Cyclodextrin glucosyltransferase mutant and application of cyclodextrin glucosyltransferase mutant in preparation of trehalose

Abstract

The invention provides a cyclodextrin glucosyltransferase mutant and application thereof in preparation of trehalose, belongs to the technical field of gene engineering and enzyme engineering, and mainly provides the cyclodextrin glucosyltransferase mutant, amino acid mutation sites are 33rd and 21st amino acids of a cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2; the disproportionation characteristic enzyme activity of the cyclodextrin glucosyltransferase mutant is obviously improved compared with that of a wild type, the thermal stability of the cyclodextrin glucosyltransferase mutant has obvious advantages, the optimum temperature of the mutant is 60 DEG C and is improved by 5 DEG C compared with that of the wild type, the cyclodextrin glucosyltransferase mutant is applied to preparation of trehalose through compounding of a multi-enzyme system, the yield of trehalose is obviously increased, and the application prospect is wide. The utilization rate of a substrate is increased, and meanwhile, the application prospect of the enzyme in medicine, food and biological industries is also improved.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically 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) belongs to the α-amylase family (glycoside hydrolase 13_2, GH13_2), and is a multifunctional enzyme that can catalyze disproportionation, cyclization, coupling and hydrolysis reactions using starch, maltodextrin and other substrates. Among them, the disproportionation reaction is a transglycosidation reaction that occurs between two different molecules, which can transfer the cut part of the linear oligosaccharide to another receptor; the cyclization reaction is a transglycosidation reaction that occurs within the molecule, which is a characteristic reaction of CGTase and is often used in industry to produce cyclodextrin; the coupling reaction is the reverse reaction of the cyclization reaction, which can open the ring of cyclodextrin and transfer the glycoside to the linear malto-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 formed by two molecules of α-d-glucose linked by α,α-1,1-glycosidic bonds. It is usually in the form of dihydrate (C 12 H 22 O 11 ·2H 2 O). Trehalose has three optical isomers, the most common α,α-structure in nature and the rarely existing α,β- and β,β-structures in nature. Trehalose is widely present in various organisms. Due to its broad application prospects, the production of trehalose has made a breakthrough after the unremitting efforts of countless scientists.

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

[0005] Currently, CGTase is widely used, but there are few studies on improving the disproportionation characteristics and improving thermal stability at the same time. 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 firstly finds that a mutant N33K / S211G is obtained by performing superimposed mutation modification on beta-CGTase derived from Bacillus sp. G1, that is, the mutation sites of the mutant are asparagine (N) at position 33 mutated to lysine (K), and serine (S) at position 211 mutated to glycine (G); the dismutation property and thermal stability of the mutant 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 55°C of the wild type; the half-life of N33K / S211G relative to the mutant N33K is greatly improved, and the half-life of the mutant N33K / S211G at 60°C is 2.3 times that of the mutant N33K.

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

[0009] The technical solution of the present invention is 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] Preferably according to the present invention, 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 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 the coding gene of the mutant.

[0016] The 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, and 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 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. When applied to the preparation of trehalose, the substrate utilization rate and the yield of trehalose can be significantly improved. At the same time, the present invention has certain significance for the industrial production of cyclodextrin glucosyltransferase, and improves the application prospects of the enzyme in the pharmaceutical, food and biological 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 The 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] The drugs and reagents used in the examples are common products on the market unless otherwise specified. Any contents not described in detail in the examples are based on the prior art 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, and the balance water.

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

[0033] 12 mM EPS (4,6-ethylene-p-nitrobenzene-α-D-maltoheptaglycoside) and 20 mM maltose solutions were prepared with 50 mmol / L pH 6.0 phosphate buffer as solvent, 300 μL of 12 mM EPS and 20 mM maltose solutions were placed in a 45-65 °C water bath for preheating, 100 μL of diluted enzyme solution was added, and after reacting for 10 min, 50 μL of 3 M HCl was added, and 3 M NaOH was added for neutralization after 5 min, and then 100 μL of α-glucosidase was added and allowed to react in a 37 °C water bath for more than 60 min, and 100 μL of 1 M NaOH was added. 2 CO 3 The solution is adjusted to a pH value above 8.0, and finally the absorbance is measured at 401 nm. The dismutation activity of cyclodextrin glucosyltransferase is defined as the amount of enzyme required to convert 1 μmol 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 of cyclodextrin glucosyltransferase (Cgt) from Bacillus sp.G1 is artificially synthesized, the nucleotide sequence is shown in SEQ ID NO.1, the amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO.2, and an expression vector pET-28a(+) / Cgt is constructed, introduced into Escherichia coli BL21(DE3) for expression, and a wild-type cyclodextrin glucosyltransferase is obtained. The wild-type cyclodextrin glucosyltransferase is obtained by culturing in LB liquid medium (containing 100 mg / L kanamycin) at 37°C and 200 rpm for 10 hours, and a seed solution is obtained. The seed solution is inoculated with TB liquid fermentation medium (containing 100 mg / L kanamycin) at a volume ratio of 5% inoculation amount, and a final concentration of 0.2 mM IPTG is added after culturing at 37°C and 200 rpm for 2 hours, and the temperature is adjusted to 25°C, and the fermentation is carried out at 200 rpm for 8 hours. A certain volume of fermentation liquid is centrifuged at 4°C and 12000 rpm for 10 minutes, and the fermentation supernatant is taken, which is the crude enzyme solution of the wild enzyme.

[0039] Example 2

[0040] Preparation and expression of single mutant cyclodextrin glycosyltransferase

[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 the single mutant cyclodextrin glucosyltransferase.

[0042] PCR amplification of site-directed mutant encoding gene: Rapid PCR technology was used with the expression vector pET-28a(+) / Cgt carrying the gene encoding 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 with nucleotide sequence as shown in SEQ ID NO.4:

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

[0048] The PCR reaction system was: 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: 95°C pre-denaturation for 5 min; followed by 25 cycles (95°C for 15 s, 55°C for 15 s, 72°C for 3 min and 50 s) and 72°C extension for 5 min; and finally stored at 4°C. The PCR products were detected by 1% agarose gel electrophoresis. The mutant gene PCR verification results are shown in Figure 1 .

[0050] The above-verified correctly PCR product was digested with DpnⅠ and transferred into Escherichia coli DH5α competent cells. The transformation product was spread on an LB plate containing 100 mg / L kanamycin and cultured overnight at 37°C. Ten single colonies were picked from the plate 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 transferred into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing the single mutant N33K.

[0051] Example 3

[0052] Preparation and expression of double mutant of cyclodextrin glycosyltransferase

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

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

[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 with nucleotide sequence as shown in SEQ ID NO.6:

[0058] 5'-TATAAAT TCCATCTTCATATGATGAAAAATCTGTGCCGCC-3' (the underline indicates the mutant base). 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 2.5min. After 30 cycles, it was stored at 4°C. After DNA gel electrophoresis verification, subsequent operations were performed. The mutant gene PCR verification results are shown in Figure 1 The cyclodextrin glucosyltransferase N33K / S211G double mutant was obtained, and the 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 culture 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% inoculation, and cultured at 37°C and 200 rpm for 2 h. After that, a final concentration of 0.2 mM IPTG was added, the temperature was adjusted to 25°C, and the culture was carried out 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 taken, which was the crude enzyme solution of the double mutant (the preparation method of the crude enzyme solution is 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, the optimum pH was determined to be 6.0 and the pH stability was Figure 3 As shown, there is no significant difference compared with 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, it is 60℃, which is 5℃ higher than the wild type. The half-life of the mutant N33K / S211G at 50℃, 55℃, 60℃, and 65℃ was 2.4h, 2.0h, 55.4min, and 38.4min, respectively. The half-life of N33K / S211G relative to N33K at 50℃, 55℃, 60℃, and 65℃ was 2.0h, 1.5h, 24.0min, and 16.8min, respectively, which was greatly improved. The half-life of the mutant N33K / S211G at 60℃ was 2.3 times that of N33K. And the enzyme activity data measured by the above-mentioned method for determining the activity of cyclodextrin glucosyltransferase catalyzing dismutation reaction was found through experiments that the enzyme activity of the mutant N33K / S211G was 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, and the results were measured at 50°C for 10 minutes as 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 produce trehalose

[0068] Taking 200g / L maltodextrin as substrate, 5μg of assembled maltooligosaccharide trehalose synthase (MTSase) and maltooligosaccharide trehalose hydrolase (MTHase) prepared in the laboratory were added (reference patent: A preparation method of an immobilized MTSase enzyme and an immobilized MTHase ​​enzyme and its application in trehalose production), 5U / g of pullulanase were reciprocated in a constant temperature water bath at 60℃, and samples were taken after 48h 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. The samples were boiled for 10min to inactivate the enzyme, and saccharifying enzyme was added and reciprocated in a constant temperature water bath at 60℃. After 6h of reaction, the samples were boiled for 10min to inactivate the enzyme. After the reaction system was completed, the obtained reaction solution was placed in an ultracentrifuge and centrifuged at 12000 rpm for 10 min. The supernatant was diluted to a certain multiple and 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. Figure 5The figure shows a comparison of the amount of product generated, with no CGTase added as the 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%, and the yield of adding N33K / S211G is higher than that of BsCGT, proving that the obtained mutant can increase the yield of trehalose.

[0069] The mutant N33K / S211G constructed by the present invention has significantly improved dismutase activity and thermal stability compared with the wild enzyme. It can be applied to industrial production to prolong the reaction time and reduce the production cost. 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, which effectively improves its application prospects.

Claims

1. A mutant of cyclodextrin glucosyltransferase, It is characterized in that The amino acid mutation sites are the 33rd and 211th amino acids of the cyclodextrin glycosyltransferase amino acid sequence SEQ ID NO.

2.

2. The mutant according to claim 1, It is characterized in that The mutants are: The asparagine (N) at position 33 was mutated to lysine (K), and the serine (S) at position 211 was mutated to glycine (G), and was named N33K / S211G.

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

4. A recombinant expression vector, It is characterized in that Comprising the coding gene according to claim 3.

5. A recombinant strain, It is characterized in that Comprising the coding gene according to claim 3.

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

7. Use of the mutant according to claim 2 in the preparation of trehalose.

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