An α-glucosidase mutant and its application in the synthesis of α-arbutin

Abstract

The present invention provides an α-glucosidase mutant and its application in the synthesis of α-arbutin, including the following steps: using maltose and hydroquinone as substrates, and using the recombinant bacterium as a catalyst, carrying out a catalytic reaction under heating conditions to obtain the α-arbutin. The synthesis method of the present invention uses maltose and hydroquinone as reaction substrates and uses whole-cell catalysis to generate α-arbutin; at the same time, the sequence of the α-glucosidase mutant is integrated into the pRSEDuet-1 expression vector, transferred into Escherichia coli BL21(DE3) for expression, and the reaction system of the recombinant bacterium is optimized to determine the optimal catalytic conditions. Under the optimal conditions, the yield of α-arbutin is 24.8 g / L, and the yield can reach 95.6%, which is 1.2 times higher than that of the original strain, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to an α-glucosidase mutant and its application in the synthesis of α-arbutin. Background Art

[0002] Arbutin is a naturally active substance isolated from various plants. It is a glycoside compound derived from hydroquinone and has two isomers, namely α-arbutin and β-arbutin. The oxygen glycosidic bonds of the two are exactly opposite in space and are epimers of each other. At present, arbutin is widely used in the cosmetics field as a safe and stable whitening agent, and it has pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. Some studies have shown that the whitening effect of α-arbutin is about 10 times that of its isomer β-arbutin, and it also has higher safety, so it is more widely used in cosmetics.

[0003] Currently, there are mainly three methods for producing arbutin: plant extraction method, chemical synthesis method, and biosynthesis method. Among them, the plant extraction method for producing arbutin has problems such as long plant growth cycle, complex extraction process, and low yield; the chemical synthesis method for synthesizing arbutin has disadvantages such as harsh reaction conditions, poor stereoselectivity, and low yield. In recent years, due to the advantages of simple and mild reaction conditions and economic and environmental protection in the production process, the biosynthesis method has gradually become a popular research direction for synthesizing arbutin. In the current industrial production of arbutin using the biosynthesis method, sucrose phosphorylase and amylosucrase are mainly used, and the highest conversion rate can reach 99.05% (Chinese invention patent with application number CN202210959740.2), while the conversion rate of α-glucosidase using maltose as a glycosyl donor is 55.6%, which is still at a relatively low level. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and provides an α-glucosidase mutant and its application in the synthesis of α-arbutin.

[0005] To achieve the above object, the technical solution of the present invention is realized as follows:

[0006] The present invention provides an α-glucosidase mutant, and the amino acid sequence of the α-glucosidase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO: 1. The α-glucosidase mutant is one of the following:

[0007] (1) Threonine at position 279 is mutated to glutamic acid (mutant T279E), and the amino acid sequence is as shown in SEQ ID NO: 3;

[0008] (2)The threonine at position 279 is mutated to lysine (mutant T279K), and the amino acid sequence is as shown in SEQ ID NO: 4.

[0009] The present invention also provides a nucleic acid molecule that encodes the α-glucosidase mutant.

[0010] The present invention also provides a recombinant vector that contains the nucleic acid molecule.

[0011] The present invention also provides a recombinant bacterium that contains the recombinant vector.

[0012] Furthermore, the expression vector of the recombinant bacterium is pRSEDuet-1, and the starting strain of the recombinant bacterium is Escherichia coli BL21(DE3).

[0013] The present invention also provides an application of the α-glucosidase mutant in the synthesis of α-arbutin.

[0014] The present invention also provides a method for synthesizing α-arbutin using the α-glucosidase mutant, which includes the following steps:

[0015] Using maltose and hydroquinone as substrates, and using the recombinant bacterium as a catalyst, a catalytic reaction is carried out under heating conditions to obtain the α-arbutin.

[0016] Furthermore, the temperature of the heating step is 37 °C, the time of the catalytic reaction is 3 hours, and the OD 600 is 18 - 25.

[0017] Furthermore, the molar ratio of hydroquinone to maltose is 0.5 - 1.5:12.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The synthesis method of the present invention uses maltose and hydroquinone as reaction substrates, and uses whole-cell catalysis to generate α-arbutin; at the same time, the sequence of the α-glucosidase mutant is integrated into the pRSEDuet-1 expression vector, transferred into Escherichia coli BL21(DE3) for expression, and the reaction system of the recombinant bacterium is optimized to determine the optimal catalytic conditions. Under the optimal conditions, the yield of α-arbutin is 24.8 g / L, and the yield rate can reach 95.6%, which is 1.2 times higher than that of the original strain, and has good application prospects. Brief Description of the Drawings

[0020] Figure 1 It is the high-performance liquid chromatography diagram of the α-glucosidase catalyzing the generation of α-arbutin described in Example 2 of the present invention;

[0021] Figure 2 The high performance liquid chromatography (HPLC) chromatogram of the mutant T279E catalyzing the production of α-arbutin described in Example 2 of the present invention;

[0022] Figure 3 The high performance liquid chromatography (HPLC) chromatogram of the mutant T279K catalyzing the production of α-arbutin described in Example 2 of the present invention. Detailed implementation manners

[0023] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0024] The present invention will be described in detail below in conjunction with examples.

[0025] Example 1

[0026] The gene sequence SEQ ID NO.2 of α-glucosidase was cloned into the plasmid pRSEDuet-1 to construct a recombinant plasmid. The upstream primer and downstream primer are shown in Table 1; the spatial structure of α-glucosidase (AglA) was obtained by simulating it with the Swiss Model online software; through substrate channel simulation and molecular docking, relevant sites that may affect the enzymatic catalytic performance were screened near the catalytic pocket of the enzyme. Finally, the neutral amino acid threonine at the 279th position of the amino acid sequence was mutated into the acidic amino acid E or the basic amino acid K to obtain the α-glucosidase mutants T279E and T279K.

[0027] Table 1 Primer sequences

[0028]

[0029] The restriction enzyme sites are BamH Ⅰ and Hind Ⅲ.

[0030] The PCR system is shown in Table 2.

[0031] Table 2 PCR system

[0032]

[0033] PCR amplification conditions: pre-denaturation at 98 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 64 °C for 1 min, extension at 68 °C for 10 min, 15 cycles, and incubation at 8 °C.

[0034] The obtained recombinant plasmid was digested with DpnI restriction endonuclease to remove the plasmid template. 10 μL of the digested product was directly transformed into competent Escherichia coli DH5α cells. The recombinant cells carrying the mutant plasmid were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing. The mutant plasmid with correct sequencing was transformed into Escherichia coli BL21(DE3) for expression.

[0035] Example 2

[0036] The mutant was used to catalyze the production of α-arbutin.

[0037] LB medium: Tryptone: 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0038] TB liquid medium: Solution A: Tryptone 12 g / L, yeast extract 24 g / L, glycerol 0.4%, Solution B: 2.31 g of dipotassium hydrogen phosphate and 12.54 g of potassium dihydrogen phosphate dissolved in 100 mL of deionized water. After sterilization, Solution A and Solution B were mixed.

[0039] 1. Strain activation

[0040] The cryopreserved recombinant Escherichia coli was inoculated into 10 mL of LB medium, and 5 μL of 50 mg / L kanamycin (Kana) was added. The culture was incubated overnight at 37 °C and 180 rpm for 16 h. 2 mL of the activated bacterial solution was transferred into a conical flask containing 400 mL of TB liquid medium, and an appropriate amount of 50 mg / L kanamycin (Kana) was added to the medium. The culture was placed in a constant temperature shaking incubator at 37 °C and 180 rpm for 2 - 3 hours.

[0041] 2. Induced expression

[0042] After 3 hours of culture, 1 M inducer (IPTG) with a final concentration of 20 mM was added to the bacterial solution to induce protein production in the bacteria. The culture was induced overnight at 18 °C and 120 rpm for 16 h.

[0043] 3. Bacterial solution collection

[0044] The bacteria after expanded culture were poured into a centrifuge cup and placed in a refrigerated centrifuge. The centrifuge was set to centrifuge at 8000 rpm for 20 min at 4 °C. The supernatant was discarded, and the bacterial cells precipitated at the bottom of the centrifuge tube were collected.

[0045] 4. Whole-cell catalysis

[0046] The bacterial pellet was resuspended with 50 mM phosphate buffer, and the OD was adjusted using a spectrophotometer 600To 20, with a final volume of 20 mL, then hydroquinone and maltose were added in a molar ratio of 1:12. Specifically, 0.22 g of hydroquinone and 8.64 g of maltose were added. Hydroquinone was added in two portions with an interval of one hour. The reaction conditions were 37 °C and 200 r / min. Samples were taken every hour, and the reaction was stopped after 3 h.

[0047] 5. High-performance liquid chromatography analysis

[0048] 100 μL of the reaction solution was taken, diluted ten times with deionized water, and a C18 chromatographic column was used. Column temperature: 40 °C; the mobile phase was 8% acetonitrile and 92% aqueous phosphoric acid solution containing 4‰; flow rate: 0.8 mL / min.

[0049] Calculation of the yield of α-arbutin, the yield of α-arbutin

[0050] In the formula: M1 is the molar amount of α-arbutin generated; M2 is the molar amount of hydroquinone added to the reaction solution.

[0051] As Figures 1 - 3 It can be seen that the catalytic conversion efficiency of the α-glucosidase mutants T279E and T279K provided by the present invention. Through calculation, compared with the original enzyme, the yield of T279E is 95.6%, the yield of the original enzyme is 75.85%, and the yield of T279K is 78.97%, which is similar to the original enzyme. Figure 2 The peak area of the substrate hydroquinone in Figure 1 And Figure 3 Significantly decreased, which can also illustrate the significant increase in the yield of T279E. It can be seen that it has industrial application value.

[0052] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An α-glucosidase mutant, characterized in that: The amino acid sequence of the α-glucosidase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the α-glucosidase mutant is one of the following: (1) Threonine at position 279 mutated to glutamic acid; (2) Threonine at position 279 mutated to lysine.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the α-glucosidase mutant according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant vector according to claim 3.

5. The recombinant bacterium according to claim 4, characterized in that: The expression vector of the recombinant bacteria is pRSEDuet-1, and the starting strain of the recombinant bacteria is Escherichia coli BL21 (DE3).

6. Use of the α-glucosidase mutant according to claim 1 in synthesizing α-arbutin.

7. A method for synthesizing α-arbutin using the α-glucosidase mutant of claim 1, characterized in that: The steps include: Using maltose and hydroquinone as substrates and the recombinant bacteria according to claim 4 or 5 as a catalyst, a catalytic reaction is carried out under heating conditions to obtain the α-arbutin.

8. The synthesis method according to claim 7, characterized in that: The temperature of the heating step was 37°C, the catalytic reaction time was 3 hours, and the OD 600 For 18-25 years.

9. The synthesis method according to claim 7, characterized in that: The molar ratio of hydroquinone to maltose is 0.5-1.5:12.

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