacteria:

[0032] Through library mining, a sucrose phosphorylase SPase-Sp derived from Streptococcus parasanguini was screened, with NCBI accession number WP_049499206.1. The whole genome was synthesized 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.

[0033] Sucrose phosphorylase SPase-Sp and pET All 28a(+) plasmids were digested with NcoI and EcoRI restriction enzymes, and the products were ligated using T4 ligase to obtain the ligation products.

[0034] Stored at -80℃ E. coli BL21(DE3) competent cells were incubated on ice at 0 °C for 10 min, then 5 µL of the ligation product was added in a clean bench, followed by an ice bath at 0 °C for 30 min, a heat shock at 42 °C for 90 s, an ice bath at 0 °C for 2 min, and then 600 µL of LB medium was added. The cells were cultured at 37 °C and 200 rpm for 1 h. The culture was then spread on LB agar plates containing 50 μg / mL kanamycin resistance and cultured at 37 °C for 8–12 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant *E. coli* containing the recombinant expression plasmid pET28a-SPase-Sp were obtained. E. coli BL21(DE3) / / pET28a-SPase-Sp.

[0035] Example 2

[0036] The recombinant Escherichia coli obtained in Example 1 E. coliBL21(DE3) / / pET28a-SPase-Sp was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 ℃ and 200 rpm for 12 h. Then, it was inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 ℃ and 200 rpm until the bacterial OD reached the target cell count. 600 When the concentration reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM. After induction culture at 25 °C for 16 h, the mixture is centrifuged at 4 °C and 10,000 rpm for 10 min. The supernatant is discarded, and the precipitate is collected to obtain recombinant Escherichia coli containing sucrose phosphorylase. E. coli Wet cells of BL21(DE3) / / pET28a-SPase-Sp.

[0037] Example 3

[0038] Establishment of a sucrose phosphorylase gene mutation library:

[0039] Recombinant Escherichia coli expressing sucrose phosphorylase constructed according to Example 2 E. coli BL21(DE3) / / pET28a-SPase-Sp is the starting strain.

[0040] Modification was performed using directed evolution theory, based on the crystal structure of sucrose phosphorylase obtained through homology modeling. Based on calculations, site-directed mutagenesis was performed at sites N18, Y24, D58, R90, V128, H152, G226, Q284, A311, and I366, resulting in mutations of N18G, Y24Q, D58F, R90N, V128L, H152T, G226P, Q284V, A311V, and I366P, respectively.

[0041] The mutant PCR system (50 μL) consisted of: 25 μL of 2×PhantaMax buffer, 1 μL of dNTPs, 2 μL each of the upper and lower mutant primers, 1 μL of template (starting strain), and ddH2O added to a final volume of 50 μL.

[0042] The PCR conditions were as follows: 98 °C pre-denaturation for 5 min, followed by 30 cycles: 98 °C for 30 s, 58 °C for 15 s, 72 °C for 1 min and 30 s, and finally 72 °C final extension for 10 min.

[0043] The PCR results were verified by DNA agarose gel electrophoresis: the PCR product was digested with DpnI enzyme, inactivated at 37℃ for 1 h, 200 rpm, 65℃ for 1 min, and the PCR product was then transformed by heat shock into E. coli. E. coliBL21(DE3) was activated, incubated at 37 ℃ and 200 rpm for 1 h, and then spread on LB plates containing 50 μg / mL kanamycin resistance. The plates were incubated upside down at 37 ℃ overnight.

[0044] The primers are as follows:

[0045] Sp-N18-F (SEQ ID NO: 3), Sp-N18-R (SEQ ID NO: 4), Sp-Y24-F (SEQ ID NO: 5), Sp-Y24-R (SEQ ID NO: 6), Sp-D58-F (SEQ ID NO: 7), Sp-D58-R (SEQ ID NO: 8), Sp-R90-F (SEQ ID NO: 9), Sp-R90-R (SEQ ID NO: 10), Sp-V128-F (SEQ ID NO: 11), Sp-V128-R (SEQ ID NO: 12), Sp-H152-F (SEQ ID NO: 13), Sp-H152-R (SEQ ID NO: 14), Sp-G226-F (SEQ ID NO: 15), Sp-G226-R (SEQ ID NO: 16), Sp-Q284-F (SEQ ID NO: 17), Sp-Q284-R (SEQ ID NO: 18), Sp-A311-F (SEQ ID NO: 19), Sp-A311-R (SEQ ID NO: 20), Sp-I366-F (SEQ ID NO: 21), Sp-I366-R (SEQ ID NO: 22).

[0046] DNA sequencing revealed that the site-directed mutations N18G, Y24Q, D58F, R90N, V128L, H152T, G226P, Q284V, A311V, and I366P were completely consistent with the intended mutations.

[0047] Example 4

[0048] Screening of sucrose phosphorylase gene mutation libraries:

[0049] Single colonies were picked from the plates obtained in Example 3 and inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance. The culture was carried out at 37 °C and 200 rpm for 12 h. Then, a 1% (v / v) inoculum was added to fresh LB liquid medium containing 50 μg / mL kanamycin resistance and cultured at 37 °C and 200 rpm until the bacterial OD reached the target cell count. 600When the concentration reaches 0.6~0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. After induction culture at 25 ℃ for 16 h, centrifuge at 4 ℃ and 10000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the wet bacterial cells containing the sucrose phosphorylase gene mutant library.

[0050] (1) Initial screening:

[0051] Preparation of reaction solution (200 μL): The reaction solution has a final concentration of 0.4 mol / L hydroquinone and 1.0 mol / L sucrose. The amount of catalyst is 15 g / L based on the total weight of wet bacterial cells. The reaction solution is prepared using purified water as the reaction medium.

[0052] Reaction conditions: The reaction was carried out in a reactor at 30 ℃ and 200 rpm for 24 h. After the reaction was completed, 10 μL of the final sample was taken, diluted 10 times, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 1.

[0053] Table 1 Initial screening reaction results

[0054]

[0055] After initial screening, the α-arbutin concentrations obtained by mutants H152T and I366P in the preparation of α-arbutin from hydroquinone and sucrose were higher than those of the parent mutant, indicating that the catalytic activity of these two mutants was improved. Subsequent experiments were conducted using mutants H152T and I366P.

[0056] (2) The strains obtained from the initial screening are screened again.

[0057] The mutant H152T+I366P was constructed using the method described in Example 3. DNA sequencing showed that the mutant's DNA sequencing results were completely consistent with the intended design mutation.

[0058] Re-screening and preparation of reaction solution (200 μL): The reaction solution has a final concentration of 0.4 mol / L hydroquinone and 1.0 mol / L sucrose. The amount of catalyst is 15 g / L based on the total weight of wet bacterial cells. The reaction solution is prepared using purified water as the reaction medium.

[0059] Reaction conditions: After reacting for 24 h in a reactor at 30 ℃ and 200 rpm, 10 μL of the reaction sample was taken after the reaction was completed, diluted 10 times, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The results are shown in Table 2.

[0060] Table 2 Results of the secondary screening reaction

[0061]

[0062] After further screening, it was confirmed that the catalytic activities of all the mutants recorded in Table 2 were significantly improved. Among them, the mutant H152T+I366P exhibited the highest catalytic activity. Therefore, the mutant H152T+I366P was selected for subsequent experiments.

[0063] Example 5

[0064] The recombinant sucrose phosphorylase mutant with the highest activity obtained in Example 4 E. coli BL21(DE3) / / pET28a-SPase-Sp-H152T+I366P was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 9 h. This seed culture was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 3.5%. The prepared medium was added to the fermenter, and the inlet and outlet were sealed tightly. The inoculation port was left open, and the fermenter, along with the prepared lactose inducer, was placed in an autoclave at 115°C for 30 min for sterilization. The sterilized fermenter was then fitted with the inoculation port and attached to the operating system. Condensate and air were introduced (the inlet pipe should be fitted with a sterilizing membrane). The outlet was inserted below the liquid level in the conical flask. When the autoclave temperature dropped to 37°C, a flame ring was placed over the inoculation port, and the cultured seed culture was inoculated into the fermenter. The fermentation was carried out at 37°C and 500 rpm for approximately 3–4 h, and the bacterial density (OD) was determined. 600 To achieve the desired 6-8 saturation levels, the fermenter temperature was lowered to 25 °C, and lactose at a final concentration of 16 g / L was added as an inducer. The fermentation was then incubated at 25 °C and 500 rpm for 12 h. The fermented broth was centrifuged at 8000 rpm for 10 min to obtain the sucrose phosphorylase mutant. E. coli Wet cells of BL21(DE3) / / pET28a-SPase-Sp-H152T+I366P.

[0065] The fermenter culture medium consists of: 45 g tryptone, 36 g yeast extract, 30 g sodium chloride, 4.08 g potassium dihydrogen phosphate, 45 g glycerol, 6.84 g dipotassium hydrogen phosphate trihydrate, 15 g ammonium sulfate, 1.125 g magnesium sulfate, and 4 g defoamer. Distilled water is added to a final volume of 3 L for dissolution.

[0066] The catalyst dosage was 15 g / L based on the total weight of wet bacterial cells. The reaction solution, with a final concentration of 0.4 mol / L hydroquinone and 1.0 mol / L sucrose, and purified water as the reaction medium, comprised a total reaction volume of 1 L. Reaction conditions: The reaction was carried out at 30 ℃ and 200 rpm for 24 h. After the reaction was complete, 10 μL of the final sample was taken, diluted 10-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The reaction progress curve is shown below. Figure 1As shown, after the reaction, the concentration of α-arbutin was 104.55 g / L, the conversion rate was 96%, and the time to reach the peak yield of α-arbutin was 20 h.

[0067] Comparative Example 1

[0068] The recombinant Escherichia coli obtained in Example 1 E. coli BL21(DE3) / / pET28a-SPase-Sp (parent culture) was used as a control, and wet cells were obtained according to the same method as in Example 5. The catalyst dosage was 15 g / L based on the total weight of the wet cells before high-pressure homogenization. The reaction solution consisted of 0.4 mol / L hydroquinone and 1.0 mol / L sucrose, with purified water as the reaction medium, and a total volume of 1 L. Reaction conditions: The reaction was carried out at 30 ℃ and 200 rpm for 24 h. After the reaction, 10 μL of the final sample was taken, diluted 10-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The reaction progress curve is shown below. Figure 1 As shown, after the reaction, the concentration of α-arbutin was 51.12 g / L, the conversion rate was 47%, and the time to reach the peak yield of α-arbutin was 24 h. The reaction time of the mutant was significantly shortened compared to that of the mutant.

[0069] 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 is caused by a mutation of histidine at position 152 of the wild-type sucrose phosphorylase, as shown in the amino acid sequence SEQ ID NO: 2, to threonine.

2. The gene encoding the sucrose phosphorylase mutant of claim 1.

3. An expression box, characterized in that, It contains the gene described in claim 2.

4. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.

5. A recombinant bacterium, characterized in that, It contains the gene described in claim 2.

6. A transgenic cell line, characterized in that, It contains the gene described in claim 2.

7. The use of the sucrose phosphorylase mutant of claim 1, the encoding gene of claim 2, or the recombinant bacteria of claim 5 in the catalytic synthesis of α-arbutin.

8. A method for synthesizing α-arbutin, characterized in that, include: α-Arbutin was obtained by using wet bacterial cells obtained through induced culture of genetically engineered bacteria with sucrose phosphorylase mutant, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, or immobilized enzyme as catalysts, and sucrose and hydroquinone as substrates. The genetically engineered bacteria are constructed by introducing the sucrose phosphorylase mutant of claim 1 into a host bacterium.

9. The method as described in claim 8, characterized in that, The host bacterium is a bacterium; the bacterium is Escherichia coli.

10. The method as described in claim 8, characterized in that, The amount of catalyst used is 10-25 g / L based on the total weight of wet cells or immobilized enzymes, the final concentration of substrate sucrose is 0.8-1.2 mol / L, and the final concentration of hydroquinone is 0.3-0.5 mol / L. The reaction temperature is 28~35 ℃; the reaction time is 18~36 h.

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