Cyclodextrin glucosyltransferase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a cyclodextrin glucosyltransferase mutant and its application. The cyclodextrin glucosyltransferase mutant of the present invention is obtained by subjecting the amino acid sequence of the cyclodextrin glucosyltransferase shown in SEQ ID NO.1 to the following mutation: the aspartic acid at position 251 is mutated to alanine. The cyclodextrin glucosyltransferase mutant has a high enzymatic activity, which is 1.2 times that of the wild type. It is applied to the modification of hesperidin, and the substrate conversion rate is as high as 95.3%. After the hesperidin is modified, it can be enzymatically hydrolyzed by glucosidase to obtain a high content of α-glucosyl hesperidin.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a cyclodextrin glucosyltransferase mutant and application thereof. Background Art

[0002] Hesperidin is a flavonoid found in high concentrations in citrus peel and preharvest fruit, ranging from 5% to 8%. Hesperidin has antioxidant, anticancer, and antitumor properties, as well as anti-circulatory, anti-inflammatory, and anti-allergic effects, and enhances immunity. However, its poor water solubility (1g / 50L) limits its applications. Modification of hesperidin could expand its applications in food, cosmetics, and medicine.

[0003] α-Glucosyl hesperidin is a compound composed of glucose and hesperidin linked by a glycosidic bond. Its solubility in water is approximately 100,000 times higher than that of hesperidin, allowing for a wider range of applications, including cosmetics, food, and pharmaceuticals. α-Glucosyl hesperidin's excellent water solubility allows for easier dissolution and absorption in the human digestive system, thereby increasing its bioavailability and enabling it to better exert its physiological effects. It can also be easily added to various aqueous products, such as lotions, oral solutions, and injections, significantly broadening its application.

[0004] Currently, there are numerous reports on the modification of hesperidin both domestically and internationally, primarily categorized as chemical and enzymatic methods. Chemical modifications, such as sulfonation, metal chelation, acidolysis, and methylation, suffer from poor selectivity, difficult reaction control, the need for high temperature and high pressure treatment, difficulty in separating reaction products, and environmental pollution. Enzymatic modification, due to its high enzyme specificity, mild conditions, and the absence of "three wastes," has become the primary method for modifying hesperidin. Cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19) is a multifunctional enzyme that catalyzes three transglycosylation reactions (disproportionation, cyclization, and coupling) as well as hydrolysis. While some CGTases have been reported for the modification of hesperidin, they suffer from low substrate conversion rates. Summary of the Invention

[0005] The purpose of the present invention is to provide a cyclodextrin glucosyltransferase mutant, which has high enzyme activity and is modified with hesperidin to have a high conversion rate.

[0006] The technical solutions of the present invention are as follows:

[0007] A cyclodextrin glucosyltransferase mutant is obtained by subjecting the cyclodextrin glucosyltransferase with the amino acid sequence shown in SEQ ID NO. 1 to the following mutation: aspartic acid at position 251 is mutated to alanine, and is named Asp251Ala.

[0008] The cyclodextrin glucosyltransferase with an amino acid sequence as shown in SEQ ID NO. 1 is derived from alkalophilic Bacillus sp. G1-2004.

[0009] SEQ ID NO.1:

[0010] .

[0011] A gene encoding a cyclodextrin glucosyltransferase mutant, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0012] SEQ ID NO.2:

[0013]

[0014] The present invention provides application of the cyclodextrin glucosyltransferase mutant in the modification of hesperidin.

[0015] The present invention also provides a method for preparing α-glucosyl hesperidin using the cyclodextrin glucosyltransferase mutant, comprising the following steps:

[0016] (1) Using hesperidin as substrate, β-cyclodextrin as glycosyl donor, and a mutant cyclodextrin glucosyltransferase as catalyst, a glycosylation reaction was carried out in a bioreactor by using a countercurrent addition method of hesperidin and β-cyclodextrin to obtain an oligomeric glucosyl hesperidin mixture;

[0017] (2) Glucosidase is added to the oligosaccharide hesperidin mixture for enzymatic hydrolysis to obtain α-glucosyl hesperidin.

[0018] In step (1), the bioreactor temperature is 40-50°C, the rotation speed is 100-200 rpm, and the pH is 8-9.

[0019] The specific method for countercurrent addition of hesperidin and β-cyclodextrin is as follows: a crude enzyme solution of a mutant cyclodextrin glucosyltransferase is added to a bioreactor. The hesperidin and β-cyclodextrin reaction solutions are prepared and added to two feeding bottles connected to the bioreactor. The feeding rate is set at 100 mL / 8-12 hours. The glycosylation reaction is continued for 12-16 hours after the feeding is completed. After the reaction is completed, the solids are filtered to obtain a mixture of oligomeric glucosyl hesperidin. In addition, HCl solution is added to a third feeding bottle to maintain the pH of the bioreactor system.

[0020] The added amounts of hesperidin solution, β-cyclodextrin solution, cyclodextrin glucosyltransferase mutant crude enzyme solution and glucosidase can be adjusted according to actual conditions.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The cyclodextrin glucosyltransferase mutant Asp251Ala provided by the present invention has a high enzymatic activity, which is 1.2 times that of the wild type. When applied to hesperidin modification, the substrate conversion rate reaches as high as 95.3%. After the hesperidin is modified, it is enzymatically hydrolyzed by glucosidase to obtain a high content of α-glucosyl hesperidin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC chart of the crude enzyme solution of cyclodextrin glucosyltransferase mutant Asp251Ala catalyzing hesperidin for 24 hours;

[0024] Figure 2 The HPLC chart of oligomeric glucosylhesperidin cleaved by glucosidase for 3 h. DETAILED DESCRIPTION

[0025] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings. The experimental methods described in the following examples are all conventional methods unless otherwise specified; if no specific techniques or conditions are specified in the experiments, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; the reagents and materials described are all commercially available unless otherwise specified.

[0026] Example 1

[0027] In this example, the substrate hesperidin was first docked to cyclodextrin glucosyltransferase using Autodock Vina to identify the catalytic site of the cyclodextrin glucosyltransferase. Theoretically, the Asp251Ala mutant demonstrated higher enzymatic activity. The enzymatic activity of the Asp251Ala mutant was then measured and compared to that of the wild-type cyclodextrin glucosyltransferase.

[0028] The amino acid sequence of the cyclodextrin glucosyltransferase (CDGT) shown in SEQ ID NO: 1 (NCBI accession number AAV38118) and the CDGT mutant Asp251Ala were synthesized by a biotechnology company and cloned into the NcoI / BamHI region of the expression vector pET22b to generate recombinant plasmids. The recombinant plasmids were transformed into the expression host E. coli BL21 and verified by PCR and sequencing. The correct Asp251Ala expression and the wild-type engineered strain were then preserved.

[0029] The Asp251Ala and wild-type engineered strains were inoculated into 25 mL of LB liquid medium containing a final concentration of 50 μg / mL Kana, and cultured overnight at 37°C and 200 rpm. Then, the inoculation volume was transferred to 25 mL of TB liquid medium containing a final concentration of 50 μg / mL Kana at a 1% inoculum. When the bacterial concentration OD 600 When the pH reached 0.6, IPTG solution was added to a final concentration of 1 mmol / L, and the cells were cultured at 25°C, 200 rpm, for 20 hours. After the culture period, the cells were centrifuged at 8000 rpm at 4°C for 5 minutes, the supernatant discarded, and the cells were suspended in 0.01 mol / L PBS buffer to obtain a bacterial suspension. The suspension was then ultrasonically disrupted in an ice-water bath for 20 minutes, and the suspension was centrifuged at 8000 rpm at 4°C for 10 minutes. The supernatant was collected to obtain a crude enzyme solution containing Asp251Ala and wild-type cyclodextrin glucosyltransferase.

[0030] The crude enzyme solution was tested for enzyme activity, and the results showed that the enzyme activity of the mutant Asp251Ala crude enzyme solution was 8640 U / mL, and the enzyme activity of the wild-type cyclodextrin glucosyltransferase was 7200 U / mL. The enzyme activity of the mutant Asp251Ala crude enzyme solution was 1.2 times that of the wild-type.

[0031] The enzyme activity test method is as follows: accurately weigh 2g of soluble starch and dissolve it in 50mL of glycine-NaOH solution with a pH of 7. After heating it in a microwave to dissolve, let it cool at room temperature, take 900μL and add it to a test tube, then add 100μL of crude enzyme solution. After accurately reacting in a 50℃ water bath for 10 minutes, add 1M HCl solution to terminate the reaction. Finally, add 4mL of 4-fold diluted methyl orange solution and let it stand at room temperature for 20 minutes to develop color. The control group first added 1M HCl to terminate the reaction and then added 100μL of crude enzyme solution. The other experimental conditions were the same. The light absorbance of the reaction solution was then measured at 507nm using a microplate reader. The amount of enzyme required to produce 1μmoL cyclodextrin per unit time was defined as one unit of enzyme activity.

[0032] Example 2

[0033] In this example, a crude enzyme solution of the mutant Asp251Ala was used to prepare α-glucosyl hesperidin. The preparation method includes the following steps:

[0034] (1) Preparation of the reaction solution of hesperidin and β-cyclodextrin

[0035] Add 1g of 95% hesperidin to 100mL of ultrapure water, then add solid sodium hydroxide and stir until the hesperidin is completely dissolved. Stop adding sodium hydroxide, and the solution will turn dark brown to obtain a hesperidin solution. Add 3g of β-cyclodextrin to 100mL of ultrapure water and heat in a microwave oven until dissolved to obtain a β-cyclodextrin solution.

[0036] (2) Hesperidin modification

[0037] 800 mL of the crude enzyme solution of mutant Asp251Ala prepared in Example 1 was added to a 2 L fermentor, and the reaction temperature of the fermentor was set to 45°C, the rotation speed was set to 200 rpm, and the pH was set to 9; the hesperidin and β-cyclodextrin prepared in step (1) were added to two feeding bottles connected to the fermentor, and the feeding rate was set to 100 mL / 12 h; in addition, 1 mol / L HCl solution was added to the third feeding bottle to maintain the pH of the system in the bioreactor;

[0038] After 12 hours of feeding, the glycosylation reaction was continued for 12 hours. After the reaction was completed, the solids were filtered to remove the oligomeric glucosyl hesperidin mixture. At this time, the sample was subjected to HPLC analysis, and the mass of α-glucosyl hesperidin was measured to be 25.1% of the mass of the oligomeric glucosyl hesperidin mixture. The conversion rate of substrate hesperidin was 95.3%. Figure 1 .

[0039] (3) Preparation of α-glucosylhesperidin

[0040] 50 U of glucosidase was added to the oligomeric glucosyl hesperidin mixture obtained in step (2) and enzymolysis was performed for 3 h to obtain a mixture. At this time, the sample was subjected to HPLC determination, and the mass of α-glucosyl hesperidin was measured to be 60.5% of the mass of the mixture. Figure 2 .

Claims

1. A cyclodextrin glucosyltransferase mutant, characterized in that: The cyclodextrin glucosyltransferase is obtained by subjecting the amino acid sequence of the cyclodextrin glucosyltransferase shown in SEQ ID NO. 1 to the following mutation: aspartic acid at position 251 is mutated to alanine.

2. The cyclodextrin glucosyltransferase mutant according to claim 1, wherein: The cyclodextrin glucosyltransferase with an amino acid sequence as shown in SEQ ID NO. 1 is derived from alkalophilic Bacillus.

3. A gene encoding the cyclodextrin glucosyltransferase mutant according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

4. Use of the cyclodextrin glucosyltransferase mutant according to claim 1 in the modification of hesperidin.

5. A method for preparing α-glucosylhesperidin using the cyclodextrin glucosyltransferase mutant according to claim 1, characterized in that: The following steps are involved: (1) Using hesperidin as substrate, β-cyclodextrin as glycosyl donor, and a mutant cyclodextrin glucosyltransferase as catalyst, a glycosylation reaction was carried out in a bioreactor by using a countercurrent addition method of hesperidin and β-cyclodextrin to obtain an oligomeric glucosyl hesperidin mixture; (2) Glucosidase is added to the oligosaccharide hesperidin mixture for enzymatic hydrolysis to obtain α-glucosyl hesperidin.

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