Cyclodextrin glucosyltransferase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a cyclodextrin glucosyltransferase mutant and application thereof. The cyclodextrin glucosyltransferase mutant disclosed by the invention is obtained by performing the following mutations on cyclodextrin glucosyltransferase with an amino acid sequence shown as SEQ ID NO.1: aspartic acid at the 251st site is mutated into alanine. The cyclodextrin glucosyltransferase mutant is high in enzyme activity which is 1.2 times that of a wild type, the cyclodextrin glucosyltransferase mutant is applied to hesperidin modification, the substrate conversion rate reaches up to 95.3%, and high-content alpha-glucosyl hesperidin can be obtained after hesperidin modification and enzymolysis of glucosidase.

Description

Technical Field

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

[0002] Hesperidin is one of the flavonoid substances with relatively high content in citrus peel residues, mainly existing in citrus peels and pre-harvest citrus fruit drop, with a content of 5% - 8%. Hesperidin has functions such as antioxidant, anti-cancer, anti-tumor, preventing circulatory system diseases, anti-inflammatory, anti-allergic and enhancing the body's immune ability, etc., with relatively high added value. However, due to the poor water solubility of hesperidin (1 g / 50 L), its application fields are restricted. Modifying hesperidin can expand its applications in the fields of food, cosmetics and medicine.

[0003] α-Glucosyl hesperidin is a compound formed by connecting glucose and hesperidin through a glycosidic bond. The solubility of α-glucosyl hesperidin in water is about 100,000 times higher than that of hesperidin, and it has a wider application range, such as in the fields of cosmetics, food, medicine, etc. The good water solubility of α-glucosyl hesperidin enables it to be more easily dissolved and absorbed in the human digestive system, thereby improving its bioavailability, enabling it to better exert its physiological effects, and can also be more conveniently added to products in various aqueous systems, such as lotion, oral liquid, injection, etc., greatly broadening its application range.

[0004] At present, there are many reports on the modification of hesperidin at home and abroad, mainly divided into chemical modification and bio-enzymatic modification. Chemical modifications such as sulfonation, metal chelation, acidolysis, methylation, etc. have disadvantages such as poor selectivity, difficult reaction control, requiring high temperature and high pressure treatment, difficult separation of reaction products and environmental pollution. Bio-enzymatic modification has become the main method for modifying hesperidin due to the strong specificity of enzymes, mild conditions and no generation of "three wastes". Cyclodextrin glucosyltransferase (abbreviated as CGTase, EC2.4.1.19) is a multi-functional enzyme, which can catalyze three transglycosylation reactions (disproportionation reaction, cyclization reaction and coupling reaction) and hydrolysis reaction. Some CGTases have been reported to be used for modifying hesperidin, but there are disadvantages such as low substrate conversion rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a cyclodextrin glucosyltransferase mutant with relatively high enzyme activity and high conversion rate when applied to modify hesperidin.

[0006] The technical solution of the present invention is as follows: A cyclodextrin glucosyltransferase mutant is obtained by mutating the cyclodextrin glucosyltransferase with the amino acid sequence shown in SEQ ID NO.1 as follows: Aspartic acid at position 251 is mutated to alanine, named Asp251Ala.

[0007] The cyclodextrin glucosyltransferase with the amino acid sequence shown in SEQ ID NO.1 is derived from Bacillus sp. G1-2004.

[0008] SEQ ID NO.1: MNDLNDFLKTISLSFIFFLLLSLPTVAEADVTNKVNYSKDVIYQVVTDRFSDGNPGNNPSGAIFSQNCIDLHKYCGGDWQGIIDKINDGYLTDLGITALWISQPVENVYALHPSGYTSYHGYWARDYKKTNPYYGNFDDFDRLMSTAHSNGIKVIMDFTPNHSSPALETNPNYVENGAIYDNGTLLGNYSNDQQNLFHHNGGTDFSSYEDSIYRNLYDLADYDLNNTVMDQYLKESIKFWLDKGIDGIRVDAVKHMSEGWQTSLMSEIYSHKPVFTFGEWFLGSGEVDPQNHHFANESGMSLLDFQFGQTIRNVLKDRTSNWYDFNEMITSTEKEYNEVIDQVTFIDNHDMSRFSVGSSSNRQTDMALAVLLTSRGVPTIYYGTEQYVTGGNDPENRKPLKTFDRSTNSYQIISKLASLRQTNSALGYGTTTERWLNEDIYIYERTFGNSIVLTAVNSSNSNQTITNLNTSLPQGNYTDELQQRLDGNTITVNANGAVNSFQLRANSVAVWQVSNPSTSPLIGQVGPMMGKSGNTITVSGEGFGDERGSVLFDSTSSEIISWSNTEISVKVPNVAGGYYDLSVVTAANLKSPTYKEFEVLSGNQVSVRFGVNNATTSPGTNLYIVGNVSELGNWDADKAIGPMFNQVMYQYPTWYYDISVPAGKNLEYKYIKKDQNGNVVWQSGNNRTYTSPTTGTDTVMINW.

[0009] A coding gene of a cyclodextrin glucosyltransferase mutant, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:

[0011] The present invention provides the application of the cyclodextrin glucosyltransferase mutant in the modification of hesperidin.

[0012] The present invention also provides a method for preparing α-glucosyl hesperidin using the cyclodextrin glucosyltransferase mutant, comprising the following steps: (1) Using hesperidin as a substrate, β-cyclodextrin as a glycosyl donor, and the cyclodextrin glucosyltransferase mutant as a catalyst, a glycosylation reaction is carried out in a bioreactor by the method of reverse feeding of hesperidin and β-cyclodextrin to obtain an oligoglucosyl hesperidin mixture; (2) Adding glucosidase to the oligoglucosyl hesperidin mixture for enzymatic hydrolysis to obtain α-glucosyl hesperidin.

[0013] In step (1), the temperature of the bioreactor is 40 - 50 °C, the rotation speed is 100 - 200 rpm, and the pH is 8 - 9.

[0014] The specific method of reverse feeding of hesperidin and β-cyclodextrin: Add the crude enzyme solution of the cyclodextrin glucosyltransferase mutant into the bioreactor, prepare the reaction solutions of hesperidin and β-cyclodextrin, add them into two feeding bottles respectively and connect to the bioreactor, set the feeding rate to 100 mL / 8 - 12 h, continue the glycosylation reaction for 12 h - 16 h after the feeding ends, and filter to remove solids after the reaction ends to obtain the oligoglucosyl hesperidin mixture. In addition, an HCl solution is added to a third feeding bottle to maintain the pH of the system in the bioreactor.

[0015] The addition amounts of the hesperidin solution, β-cyclodextrin solution, the crude enzyme solution of the cyclodextrin glucosyltransferase mutant, and glucosidase can be adjusted according to actual situations.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The cyclodextrin glucosyltransferase mutant Asp251Ala provided by the present invention has a relatively high enzyme activity, which is 1.2 times that of the wild type. Its application in the modification of hesperidin has a substrate conversion rate as high as 95.3%. After the modification of hesperidin, high-content α-glucosyl hesperidin can be obtained through enzymatic hydrolysis with glucosidase. Description of the Drawings

[0017] Figure 1 It is the HPLC chart of the crude enzyme solution of the cyclodextrin glucosyltransferase mutant Asp251Ala catalyzing hesperidin for 24 h; Figure 2 It is the HPLC chart of glucosidase cleaving oligoglucosyl hesperidin for 3 h. Detailed Embodiments

[0018] To make the objectives and technical solutions of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; for those technologies or conditions not specified in the experiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0019] Example 1 In this example, autodock vina was first used to dock the substrate hesperidin with cyclodextrin glucosyltransferase to determine the catalytic site of cyclodextrin glucosyltransferase. Theoretically, the mutant Asp251Ala has higher enzyme activity. Then, the enzyme activity of the mutant Asp251Ala was further measured and compared with the wild-type enzyme activity of cyclodextrin glucosyltransferase.

[0020] The cyclodextrin glucosyltransferase sequence (NCBI accession number NO. AAV38118) with the amino acid sequence shown in SEQ ID NO: 1 and the cyclodextrin glucosyltransferase mutant Asp251Ala sequence were submitted to a biological company to synthesize their encoding genes respectively, and were cloned between NcoI / BamHI of the expression vector pET22b to obtain recombinant plasmids. The recombinant plasmids were transformed into the expression host E. coli BL21. Through colony PCR verification and sequencing, the correctly verified Asp251Ala and wild-type engineering strains were preserved.

[0021] The Asp251Ala and wild-type engineering strains were respectively inoculated into 25 mL of LB liquid medium containing Kana with a final concentration of 50 μg / mL and cultured overnight at 37°C with 200 r / min. Then, they were transferred to 25 mL of TB liquid medium containing Kana with a final concentration of 50 μg / mL at an inoculation amount of 1%. When the cell concentration OD 600 reached 0.6, an IPTG solution with a final concentration of 1 mmol / L was added, and the cells were cultured at 25°C with 200 r / min for 20 h. After the culture was completed, the cells were centrifuged at 8000 r / min for 5 min at 4°C, the supernatant was discarded, and the cells were suspended with 0.01 mol / L PBS buffer to obtain a cell suspension. The cell suspension was ultrasonically disrupted in an ice-water bath for 20 min and centrifuged at 8000 r / min for 10 min at 4°C, and the supernatant was collected to obtain the crude enzyme solutions of Asp251Ala and wild-type cyclodextrin glucosyltransferase.

[0022] The crude enzyme solutions were detected for enzyme activity. The enzyme activity of the crude enzyme solution of the mutant Asp251Ala was 8640 U / mL, and the enzyme activity of the wild-type cyclodextrin glucosyltransferase was 7200 U / mL. The enzyme activity of the crude enzyme solution of the mutant Asp251Ala was 1.2 times that of the wild type.

[0023] The method for detecting enzyme activity is as follows: Accurately weigh 2 g of soluble starch and dissolve it in 50 mL of glycine-NaOH solution with a pH of 7. After heating and dissolving it in a microwave oven and cooling it to room temperature, take 900 μL and add it to a test tube. Then add 100 μL of the crude enzyme solution, and react precisely in a water bath at 50 °C for 10 min. Then add 1 M HCl solution to terminate the reaction. Finally, add 4 mL of methyl orange solution diluted 4 times and let it stand at room temperature for 20 min for color development. In the control group, 1 M HCl is added first to terminate the reaction, and then 100 μL of the crude enzyme solution is added, and the other experimental conditions are the same. Then use a microplate reader to measure the light absorption value of the reaction solution at 507 nm. The amount of enzyme required to generate 1 μmoL of cyclodextrin per unit time is defined as one enzyme activity unit.

[0024] Example 2 In this example, the crude enzyme solution of the mutant Asp251Ala is used to prepare α-glucosyl hesperidin, and the preparation method includes the following steps: (1) Prepare the reaction solution of hesperidin and β-cyclodextrin First, add 1 g of 95% hesperidin to 100 mL of ultrapure water, then add solid sodium hydroxide and stir until the hesperidin is completely dissolved. Stop adding sodium hydroxide. At this time, the solution is dark brown, and the hesperidin reaction solution to be reacted is obtained. Add 3 g of β-cyclodextrin to 100 mL of ultrapure water and heat it to dissolve in a microwave oven to obtain the β-cyclodextrin reaction solution to be reacted.

[0025] (2) Modification of hesperidin Add 800 mL of the crude enzyme solution of the mutant Asp251Ala prepared in Example 1 to a 2 L fermenter. Set the reaction temperature of the fermenter to 45 °C, the rotation speed to 200 rpm, and the pH to 9. Add the prepared reaction solutions of hesperidin and β-cyclodextrin in step (1) to two feeding bottles respectively and connect them to the fermenter. Set the feeding speed to 100 mL / 12 h. In addition, add 1 mol / L HCl solution to the third feeding bottle to maintain the pH of the system in the bioreactor. After feeding for 12 h, continue the glycosylation reaction for 12 h. After the reaction is completed, filter to remove solids to obtain an oligoglucosyl hesperidin mixture. At this time, the sample is measured by HPLC. The mass of α-glucosyl hesperidin is 25.1% of the mass of the oligoglucosyl hesperidin mixture, and the conversion rate of the substrate hesperidin is 95.3%, as Figure 1 .

[0026] (3) Prepare α-glucosyl hesperidin Add 50 U of glucosidase to the oligomeric glucosyl hesperidin mixture obtained in step (2), enzymatically hydrolyze for 3 h to obtain a mixture. At this time, the sample is measured by HPLC, and the mass of α-glucosyl hesperidin is 60.5% of the mass of the mixture, as Figure 2 .

Claims

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

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

3. The coding gene of 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-glucosyl hesperidin using the cyclodextrin glucosyltransferase mutant described in claim 1, characterized in that: It includes the following steps: (1) Using hesperidin as a substrate, β-cyclodextrin as a glycosyl donor, and the cyclodextrin glucosyltransferase mutant as a catalyst, a glycosylation reaction is carried out in a bioreactor by the method of reverse feeding of hesperidin and β-cyclodextrin to obtain an oligoglucosyl hesperidin mixture; (2) Adding glucosidase to the oligoglucosyl hesperidin mixture for enzymatic hydrolysis to obtain α-glucosyl hesperidin.

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