A cyclodextrin glucosyltransferase mutant and use thereof in preparing trehalose

Abstract

The application provides a cyclodextrin glucosyltransferase mutant and application thereof in preparation of trehalose, belongs to the technical field of genetic engineering and enzyme engineering, and mainly provides a cyclodextrin glucosyltransferase mutant, and amino acid mutation sites are the 33th and 211th amino acids in the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2; the disproportionation characteristic enzyme activity of the cyclodextrin glucosyltransferase mutant provided by the application is obviously improved compared with the wild type, the thermal stability is obviously superior, the optimal temperature of the mutant is 60 DEG C, which is 5 DEG C higher than the optimal temperature 55 DEG C of the wild type, the trehalose yield is obviously improved when the mutant is applied to a multi-enzyme system for preparing trehalose, the substrate utilization rate is improved, and meanwhile, the application prospect of the enzyme in the medical, food and biological industries is improved.

Description

Technical Field

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a cyclodextrin glucosyltransferase mutant and its application in the preparation of trehalose. Background Art

[0002] Cyclodextrin glycosyl transferase (CGTase, EC2.4.1.19), a member of the α-amylase family (glycoside hydrolase 13_2, GH13_2), is a multifunctional enzyme that catalyzes disproportionation, cyclization, coupling, and hydrolysis reactions on substrates such as starch and maltodextrin. Disproportionation is a transglycosidic reaction between two different molecules, transferring the cleaved portion of a linear oligosaccharide to another receptor. Cyclization is an intramolecular transglycosidic reaction, a characteristic reaction of CGTase and widely used in industrial production of cyclodextrins. Coupling is the reverse reaction of cyclization, opening the cyclodextrin ring and transferring the glycoside to a linear maltodextrin oligomer.

[0003] Trehalose is also known as Trehalose. The molecular formula of trehalose is C 12 O 22 H 11 , molecular weight 378.33, is composed of two molecules of α-d-glucose linked by α,α-1,1-glycosidic bond. It is usually in the form of dihydrate (C 12 H 22 O 11 Trehalose exists in the form of 2H2O). Trehalose has three optical isomers: the most common α,α-structure in nature and the less common α,β- and β,β-structures. Trehalose is widely present in various organisms. Due to its broad application prospects, the tireless efforts of countless scientists have led to breakthroughs in its production.

[0004] Trehalose can be synthesized by chemical methods, but there are disadvantages such as low trehalose production efficiency, many by-products, and difficulty in purification, making it difficult to apply to industrial production. In addition, although trehalose is widely distributed, its content in nature is low. Traditional biological extraction methods mainly obtain it from yeast. This method is complex and has high production costs, which limits its large-scale production. Microbial fermentation methods have disadvantages such as low conversion rate, many by-products, and difficulty in separation and purification. Currently, enzymatic conversion is widely used in the production of trehalose. This method has the characteristics of high conversion rate, simple components in the conversion liquid, and easy separation. This method has been widely used in industrial production, but it also requires the use of multiple enzymes such as α-amylase, isoamylase, pullulanase, cyclodextrin glucosyltransferase, and saccharifying enzyme for compounding. The preparation process is relatively complicated, and the efficiency of enzyme utilization for substrates needs to be further improved.

[0005] Currently, CGTase is widely used, but there are few studies on improving its disproportionation properties and simultaneously improving its thermal stability. Due to its own poor thermal stability, CGTase has greatly limited its industrial application scope. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a cyclodextrin glucosyltransferase mutant and its application in the preparation of trehalose.

[0007] The present invention is the first to discover that by subjecting β-CGTase from Bacillus sp. G1 to superimposed mutation modification, that is, the mutation sites of the mutant are asparagine (N) at position 33 to lysine (K), and serine (S) at position 211 to glycine (G), to obtain a mutant N33K / S211G; the dismutation characteristics and thermal stability of the mutant are significantly improved compared with the wild type, and the optimum temperature of the mutant is 60°C, which is 5°C higher than the optimum temperature of 55°C of the wild type; the half-life of N33K / S211G is greatly improved relative to that of mutant N33K, and the half-life of mutant N33K / S211G at 60°C is 2.3 times that of mutant N33K.

[0008] The nucleotide sequence encoding the cyclodextrin glucosyltransferase from Bacillus sp. G1 in the present invention is shown in SEQ ID NO. 1, and the amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO. 2.

[0009] The technical solutions of the present invention are as follows:

[0010] A mutant of cyclodextrin glucosyltransferase, wherein the amino acid mutation sites are the 33rd and 211th amino acids of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2.

[0011] According to the present invention, preferably, the mutant is:

[0012] The asparagine (N) at position 33 was mutated to lysine (K), and the serine (S) at position 211 was mutated to glycine (G), and the gene was named N33K / S211G.

[0013] The coding gene of the mutant is obtained by performing site-directed mutagenesis on the coding nucleotide sequence SEQ ID NO. 1 of cyclodextrin glucosyltransferase according to the mutation site of the amino acid.

[0014] A recombinant expression vector comprises the coding gene of the mutant.

[0015] A recombinant strain comprising a gene encoding the mutant.

[0016] Application of the coding gene, recombinant expression vector or recombinant strain of the mutant in the preparation of cyclodextrin glucosyltransferase.

[0017] Application of the mutant in the preparation of trehalose.

[0018] Beneficial effects

[0019] The dismutase activity and thermal stability of the cyclodextrin glucosyltransferase mutant provided by the present invention are significantly improved compared with the wild type. The optimum temperature of the mutant is 60°C, which is 5°C higher than the optimum temperature of the wild type of 55°C.

[0020] The half-life of N33K / S211G mutant is significantly improved compared to that of N33K, reaching 2.3 times that of N33K at 60°C. Application in the preparation of trehalose significantly improves substrate utilization and trehalose yield. Furthermore, this invention has implications for the industrial production of cyclodextrin glucosyltransferase and enhances its potential applications in the pharmaceutical, food, and biotechnology industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result of electrophoresis detection of mutant genes;

[0022] In the figure: Lane 1 is the PCR verification band for the N33K site mutation, and Lane 2 is the PCR verification band for the N33K / S211G site mutation.

[0023] Figure 2 The optimal pH diagram of the wild type and mutants.

[0024] Figure 3 The pH stability diagram of the wild type and mutants.

[0025] Figure 4 Optimal temperature diagram for the wild type and mutants.

[0026] Figure 5 This is a comparison chart of trehalose production. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0028] Unless otherwise specified, the drugs and reagents used in the examples are common products on the market. Any matters not described in detail in the examples are based on the existing technology in the art.

[0029] The culture medium and detection method involved in the following examples are as follows:

[0030] LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and the balance water.

[0031] TB medium: tryptone 12 g / L, yeast extract powder 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L, balance water.

[0032] Method for determining the activity of cyclodextrin glucosyltransferase catalyzing dismutation reaction:

[0033] A 12 mM solution of EPS (4,6-ethylene-p-nitrophenyl-α-D-maltoheptaglycoside) and a 20 mM maltose solution were prepared in 50 mmol / L phosphate buffer (pH 6.0). 300 μL of each 12 mM EPS and 20 mM maltose solution were preheated in a 45-65°C waterbath. 100 μL of the diluted enzyme solution was added and allowed to react for exactly 10 minutes. After that, 50 μL of 3 M HCl was added. After 5 minutes, 3 M NaOH was added for neutralization. Then, 100 μL of α-glucosidase was added and the reaction was allowed to proceed in a 37°C waterbath for at least 60 minutes. The pH was adjusted to above 8.0 by adding 100 μL of 1 M Na₂CO₃ solution. Finally, the absorbance was measured at 401 nm. The dismutation activity of cyclodextrin glucosyltransferase is defined as the amount of enzyme required to convert 1 μmol of EPS per minute.

[0034] Trehalose product detection method:

[0035] The trehalose content in the conversion solution was determined by HPLC using an NH2 column, a mobile phase of acetonitrile:water = 4:1, a flow rate of 1.0 mL / min, and a column temperature of 40°C.

[0036] Example 1

[0037] Preparation and expression of wild-type cyclodextrin glucosyltransferase

[0038] The gene for cyclodextrin glucosyltransferase (Cgt) from Bacillus sp. G1 was artificially synthesized, with the nucleotide sequence shown in SEQ ID NO. 1 and the amino acid sequence shown in SEQ ID NO. 2. An expression vector pET-28a(+) / Cgt was constructed and introduced into Escherichia coli BL21(DE3) for expression to obtain wild-type cyclodextrin glucosyltransferase. The culture was cultured in LB liquid medium (containing 100 mg / L kanamycin) at 37° C. and 200 rpm for 10 hours to obtain a seed solution. The seed solution was inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a volume ratio of 5% inoculum, cultured at 37° C. and 200 rpm for 2 hours, and then IPTG was added to a final concentration of 0.2 mM. The temperature was adjusted to 25° C. and cultured at 200 rpm for 8 hours. A certain volume of the fermentation broth was centrifuged at 4° C. and 12,000 rpm for 10 minutes, and the fermentation supernatant was obtained as a crude enzyme solution of the wild-type enzyme.

[0039] Example 2

[0040] Preparation and expression of a single mutant of cyclodextrin glucosyltransferase

[0041] (1) Based on the nucleotide sequence of Bacillus sp. G1 cyclodextrin glucosyltransferase, as shown in SEQ ID NO. 1, primers for introducing single mutations were designed and synthesized, and site-directed mutagenesis of cyclodextrin glucosyltransferase Cgt was performed. Sequencing was performed to confirm whether the coding gene of the cyclodextrin glucosyltransferase mutant was correct; the vector carrying the mutant gene was introduced into Escherichia coli for expression to obtain a single mutant cyclodextrin glucosyltransferase.

[0042] PCR amplification of the site-directed mutant encoding gene: Rapid PCR technology was used with the expression vector pET-28a(+) / Cgt carrying the gene encoding the wild-type cyclodextrin glucosyltransferase as a template.

[0043] The site-directed mutagenesis primers for introducing the N33K mutation are:

[0044] The nucleotide sequence of the forward primer is shown in SEQ ID NO.3:

[0045] 5'-GTTACA AAG AAAGTCAATTATTCTAAGGATGTGATTTACCA-3' (the mutated base is underlined);

[0046] The reverse primer has a nucleotide sequence as shown in SEQ ID NO.4:

[0047] 5'-TGACTTT CTTTGTAACATCTGCTTCAGCTACTG-3' (the mutated base is underlined).

[0048] The PCR reaction system was as follows: 2.5 μL each of 10 μM forward primer and reverse primer, 25 μL of 2× Phanta Max MasterMix, 2.5 μL of template, and double-distilled water to make up to 50 μL.

[0049] The PCR conditions were as follows: 95°C denaturation for 5 min; followed by 25 cycles (95°C for 15 s, 55°C for 15 s, 72°C for 3 min 50 s) and extension at 72°C for 5 min; and finally storage at 4°C. PCR products were detected by 1% agarose gel electrophoresis. The mutant gene PCR verification results are shown in Figure 1 .

[0050] The above-mentioned correctly verified PCR product was digested with DpnⅠ and transformed into Escherichia coli DH5α competent cells. The transformation product was spread on LB plates containing 100 mg / L kanamycin and cultured overnight at 37°C. Ten single colonies were picked from the plates and transferred into LB liquid culture medium containing 100 mg / L kanamycin. After 8 hours, the plasmid was extracted and verified. Three correctly verified plasmids were selected for sequencing, and the correctly sequenced plasmids were transformed into Escherichia coli BL21 (DE3) to obtain recombinant E. coli expressing the single mutant N33K.

[0051] Example 3

[0052] Preparation and expression of a double mutant of cyclodextrin glucosyltransferase

[0053] (1) Preparation of double mutants of cyclodextrin glucosyltransferase

[0054] The plasmid carrying the gene encoding the mutant N33K constructed in Example 2 was used as a template for the double mutant, and site-directed mutagenesis of the S211G mutation was introduced using the following primers:

[0055] The nucleotide sequence of the forward primer is shown in SEQ ID NO.5:

[0056] 5'-GAAGAT GGA ATTTATAGAAATCTGTATGATTTGGCAGACTACG-3' (the mutated base is underlined);

[0057] The reverse primer has a nucleotide sequence as shown in SEQ ID NO.6:

[0058] 5'-TATAAAT TCCATCTTCATATGATGAAAAATCTGTGCCGCC-3' (the underlined base is the mutant). Rapid PCR technology was used to perform site-directed mutagenesis on the plasmid carrying the gene encoding the mutant N33K. The mutant N33K gene was used as a template and the above primers were used as amplification primers for PCR amplification. The annealing temperature involved was 55°C; the fragment extension time was 2.5 min, and after 30 cycles, it was stored at 4°C. After DNA gel electrophoresis verification, subsequent operations were carried out. The mutant gene PCR verification results are shown in Figure 1 A cyclodextrin glucosyltransferase N33K / S211G double mutant was obtained, and recombinant Escherichia coli expressing the double mutant was prepared according to the method in Example 2.

[0059] (2) Expression of mutants

[0060] The recombinant Escherichia coli expressing the mutant prepared in step (1) of this example was inoculated into LB liquid medium containing 100 mg / L kanamycin and cultured at 200 rpm for 10 h to obtain a seed solution. The seed solution was inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a volume ratio of 5% inoculum, cultured at 37°C and 200 rpm for 2 h, and then IPTG was added to a final concentration of 0.2 mM. The temperature was adjusted to 25°C and the culture was cultured at 200 rpm for 8 h. A certain volume of fermentation liquid was centrifuged at 4°C and 12000 rpm for 10 min, and the fermentation supernatant was obtained to obtain a crude enzyme solution of the double mutant (the preparation method of the crude enzyme solution was the same as the preparation method of the crude enzyme solution of the wild enzyme in Example 1).

[0061] Example 4

[0062] Analysis of basic enzymatic properties of mutant N33K / S211G

[0063] The fermentation supernatant crude enzyme solutions obtained in Example 1 and Example 3 were subjected to enzymatic property experiments, such as Figure 2 As shown in the figure, the optimum pH is 6.0 and the pH stability is as follows Figure 3 As shown, there is no significant difference compared to the wild type. When studying the optimal temperature, it was found that the optimal temperature of the mutant N33K / S211G is as follows: Figure 4As shown, the half-life of mutant N33K / S211G at 50°C, 55°C, 60°C, and 65°C was 2.4 hours, 2.0 hours, 55.4 minutes, and 38.4 minutes, respectively. This significantly improves the half-life of N33K / S211G relative to N33K, which was 2.0 hours, 1.5 hours, 24.0 minutes, and 16.8 minutes at 50°C, 55°C, 60°C, and 65°C, respectively. The half-life of mutant N33K / S211G at 60°C was 2.3 times that of N33K. Furthermore, based on the enzymatic activity data obtained using the aforementioned method for determining the dismutation reaction catalyzed by cyclodextrin glucosyltransferase, experiments revealed that mutant N33K / S211G had an enzymatic activity 2.1 times that of the wild type. At the same time, the enzyme activities of the other mutants N33K / H255W, N33K / H255F, and N33K / H255Y studied in this laboratory were compared. The results were measured at 50°C for 10 minutes and are shown in Table 1. The present invention found that the mutant N33K combined with the amino acid mutation at position 255 did not significantly improve the enzyme activity compared with the wild type.

[0064] Table 1 Comparison of dismutase activity

[0065]

[0066] Example 5

[0067] Mutant N33K / S211G is used to prepare trehalose

[0068] 200 g / L maltodextrin was used as the substrate, 5 μg of laboratory-prepared assembled maltooligosaccharide-trehalose synthase (MTSase) and maltooligosaccharide-trehalose hydrolase (MTHase) were added (reference patent: A method for preparing an immobilized MTSase enzyme and an immobilized MTHase ​​enzyme and its application in trehalose production), 5 U / g of pullulanase were reciprocally shaken in a constant temperature water bath at 60°C, and samples were taken after 48 hours of reaction. In the later stage of the reaction, wild-type BsCGT and mutant enzyme N33K / S211G were added respectively, and samples were taken during the reaction process. The samples were boiled for 10 minutes to inactivate the enzyme, and saccharifying enzyme was added and reciprocated in a constant temperature water bath at 60°C. After 6 hours of reaction, the samples were boiled for 10 minutes to inactivate the enzyme. After the reaction system is completed, the obtained reaction solution is placed in an ultracentrifuge and centrifuged at 12000 rpm for 10 minutes. The supernatant is diluted to a certain multiple and the trehalose content in the conversion solution is determined by HPLC using an NH2 column, a mobile phase of acetonitrile:water = 4:1, a flow rate of 1.0 mL / min, and a column temperature of 40°C. Figure 5The figure shows a comparison of the amount of product generated, with no CGTase added as a blank control. It can be seen from the figure that the addition of CGTase can significantly increase the yield of trehalose by more than 20%. The yield of adding N33K / S211G is higher than that of BsCGT, proving that the obtained mutant can increase trehalose production.

[0069] The mutant N33K / S211G constructed by the present invention has significantly improved dismutase activity and thermal stability compared with the wild enzyme. Its application in industrial production can prolong the reaction time and reduce production costs. Its dismutation reaction activity can be utilized for its application in transglycoside compounds, such as in the production of trehalose, and in the modification of substances such as L-ascorbic acid and stevioside, effectively improving its application prospects.

Claims

1. A mutant of cyclodextrin glucosyltransferase, characterized in that: The amino acid mutation sites are amino acids 33 and 211 of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2; The mutant is as follows: asparagine (N) at position 33 is mutated to lysine (K), and serine (S) at position 211 is mutated to glycine (G), and is named N33K / S211G.

2. The gene encoding the mutant according to claim 1, characterized in that According to the mutation site of the amino acid, site-directed mutagenesis is performed on the nucleotide sequence encoding SEQ ID NO.1 of cyclodextrin glucosyltransferase.

3. A recombinant expression vector, characterized in that: Comprising the coding gene according to claim 2.

4. A recombinant strain, characterized in that Comprising the coding gene according to claim 2.

5. Use of the encoding gene according to claim 2, the recombinant expression vector according to claim 3 or the recombinant strain according to claim 4 in the preparation of cyclodextrin glucosyltransferase.

6. Use of the mutant according to claim 1 in the preparation of trehalose.

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