A cyclodextrin glucanotransferase mutant, a construction method and application thereof

Abstract

The application relates to the technical field of genetic engineering and enzyme engineering, and discloses a cyclodextrin glucosyltransferase mutant and a construction method and application thereof, the mutant being that tyrosine at the 199th position of cyclodextrin glucosyltransferase with an amino acid sequence as shown in SEQ ID NO. 1 is mutated into phenylalanine, named Y199F, and the amino acid sequence is as shown in SEQ ID NO. 2. The cyclodextrin glucosyltransferase mutant prepared by the application can improve the conversion rate of RebA from 64.8% to 72.5% in the reaction of converting RebA into glucosyl steviol glycoside.

Description

Technical Field

[0001] This invention relates to the technical fields of genetic engineering and enzyme engineering, and in particular to a cyclodextrin glucosyltransferase mutant, its construction method, and its application. Background Technology

[0002] Steviosides are high-sweetness, zero-calorie natural sweeteners extracted from the stevia plant. They boast ten major advantages: all-natural, zero-calorie, heat-stable, no effect on blood sugar, non-fermentable, acid-base stable, 150-300 times sweeter than regular sugar, anti-caries, no browning reaction, and no fat or carbohydrates. These advantages have made them popular with consumers and developers worldwide. The main components of steviol glycosides are steviol glycoside (Stev) and rebaudioside A (RebA). However, Stev and RebA have a noticeable bitter aftertaste, which is usually improved through transglycosylation.

[0003] Cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) belongs to the α-amylase family (glycoside hydrolase 13_2, GH13_2). It is an extracellular enzyme and also a multifunctional enzyme that can catalyze the transglycosylation reaction using starch and steviol glycosides as substrates, converting steviol glycosides into glucosyl steviol glycosides. In comparison, glucosyl steviol glycosides converted using RebA as a substrate have a better taste.

[0004] Therefore, improving the conversion rate of RebA is of great significance for the production and quality improvement of glucosylsteviosides. Currently, there are few cyclodextrin glucosyltransferases specifically designed for steviol glycoside modification on the market, and methods for improving RebA conversion rate are even rarer. Summary of the Invention

[0005] To address the technical challenges of improving the conversion rate of RebA and enhancing the flavor of glucosylstevioside, this invention provides a cyclodextrin glucosyltransferase mutant, its construction method, and its application. By mutating tyrosine at position 199 of the cyclodextrin glucosyltransferase to phenylalanine, the conversion rate of RebA to glucosylstevioside can be improved, and the flavor of glucosylstevioside can be enhanced.

[0006] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a cyclodextrin glucosyltransferase mutant, wherein the mutant is formed by mutating the tyrosine at position 199 of the cyclodextrin glucosyltransferase as shown in SEQ ID NO.1 to phenylalanine, named Y199F, and the amino acid sequence is shown in SEQ ID NO.2.

[0007] The cyclodextrin glucosyltransferase is derived from Alkalihalobacillus oshimensis.

[0008] In a second aspect, the gene encoding the cyclodextrin glucosyltransferase mutant is characterized in that the nucleotide sequence of the gene is as shown in SEQ ID NO.3.

[0009] Thirdly, the expression vector carrying the gene.

[0010] Preferably, the expression vector is a pGEX plasmid, pHY300 plasmid, pPIC3K plasmid, pPIC9K plasmid, pHY300PLK plasmid, pET plasmid, or Duet plasmid.

[0011] Preferably, the pET plasmid is selected from pET-15, pET-19, pET-20, pET-24, pET-28, or pET-32; the Duet plasmid is selected from pRSFDuet-1 or pACYCDuet-1; and the pGEX plasmid is selected from pGEX-4T-2 or pGEX-6P.

[0012] Fourthly, microbial cells carrying the gene or the expression vector.

[0013] Preferably, the microbial cells are bacterial cells or fungal cells.

[0014] Preferably, the microbial cells are recombinant prokaryotic cells or eukaryotic cells; the prokaryotic cells are Gram-negative bacteria or Gram-positive bacteria.

[0015] Preferably, the method for constructing the microbial cells is as follows: a recombinant expression vector carrying the gene is transferred into the host cell by electroporation or chemical transformation.

[0016] Fifthly, a method for constructing a cyclodextrin glucosyltransferase mutant involves inoculating microbial cells into a fermentation medium for fermentation, collecting the fermentation broth after fermentation and centrifuging it, and separating the cyclodextrin glucosyltransferase mutant from the fermentation supernatant or bacterial cells obtained by centrifugation.

[0017] Sixthly, the application of the cyclodextrin glucosyltransferase mutant in the preparation of glucosylsteviosides.

[0018] The seventh aspect is the application of the cyclodextrin glucosyltransferase mutant in improving the conversion rate of cyclodextrin glucosyltransferase in converting leboside A to glucosylstevioside.

[0019] Preferably, the conversion rate of RebA by the cyclodextrin glucosyltransferase mutant is determined by the amount of RebA reduction. Specifically, the cyclodextrin glucosyltransferase mutant or the microbial cells are added to a reaction system containing starch and steviol glycosides for reaction.

[0020] Compared with the prior art, the beneficial effects of the present invention are: Figure 4 and Figure 5 As shown, molecular docking results indicate that, compared to the unmutated form, the Y199F mutant can bind to RebA to form a better conformation with a lower binding energy. Furthermore, this conformation involves more amino acids participating in hydrogen bond formation, suggesting a dual effect of further promoting RebA conversion. Experimental results also show that by mutating tyrosine at position 199 of the cyclodextrin glucosyltransferase to phenylalanine, a cyclodextrin glucosyltransferase mutant was prepared, which increased the RebA conversion rate from 64.8% to 72.5% in the reaction of RebA to glucosylstevioside. Attached Figure Description

[0021] Figure 1 This is a graph showing the RebA conversion rate of cyclodextrin glucosyltransferase before and after mutation in this invention; Figure 2 This is a sequencing peak diagram at the mutation site of the cyclodextrin glucosyltransferase mutant in this invention; Figure 3 This is an SDS-PAGE image of cyclodextrin glucosyltransferase before and after mutation in this invention; Figure 4 This is a schematic diagram of the interaction between cyclodextrin glucosyltransferase (CGTase-15) and RebA. Figure 5 This is a schematic diagram illustrating the interaction between the cyclodextrin glucosyltransferase mutant (CGTase-15-Y199F) and RebA in this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments.

[0023] Example 1 Preparation of site-directed mutagenesis of cyclodextrin glucosyltransferase mutant Using the recombinant plasmid containing SEQ ID NO.1 as a template plasmid, the site-directed mutant sequence of CGTase was amplified by PCR using the Fast Mutagenesis System mutagenesis kit and the designed corresponding mutation primer pairs. The specific operation steps are as follows: (1) Mutation primers The forward primer 15#-E-Y199F-F is 5'-CATTTATAAAAACCTGTTTGATCTGGCGG-3'. The reverse primer 15#-E-Y199F-R is 5'-AACAGGTTTTTATAAATGCCGTTTTCCAG-3'.

[0024] (2) Reaction system (as shown in Table 1) Table 1 Plasmid 1 μL (concentration 10 ng / μL) Forward Primer 1μL Reverse Primer 1μL 2*TransStart FastPfu Fly PCR SuperMix 25μL Nuclease-free Water 22μL (3) PCR conditions (as shown in Table 2) Table 2 (4) Digestion of PCR products Add 1 μL of DMT enzyme to the PCR product verified in step (3), mix well and incubate at 37°C for 1 h.

[0025] (5) Transformation ① Take 5 μL of the DMT enzyme digestion product from step (4) into 50 μL of DMT competent cells (add the ligation product when the competent cells have just thawed), gently mix, and incubate on ice for 20-30 min; ② After heat shock at 42℃ for 45 seconds, immediately place in an ice bath for 2 minutes; ③ Incubate in 250 μL of LB medium equilibrated to room temperature for 1 h at 200 rpm and 37 °C; ④ Preheat the kanamycin resistance plate (working concentration of 50 ng / μL, specifically prepared by adding 500 μL of 10 mg / mL stock solution to 100 ml of culture medium) in an incubator at 37°C; ⑤ Streak the bacterial culture obtained in step ③ onto a kanamycin-resistant plate and incubate overnight at 37°C.

[0026] ⑥ Picking colonies: On the second day, observe the overnight kanamycin-resistant plates. Colonies were observed on all plates. Pick 5 single colonies from each plate and place them in 1 mL LB (kanamycin, 2 mL tubes) and incubate at 220 rpm and 37°C. Extract plasmids and sequence them to confirm that the obtained plasmids showed mutations at the target mutation site but not at non-target mutation sites. Figure 2 The diagram shown is a sequencing peak at the mutation site, indicating that the mutant was successfully constructed. ⑦ Transformation: Transform the correctly sequenced plasmid into competent Escherichia coli BL21(DE3) cells expressing the host cell line, following the same steps as ①~⑤.

[0027] The amino acid sequence of cyclodextrin glucosyltransferase is shown in SEQ ID NO.1:Met Ala Tyr Ala Asp Pro Asp Ile Ala Val Thr Asn Lys Gln Ser Phe SerThr Asp Val Ile Tyr Gln Val Phe Thr Asp Arg Phe Leu Asp Gly Asn Pro Ser AsnAsn Pro Thr Gly Ala Ala Tyr Asp Ala Thr Cys Ser Asn Leu Lys Leu Tyr Cys GlyGly Asp Trp Gln Gly Leu Ile Asn Lys Ile Asn Asp Asn Tyr Phe Ser Asp Leu GlyVal Thr Ala Leu Trp Ile Ser Gln Pro Val Glu Asn Ile Phe Ala Thr Ile Asn TyrSer Gly Val Thr Asn Thr Ala Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys ThrAsn Pro Tyr Phe Gly Thr Met Ala Asp Phe Gln Asn Leu Ile Thr Thr Ala His AlaLys Gly Ile Lys Ile Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala Met GluThr Asp Thr Ser Phe Ala Glu Asn Gly Lys Leu Tyr Asp Asn Gly Thr Leu Val GlyGly Tyr Thr Asn Asp Thr Asn Gly Tyr Phe His His Asn Gly Gly Ser Asp Phe SerSer Leu Glu Asn Gly Ile Tyr Lys Asn Leu Tyr Asp Leu Ala Asp Phe Asn His AsnAsn Ala Thr Ile Asp Lys Tyr Phe Lys Asp Ala Ile Lys Leu Trp Leu Asp Met GlyVal Asp Gly Ile Arg Val Asp Ala Val Lys His Ile Ala Leu Gly Trp Gln Lys SerTrp Met Ser Ser Ile Tyr Val HisLys Pro Val Phe Thr Phe Gly Glu Trp Phe LeuGly Ser Ala Ala Ser Asp Ala Asp Asn Thr Asp Phe Ala Asn Lys Ser Gly Met SerLeu Leu Asp Phe Arg Phe Asn Ser Ala Val Arg Asn Val Phe Arg Asp Asn Thr SerAsn Met Tyr Ala Leu Asp Ser Met Ile Asn Ser Thr Ala Thr Asp Tyr Asn Gln ValAsn Asp Gln Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Lys Thr Ser AlaVal Asn Asn Arg Arg Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser Arg Gly ValPro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Leu Thr Gly Asn Gly Asp Pro Asp AsnArg Ala Lys Met Pro Ser Phe Ser Lys Ser Thr Thr Ala Phe Asn Val Ile Ser Lysleu Leu Ala Ala Gly Ala Thr Ala ValTrp Gln Tyr Thr Ala Ala Glu Thr Thr Pro Thr Ile Gly HisVal Gly Pro Val MetGly Lys Pro Gly Asn Val Val Thr Ile Asp Gly Arg Gly Phe Gly Ser Thr Lys GlyThr Val Tyr Phe Gly Ser Thr Lys GlyThr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Ala Ile Thr Ser Trp Glu AspThr Gln Ile Lys Val Thr Ile Pro Ser Val Ala Ala Gly Asn Tyr Ala Val Lys ValAla Ala Asn Gly Val Asn Ser Asn Ala Tyr Asn His Phe Thr Ile Leu Thr Gly AspGln Val Thr Val Arg Phe Val Ile Asn Asn Ala Ser Thr Thr Leu Gly Gln Asn IleTyr Leu Thr Gly Asn Val Ala Glu Leu Gly Asn Trp Ser Thr Gly Ser Thr Ala IleGly Pro Ala Phe Asn Gln Val Ile His Gln Tyr Pro Thr Trp Tyr Tyr Asp Val SerVal Pro Ala Gly Lys Glu Leu Glu Phe Lys Phe Phe Lys Lys Asn Gly Ser Thr IleThr Trp Glu Gly Gly Ser Asn His Lys Phe Thr Thr Pro Ala Ser Gly Thr Ala ThrVal Thr Val Asn Trp Gln The amino acid sequence of the cyclodextrin glucosyltransferase mutant (Y199F) is shown in SEQ ID NO.2: Met Ala Tyr Ala Asp Pro Asp Ile Ala Val Thr Asn Lys Gln Ser Phe SerThr Asp Val Ile Tyr Gln Val Phe Thr Asp Arg Phe Leu Asp Gly Asn Pro Ser AsnAsn Pro Thr Gly Ala Ala Tyr Asp Ala Thr Cys Ser Asn Leu Lys Leu Tyr Cys GlyGly Asp Trp Gln Gly Leu Ile Asn Lys Ile Asn Asp Asn Tyr Phe Ser Asp Leu GlyVal Thr Ala Leu Trp Ile Ser Gln Pro Val Glu Asn Ile Phe Ala Thr Ile Asn TyrSer Gly Val Thr Asn Thr Ala Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys ThrAsn Pro Tyr Phe Gly Thr Met Ala Asp Phe Gln Asn Leu Ile Thr Thr Ala His AlaLys Gly Ile Lys Ile Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala Met GluThr Asp Thr Ser Phe Ala Glu Asn Gly Lys Leu Tyr Asp Asn Gly Thr Leu Val GlyGly Tyr Thr Asn Asp Thr Asn Gly Tyr Phe His His Asn Gly Gly Ser Asp Phe SerSer Leu Glu Asn Gly Ile Tyr Lys Asn Leu Phe Asp Leu Ala Asp Phe Asn His AsnAsn Ala Thr Ile Asp Lys Tyr Phe Lys Asp Ala Ile Lys Leu Trp Leu Asp Met GlyVal Asp Gly Ile Arg Val Asp Ala Val Lys His Ile Ala Leu Gly Trp Gln Lys SerTrp Met Ser Ser Ile Tyr Val HisLys Pro Val Phe Thr Phe Gly Glu Trp Phe LeuGly Ser Ala Ala Ser Asp Ala Asp Asn Thr Asp Phe Ala Asn Lys Ser Gly Met SerLeu Leu Asp Phe Arg Phe Asn Ser Ala Val Arg Asn Val Phe Arg Asp Asn Thr SerAsn Met Tyr Ala Leu Asp Ser Met Ile Asn Ser Thr Ala Thr Asp Tyr Asn Gln ValAsn Asp Gln Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Lys Thr Ser AlaVal Asn Asn Arg Arg Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser Arg Gly ValPro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Leu Thr Gly Asn Gly Asp Pro Asp AsnArg Ala Lys Met Pro Ser Phe Ser Lys Ser Thr Thr Ala Phe Asn Val Ile Ser Lysleu Leu Ala Ala Gly Ala Thr Ala ValTrp Gln Tyr Thr Ala Ala Glu Thr Thr Pro Thr Ile Gly HisVal Gly Pro Val MetGly Lys Pro Gly Asn Val Val Thr Ile Asp Gly Arg Gly Phe Gly Ser Thr Lys GlyThr Val Tyr Phe Gly Ser Thr Lys GlyThr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Ala Ile Thr Ser Trp Glu AspThr Gln Ile Lys Val Thr Ile Pro Ser Val Ala Ala Gly Asn Tyr Ala Val Lys ValAla Ala Asn Gly Val Asn Ser Asn Ala Tyr Asn His Phe Thr Ile Leu Thr Gly AspGln Val Thr Val Arg Phe Val Ile Asn Asn Ala Ser Thr Thr Leu Gly Gln Asn IleTyr Leu Thr Gly Asn Val Ala Glu Leu Gly Asn Trp Ser Thr Gly Ser Thr Ala IleGly Pro Ala Phe Asn Gln Val Ile His Gln Tyr Pro Thr Trp Tyr Tyr Asp Val SerVal Pro Ala Gly Lys Glu Leu Glu Phe Lys Phe Phe Lys Lys Asn Gly Ser Thr IleThr Trp Glu Gly Gly Ser Asn His Lys Phe Thr Thr Pro Ala Ser Gly Thr Ala ThrVal Thr Val Asn Trp Gln The nucleotide sequence of the cyclodextrin glucosyltransferase mutant (Y199F) is shown in SEQ ID NO.3: Example 2 Expression and purification of cyclodextrin glucosyltransferase mutant Protein-induced expression: (1) Strains and conditions ① Two proteins need to be induced to express: CGTase#15 and CGTase#15-Y199F. E. coli BL21(DE3) competent cells containing mutant plasmids were seeded into 10 mL LB medium (containing 50 ng / μL kanamycin) and cultured overnight at 220 rpm and 30 °C. ② Transfer all the overnight cultured bacterial suspensions to 1000 mL LB medium (containing 50 ng / μL kanamycin) and incubate at 220 rpm and 30°C for 4 h; ③ Add IPTG to a final concentration of 0.1 mM and incubate at 220 rpm and 16 ℃ for 20 h.

[0028] (2) Ultrasonic cell disruption ① After inducing protein expression in (1), the bacterial culture was centrifuged at 10000×g for 5 min at room temperature, and the cells were collected; ② After resuspending the cells in 30 mL of 20 mM PBS, place them in an ice-water bath; ③Use an amplitude transformer with a diameter of 6mm and ultrasonically break it up for 15 minutes under the conditions of 20% intensity and 2 seconds of ultrasonication followed by 2 seconds of pause. ④ Centrifuge at 4℃ and 22000rpm for 20min, filter the supernatant through a 0.22μm filter membrane, and store at 4℃ for later use.

[0029] (3) Protein purification ①Filter the supernatant obtained in (2) through a 0.22μm filter membrane and then load it into the AKTA system; ② After the sample loading is completed, wash with 20mM imidazole PBS buffer and observe the UV absorption until the UV absorption value drops to the initial level; ③ Replace with 40mM imidazole PBS buffer and continue washing. After an absorption peak appears, the UV absorbance value decreases to the initial level. ④ Replace with 250mM imidazole PBS buffer, elute, and start collecting the eluent when the UV absorbance index rises, one tube per 2mL, until the UV absorption level flattens out; replace with 500mM imidazole PBS buffer, elute, and start collecting the eluent when the UV absorbance index rises, one tube per 2mL, until the UV absorption level flattens out.

[0030] (4) SDS-PAGE ① Take 20 μL of the protein purification collection solution from (3) above, add it to 10 μL of 6× protein loading buffer, mix well, and mix at 95℃ for 10 min; ② Take two pieces of pre-prepared 12% polyacrylamide gel and put them into the electrophoresis tank; ③ Load the samples in sequence, such as Figure 3 The image shows SDS-PAGE images of cyclodextrin glucosyltransferase before and after mutation (E. coli host, nickel column purification), indicating that the mutant protein was successfully expressed.

[0031] Example 3 Cyclodextrin glucosyltransferase transglycosylation activity analysis (1) Using a solution of 20 g / L soluble starch and 20 g / L steviol glycosides prepared with water as a substrate, the soluble starch was completely dissolved in a boiling water bath for 10 min. (2) After the substrate has cooled, it is divided into two groups: the experimental group and the control group. 20 mg / L of enzyme solution (containing cyclodextrin glucosyltransferase mutant) is added to the experimental group, and the same volume of 50 mM pH 7.0 PBS is added to the control group. (3) Place the experimental group and the control group in centrifuge tubes respectively, place the centrifuge tubes in 3L Erlenmeyer flasks, place the Erlenmeyer flasks in a shaker at 40℃, react at 220rpm for 22h, and then centrifuge at 12000rpm for 2min at room temperature. (4) 100 μL of supernatant was taken from both the experimental and control groups, added to 900 μL of water, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC. Figure 1 The figure shows the RebA conversion rate of cyclodextrin glucosyltransferase before and after the mutation, indicating that the cyclodextrin glucosyltransferase mutant can significantly improve the RebA conversion rate compared with the unmutated one.

[0032] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A cyclodextrin glucosyltransferase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. The application of the cyclodextrin glucosyltransferase mutant of claim 1 in the preparation of glucosylsteviosides.

3. A method for preparing glucosylstevioside, characterized in that, The cyclodextrin glucosyltransferase mutant as described in claim 1 was used.

4. The application of the cyclodextrin glucosyltransferase mutant of claim 1 in improving the conversion rate of cyclodextrin glucosyltransferase to leboside A into glucosylstevioside.

5. The application as described in claim 4, characterized in that, The conversion rate of RebA by the cyclodextrin glucosyltransferase mutant was determined by the amount of RebA reduction. The specific method was as follows: the above-mentioned cyclodextrin glucosyltransferase mutant was added to a reaction system containing starch and steviol glycosides for reaction.

6. A method for improving the conversion rate of cyclodextrin glucosyltransferase in converting leboside A to glucosylstevioside, characterized in that, The cyclodextrin glucosyltransferase mutant as described in claim 1 was used.

7. The method as described in claim 6, characterized in that, The conversion rate of RebA by the cyclodextrin glucosyltransferase mutant was determined by the amount of RebA reduction. The specific method was as follows: the above-mentioned cyclodextrin glucosyltransferase mutant was added to a reaction system containing starch and steviol glycosides for reaction.

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