Gamma-cyclodextrin glucosyltransferase mutant with improved thermal stability and application of gamma-cyclodextrin glucosyltransferase mutant

Abstract

The invention discloses a gamma-cyclodextrin glucosyltransferase mutant with improved thermal stability and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The 165 site, the 398 site, the 478 site and the 522 site of wild type gamma-cyclodextrin glucosyltransferase are mutated, the thermal stability of the gamma-cyclodextrin glucosyltransferase mutant obtained through construction is obviously improved compared with the thermal stability of the wild type gamma-cyclodextrin glucosyltransferase mutant, the half-life period at 50 DEG C is 101.93 min, and the thermal stability is improved by 7.46 times compared with the thermal stability of the wild type gamma-cyclodextrin glucosyltransferase mutant. When the strain is applied to enzyme production of gamma-cyclodextrin, the yield of gamma-cyclodextrin is increased by 46.3% and the yield of gamma-cyclodextrin is increased by 40.3% compared with that of wild type gamma-cyclodextrin. The production efficiency of continuous production of the gamma-cyclodextrin glucosyltransferase in actual industrial application is improved, and the application prospect of the glucosyltransferase in the fields of food, medicine, biology, materials and the like is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a gamma-cyclodextrin glucosyltransferase mutant with improved thermal stability and its application, and belongs to the technical field of genetic engineering and enzyme engineering. BACKGROUND

[0002] Gamma-cyclodextrin is a cyclic oligomer composed of 8 D-glucopyranose units connected by α-1, 4-glycosidic bonds, and its spatial structure is a hollow cylindrical structure. Therefore, it has both hydrophilic surface and hydrophobic cavity structure, the best solubility, the largest cavity, and can be completely degraded by α-amylase in vivo. It can be rapidly degraded and absorbed in the gastrointestinal tract and metabolized and excreted. Therefore, gamma-cyclodextrin has the lowest toxicity, and there is no limit on the minimum daily intake. It has been used in the fields of medicine, food, cosmetics, materials, etc.

[0003] The production of gamma-cyclodextrin mainly depends on the conversion of starch by gamma-cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19). Gamma-cyclodextrin glucosyltransferase belongs to the α-amylase family and has four catalytic abilities of hydrolysis, disproportionation, cyclization and coupling. The cyclization reaction is its characteristic reaction and is mainly used for the production of cyclodextrin. The current domestic gamma-cyclodextrin industrial production system is not mature, mainly due to the lack of gamma-cyclodextrin glucosyltransferase with high specificity, high stability and good catalytic efficiency. The source of gamma-cyclodextrin glucosyltransferase is scarce. There are only three gamma-cyclodextrin glucosyltransferases with application value reported so far, and all of them have the disadvantage of poor thermal stability, which makes it difficult to meet the industrial application and greatly limits the development of gamma-cyclodextrin industry. Therefore, it is urgent to find a gamma-cyclodextrin glucosyltransferase with high thermal stability for the production of gamma-cyclodextrin.

[0004] Cyclodextrin glucosyltransferase derived from Bacillus sp. G-825-6STB17 is a gamma-cyclodextrin glucosyltransferase with great application potential. Its catalytic activity per unit of protein is much higher than that of other sources of gamma-cyclodextrin glucosyltransferase and can be used for the industrial production of gamma-cyclodextrin. However, the thermal stability of the enzyme is poor, with a half-life of only about 15 min at 50℃, which increases the use cost of gamma-CGTase. Therefore, it is imperative to improve the thermal stability of the enzyme. SUMMARY

[0005] In order to solve the existing technical problems, the present application provides a gamma-cyclodextrin glucosyltransferase mutant with improved thermal stability and its application.

[0006] The first object of the present application is to provide a γ-cyclodextrin glucosyltransferase mutant, which is obtained by mutating one or more of the following positions: position 165, position 398, position 478, and position 522 of a wild-type cyclodextrin glucosyltransferase having an amino acid sequence as shown in SEQ ID NO. 1.

[0007] In an embodiment, the mutant is one or a combination of the following (a) to (d):

[0008] (a) the threonine at position 165 of the wild-type γ-cyclodextrin glucosyltransferase is mutated to histidine, and the obtained mutant is named T165H;

[0009] (b) the glutamic acid at position 398 of the wild-type γ-cyclodextrin glucosyltransferase is mutated to isoleucine or valine, and the obtained mutant is named E398I or E398V;

[0010] (c) the glutamine at position 478 of the wild-type γ-cyclodextrin glucosyltransferase is mutated to isoleucine, and the obtained mutant is named Q478I;

[0011] (d) the serine at position 522 of the wild-type γ-cyclodextrin glucosyltransferase is mutated to valine, and the obtained mutant is named S522V.

[0012] In an embodiment, the γ-cyclodextrin glucosyltransferase mutant has an amino acid sequence as shown in SEQ ID NO. 2.

[0013] The second object of the present application is to provide a gene encoding the γ-cyclodextrin glucosyltransferase mutant.

[0014] In an embodiment, the gene has a nucleotide sequence as shown in SEQ ID NO. 3.

[0015] The third object of the present application is to provide a recombinant expression vector carrying the gene sequence.

[0016] In an embodiment, the vector can be selected according to the need (e.g., host species); the vector of the recombinant plasmid includes, but is not limited to, a pET series vector, a pP43NMK series vector, a pHT series vector, or a pPIC9K series vector.

[0017] The fourth object of the present application is to provide a recombinant microbial cell expressing the γ-cyclodextrin glucosyltransferase mutant.

[0018] In one embodiment, the microorganism is a fungus or a bacterium, including but not limited to Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris.

[0019] In one embodiment, the microorganism is Bacillus subtilis.

[0020] A fifth object of the present application is to provide a method for preparing the mutant of γ-cyclodextrin glucosyltransferase, comprising the steps of constructing the expression vector and fermentative production using the recombinant cell.

[0021] The present application also provides a method for improving the thermal stability of γ-cyclodextrin glucosyltransferase, which is to mutate the γ-cyclodextrin glucosyltransferase with one or more of the following mutations:

[0022] (a) mutating the threonine at position 165 to histidine;

[0023] (b) mutating the glutamic acid at position 398 to isoleucine or valine;

[0024] (c) mutating the glutamine at position 478 to isoleucine;

[0025] (d) mutating the serine at position 522 to valine.

[0026] The present application also provides the use of the mutant of γ-cyclodextrin glucosyltransferase, the genetic sequence, the recombinant expression vector, the recombinant cell, or the mutant of γ-cyclodextrin glucosyltransferase prepared by the method in food, medicine, biology, or material.

[0027] In one embodiment, the use includes hydrolyzing α-1,4 glycosidic bonds or hydrolyzing substances containing α-1,4 glycosidic bonds.

[0028] In one embodiment, the use includes the production of cyclodextrin.

[0029] In one embodiment, the use includes the transglycosylation modification of substances.

[0030] The present application also provides a method for producing γ-cyclodextrin, which utilizes the mutant of γ-cyclodextrin glucosyltransferase to convert starch into γ-cyclodextrin.

[0031] In one embodiment, the method is carried out at 50-60°C.

[0032] In one embodiment, the starch includes but is not limited to cassava starch.

[0033] In one embodiment, the method is that the preheated cassava starch solution is used as a substrate, and the γ-cyclodextrin glucosyltransferase mutant is added at ≥1 U / g of the substrate, and the reaction is carried out at 50-55°C for at least 30 min.

[0034] In one embodiment, the reaction time is ≥10 h.

[0035] The application also provides the use of the γ-cyclodextrin glucosyltransferase mutant or the method in the preparation of γ-cyclodextrin or a product containing γ-cyclodextrin.

[0036] Advantages

[0037] (1) The γ-cyclodextrin glucosyltransferase provided by the application has significantly improved thermal stability, and can retain more than 80% of the activity after incubation at 50°C for 30 min, with a half-life of 101.93 min, and the thermal stability is increased by 7.46 times compared with the wild type.

[0038] (2) The optimal mutant of the γ-cyclodextrin glucosyltransferase provided by the application can be used to improve the yield of γ-cyclodextrin, and when 10% (w / w) of starch is used as a substrate, the yield of γ-cyclodextrin can reach 6.32 g / L, and the proportion of γ-cyclodextrin is 63.7%, which is increased by 46.3% and 40.3% respectively compared with the yield and proportion of the wild type. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is an agarose gel electrophoresis map of the wild type γ-cyclodextrin glucosyltransferase and its mutants, wherein M: DNA standard molecular weight, 1: wild type plasmid, 2-6: mutant plasmid, respectively T165H, E398V, E398I, Q478I, S522V.

[0040] Figure 2 It is an SDS-PAGE map of the wild type γ-cyclodextrin glucosyltransferase and its mutants, wherein M: protein standard molecular weight, 1: wild type, 2-6: mutant, respectively T165H, E398V, E398I, Q478I, S522V.

[0041] Figure 3 It is a characterization of the thermal stability of the wild type γ-cyclodextrin glucosyltransferase and its mutants at 50°C. DETAILED DESCRIPTION

[0042] The examples described below are only a part of the examples of the application, but not all the examples. Based on the examples in the application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0043] (1) Method for measuring γ-cyclization activity:

[0044] The γ-cyclization activity was measured by the bromocresol green (BCG) method. 0.1 mL of appropriately diluted enzyme solution was added to a centrifuge tube containing 0.9 mL of 1% maltodextrin (DE = 4) prepared in advance with a pH 9.0, 20 mM Tris-HCl buffer, preheated for 5 min, and reacted at 55°C for 10 min. The reaction was terminated by adding 100 μL of 1.0 M hydrochloric acid solution, followed by the addition of 2 mL of 0.2 M, pH 4.2 citric acid buffer and 100 μL of 5 mM bromocresol green solution (BCG). Color development was performed at room temperature for 20 min, and the absorbance was measured at a wavelength of 630 nm. Inactivated enzyme solution was used as a blank.

[0045] One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of γ-cyclodextrin per minute under the above conditions.

[0046] (2) Method for analyzing reaction products:

[0047] The detection method for cyclodextrin used high performance liquid chromatography (HPLC). The reaction solution was inactivated by boiling in a water bath for 30 min, centrifuged (10,000 x g, 10 min), and the supernatant was passed through a 0.22 μm water filter membrane before being analyzed for products using HPLC. The HPLC conditions were as follows: Waters 600 HPLC system (with a differential refractive index detector), chromatographic column Hypersil GOLD Amino HPLC (4.6 mm x 250 mm), column temperature 30°C, mobile phase 70% (v / v) acetonitrile aqueous solution, flow rate 1 mL / min. TM

[0048] Example 1: Method for preparing mutants

[0049] (1) The vector pP43NMK-γ-cgt carrying the wild-type γ-cyclodextrin glucosyltransferase gene sequence was used as a template, and the primers in Table 1 were used for site-directed mutagenesis. PCR amplification was performed according to the method described in the TaKaRa Primer STAR GXL kit manual. The PCR product was digested with Dpn I, and then transformed into E. coli JM109 super-competent cells to obtain genetically engineered bacteria carrying the mutant recombinant plasmid.

[0050] The PCR reaction amplification conditions were as follows: pre-denaturation at 98°C for 3 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 15 s, and extension at 68°C for 7 min, and finally 68°C for 10 min.

[0051] Table 1 Mutant primer sequences

[0052] ​

[0053]

[0054] Note: The underlined base corresponds to the corresponding mutant amino acid.

[0055] (2) The transformants were coated on ampicillin (20 μg / mL) LB solid medium, and incubated at 37°C in an inverted incubator. Positive monoclonal was picked up and inoculated into LB liquid medium containing ampicillin (20 μg / mL) and incubated at 37°C, 200 rpm for 10-12 h. The plasmid was extracted and sequenced.

[0056] (3) The correctly sequenced vector plasmid was transformed into B. subtilis WB600 to obtain the corresponding mutant host bacteria.

[0057] Example 2: Preparation and purification of γ-cyclodextrin glucosyltransferase mutants

[0058] (1) The recombinant genetically engineered bacteria constructed in Example 1 were streaked on LB solid medium containing kanamycin (20 μg / mL) and incubated at 37°C in an incubator for 12 h. Single colonies were picked up and inoculated into LB liquid medium containing kanamycin (20 μg / mL) and incubated at 37°C, 200 rpm for 10-12 h to prepare seed liquid.

[0059] (2) The seed liquid prepared in step (1) was inoculated into fermentation liquid medium containing kanamycin (20 μg / mL) at an inoculation amount of 2% (v / v), and fermented at 30°C, 200 rpm for 72 h. The fermentation liquid was centrifuged at 4°C, 10,000 rpm for 20 min, and the supernatant was collected to obtain crude enzyme liquid.

[0060] (3) The crude enzyme liquid was purified by nickel column affinity chromatography: ① Enzyme liquid pretreatment: 50 mL of crude enzyme liquid was added with 500 mM NaCl and 20 mM imidazole, and filtered through a 0.45 μm water system filter to remove macromolecular impurities; ② The equilibrium liquid (A liquid, pH 7.5) was 500 mM NaCl + 50 mM Tris-HCl + 20 mM imidazole, and the elution liquid (B liquid, pH 7.5) was 500 mM NaCl + 50 mM Tris-HCl + 500 mM imidazole. The crude enzyme liquid was adjusted to pH 7.5 and filtered through a 0.45 μm water system membrane before purification. Gradient elution was performed with 60% elution liquid B at a flow rate of 1 mL / min, and the corresponding elution liquid was collected according to the elution peak, which was the pure enzyme.

[0061] (4) The pure enzyme was added to an ultrafiltration centrifuge tube and centrifuged at low speed. After the buffer was replaced with water to desalt, the mutant pure enzyme enzyme liquid was obtained. The specific enzyme activity of the pure enzyme was measured at 55°C, and the results are shown in Table 2.

[0062] Table 2 Comparison of specific enzyme activities of wild-type enzyme and mutants

[0063]

[0064]

[0065] Example 3: Determination of optimal reaction temperature and thermal stability of γ- cyclodextrin glucosyltransferase mutants

[0066] (1) Determination of optimal reaction temperature:

[0067] The cyclization activity of γ-cyclodextrin glucosyltransferase, mutant T165H and mutant E398V, diluted to an appropriate concentration, was determined at different temperatures (40-70°C). The highest enzyme activity was defined as 100%, and the corresponding reaction temperature at the highest enzyme activity was the optimal reaction temperature.

[0068] The results showed that the optimal reaction temperature of mutant T165H could be increased to 60°C, and the enzyme activity at 60°C could reach 31.20 U / mg.

[0069] (2) Determination of thermal stability:

[0070] The residual activity of γ-cyclodextrin glucosyltransferase, mutant T165H and mutant E398V, diluted to an appropriate concentration, was determined at different time points after incubation at 50°C. The original enzyme activity without incubation was defined as 100%, and the change trend of relative residual enzyme activity with time at different temperatures was determined.

[0071] The results are shown in Table 2. Figure 3 The half-life of wild-type γ-cyclodextrin glucosyltransferase was 13.67 min, while mutant T165H retained more than 80% of its activity after incubation at 50°C for 30 min, and more than 40% of its activity after incubation for 135 min. The half-life of this mutant was 101.93 min, which was 7.46 times that of the wild type.

[0072] Example 4: Application of γ-cyclodextrin glucosyltransferase mutants in γ- cyclodextrin production

[0073] (1) Prepare a cassava starch solution with a mass fraction of 10% (w / w) and place it in a 55°C water bath for 15 min. Stir the solution at a speed of 200-300 rpm to obtain the reaction system.

[0074] (2) Add the wild-type γ-cyclodextrin glucosyltransferase and mutant T165H enzyme solution prepared in Example 2 to the reaction system, respectively, at an addition amount of 2 U / g starch, and heat to 90°C for liquefaction. The liquefaction time is 20 min.

[0075] (3) After liquefaction is completed, respectively, temperature is reduced to 50 DEG C, 55 DEG C, and then gamma-cyclodextrin glucosyltransferase enzyme solution is added again, the adding amount is 2 U / g starch, and cyclization reaction is carried out, and the reaction time is 10 h.

[0076] (4) After reaction is completed, the system is heated to 90 DEG C to carry out enzyme inactivation, sampling is carried out, and after dilution of a proper multiple, HPLC is used to analyze reaction products. The results show that the yield of gamma-cyclodextrin prepared by the mutant enzyme T165H at 50 DEG C reaction can reach 6.18±0.02 g / L, and the proportion of gamma-cyclodextrin is 57.88%±0.17%, which is increased by 27.2% and 18.51% respectively compared with the yield and proportion of the wild type; the yield of gamma-cyclodextrin prepared by the mutant enzyme T165H at 55 DEG C reaction can reach 6.32±0.07 g / L, and the proportion of gamma-cyclodextrin is 63.7%±0.02%, which is increased by 46.3% and 40.3% respectively compared with the yield and proportion of the wild type.

[0077] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A mutant of a γ-cyclodextrin glucosyltransferase characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 1, one or more of the following mutations at positions 165, 398, 478, and 522 are made.

2. The γ-cyclodextrin glucanotransferase mutant according to claim 1, characterized in that, The mutant is one or more combinations of the following (a) to (d): (a) the threonine at position 165 of the γ-cyclodextrin glucosyltransferase shown in SEQ ID NO. 1 is mutated to histidine; (b) the glutamic acid at position 398 of the γ-cyclodextrin glucosyltransferase shown in SEQ ID NO. 1 is mutated to isoleucine or valine; (c) the glutamine at position 478 of the γ-cyclodextrin glucosyltransferase shown in SEQ ID NO. 1 is mutated to isoleucine; (d) the serine at position 522 of the γ-cyclodextrin glucosyltransferase shown in SEQ ID NO. 1 is mutated to valine.

3. A gene encoding the γ-cyclodextrin glucosyltransferase mutant of claim 1 or 2.

4. A recombinant expression vector carrying the gene of claim 3.

5. A recombinant microbial cell expressing the γ-cyclodextrin glucosyltransferase mutant of claim 1 or 2.

6. A recombinant Bacillus subtilis characterized in that, The γ-cyclodextrin glucosyltransferase mutant of claim 1 or 2 is expressed in Bacillus subtilis WB600 using the pP43NMK plasmid as a vector.

7. A method for improving the thermostability of a γ-cyclodextrin glucosyltransferase, characterized in that, The γ-cyclodextrin glucosyltransferase is mutated as follows: (a) the threonine at position 165 is mutated to histidine; (b) the glutamic acid at position 398 is mutated to isoleucine or valine; (c) the glutamine at position 478 is mutated to isoleucine; (d) the serine at position 522 is mutated to valine.

8. A process for the preparation of γ-cyclodextrin, characterized in that, The γ-cyclodextrin glucosyltransferase mutant of claim 1 or 2 is used to convert a substrate to γ-cyclodextrin using starch as the substrate.

9. The method of claim 8, wherein, The method is carried out at 50 to 60°C; the starch includes but is not limited to cassava starch.

10. The γ-cyclodextrin glucosyltransferase mutant of claim 1 or 2, or the gene of claim 3, or the recombinant expression vector of claim 4, or the recombinant microbial cell of claim 5, or the recombinant Bacillus subtilis of claim 6, or the method of any one of claims 8 to 9, for use in the food, pharmaceutical, biological, or material fields.

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