Cyclodextrin glucosyltransferase mutant with improved disproportionation activity and application of cyclodextrin glucosyltransferase mutant

Abstract

The invention provides a cyclodextrin glucosyltransferase mutant with improved disproportionation activity, belongs to the technical field of gene engineering and enzyme engineering, and mainly provides a cyclodextrin glucosyltransferase mutant, amino acid mutation sites are 62,153,481th amino acids of a cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2; the dismutase activity of the cyclodextrin glucosyltransferase mutant provided by the invention is obviously improved compared with that of a wild type, and the cyclodextrin glucosyltransferase mutant has certain significance for industrial production of cyclodextrin glucosyltransferase, and can obviously improve the yield of trehalose when being applied to preparation of trehalose by compounding a multi-enzyme system; meanwhile, the application prospects of the enzyme in medicine, food and biological industries are also widened.

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

[0002] Cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) 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 transglycosidic 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 transglycosidic reaction that occurs within the molecule, which is the characteristic reaction of CGTase and is often used in the 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 ·2H2O). 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 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 is the first to discover that by performing superimposed mutation modification on CGTase derived from Bacillus firmus strain 37, that is, the mutation sites of the mutant are 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), thereby obtaining a mutant V62Y / N153E / T481K; the dismutase activity of the mutant is increased by 84.9% compared with the wild type.

[0008] The encoding nucleotide sequence of the cyclodextrin glucosyltransferase derived from Bacillus firmus strain 37 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 solution of the present invention is 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] Preferably according to the present invention, 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 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 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] The use of the mutant, the coding gene, the recombinant expression vector or the recombinant strain in the preparation of coupling sugar.

[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 the 62nd and 153rd amino acids of the cyclodextrin glucosyltransferase amino acid sequence SEQ ID NO.2, the valine (V) at the 62nd position is mutated to tyrosine (Y), and the asparagine (N) at the 153rd position is mutated to glutamic acid (E), and is named V62Y / N153E.

[0020] The coding gene of the mutant I 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.

[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 coupling sugars.

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

[0026] Beneficial Effects

[0027] 1. The dismutase activity of the cyclodextrin glucosyltransferase mutant V62Y / N153E / T481K provided by the present invention is increased by 84.9% compared with the wild type, and 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.

[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. The cyclodextrin glucosyltransferase mutant provided by the present invention can not only increase the yield of trehalose, but 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, and it 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 This is a 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] 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.

[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, and the balance water.

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

[0038] Using 50mmol / L pH 6.0 phosphate buffer as solvent, 12mM EPS (4,6-ethylene-p-nitrobenzene-α-D-maltoheptaglycoside) and 20mM maltose solutions were prepared respectively, 300μL of 12mM EPS and 20mM maltose solutions were taken and placed in a 45-65℃ water bath for preheating, 100μL of diluted enzyme solution was added, and after accurate reaction for 10min, 50μL 3MHCl was added, and 3M NaOH was added for neutralization after 5min, and then 100μL α-glucosidase was added and allowed to react in a 37℃ water bath for more than 60min, 100μL 1M Na2CO3 solution was added to adjust the pH to above 8.0, and finally the absorbance was measured at 401nm. The dismutation activity of cyclodextrin glucosyltransferase is defined as the amount of enzyme required to convert 1μmol EPS per minute.

[0039] Method for determining the optimum temperature of cyclodextrin glycosyltransferase:

[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 obtained enzyme solution appropriately, measure the enzyme activity, and define the highest enzyme activity measured as 100%.

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

[0042] After the enzyme solution was appropriately diluted, it was placed in a constant temperature water bath at 35-75°C (every 5°C as a 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: aqueous 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 glycosyltransferase

[0048] The cyclodextrin glucosyltransferase (Cgt) gene derived from Bacillus firmus strain 37 is 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 is constructed and introduced into Escherichia coli BL21 (DE3) for expression to obtain a wild-type cyclodextrin glucosyltransferase. In LB liquid medium (containing 100 mg / L kanamycin), culture at 37°C, 200 rpm for 10 h to obtain seed solution, and the seed solution was inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a volume ratio of 5% inoculation amount, and cultured at 37°C, 200 rpm for 2 h. After that, a final concentration of 0.2 mM IPTG was added, and the temperature was adjusted to 25°C, and cultured at 200 rpm for 8 h. A certain volume of fermentation liquid was centrifuged at 4°C, 12000 rpm for 10 min, and the fermentation supernatant was taken, which was the crude enzyme solution of the wild enzyme. 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 mutant T481K is higher than that of mutant N153E, but the enzyme activity of mutant V62Y / T481K is lower than that of mutant V62Y / N153E. The enzyme activity of mutant V62Y / N153E reaches 11.0U / 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 single mutant 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 the single mutant cyclodextrin glucosyltransferase.

[0057] PCR amplification of site-directed mutant encoding gene: Rapid PCR technology was used with the expression vector pRSFDuet-1 / Cgt carrying the gene encoding 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' (the mutated base is underlined);

[0061] The reverse primer with 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: 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] The 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-verified correctly PCR product was digested with DpnⅠ and transferred 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 verified correctly plasmids were selected for sequencing, and the correctly sequenced plasmids were transferred into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing single mutants.

[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 a 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 glycosyltransferase

[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, and the primers were:

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

[0074] 5'-CATCAAA CGT CCCATAGTAAGGGTTTGT-3' (the 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' (the mutated base is underlined);

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

[0078] 5'-AGTGAT ATA CTGGTTACTATTACTGCT-3' (the mutated base is underlined);

[0079] Using the rapid PCR technique, the plasmid carrying the gene encoding the mutant V62Y was subjected to site-directed mutagenesis. The mutant V62Y gene was used as a template, and the primers shown in the sequences of SEQ ID NO.5 and SEQ ID NO.6 were used for PCR amplification, wherein the annealing temperature involved was 55°C; the fragment extension time was 5min, and the fragment was stored at 4°C after 30 cycles, and DNA gel electrophoresis was performed to verify it, and DNA sequencing was performed to obtain the cyclodextrin glucosyltransferase V62Y / N153E double mutant. Using this double mutant as a template, the primers shown in the sequences of SEQ ID NO.7 and SEQ ID NO.8 were used for PCR amplification, wherein the annealing temperature involved was 55°C; the fragment extension time was 5min, and the fragment was stored at 4°C after 30 cycles, and DNA gel electrophoresis was performed to verify it, and DNA sequencing was performed to obtain the cyclodextrin glucosyltransferase V62Y / N153E / T481K mutant. The 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 Escherichia 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% inoculation, and cultured at 37°C, 200 rpm for 2 h, and then a final concentration of 0.2 mM IPTG was added, the temperature was adjusted to 25°C, and cultured at 200 rpm for 8 h. A certain volume of fermentation liquid was centrifuged at 4°C, 12000 rpm for 10 min, and the fermentation supernatant was taken, which was the 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] With 200g / L maltodextrin as substrate, 5μg of laboratory-prepared maltooligosaccharide trehalose synthase (MTSase) and maltooligosaccharide trehalose hydrolase (MTHase) were added, and 5U / g of pullulanase were reciprocated in a 60℃ constant temperature water bath. In the later stage of the reaction, wild-type CGT and mutant enzyme V62Y / N153E / T481K were added respectively, and samples were taken during the reaction. The samples were boiled for 10min to kill the enzyme, and saccharifying enzyme was added and reciprocated in a 60℃ constant temperature water bath. After 6h of reaction, the samples were boiled for 10min to kill the enzyme. After the above reaction system was completed, the obtained reaction solution was placed in a centrifuge at 12000rpm for 10min, and the supernatant was diluted to a certain multiple, and the trehalose content in the conversion solution was determined by HPLC, using an NH2 chromatographic column, the mobile phase was acetonitrile: water = 4:1, the flow rate was 1.0mL / min, and the column temperature was 40℃. 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 the trehalose yield.

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

[0085] With 10 g / L hydroquinone and 60 g / L maltodextrin as substrates, wild-type CGT and mutant enzyme V62Y / N153E / T481K were added respectively, and reacted in a constant temperature water bath shaker at 100 r / min and 40℃ at pH 6.0 for 24 hours, and then the sample was put into a boiling water bath for 10 minutes to inactivate, and then amyloglucosidase was added, and reacted in a water bath shaker at 40℃ and 100 r / min for 4 hours, and then boiled in a boiling water bath for 10 minutes to inactivate, and the sample was centrifuged and diluted with mobile phase, and analyzed by high performance liquid chromatography (HPLC). A C18 column (4.6 mm×25 cm, 5 μm) was used; the mobile phase consisted of methanol: aqueous solution with a volume ratio of 20:80; the flow rate was 0.6 mL / min; the column temperature was 30℃; the UV detection wavelength was 287 nm; and the injection volume was 10 μL. The α-arbutin production of V62Y / N153E / T481K-added enzyme reached 8.9 g / L, which was 14.1% higher than that of the original CGTase (7.8 g / L). This indicates that the mutant obtained not only has the ability to increase the production of trehalose, but also has the ability to increase the production of α-arbutin.

[0086] The dismutase activity of the mutants V62Y / N153E / T481K and V62Y / N153E constructed by the invention is significantly improved compared with the wild enzyme, and the dismutase activity of the mutant V62Y / N153E / T481K is improved more significantly. The application of the mutants to industrial production can reduce the production cost, and the dismutation reaction activity thereof can be utilized to be applied to the modification of substances such as L-ascorbic acid and stevioside and the preparation of alpha-arbutin, and the mutants have broad application prospects.

Claims

1. A mutant of cyclodextrin glucosyltransferase, characterized in that: The amino acid mutation sites are the 62nd, 153rd and 481st amino acids of the cyclodextrin glycosyltransferase amino acid sequence SEQ ID NO.

2.

2. The mutant according to claim 1, characterized in that The mutants are: 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 was named V62Y / N153E / T481K.

3. The gene encoding the mutant according to any one of claims 1 to 2, 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, characterized in that: A gene encoding the mutant according to claim 3.

5. A recombinant strain comprising a gene encoding the mutant according to claim 3.

6. Use of the coding gene of the mutant 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 any one of claims 1 to 2, the gene encoding the mutant according to claim 3, the recombinant expression vector according to claim 4 or the recombinant strain according to claim 5 in the preparation of coupling sugars.

8. Use of the mutant according to any one of claims 1 to 2, the encoding gene of the mutant according to claim 3, the recombinant expression vector according to claim 4 or the recombinant strain according to claim 5 in the preparation of trehalose.

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

10. The gene encoding mutant I according to claim 9, 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; Preferably, a recombinant expression vector comprises a gene encoding the mutant I; Preferably, a recombinant strain comprises a gene encoding the mutant I; Preferably, the coding gene, recombinant expression vector or recombinant strain of the mutant I is used in the preparation of cyclodextrin glucosyltransferase; Preferably, the use of the mutant I, the encoding gene of the mutant I, the recombinant expression vector or the recombinant strain in the preparation of coupling sugars; Preferably, the mutant I, the encoding gene of mutant I, the recombinant expression vector or the recombinant strain is used in the preparation of trehalose and α-arbutin.

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