A cyclodextrin glucosyltransferase mutant with improved dismutation activity and its application

Abstract

The present invention provides a cyclodextrin glucosyltransferase mutant with improved dismutation activity, belonging to the technical fields of genetic engineering and enzyme engineering. The invention mainly provides a cyclodextrin glucosyltransferase mutant, wherein the amino acid mutation sites are amino acids 62, 153, and 481 of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2. The dismutase activity of the cyclodextrin glucosyltransferase mutant provided by the present invention is significantly improved compared with the wild type, which is of certain significance for the industrial production of cyclodextrin glucosyltransferase. The application of the mutant in the preparation of trehalose in a multi-enzyme system can significantly increase the yield of trehalose, and also broadens the application prospects of the enzyme in the pharmaceutical, food, and biological industries.

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 with improved dismutation activity and application thereof in preparing trehalose. Background Art

[0002] Cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19), 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 the 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. Due to its low disproportionation properties, the industrial application scope of CGTase is greatly limited. Summary of the Invention

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

[0007] The present invention discovered for the first time that by subjecting CGTase from Bacillus firmus strain 37 to a stacked mutation modification, the mutant's mutation sites were valine (V) at position 62 mutated to tyrosine (Y), asparagine (N) at position 153 mutated to glutamate (E), and threonine (T) at position 481 mutated to lysine (K), resulting in a mutant V62Y / N153E / T481K. The dismutase activity of the mutant was increased by 84.9% compared to the wild type.

[0008] The nucleotide sequence encoding the cyclodextrin glucosyltransferase derived from Bacillus firmus strain 37 of 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 62nd, 153rd and 481st 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 valine (V) at position 62 mutated to tyrosine (Y), the asparagine (N) at position 153 mutated to glutamic acid (E), and the threonine (T) at position 481 mutated to lysine (K), and the strain was named V62Y / N153E / T481K.

[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] Use of the mutant, the encoding gene of the mutant, the recombinant expression vector or the recombinant strain in the preparation of coupled sugars.

[0018] Application of the mutant, the encoding gene of the mutant, the recombinant expression vector or the recombinant strain in the preparation of trehalose and α-arbutin.

[0019] A mutant I of cyclodextrin glucosyltransferase, wherein the amino acid mutation sites are amino acids 62 and 153 of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2, wherein the valine (V) at position 62 is mutated to tyrosine (Y), and the asparagine (N) at position 153 is mutated to glutamic acid (E), and is named V62Y / N153E.

[0020] The coding gene of the above mutant I was 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.

[0021] A recombinant expression vector comprises the coding gene of the mutant I.

[0022] A recombinant strain comprising the coding gene of the mutant I.

[0023] The application of the coding gene, recombinant expression vector or recombinant strain of the mutant I in the preparation of cyclodextrin glucosyltransferase.

[0024] The use of the mutant I, the coding gene of the mutant I, the recombinant expression vector or the recombinant strain in the preparation of coupled sugars.

[0025] Application of the mutant I, the coding gene, recombinant expression vector or recombinant strain of the mutant I in the preparation of trehalose and α-arbutin.

[0026] Beneficial effects

[0027] The cyclodextrin glucosyltransferase mutant V62Y / N153E / T481K provided by the present invention exhibits an 84.9% increase in dismutase activity compared to the wild-type. When used in the preparation of trehalose, it significantly improves substrate utilization and trehalose yield. Furthermore, the present invention has significant implications for the industrial production of cyclodextrin glucosyltransferase and enhances its potential applications in the pharmaceutical, food, and biotechnology industries.

[0028] 2. The mutant provided by the present invention not only has significantly improved dismutase activity, but also has no effect on the thermal stability of the mutant.

[0029] 3. In addition to increasing the yield of trehalose, the cyclodextrin glucosyltransferase mutant provided by the present invention also has the ability to increase the yield of α-arbutin.

[0030] 4. The dismutase activity of the cyclodextrin glucosyltransferase mutant V62Y / N153E provided by the present invention is increased by 50.7% compared with the wild type, which also has certain advantages and can be applied to the preparation of trehalose, which is of certain significance for the industrial production of cyclodextrin glucosyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Graph showing the optimal temperature and temperature stability experimental results for the mutant V62Y / N153E / T481K and the wild type. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The culture medium and detection method involved in the following examples are as follows:

[0035] LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and the balance water.

[0036] 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.

[0037] Method for determining the activity of cyclodextrin glucosyltransferase catalyzing dismutation reaction:

[0038] A 12 mM EPS (4,6-ethylene-p-nitrobenzene-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.

[0039] Method for determining the optimal temperature of cyclodextrin glucosyltransferase:

[0040] Prepare the reaction substrate in advance and preheat it in a constant temperature water bath at 35-75°C (5°C per gradient). Dilute the resulting enzyme solution appropriately and measure the enzyme activity. The highest enzyme activity measured is defined as 100%.

[0041] Method for determining the temperature stability of cyclodextrin glucosyltransferase:

[0042] After the enzyme solution was appropriately diluted, it was placed in a constant temperature water bath at 35-75°C (5°C per gradient) for 1 hour. After sampling, the enzyme activity was measured, and the highest enzyme activity measured was defined as 100%.

[0043] Trehalose product detection method:

[0044] 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.

[0045] α-Arbutin product detection method:

[0046] The content of α-arbutin in the conversion solution was determined by HPLC using a C18 column, a mobile phase consisting of methanol:water solution in a volume ratio of 20:80, a flow rate of 0.6 mL / min, a column temperature of 30 °C, and an ultraviolet detection wavelength of 287 nm.

[0047] Example 1 Preparation and expression of wild-type cyclodextrin glucosyltransferase

[0048] The cyclodextrin glucosyltransferase (Cgt) gene from Bacillus firmus strain 37 was artificially synthesized, and 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. An expression vector pRSFDuet-1 / Cgt was constructed and introduced into Escherichia coli BL21 (DE3) for expression to obtain wild-type cyclodextrin glucosyltransferase. The seed solution was prepared by culturing in LB liquid medium (containing 100 mg / L kanamycin) at 37°C and 200 rpm for 10 h. The seed solution was inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a volume ratio of 5%. After culturing at 37°C and 200 rpm for 2 h, IPTG was added at a final concentration of 0.2 mM. The temperature was adjusted to 25°C and the culture was carried out at 200 rpm for 8 h. A certain volume of fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min. The fermentation supernatant was obtained as the crude enzyme solution of the wild enzyme, and the dismutase activity was determined to be 7.3 U / mL.

[0049] When studying the cyclodextrin glucosyltransferase mutant, the inventors found that among multiple mutants, the cyclodextrin glucosyltransferase mutant V62Y / N153E / T481K had the highest dismutase activity compared to the wild-type enzyme, which had a dismutase activity of 7.3 U / mL. The dismutase activity of the mutant V62Y / N153E / T481K reached 13.5 U / mL, as shown in Table 1.

[0050] As can be seen from Table 1, different mutation sites of the enzyme have different effects on the enzyme activity. For example, the enzyme activity of the mutant T481K is higher than that of the mutant N153E, but the enzyme activity of the mutant V62Y / T481K is lower than that of the mutant V62Y / N153E. The enzyme activity of the mutant V62Y / N153E reaches 11.0 U / mL.

[0051] Table 1

[0052]

[0053]

[0054] The inventors compared the optimal temperature and temperature stability of the mutant V62Y / N153E / T481K and the wild type and found that while the dismutase activity of the mutant was improved, the optimal temperature and temperature stability were not significantly affected. Figure 1 .

[0055] Example 2 Preparation and expression of a single mutant of cyclodextrin glycosyltransferase

[0056] (1) Based on the nucleotide sequence of Bacillus firmus strain 37 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.

[0057] PCR amplification of the site-directed mutant encoding gene: Rapid PCR technology was used with the expression vector pRSFDuet-1 / Cgt carrying the gene encoding the wild-type cyclodextrin glucosyltransferase as a template.

[0058] The site-directed mutagenesis primers for introducing the V62Y mutation are:

[0059] The nucleotide sequence of the forward primer is shown in SEQ ID NO.3:

[0060] 5'-GATTTACCAG TAT GTTACCGATCGA-3' (mutated bases are underlined);

[0061] The reverse primer has a nucleotide sequence as shown in SEQ ID NO.4:

[0062] 5'-CGGTAAC ATA CTGGTAAATCACATCT-3' (the mutated base is underlined).

[0063] 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 was added to make up to 50 μL.

[0064] PCR conditions were as follows: pre-denaturation at 95°C for 5 min; followed by 25 cycles (95°C for 15 s, 55°C for 15 s, 72°C for 5 min) and extension at 72°C for 5 min; and finally storage at 4°C.

[0065] 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. Three single colonies were picked from the plates and transferred into LB liquid culture medium containing 100 mg / L kanamycin. After culturing for 8 hours, the plasmids were extracted and verified. Two 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.

[0066] (2) Expression of mutants

[0067] The recombinant Escherichia coli expressing the single mutant prepared in step (1) of this example was used to prepare a crude enzyme solution of the single mutant according to the method for preparing the crude enzyme solution of the wild enzyme in Example 1. The enzyme activity was measured to be 8.7 U / mL.

[0068] Example 3 Preparation and expression of cyclodextrin glycosyltransferase mutant V62Y / N153E / T481K

[0069] (1) Preparation of double mutants of cyclodextrin glucosyltransferase

[0070] Using the plasmid carrying the gene encoding mutant V62Y constructed in Example 2 as a template, site-directed mutagenesis of the N153E / T481K mutation was introduced using the following primers:

[0071] The nucleotide sequence of the forward primer is shown in SEQ ID NO.5:

[0072] 5'-CTATGGG ACG TTTGATGACTTTGATCGT-3' (the mutated base is underlined);

[0073] The reverse primer has a nucleotide sequence as shown in SEQ ID NO.6:

[0074] 5'-CATCAAA CGT CCCATAGTAAGGGTTTGT-3′ (mutated bases are underlined);

[0075] The nucleotide sequence of the forward primer is shown in SEQ ID NO.7:

[0076] 5'-CCAG TAT ATCACTAATTTAAACACCTCT-3' (mutated bases are underlined);

[0077] The reverse primer has a nucleotide sequence as shown in SEQ ID NO.8:

[0078] 5'-AGTGAT ATA CTGGTTACTATTACTGCT-3′ (mutated bases are underlined);

[0079] Using rapid PCR technology, site-directed mutagenesis was performed on a plasmid carrying the gene encoding mutant V62Y. Using the mutant V62Y gene as a template, PCR amplification was performed using primers represented by SEQ ID NO. 5 and SEQ ID NO. 6. The annealing temperature was 55°C, and the fragment extension time was 5 minutes. After 30 cycles, the fragment was stored at 4°C. After verification by DNA gel electrophoresis, it was verified by DNA sequencing. A cyclodextrin glucosyltransferase V62Y / N153E double mutant was obtained. Using this double mutant as a template, PCR amplification was performed using primers represented by SEQ ID NO. 7 and SEQ ID NO. 8. The annealing temperature was 55°C, and the fragment extension time was 5 minutes. After 30 cycles, the fragment was stored at 4°C. After verification by DNA gel electrophoresis, it was verified by DNA sequencing. The cyclodextrin glucosyltransferase V62Y / N153E / T481K mutant was obtained. Recombinant Escherichia coli expressing the V62Y / N153E / T481K mutant was prepared according to the method in Example 2.

[0080] (2) Expression of mutants

[0081] The recombinant E. coli expressing the V62Y / N153E / T481K mutant prepared in step (1) of this embodiment 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, 200 rpm for 2 h, and then added with a final concentration of 0.2 mM IPTG. The temperature was adjusted to 25°C and cultured at 200 rpm for 8 h. A certain volume of fermentation broth was centrifuged at 4°C, 12000 rpm for 10 min, and the fermentation supernatant was taken to obtain a crude enzyme solution of the V62Y / N153E / T481K 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). The crude enzyme solution was subjected to enzyme activity assay, and the dismutase activity was 13.5 U / mL.

[0082] Example 4 Preparation of trehalose using mutant V62Y / N153E / T481K

[0083] Using 200 g / L maltodextrin as the substrate, 5 μg each of laboratory-prepared maltooligosaccharyl trehalose synthase (MTSase) and maltooligosaccharyl trehalose hydrolase (MTHase) were added, along with 5 U / g of pullulanase, and the mixture was shaken back and forth in a 60°C constant temperature water bath. Wild-type CGT and mutant enzyme V62Y / N153E / T481K were added at the end of the reaction. Samples were taken during the reaction and inactivated by boiling for 10 minutes. Saccharifying enzyme was added and shaken back and forth in a 60°C constant temperature water bath. After 6 hours of reaction, the sample was boiled for 10 minutes to inactivate the enzyme. After the reaction system was completed, the reaction solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was diluted to a certain multiple and the trehalose content in the conversion solution was determined by HPLC using an NH2 column with a mobile phase of acetonitrile:water = 4:1, a flow rate of 1.0 mL / min, and a column temperature of 40°C. The trehalose yield after adding V62Y / N153E / T481K reached 138 g / L, which was 10.4% higher than the yield of the original CGTase (125 g / L), proving that the obtained mutant can increase trehalose yield.

[0084] Example 5 Preparation of α-arbutin using mutant V62Y / N153E / T481K

[0085] Using 10 g / L hydroquinone and 60 g / L maltodextrin as substrates, wild-type CGT and mutant enzyme V62Y / N153E / T481K were added, respectively. The reaction was allowed to proceed at pH 6.0 in a shaker at 100 rpm and 40°C for 24 h. The sample was then inactivated by boiling in a water bath for 10 min. Amyloglucosidase was then added and the reaction was continued in a shaker at 40°C and 100 rpm for 4 h. The sample was then inactivated by boiling in a water bath for another 10 min. The sample was centrifuged and diluted with mobile phase for analysis by high-performance liquid chromatography (HPLC). A C18 column (4.6 mm × 25 cm, 5 μm) was used; the mobile phase consisted of methanol:water (volume ratio, 20:80); the flow rate was 0.6 mL / min; the column temperature was 30°C; the UV detection wavelength was 287 nm; and the injection volume was 10 μL. The α-arbutin production of the mutant with V62Y / N153E / T481K added reached 8.9 g / L, which was 14.1% higher than the production of the original CGTase (7.8 g / L). This demonstrates that the mutant can not only increase the production of trehalose, but also has the ability to increase the production of α-arbutin.

[0086] The dismutase activities of the mutants V62Y / N153E / T481K and V62Y / N153E constructed by the present invention are significantly improved compared with the wild-type enzyme, and the dismutase activity of the mutant V62Y / N153E / T481K is even more significantly improved. Application in industrial production can reduce production costs, and the dismutation reaction activity can be utilized for application in the modification of substances such as L-ascorbic acid and stevioside, as well as in the preparation of α-arbutin, thereby having broad application prospects.

Claims

1. A mutant of cyclodextrin glucosyltransferase, characterized in that: The amino acid mutation sites are valine (V) at position 62 of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2 mutated to tyrosine (Y), asparagine (N) at position 153 mutated to glutamic acid (E), and threonine (T) at position 481 mutated to lysine (K), and the enzyme was named V62Y / N153E / T481K.

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: A gene encoding the mutant according to claim 2. A recombinant strain comprising a gene encoding the mutant according to claim 2.

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

7. Use of the mutant according to claim 1, the gene encoding the mutant according to claim 2, the recombinant expression vector according to claim 3 or the recombinant strain according to claim 4 in the preparation of trehalose.

8. A mutant I of cyclodextrin glucosyltransferase, wherein the amino acid mutation sites are at amino acids 62 and 153 of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO. 2; valine (V) at position 62 is mutated to tyrosine (Y), and asparagine (N) at position 153 is mutated to glutamic acid (E); the mutant is designated V62Y / N153E.

9. The gene encoding mutant I according to claim 8, 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.

10. A recombinant expression vector, characterized in that: A gene encoding the mutant I according to claim 9.

11. A recombinant strain, characterized in that A gene encoding the mutant I according to claim 9.

12. Use of the gene encoding mutant I according to claim 9, the recombinant expression vector according to claim 10, or the recombinant strain according to claim 11 in the preparation of cyclodextrin glucosyltransferase.

13. Use of the mutant I according to claim 8, the gene encoding the mutant I according to claim 9, the recombinant expression vector according to claim 10 or the recombinant strain according to claim 11 in the preparation of coupling sugars.

14. Use of the mutant I according to claim 8, the gene encoding the mutant I according to claim 9, the recombinant expression vector according to claim 10, or the recombinant strain according to claim 11 in the preparation of trehalose and α-arbutin.

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