Cyclodextrin transferase mutant gene and application thereof

Abstract

The application discloses a cyclodextrin transferase mutant gene and application thereof, wherein the gene encodes a cyclodextrin transferase mutant with an amino acid sequence as shown in SEQ ID NO. 2. The mutant gene is applied to synthesis of vitamin C ethyl ether. An expression frame of the cyclodextrin transferase mutant gene is constructed to obtain the cyclodextrin transferase mutant, and then vitamin C and ethanol are used as substrates to realize one-step enzymatic catalysis and efficient synthesis of vitamin C ethyl ether. The mutant can be combined with the substrates vitamin C and ethanol to realize the biological enzyme method for synthesizing vitamin C ethyl ether.

Description

Technical Field

[0001] This invention belongs to the field of vitamin C ethyl ether synthesis, and particularly relates to a cyclodextrin transferase mutant gene and its application. Background Technology

[0002] Vitamin C (VC), also known as L-ascorbic acid, is an essential water-soluble vitamin for maintaining life, widely distributed in fresh fruits, vegetables, and the green parts of plants. Modern research shows that VC promotes collagen synthesis, participates in the metabolism of aromatic amino acids and fats, improves the utilization of iron, calcium, and folic acid, catalyzes the redox of melanin, enhances the body's stress resistance and immunity, and inhibits the proliferation of cancer cells. However, the human body lacks gulonolactone oxidase and cannot synthesize VC; therefore, it must be obtained from food, thus VC is listed as an essential nutrient. Clinically, VC is mainly used to prevent and treat scurvy, enhance the body's resistance, promote wound healing, and is used as an adjunct in treatment and health care products. Furthermore, in the food industry, vitamin C is an important acidulant, antioxidant, and stabilizer; in the cosmetics industry, it is a reducing agent, ultraviolet absorber, and melanin synthesis inhibitor; in animal husbandry and aquaculture, vitamin C promotes collagen synthesis, growth of young animals, improves larval survival rates, and helps prevent black death disease, abnormal bone bleeding, and erosion in fish. Therefore, vitamin C is widely used in pharmaceuticals, animal feed, cosmetics, and food additives (27 yuan / kg), with its application in food additives being the most significant, accounting for approximately 70%. In addition, vitamin C has consistently been one of the world's largest raw materials for pharmaceuticals.

[0003] However, the unique allenyldiol structure in the vitamin C molecule is easily oxidized and degraded into dehydrogenated vitamin C in aqueous solution and air, thus losing its physiological activity. Since the 1970s, researchers have been dedicated to studying various vitamin C derivatives, hoping to find a novel vitamin C derivative that can overcome the instability of vitamin C while still fulfilling its physiological functions. Currently, vitamin C derivatives mainly include three forms: vitamin C salts, vitamin C esters, and vitamin C glycosides. Among these, vitamin C glycoside derivatives have high stability, physiological activity closest to that of vitamin C, and are mostly biosynthesized, attracting considerable attention in recent years. Therefore, developing a vitamin C derivative that is easy to produce and possesses good physicochemical properties and physiological functions remains a future direction. Summary of the Invention

[0004] Purpose of the invention: The first objective of this invention is to provide a cyclodextrin transferase mutant and its encoding gene; another technical problem to be solved by this invention is to provide the application of the cyclodextrin transferase mutant in the catalytic ethyl etherification process of vitamin C.

[0005] Technical Solution: To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a cyclodextrin transferase mutant gene, wherein the gene encodes a cyclodextrin transferase mutant with the amino acid sequence shown in SEQ ID NO.2.

[0007] As one embodiment, the nucleotide sequence of the cyclodextrin transferase mutant gene is shown in SEQ ID NO.1.

[0008] Secondly, the present invention provides a cyclodextrin transferase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Thirdly, the present invention provides a recombinant expression vector comprising the cyclodextrin transferase mutant gene described above.

[0010] Fourthly, the present invention provides a recombinant microorganism, the microorganism comprising the cyclodextrin transferase mutant gene, or expressing the cyclodextrin transferase mutant.

[0011] Preferably, the microorganism is Escherichia coli or Pichia pastoris.

[0012] Fifthly, the present invention provides a method for preparing vitamin C ethyl ether, comprising the following steps: using vitamin C as a substrate and ethanol as an ethyl ether donor, vitamin C ethyl ether is formed by a cyclodextrin transferase mutant catalytic reaction.

[0013] As a preferred embodiment, the system for the cyclodextrin transferase mutant catalyzed reaction is as follows: 0.1-50 mM vitamin C, 0.1-100 μM cyclodextrin transferase mutant, and 0.1-50 mM ethanol are added to a 1-200 mM Tris-HCl solution; the catalytic reaction temperature is 10-50 °C, the reaction pH is 5-9, and the reaction time is 1-50 h.

[0014] As a specific implementation scheme, the preparation method of the vitamin C ethyl ether includes the following steps:

[0015] (1) Construct an expression cassette of the cyclodextrin transferase mutant, including the cyclodextrin transferase mutant gene, promoter and terminator, on an expression vector, and construct a recombinant expression vector of the cyclodextrin transferase mutant;

[0016] (2) Using the recombinant expression vector in the host cell transformation step (1), a recombinant strain of cyclodextrin transferase mutant was obtained, induced and cultured to obtain crude enzyme solution of cyclodextrin transferase mutant, and vitamin C ethyl ether was obtained in vitro through enzyme catalysis.

[0017] As one implementation scheme, in step (1), Escherichia coli is used as the host cell to construct an expression cassette carrying the cyclodextrin transferase mutant gene, T7 promoter and T7 terminator on the Escherichia coli expression vector, that is, to construct the Escherichia coli recombinant expression cassette of the cyclodextrin transferase mutant; preferably, the Escherichia coli expression vector is a pET series vector;

[0018] More preferably, after the expression vector is transformed into Escherichia coli, positive clones are selected and transferred to the fermentation medium, and fermentation is carried out at 20-37℃ until the OD600 nm is 0.6-0.8. Then, IPTG with a final concentration of 0.01-1mM is added, and the supernatant is collected by centrifugation after induction.

[0019] As another implementation, in step (1), Pichia pastoris is used as the host cell, and an expression cassette carrying a cyclodextrin transferase mutant, an AOX promoter and an AOX terminator is constructed on the Pichia pastoris expression vector, that is, the Pichia pastoris recombinant expression cassette of the cyclodextrin transferase mutant is constructed; preferably, the Pichia pastoris expression vector is a pPIC series vector.

[0020] More preferably, after the expression vector is transformed into Pichia pastoris, positive clones are selected and transferred to the fermentation medium, and fermentation is carried out at 20-37℃. After 24 hours of culture, methanol with a final concentration of 0.5% is added, and the supernatant is collected by centrifugation after induction.

[0021] In step (2), the crude enzyme solution can be further filtered, eluted by column, the enzyme active portion can be collected, concentrated, and finally freeze-dried before the enzyme catalytic reaction can be carried out.

[0022] In a sixth aspect, the present invention provides the application of the cyclodextrin transferase mutant gene, the cyclodextrin transferase mutant, the recombinant expression vector, or the recombinant microorganism in the biosynthesis of vitamin C ethyl ether.

[0023] Beneficial effects: Compared with the prior art, the cyclodextrin transferase mutant encoded by the gene of the present invention is obtained by molecular docking and virtual mutation using bioinformatics software based on the crystal structure of cyclodextrin transferase and the chemical structures of the substrates vitamin C and ethanol. The mutant has a high binding force with the substrates vitamin C and ethanol, and its application in the synthesis of vitamin C ethyl ether provides the possibility of synthesizing vitamin C ethyl ether by bioenzymatic method. Attached Figure Description

[0024] Figure 1This diagram illustrates the construction of a recombinant *E. coli* cell factory and the synthesis of a cyclodextrin transferase mutant. In the diagram, A represents the construction of recombinant *E. coli* BL21 pET28a(+)-CGTasem; B shows the SDS-PAGE analysis of the synthesis of the cyclodextrin transferase mutant in the *E. coli* cell factory. Lane M represents the marker, lane 1 is the intracellular supernatant sample of recombinant *E. coli* BL21 pET28a(+)-CGTasem, and lane 2 is the intracellular supernatant sample of recombinant *E. coli* BL21 pET28a(+).

[0025] Figure 2 This diagram illustrates the construction of a recombinant Pichia pastoris cell factory and the synthesis of a cyclodextrin transferase mutant. In the diagram, A represents the construction of recombinant Pichia pastoris GS115 pPIC9K-CGTasem; B shows the SDS-PAGE analysis of the synthesis of the cyclodextrin transferase mutant in the Pichia pastoris cell factory. Lane M represents the marker, lane 1 is the recombinant Pichia pastoris GS115 pPIC9K-CGTasem sample, and lane 2 is the recombinant Pichia pastoris GS115 pPIC9K sample.

[0026] Figure 3 The catalytic products of the cyclodextrin transferase mutant enzyme were analyzed by LC-TOF-MS. Among them, A is the MS spectrum of the cyclodextrin transferase expressed by recombinant Escherichia coli after catalysis, B is the LC spectrum of the cyclodextrin transferase mutant enzyme expressed by recombinant Escherichia coli and recombinant Pichia pastoris after catalysis, and C is the MS spectrum of the cyclodextrin transferase mutant enzyme expressed by recombinant Escherichia coli and recombinant Pichia pastoris after catalysis. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the host cells, expression vectors, enzymes, and other reagents used in the present invention are all commercially available. Specifically, the catalog number of pET28a(+) is 69864-3 (Novagen).

[0028] Example 1: Preparation of Cyclodextrin Transferase Mutant using Microbial Cells

[0029] 1. Preparation of cyclodextrin transferase mutants using recombinant Escherichia coli

[0030] Based on the crystal structure (PDB: 1 pam) of the candidate gene cyclodextrin transferase mutant and the chemical structures of the substrates vitamin C and ethanol, the amino acid sequence of the mutant (CGTasem) with enhanced binding affinity to the substrates vitamin C and ethanol was obtained by molecular docking and virtual mutation using the bioinformatics software Discovery Studio, as shown in SEQ ID NO.2. The nucleotide sequence of CGTasem synthesized after codon optimization is shown in SEQ ID NO.1 (synthesized by Shanghai Sangon Biotech Co., Ltd.).

[0031] Primers F1 and R1, F2 and R2 were designed respectively. Using the high-fidelity PCR polymerase Prime Star, the CGTasem and CGTase gene (non-mutated gene) fragments were amplified using SEQ ID NO.1 and GenBank: M17366.1 as templates. The fragments were then integrated into the BamHI and SacI sites of the expression vector pET28a(+) plasmid (Invitrogen) via Gibbson assembly to obtain the recombinant expression vectors of CGTasem and CGTase. The recombinant expression vectors pET28a(+)-CGTasem and pET28a(+)-CGTase constructed above were incubated with E. coli BL21 competent cells on ice for 30 min, and then transformed into E. coli BL21 by heat shock at 42℃ for 90 s to obtain recombinant E. coli expression strains E. coli BL21pET28a(+)-CGTasem and E. coli BL21 pET28a(+)-CGTasem and E. coli BL21 pET28a(+)-CGTase expressing CGTasem and CGTase, respectively.

[0032] Single colonies of recombinant E. coli expression strains E. coli BL21 pET28a(+)-CGTasem and E. coli BL21 pET28a(+)-CGTase were picked and inoculated into LB medium containing 50 mg / L kanamycin. After overnight incubation at 37°C, they were transferred at a ratio of 10% (V / V) to 50 mL of LB medium containing 50 mg / L kanamycin. The colonies were cultured until the OD reached... 600 After adjusting the concentration to 0.6-0.8, IPTG was added to a final concentration of 1 mM to induce the expression of CGTasem and CGTase. After 5 h of induction culture, the cells were collected by centrifugation at 8000 rpm and 4°C for 5 min. The cells were washed with pre-cooled Tris-HCl (pH 7.0), resuspended in 50 mL, and sonicated on ice under the following conditions: 400 W, 2 s on, 3 s off, 200 cycles until the bacterial solution was clear. The solution was then centrifuged at 14000 rpm and 4°C for 20 min, and the supernatant was collected as the crude enzyme solution. Figure 1 ).

[0033] 2. Construction of recombinant Pichia pastoris cell factory and preparation of cyclodextrin transferase mutant

[0034] The artificially synthesized cyclodextrin transferase mutant candidate gene CGTasem was amplified using primers F3 and R3, then assembled via Gibbon assembly, and homologously integrated into the EcoRI and NotI sites of pPIC9K to construct the recombinant expression vector pPIC9K-CGTasem. The sequence of CGTasem is shown in SEQ.ID NO.1. The recombinant expression vector was transformed into Pichia pastoris to obtain the transformant P. patoris GS115pPIC9K-CGTasem, which will be used in subsequent Pichia pastoris cell factory production of cyclodextrin transferase mutants. Figure 2 A).

[0035] Single colonies of *Pichia pastoris* transformant *P. patoris* GS115 pPIC9K-CGTasem were picked from plates and inoculated into 3 mL of YPD medium. The culture was incubated overnight at 30°C and 200 rpm. The culture was then transferred to 50 mL of BMMY medium (with methanol added to a final concentration of 0.5 g / L) at a ratio of 1% (v / v) and induced at 20°C and 200 rpm for 5 days. The culture was centrifuged at 8000 rpm and 4°C for 5 min, and the supernatant was collected as the crude enzyme solution of the cyclodextrin transferase mutant. Figure 2 B).

[0036] Example 2: Establishment of the catalytic system of the cyclodextrin transferase mutant

[0037] 1. Purification of cyclodextrin transferase mutant

[0038] After equilibrating the Ni-NTA Agarose Fast Flow column with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.4), crude enzyme solutions of cyclodextrin transferase expressed by *E. coli*, crude enzyme solutions of cyclodextrin transferase mutant expressed by *E. coli*, and crude enzyme solutions of cyclodextrin transferase mutant expressed by *Pichia pastoris* were passed through a 0.22 μm filter membrane. The supernatant was then passed through the Ni-NTA Agarose Fast Flow column at a rate of 3 mL / min. After binding for 10 min, the enzyme active fraction was collected by elution with Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.4), concentrated, and freeze-dried.

[0039] 2. Preparation of Vitamin C Ethyl Ether

[0040] Vitamin C ethyl ether was prepared using vitamin C and ethanol as raw materials. The reaction solution included 1-200 mM Tris-HCl (pH 7.0), 0.001-1 g / L cyclodextrin transferase mutant (or 0.001-1 g / L cyclodextrin transferase), 50 g / L vitamin C, 1-50 g / L ethanol, and reacted at 10-50 °C for 1-50 h. The reaction was then terminated by heating at 70 °C for 5 min.

[0041] In this embodiment, vitamin C and ethanol were used as raw materials. The reaction solution included 20 mM Tris-HCl (pH 7.0), 0.01 g / L cyclodextrin transferase mutant (or 0.01 g / L cyclodextrin transferase), 50 g / L vitamin C, and 5 g / L ethanol. The reaction was carried out at 37°C for 1 h, and then heated at 70°C for 5 min to terminate the reaction, thus preparing the enzyme-catalyzed product of vitamin C and ethanol.

[0042] Example 3: Detection of Vitamin C Ethyl Ether

[0043] Detection of vitamin C ethyl ether using high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0044] The HPLC-MS conditions used were as follows: column: Agilent AdvanceBio MS SpentMedia 2.1 × 150 mm; mobile phase: 10 mM ammonium acetate aqueous solution (A), 10 mM ammonium acetate acetonitrile solution (B); detection wavelength: flow rate: 0.25 mL / min. The elution program was as follows: time (min) 0: pump 10% + pump B 90%; time (min) 2: pump A 10% + pump B 90%; time (min) 12: pump A 60% + pump B 40%; time (min) 13: pump A 80% + pump B 20%; time (min) 16: pump A 80% + pump B 20%; time (min) 17: pump A 10% + pump B 90%. The corresponding mass spectrometry conditions used were: ion source: ESI; nebulizer flow rate: 1.5 L / min; drying gas flow rate: 5 L / min; ion source temperature: 200℃; transfer line temperature: 250℃.

[0045] The results showed that the cyclodextrin transferase mutant could catalyze the synthesis of vitamin C ethyl ether from vitamin C and ethanol, while the cyclodextrin transferase itself did not exhibit the corresponding function. Under the conditions described in this test, the retention time of vitamin C ethyl ether was 4.786 minutes. Figure 3 ).

[0046] Table 1 Primer sequences

[0047]

Claims

1. A cyclodextrin transferase mutant gene, characterized in that, The gene encodes a cyclodextrin transferase mutant with the amino acid sequence shown in SEQ ID NO.

2.

2. The cyclodextrin transferase mutant gene according to claim 1, characterized in that, The nucleotide sequence of the cyclodextrin transferase mutant gene is shown in SEQ ID NO.

1.

3. A cyclodextrin transferase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, Includes the cyclodextrin transferase mutant gene as described in claim 1 or 2.

5. A recombinant microorganism, characterized in that, The microorganism contains the cyclodextrin transferase mutant gene as described in claim 1 or 2, or expresses the cyclodextrin transferase mutant as described in claim 3.

6. The recombinant microorganism according to claim 5, characterized in that, The microorganisms mentioned are Escherichia coli or Pichia pastoris.

7. A method for preparing vitamin C ethyl ether, characterized in that, The method includes the following steps: using vitamin C as a substrate and ethanol as an ethyl ether donor, vitamin C ethyl ether is formed by a catalytic reaction catalyzed by the cyclodextrin transferase mutant described in claim 3; the system for the cyclodextrin transferase mutant catalytic reaction is as follows: 50 g / L vitamin C, 0.001-1 g / L cyclodextrin transferase mutant, and 1-50 g / L ethanol are added to a 1-200 mM Tris-HCl solution; the temperature of the catalytic reaction is 10-50℃, the pH is 7.0, and the reaction time is 1-50 h.

8. The method for preparing vitamin C ethyl ether according to claim 7, characterized in that, Includes the following steps: (1) Construct an expression cassette of a cyclodextrin transferase mutant including the cyclodextrin transferase mutant gene, promoter and terminator as described in claim 1 or 2 on an expression vector, and construct a recombinant expression vector of the cyclodextrin transferase mutant; (2) Using the recombinant expression vector in the host cell transformation step (1), a recombinant strain of cyclodextrin transferase mutant was obtained, induced and cultured to obtain crude enzyme solution of cyclodextrin transferase mutant, and vitamin C ethyl ether was obtained in vitro through enzyme catalysis.

9. The application of the cyclodextrin transferase mutant gene of claim 1 or 2, the cyclodextrin transferase mutant of claim 3, the recombinant expression vector of claim 4, or the recombinant microorganism of claim 5 or 6 in the biosynthesis of vitamin C ethyl ether.

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