Alpha-glucosidase mutant and application thereof in synthesis of alpha-arbutin

Abstract

The invention provides an alpha-glucosidase mutant and application thereof in synthesis of alpha-arbutin, and the alpha-glucosidase mutant comprises the following steps: maltose and hydroquinone are taken as substrates, the recombinant bacteria are taken as a catalyst, catalytic reaction is carried out under a heating condition, and the alpha-arbutin is obtained. According to the synthesis method, maltose and hydroquinone are used as reaction substrates, and alpha-arbutin is generated through whole-cell catalysis; meanwhile, the sequence of the alpha-glucosidase mutant is integrated onto a pRSEDuet-1 expression vector and transferred into escherichia coli BL21 (DE3) for expression, a reaction system of recombinant bacteria is optimized, the optimal catalytic condition is determined, the yield of alpha-arbutin under the optimal condition is 24.8 g / L, the yield can reach 95.6% and is improved by 1.2 times compared with that of an original strain, and the alpha-glucosidase mutant has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to an alpha-glucosidase mutant and application thereof in synthesizing alpha-arbutin. Background Art

[0002] Arbutin is a natural active substance isolated from various genera of 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 opposite in space and are diastereomers of each other. At present, arbutin is widely used in the field of cosmetics as a safe and stable whitening agent. It has antioxidant, antibacterial, anti-inflammatory, anti-tumor and other pharmacological activities. Studies have shown that the whitening effect of α-arbutin is about 10 times that of the isomer β-arbutin, and it has higher safety, so it is more widely used in cosmetics.

[0003] At present, there are three main methods for producing arbutin, plant extraction, chemical synthesis and biosynthesis. Among them, the production of arbutin by plant extraction has problems such as long plant growth cycle, complex extraction process and low yield; the synthesis of arbutin by chemical synthesis has the disadvantages of harsh reaction conditions, poor stereoselectivity and low yield. In recent years, biosynthesis has gradually become a popular research direction for the synthesis of arbutin due to its advantages such as simple and mild reaction conditions and economic and environmentally friendly production process. Among the schemes currently using biosynthesis to industrially produce arbutin, there are mainly sucrose phosphorylase and amylosucrase, with a conversion rate of up to 99.05% (Chinese invention patent with application number CN202210959740.2), while the conversion rate of α-glucosidase using maltose as the 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 proposes 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 achieved as follows: The present invention provides an α-glucosidase mutant, wherein 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 is mutated to glutamic acid (mutant T279E), and the amino acid sequence is shown in SEQ ID NO: 3; (2) The threonine at position 279 is mutated to lysine (mutant T279K), and the amino acid sequence is shown in SEQ ID NO: 4.

[0006] The present invention also provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the α-glucosidase mutant.

[0007] The present invention also provides a recombinant vector, which contains the nucleic acid molecule.

[0008] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant vector.

[0009] Furthermore, the expression vector of the recombinant bacteria is pRSEDuet-1, and the starting strain of the recombinant bacteria is Escherichia coli BL21 (DE3).

[0010] The present invention also provides an application of an alpha-glucosidase mutant in synthesizing alpha-arbutin.

[0011] The present invention also provides a method for synthesizing α-arbutin using the α-glucosidase mutant, comprising the following steps: Maltose and hydroquinone are used as substrates, the recombinant bacteria are used as catalysts, and a catalytic reaction is carried out under heating conditions to obtain the alpha-arbutin.

[0012] Furthermore, the temperature of the heating step is 37°C, the catalytic reaction time is 3 hours, and the OD 600 For 18-25 years.

[0013] Furthermore, the molar ratio of hydroquinone to maltose is 0.5-1.5:12.

[0014] Compared with the prior art, the present invention has the following advantages: The synthesis method described in 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, and transferred into Escherichia coli BL21 (DE3) for expression, and the reaction system of the recombinant bacteria is optimized to determine the optimal catalytic conditions. Under the optimal conditions, the output 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The HPLC chromatogram of α-arbutin produced by α-glucosidase as described in Example 2 of the present invention; Figure 2 This is a high performance liquid chromatogram of the production of α-arbutin catalyzed by the mutant T279E described in Example 2 of the present invention; Figure 3This is a high performance liquid chromatogram of the production of α-arbutin catalyzed by the mutant T279K described in Example 2 of the present invention. DETAILED DESCRIPTION

[0016] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0017] The present invention will be described in detail below with reference to the embodiments.

[0018] Example 1

[0019] The gene sequence of α-glucosidase SEQ ID NO.2 was cloned into plasmid pRSEDuet-1 to construct a recombinant plasmid. The upstream primers and downstream primers are shown in Table 1. The α-glucosidase (AglA) was simulated using Swiss Model online software to obtain the spatial structure of α-glucosidase. Through substrate channel simulation and molecular docking, relevant sites that may affect the catalytic performance of the enzyme were screened near the catalytic pocket of the enzyme. Finally, the neutral amino acid threonine at position 279 of the amino acid sequence was mutated into acidic amino acid E or basic amino acid K to obtain α-glucosidase mutants T279E and T279K.

[0020] Table 1 Primer sequences

[0021] The restriction site is Bm Ⅰ and Hind III.

[0022] The PCR system is shown in Table 2.

[0023] Table 2 PCR system

[0024] 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 insulation at 8 °C.

[0025] The obtained recombinant plasmid was digested with DpnI restriction endonuclease. 10 μL of the digested product was directly transformed into E. coli DH5α competent 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 E. coli BL21 (DE3) for expression.

[0026] Example 2

[0027] The mutant was used to catalyze the production of α-arbutin.

[0028] LB medium: tryptone: 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L; TB liquid culture medium: Liquid A: 12 g / L tryptone, 24 g / L yeast powder, 0.4% glycerol; Liquid B: 2.31 g dipotassium hydrogen phosphate and 12.54 g potassium dihydrogen phosphate dissolved in 100 mL deionized water. After sterilization, liquid A and liquid B are mixed.

[0029] 1. Bacteria activation Inoculate the frozen recombinant E. coli into 10 mL LB medium, add 5 μL 50 mg / L kanamycin (Kana), and culture overnight at 37 ℃ and 180 rpm for 16 h. Take 2 mL of activated bacterial solution and transfer it into a conical flask containing 400 mL TB liquid medium, add an appropriate amount of 50 mg / L kanamycin (Kana) to the medium, and culture it in a constant temperature shaking incubator at 37 ℃ and 180 rpm for 2-3 hours.

[0030] 2. Inducible expression After 3 hours of cultivation, 1 M inducer (IPTG) with a final concentration of 20 mM was added to the bacterial solution to induce bacteria to produce protein, and the induction was carried out overnight in a constant temperature shaking incubator at 18°C ​​and 120 rpm for 16 hours.

[0031] 3. Bacterial liquid collection Pour the expanded cultured bacteria into a centrifuge cup, place in a refrigerated centrifuge, set to 8000 rpm, 20 min, 4°C centrifuge, pour out the supernatant, and collect the bacteria precipitated at the bottom of the centrifuge tube.

[0032] 4. Whole-cell catalysis Resuspend the bacterial slurry in 50 mM phosphate buffer and adjust the OD by spectrophotometer. 600 to 20, the final volume is 20 mL, and then hydroquinone and maltose are added in a molar ratio of 1:12, specifically 0.22 g of hydroquinone and 8.64 g of maltose. The hydroquinone is added twice with an interval of one hour. The reaction conditions are 37 °C and 200 r / min. Samples are taken every hour and the reaction is stopped after 3 h.

[0033] 5. High performance liquid chromatography analysis Take 100 μL of the reaction solution, dilute it ten times with deionized water, use a C18 chromatographic column, column temperature: 40 °C; mobile phase: 8% acetonitrile and 92% aqueous phosphoric acid containing 4‰; flow rate: 0.8 mL / min.

[0034] Calculation of the yield of α-arbutin, the yield of α-arbutin

[0035] Wherein: M1 is the molar amount of α-arbutin generated; M2 is the molar amount of hydroquinone added to the reaction solution.

[0036] like Figure 1-3 It can be seen that the catalytic conversion efficiency of the α-glucosidase mutants T279E and T279K provided by the present invention is calculated. 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 close to the original enzyme. Figure 2 The peak area of ​​hydroquinone in Figure 1 and Figure 3 It can also be explained that the yield of T279E is significantly improved, which shows that it has industrial application value.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should 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 18-25.

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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