Alpha-cyclodextrin glucosyltransferase mutants and their use in the preparation of short-chain glucosylated steviol glycosides

Abstract

The application discloses alpha-cyclodextrin glucosyltransferase mutants and application thereof in preparation of short-chain glucosylated steviol glycoside, and belongs to the technical field of genetic engineering and enzyme engineering. The double mutants Y100I / F183L, Y100I / H328L and F183L / H328L obtained by the application have the advantages of keeping the dismutation activity, C-19 and C-13 region selectivity equivalent to WT, and further improving the yield of short-chain glucosylated steviol glycoside. When maltose is used as a glycosyl donor, the yield reaches more than 26%; when corn starch is used as a glycosyl donor, the yield reaches more than 41%, and the application has extremely high practical value and economic value.

Description

Technical Field

[0001] This invention relates to α-cyclodextrin glucosyltransferase mutants and their application in the preparation of short-chain glucosylated steviol glycosides, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Glucosylsteviosides (GSGs) are derivatives of steviol glycosides that introduce additional glucose residues through glycosylation. They can mask bitterness and astringency, and improve water solubility, surface activity, and processing adaptability, thus showing promising potential in the food, beverage, and pharmaceutical industries. Their sweetness quality is regulated by the number and linkage of glycosyl groups at C-13 and C-19: adding 1-2 glucose groups at C-19 usually optimizes the taste, but excessive glycosylation reduces sweetness intensity and prolongs unpleasant aftertastes. Therefore, the highly selective synthesis of short-chain glucosylated products at C-19 has become an important direction for steviol glycoside modification.

[0003] Currently, GSGs are synthesized via chemical and enzymatic methods. Enzymatic methods have become the mainstream due to their mild conditions, high selectivity, and environmental friendliness. Commonly used enzymes include cyclodextrin glucosyltransferase (CGTase), uridine diphosphate glucosyltransferase (UGT), galactosidase, glucosidase, and β-fructofuranosidase. Among these, CGTase can use inexpensive starch or maltodextrin as glycosyl donors without requiring high-cost uridine diphosphate glucose, making it highly valuable for industrial applications in the modification of rebaudioside A (RA).

[0004] However, existing CGTase-catalyzed RA glycosylation has shortcomings: conventional CGTases have poor regioselectivity, modifying both C-13 and C-19 positions simultaneously; their substrate-binding regions contain multiple glucose-binding subsites, tending to bind long-chain donors. When using long-chain donors such as starch, the enzyme tends to continue extending the sugar chain after initial transglycosylation, generating a mixture of different sites and chain lengths. This not only makes separation difficult but also results in a high proportion of long-chain byproducts and a low content of the desired C-19 short-chain product, affecting quality stability.

[0005] To enrich short-chain products, current strategies mainly fall into two categories: one is to introduce glycoside hydrolases after the reaction to hydrolyze long chains into short chains, but this requires additional enzymes and steps, increasing costs and process complexity, and making it difficult to precisely control the degree of hydrolysis, often leading to product fluctuations or target degradation; the other is to directly use short-chain donors such as maltose for the reaction, which can inhibit chain elongation and simplify the process. However, most CGTases have low affinity and transglycosylation efficiency for maltose because they cannot effectively occupy multiple binding subsites. Therefore, enhancing the utilization capacity of CGTases for maltose while maintaining high C-19 regioselectivity is key to obtaining high-yield C-19 short-chain glycosylated RA.

[0006] From Paenibacillus maceransα-CGTase can regioselectively transglycosylate at the C-19 position of RA to generate a C19 glucosylated derivative. When using long-chain donors such as starch, the conversion rate of RA is high, but the products are mostly long-chain compounds with a low proportion of short chains. Although using maltose can increase the proportion of short chains, the overall conversion rate and target yield are still not ideal because the enzyme's utilization efficiency of maltose is poor.

[0007] Therefore, it is necessary to perform molecular modification on this α-CGTase to retain its high regioselectivity at C-19 while significantly improving the transglycosylation activity using maltose as a donor, thereby achieving the preparation of C-19 short-chain glucosylated steviol glycosides with low long-chain byproducts and high yield. Summary of the Invention

[0008] To address the aforementioned problems, this invention utilizes genetic engineering and enzyme engineering techniques to [address the issues arising from...]. Paenibacillus macerans The α-cyclodextrin glucosyltransferase was molecularly modified to retain its high regioselectivity at C-19 while improving its catalytic utilization efficiency of maltose in the transglycosylation reaction, thereby increasing the yield of C-19 short-chain glucosylated rebaudioside A.

[0009] The first objective of this invention is to provide an α-cyclodextrin glucosyltransferase mutant, wherein the α-cyclodextrin glucosyltransferase mutant has a mutation at any two sites, namely, position 100, position 183, or position 328, based on the amino acid sequence as shown in SEQ ID NO.1.

[0010] In one embodiment, the α-cyclodextrin glucosyltransferase mutant is based on the amino acid sequence as shown in SEQ ID NO.1, with tyrosine at position 100 mutated to isoleucine and phenylalanine at position 183 mutated to leucine, and is named Y100I / F183L (amino acid sequence as shown in SEQ ID NO.5). Alternatively, the tyrosine at position 100 is mutated to isoleucine, and the histidine at position 328 is mutated to leucine, named Y100I / H328L (amino acid sequence as shown in SEQ ID NO.6). Alternatively, phenylalanine at position 183 is mutated to leucine, and histidine at position 328 is mutated to leucine, named F183L / H328L (amino acid sequence as shown in SEQ ID NO.7).

[0011] The present invention also provides an α-cyclodextrin glucosyltransferase mutant, wherein the α-cyclodextrin glucosyltransferase mutant is based on the amino acid sequence as shown in SEQ ID NO.1, with tyrosine at position 100 mutated to isoleucine, phenylalanine at position 183 mutated to leucine, and histidine at position 328 mutated to leucine, and is named Y100I / F183L / H328L (amino acid sequence as shown in SEQ ID NO.8).

[0012] The present invention also provides an α-cyclodextrin glucosyltransferase mutant, wherein the α-cyclodextrin glucosyltransferase mutant is based on the amino acid sequence as shown in SEQ ID NO.1, with the tyrosine at position 100 mutated to isoleucine, and the mutant is named Y100I (amino acid sequence as shown in SEQ ID NO.2). Alternatively, the phenylalanine at position 183 is mutated to leucine, and the mutant is named F183L (amino acid sequence as shown in SEQ ID NO. 3). Alternatively, histidine at position 328 can be mutated to leucine, and the mutant can be named H328L (amino acid sequence as shown in SEQ ID NO. 4).

[0013] A second objective of this invention is to provide a polynucleotide of any of the above-described α-cyclodextrin glucosyltransferase mutants.

[0014] A third object of the present invention is to provide a plasmid carrying the aforementioned polynucleotides.

[0015] Optionally, the plasmid includes pET series vectors, pBAD series vectors, pGEX series vectors, or pCold series vectors.

[0016] A fourth object of the present invention is to provide cells expressing any of the above-described α-cyclodextrin glucosyltransferase mutants.

[0017] Alternatively, the cells may be fungi or bacteria; Optionally, the cells include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris.

[0018] The fifth objective of this invention is to provide a genetically engineered Escherichia coli strain that expresses any of the above-mentioned α-cyclodextrin glucosyltransferase mutants.

[0019] The sixth object of the present invention is to provide a method for preparing C-19 short-chain glucose steviol glycoside, using maltose or corn starch as a glycosyl donor, rebaudioside A as a glycosyl acceptor, and using any of the above-mentioned α-cyclodextrin glucosyltransferase mutants to catalyze the generation of C-19 short-chain glucose steviol glycoside.

[0020] In one embodiment, the catalytic temperature is 30-60°C and the catalytic time is 10-20 h; wherein, the C-19 short-chain glucose steviol glycoside is a glycosylated product in which one or two glucose groups are attached to the C-19 region of rebaudioside A.

[0021] In one embodiment, when maltose is used as the glycosyl donor and rebaudioside A is used as the glycosyl acceptor, the mass ratio of maltose to rebaudioside A is 1~2:1~2; the amount of enzyme added is 0.3~1 U / mL, and the reaction time is 10~15 h; The concentrations of maltose and rebaudioside A were 5–10 g / L.

[0022] In one embodiment, when corn starch is used as the glycosyl donor and rebaudioside A is used as the glycosyl acceptor, the mass ratio of corn starch to rebaudioside A is 1~2:1~2; the enzyme addition amount is 0.3~1 U / mL, and the reaction time is 10~15 h; The corn starch needs to be gelatinized first to obtain a corn starch gelatinized solution with a concentration of 0.01 g / mL; the concentration of lebodiin A is 5~10 g / L.

[0023] The seventh object of the present invention is to provide a method for improving the utilization of maltose by α-cyclodextrin glucosyltransferase, wherein the α-cyclodextrin glucosyltransferase with the amino acid sequence shown in SEQ ID NO.1 has a mutation at position 100, position 183 or position 328; Among them, the tyrosine at position 100 is mutated to isoleucine, and the phenylalanine at position 183 is mutated to leucine, named Y100I / F183L. Alternatively, the tyrosine at position 100 is mutated to isoleucine, and the histidine at position 328 is mutated to leucine, named Y100I / H328L. Alternatively, the phenylalanine at position 183 is mutated to leucine, and the histidine at position 328 is mutated to leucine, named F183L / H328L.

[0024] An eighth object of the present invention is to provide the use of any of the above-described α-cyclodextrin glucosyltransferase mutants, or the above-described polynucleotides, or the above-described plasmids, or the above-described cells, or the above-described Escherichia coli genetically engineered bacteria in the food, pharmaceutical, or biological fields.

[0025] In one embodiment, the application includes the preparation of a C-19 short-chain glucose steviol glycoside.

[0026] Beneficial effects of the present invention The invention utilizes genetic engineering and enzyme engineering techniques to [discover / process] substances derived from [organisms]. Paenibacillus maceransThe α-cyclodextrin glucosyltransferase was molecularly modified to retain its high regioselectivity at C-19 while improving its catalytic utilization efficiency of maltose in the transglycosylation reaction, thereby increasing the yield of C-19 short-chain glucosylated rebaudioside A. Specifically: (1) The dismutation activities of the double mutants Y100I / F183L, Y100I / H328L, and F183L / H328L obtained in this invention are comparable to those of WT. (2) The double mutants Y100I / F183L, Y100I / H328L, and F183L / H328L constructed in this invention achieved a rebaudioside A conversion rate of over 40% and a short-chain glucosylated steviol glycoside yield of over 26% when maltose was used as the glycosyl donor, which were significantly better than WT (8.32%). (3) The double mutants Y100I / F183L, Y100I / H328L, and F183L / H328L constructed in this invention achieved a rebaudioside A conversion rate of over 49% and a short-chain glucosylated steviol glycoside yield of over 41% when corn starch was used as the glycosyl donor, which were significantly better than WT (20.03%). (4) The double mutants Y100I / F183L, Y100I / H328L, and F183L / H328L constructed in this invention have the same C-19 and C-13 region selectivity as WT, and can still maintain the region selectivity characteristics of wild-type enzymes. The generated short-chain glycosylation product is C-19 short-chain glucosylated steviol glycoside. (5) The double mutants Y100I / F183L, Y100I / H328L and F183L / H328L constructed in this invention achieved a yield of more than 46% of short-chain glucosylated steviol glycosides at position C-19 when maltose was used as the glycosyl donor, which was significantly better than WT (23.45%). Attached Figure Description

[0027] Figure 1 When maltose is used as a glycosyl donor, α Chromatograms of glycosylation products of rebaudioside A catalyzed by cyclodextrin glucosyltransferase and its mutants; Figure 2 When corn starch is used as a glycosyl donor, α Chromatograms of glycosylation products of cyclodextrin glucosyltransferase and its mutants catalyzing rebaudioside A. Detailed Implementation

[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0029] Raw materials used in the examples: LB medium (g / L): yeast extract 5g, trypsin 10g, NaCl 10g, pH 7.0.

[0030] TB medium (g / L): yeast extract 24, trypsin 12, glycerol 5, KH2PO4 2.3135, K2HPO4·3H2O 16.4318, pH 7.0.

[0031] Experimental and testing methods: 1. Methods for preparing mutants (1) Using the vector pET-20b(+) / cgt with the wild-type α-cyclodextrin glucosyltransferase gene sequence as a template, site-directed mutagenesis was performed using the primers in Table 1. PCR amplification was performed according to the instructions of the TaKaRa PrimerSTAR GXL kit. After the PCR product was digested with DpnI, it was transformed into Escherichia coli JM109 supercompetent cells to obtain genetically engineered bacteria carrying the mutant recombinant plasmid.

[0032] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 3 min; followed by denaturation at 98℃ for 10 s, annealing at 60℃ for 15 s, extension at 68℃ for 5 min, for 30 cycles; and finally incubation at 68℃ for 10 min.

[0033] (2) The transformed recipient bacteria were spread on LB solid plates containing 100 μg / mL ampicillin and cultured overnight at 37°C. Single clones were picked and cultured overnight in LB medium containing 100 μg / mL ampicillin. Plasmids were extracted and identified by sequencing.

[0034] (3) Transform the correctly sequenced mutant plasmid into the expression host Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium containing the mutant plasmid.

[0035] 2. Methods for mutant expression and purification (1) The recombinant genetically engineered bacteria were streaked on LB solid medium containing ampicillin (100 μg / mL) and cultured in a constant temperature incubator at 37℃ until single colonies grew. Single colonies were picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium containing ampicillin (100 μg / mL) and cultured at 37℃ and 200 r / min for 8 h to prepare seed liquid.

[0036] (2) The seed culture prepared in step (1) was inoculated at an inoculation rate of 4% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of TB liquid fermentation medium containing ampicillin (100 μg / mL), and cultured at 30℃ and 200 r / min. When the cell culture reached an OD600 of 0.6, IPTG was added to a final concentration of 0.01 mM, and the culture was quickly transferred to a shaker at 25℃ and 200 r / min for 90 h of induction culture to obtain the fermentation broth. The fermentation broth was centrifuged at 4℃ and 10000 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution.

[0037] (3) The crude enzyme solution was purified in two steps by anion exchange chromatography and hydrophobic chromatography.

[0038] 1) Enzyme pretreatment: The crude enzyme solution of the recombinant enzyme was dialyzed overnight in 20 mM Tris-HCl buffer at pH 7.5 to remove small molecule impurities and replace the buffer. The dialyzed enzyme solution was then filtered through a filter membrane and used as the enzyme solution to be purified.

[0039] 2) Anion exchange chromatography purification: The enzyme solution to be purified was initially purified using a Q-Sepharose anion exchange chromatography column. First, the Q-Sepharose column was equilibrated with 5–10 column volumes of 20 mM Tris-HCl buffer (pH 7.5). Then, the dialyzed enzyme solution was loaded at a flow rate of 1 mL / min. After loading, a gradient elution was performed with 0–0.4 M KCl at a flow rate of 1 mL / min. Each eluted fraction was collected based on the elution peaks, and fractions containing α-CGT enzyme activity were screened by enzyme activity assay.

[0040] 3) Pretreatment for hydrophobic chromatography: The fraction containing α-CGT enzyme obtained in step 2) was placed in 20 mM Tris-HCl buffer and dialyzed overnight at pH 7.5. After dialysis, solid (NH4)2SO4 was slowly added to achieve a final concentration of 20%. After complete dissolution, the solution was filtered to obtain the enzyme solution to be purified by hydrophobic chromatography.

[0041] 4) Hydrophobic chromatography purification: Further purification was performed using a phenyl-Superose HR10 / 10 hydrophobic chromatography column. First, the phenyl-Superose column was equilibrated with 5-10 column volumes of 20 mM Tris-HCl buffer containing 20% ​​(NH4)2SO4 at pH 7.5. Then, the enzyme solution treated in step 3) was loaded at a flow rate of 1 mL / min. After loading, a reverse gradient elution with 20%-0% (NH4)2SO4 was used at a flow rate of 1 mL / min, and each eluted fraction was collected based on the elution peaks.

[0042] 5) Identification of pure enzyme: SDS-PAGE analysis and enzyme activity determination were performed on each collected elution fraction. Fractions containing α-CGT enzyme with high purity were screened as purified α-CGT enzyme solution.

[0043] 3. Methods for determining disproportionation activity The glycosylation activity of CGTase was analyzed using 4,6-ethylidene-p-nitrophenyl-α-D-maltoheptaoside (EPS) as a substrate. EPS served as the glycosyl donor, and maltose as the glycosyl acceptor, facilitating the transfer of glucose from EPS to maltose via CGTase. First, 300 μL of EPS (4 mmol / L) and 300 μL of maltose (80 mmol / L) were dissolved in PBS buffer (pH 6.0, 50 mmol / L) and preheated at 50°C. These were then mixed and 100 μL of diluted enzyme solution was added, and the mixture was incubated at 50°C for 10 min. Subsequently, the sample was incubated with 200 μL of α-glucosidase at 60°C for 1 h to release p-nitrophenol and form a non-blocked linear oligosaccharide. Then, 100 μL of Na₂CO₃ (1 mol / L) was added to adjust the pH to 8.0. The absorbance of p-nitrophenol was measured at 400 nm. One unit of disproportionation activity was defined as the amount of enzyme that converts 1 μmol of EPS per minute.

[0044] 4. Product determination method: The products of α-CGTase-catalyzed RA were analyzed by HPLC. A ChromCore NH2 column (4.6 mm × 250 mm, 5 μm) was used at a column temperature of 30 °C, and the product and substrate peaks were analyzed at 210 nm. The mobile phase was a mixture of acetonitrile and water. The elution gradient was 0–2 min: V(acetonitrile):V(water) = 80:20; 2–26 min: V(acetonitrile):V(water) = 50:50; 26.1–30 min: V(acetonitrile):V(water) = 80:20, with a flow rate of 1 mL / min.

[0045] The formulas for calculating the conversion rate of RA and the yield of short-chain glucosyl steviol glycosides are as follows: RA conversion rate = (RA content before reaction - RA content after reaction) / RA content before reaction × 100% Short-chain glucosylstevioside yield = Short-chain glucosylstevioside content / RA content before reaction × 100% 5. Regional selectivity determination method 100 mL of the glycosylated product was placed in a round-bottom flask, and 10 g of sodium hydroxide was added. The mixture was reacted under reflux for 1 h. After cooling, a certain amount of glacial acetic acid was added to neutralize to pH 7, resulting in the appearance of a large amount of white flocculent precipitate. The precipitate was filtered and washed three times with distilled water to obtain the hydrolysis product. The hydrolysis product was dissolved in 75% acetonitrile, and its content was determined by liquid chromatography under the following conditions: ChromCore C18 column (4.6 mm × 250 mm, 5 μm), column temperature 30°C, analysis of product and substrate peaks at 210 nm, mobile phase isocratic with 75% acetonitrile aqueous solution, 30 min. The C-19 regioselectivity of α-CGTase and its mutants for RA was calculated using the following formula: RA hydrolysis product content (%) = RA hydrolysis product peak area / total peak area of ​​hydrolysis products × 100%; C-19 regional selectivity (%) = [1 (1 [(1 + RA hydrolysis product content) / (RA conversion rate × (1 + RA hydrolysis product content)) × 100%] C-13 Region Selectivity (%) = 100% C-19 is region-selective.

[0046] Example 1: Construction and purification of mutants 1. Mutant Construction The wild-type α-cyclodextrin glucosyltransferase (α-CGT) gene (amino acid sequence as shown in SEQ ID NO.1) and its nucleotide sequence as shown in SEQ ID NO.9 were inserted between the Nco I and EcoRI sites of plasmid pET-20b(+) to construct plasmid pET-20b(+) / cgt.

[0047] Based on plasmid pET-20b(+) / cgt, site-directed mutagenesis and combinatorial mutagenesis were performed to construct mutant plasmids, and the primers are shown in Table 1. The mutant plasmids were amplified and verified, and then transformed into the expression host Escherichia coli BL21(DE3) to construct genetically engineered bacteria containing mutant plasmids, which were named BL21(DE3)-Y100I, BL21(DE3)-F183L, BL21(DE3)-H328L, BL21(DE3)-Y100I / F183L, BL21(DE3)-Y100I / H328L, BL21(DE3)-F183L / H328L, and BL21(DE3)-Y100I / F183L / H328L, respectively.

[0048] Table 1 Primers

[0049] Note: The underlined bases correspond to the corresponding mutant amino acids.

[0050] 2. Mutant purification and performance testing The genetically engineered bacteria constructed in step 1 were fermented to prepare crude enzyme solution, which was then purified and dialyzed to obtain mutant pure enzyme solution. The dismutation activity of the mutant pure enzyme solution was detected, and the results are shown in Table 2.

[0051] Table 2. Superoxide dismutation activity of pure enzymes from mutants

[0052] The results showed that single-point mutations and combined mutations at amino acid sites 100, 183 and 328 of α-CGT enzyme did not significantly affect its dismutation activity, indicating that the above-mentioned site mutations had little effect on the overall enzyme activity of α-CGT enzyme, and the mutants could still maintain dismutation activity comparable to that of the wild type.

[0053] Example 2: Catalytic effect of α-cyclodextrin glucosyltransferase mutant with maltose as glycosyl donor The mutant prepared in Example 1 was used to test the catalytic effect when maltose was used as the glycosyl donor. The steps are as follows: (1) Prepare 10 mL of RA (rebadiol A) and maltose into 10 g / L solutions with pure water, mix them evenly, and keep them in a water bath at 50℃ for 30 min. (2) Add α-cyclodextrin glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1, WT) and its mutant to the preheated mixed solution at a concentration of 0.3 U / mL and react for 12 h to obtain the glycosylated product.

[0054] Qualitative and quantitative analysis of glycosylated products was performed by HPLC. The utilization efficiency of α-cyclodextrin glucosyltransferase for the short-chain glycosylated product before and after mutation was evaluated by calculating the conversion rate of RA and the yield of short-chain glycosylated products.

[0055] HPLC analysis results are as follows Figure 1 As shown in Table 3, the calculated results of RA conversion rate and short-chain glycosylation product yield are presented.

[0056] Table 3 RA conversion rate and yield of short-chain glycosylation products

[0057] The results showed that when maltose was used as the glycosyl donor, α-cyclodextrin glucosyltransferase could catalyze the glycosylation reaction of RA, and the products were mainly short-chain glucosylated steviol glycosides with a relatively concentrated product composition and fewer long-chain glycosylated products.

[0058] Since maltose itself is a short-chain glycosyl donor, it can effectively avoid the continuous elongation of the sugar chain, thus facilitating the formation of the target short-chain product. However, the wild-type enzyme (WT) has a weak ability to utilize maltose, resulting in a RA conversion rate of only 10.28% and a short-chain glucosylated steviol glycoside yield of only 8.32%, indicating that the wild-type enzyme has low catalytic efficiency for maltose as a glycosyl donor.

[0059] Compared with WT, all mutants exhibited significantly enhanced catalytic performance. Specifically, the single mutants Y100I, F183L, and H328L showed increased RA conversion rates of 32.17%, 39.26%, and 39.66%, respectively, and increased short-chain glucosylated steviol glycoside yields of 17.72%, 23.95%, and 22.82%, respectively. The double mutant further improved to 40.78%~43.45% and 26.05%~28.46%; the triple mutant Y100I / F183L / H328L showed the best catalytic effect, with RA conversion reaching 51.03%, which is about 3.96 times higher than WT, and short-chain glucosylated steviol glycoside yield reaching 36.37%, which is about 4.37 times higher than WT.

[0060] The results showed that each mutation site could improve the enzyme's utilization of maltose, and the combination of multiple sites had a synergistic effect, thereby further improving the RA conversion rate and the yield of the target product.

[0061] Example 3: Catalytic effect of α-cyclodextrin glucosyltransferase mutant when starch is used as a glycosyl donor The mutant prepared in Example 1 was used to test the catalytic effect when corn starch was used as the glycosyl donor. The steps are as follows: (1) Weigh 1.0 g of corn starch and slowly add it to 70 mL of boiling water under stirring to disperse it fully. Then rinse the weighing container or container with residual starch three times with 30 mL of pure water and combine the washing liquid into the above system. Continue heating and stirring until the system is completely gelatinized and transparent. After cooling to room temperature, make up to 100 mL with pure water to obtain corn starch gelatinized liquid.

[0062] (2) Prepare 10 mL of RA solution with pure water to a 10 g / L concentration, mix it with 10 mL of corn starch gelatinized liquid, and keep it in a water bath at 50℃ for 30 min. (3) α-cyclodextrin glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1, WT) and its mutant were added to the preheated mixed solution at an enzyme amount of 0.3 U / mL and reacted for 12 h to obtain the glycosylated product.

[0063] Qualitative and quantitative analysis of glycosylation products was performed by HPLC, and the conversion rate of RA and the yield of short-chain glycosylation products were calculated to evaluate the utilization effect of α-cyclodextrin glucosyltransferase on long-chain glycosylation donor corn starch before and after mutation.

[0064] HPLC analysis results are as follows Figure 2 As shown in Table 4, the calculated results of RA conversion rate and short-chain glycosylation product yield are presented.

[0065] Table 4 RA conversion rate and yield of short-chain glycosylation products

[0066] The results showed that when corn starch was used as a long-chain glycosyl donor, the α-cyclodextrin glucosyltransferase catalytic system exhibited typical long-chain glycosyl transfer characteristics, and the products had a wider distribution range, including a variety of steviol glycoside derivatives with different degrees of glycosylation.

[0067] The wild-type enzyme achieved a RA conversion rate of 35.68% and a short-chain glucosylation product yield of 20.03%. The three single-point mutants, Y100I, F183L, and H328L, showed increased RA conversion rates of 42.14%, 45.68%, and 43.17%, respectively, representing increases of 18.1%–28.0% compared to the wild-type; their short-chain product yields reached 26.18%, 30.15%, and 31.40%, respectively, representing increases of 30.7%–56.8% compared to the wild-type.

[0068] This performance improvement stems from the mutant's enhanced affinity for maltose, enabling it to utilize maltose generated from the partial degradation of starch in the reaction system, as well as short branched chains in starch molecules, as glycosyl donors, thereby increasing RA conversion rate and short-chain product yield.

[0069] The double mutants Y100I / F183L, Y100I / H328L, and F183L / H328L exhibited significant synergistic effects, with RA conversion rates reaching 49.90%–53.06% and short-chain product yields of 41.30%–43.12%, which were 9.2%–16.2% and 31.5%–37.3% higher than the optimal single mutant, respectively.

[0070] The three mutants Y100I / F183L / H328L achieved optimal catalytic performance, with a RA conversion rate as high as 61.10% and a short-chain product yield of 54.45%, which are 71.3% and 171.9% higher than the wild type, respectively. This fully demonstrates that by enhancing the recognition ability of short-chain substrates such as maltose, the mutants can efficiently utilize starch degradation products for glycosyl transfer, thus achieving a significant increase in the yield of short-chain glucosylated steviol glycosides.

[0071] Example 4: Determination of regioselectivity of α-cyclodextrin glucosyltransferase mutant The regioselectivity of α-cyclodextrin glucosyltransferase and its mutants was determined by a specific alkaline hydrolysis method, as shown in Table 5.

[0072] Table 5 Regional Selectivity

[0073] The results showed that after mutations at amino acid sites 100, 183 and 328 of α-cyclodextrin glucosyltransferase, the regional selectivity of each mutant was basically the same as that of the wild type, and no significant differences were observed.

[0074] This indicates that the above-mentioned site mutation has little effect on the regioselectivity of α-cyclodextrin glucosyltransferase. The mutant can still maintain the regioselectivity characteristics of the wild-type enzyme. Therefore, the short-chain glycosylation product generated is a short-chain glucosylated steviol glycoside at C-19.

[0075] Example 4: Application of α-cyclodextrin glucosyltransferase mutant in the preparation of C-19 short-chain glucosylated steviol glycosides A method for preparing a C-19 short-chain glucosylated steviol glycoside, comprising the following steps: (1) Prepare 10 mL of RA and maltose into 50 g / L solutions with pure water, mix them evenly, and keep them in a water bath at 50℃ for 30 min. (2) α-cyclodextrin glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1, WT) and its mutant were added to the preheated mixed solution respectively. The amount of enzyme added was 0.6 U / mL, and the reaction was carried out for 24 h to obtain the glycosylated product. (4) After the reaction is completed, the reaction solution is spray-dried to obtain short-chain glucosylated steviol glycoside at C-19.

[0076] Qualitative and quantitative analysis of the glycosylated products was performed by HPLC, and the results are shown in Table 6.

[0077] Table 6 Yields of short-chain glucosylated steviol glycosides

[0078] The results showed that the mutants obtained in this invention could effectively improve the production efficiency of short-chain glucosylated steviol glycosides at position C-19, and there was a significant synergistic effect among multiple favorable mutation sites. The yield of the target product continuously increased with the increase in the number of mutation sites, with the three-mutant mutant showing the best effect.

[0079] The results from Examples 2 and 3 show that the mutant of this invention not only improves the enzyme's utilization of short-chain glycosyl donors such as maltose, but also enhances the enzyme's catalytic efficiency for the target short-chain glycosylation reaction, thereby promoting the targeted accumulation of the target product. Because the yield of the target product is significantly increased, interference from byproducts during subsequent separation and purification processes can be reduced, improving raw material utilization and production efficiency.

[0080] Therefore, the α-cyclodextrin glucosyltransferase mutant provided by this invention is suitable for the efficient preparation of short-chain glucosylated steviol glycosides at position C-19 and has good prospects for industrial application.

[0081] The sequences used in the examples: The amino acid sequence of α-cyclodextrin glucosyltransferase WT (SEQ ID NO.1): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDT AGLFHHNGGTDFSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTRAT EQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNI VTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN Mutant amino acid sequence Y100I(SEQ ID NO.2): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGIWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDFSTIEDGIIKNLYDLADINNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTRAT EQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTWEGGGNHTYTTPASGVGTVTVDWQN Mutant amino acid sequence F183L(SEQ ID NO.3): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDLSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN Amino acid sequence of mutant H328L (SEQ ID NO.4): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDFSTIEDGIIKNLYDLADINNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNLDMDRFQVAGSGTRAT EQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTWEGGGNHTYTTPASGVGTVTVDWQN Mutant amino acid sequence Y100I / F183L (SEQ ID NO.5): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGIWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDLSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN Amino acid sequence of mutant Y100I / H328L (SEQ ID NO.6): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGIWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDFSTIEDGIIKNLYDLADINNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNLDMDRFQVAGSGTRAT EQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTWEGGGNHTYTTPASGVGTVTVDWQN Mutant amino acid sequence F183L / H328L(SEQ ID NO.7): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDLSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNLDMDRFQVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN Amino acid sequence of mutant Y100I / F183L / H328L (SEQ ID NO.8): SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGIWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDLSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNLDMDRFQVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN Nucleotide sequence of α - cyclodextrin glucanotransferase WT (SEQ ID NO.9): Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An α-cyclodextrin glucosyltransferase mutant, characterized in that, The α-cyclodextrin glucosyltransferase mutant has a mutation at any two sites, namely positions 100, 183, or 328, based on the amino acid sequence as shown in SEQ ID NO.

1.

2. The α-cyclodextrin glucosyltransferase mutant according to claim 1, characterized in that, The α-cyclodextrin glucosyltransferase mutant has the following amino acid sequence as shown in SEQ ID NO.1: tyrosine at position 100 is mutated to isoleucine, and phenylalanine at position 183 is mutated to leucine. Alternatively, the tyrosine at position 100 may be mutated to isoleucine, and the histidine at position 328 may be mutated to leucine. Alternatively, phenylalanine at position 183 may mutate into leucine, and histidine at position 328 may mutate into leucine.

3. A polynucleotide encoding the α-cyclodextrin glucosyltransferase mutant of any one of claims 1 to 2.

4. A plasmid carrying the polynucleotide of claim 3; Optionally, the plasmid includes pET series vectors, pBAD series vectors, pGEX series vectors, or pCold series vectors.

5. Cells expressing the α-cyclodextrin glucosyltransferase mutant according to any one of claims 1 to 2; Alternatively, the cells may be fungi or bacteria; Optionally, the cells include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris.

6. A genetically engineered Escherichia coli bacterium, characterized in that, The α-cyclodextrin glucosyltransferase mutant according to any one of claims 1 to 2.

7. A method for preparing C-19 short-chain glucose steviol glycoside, characterized in that, Using maltose or corn starch as the glycosyl donor and rebaudioside A as the glycosyl acceptor, utilizing any one of the α-glycosyl groups of claims 1-2 Cyclodextrin glucosyltransferase mutants catalyze the production of C-19 short-chain glucose steviol glycoside.

8. The method according to claim 7, characterized in that, The catalytic temperature is 30~60°C and the catalytic time is 10~20h; among them, the short-chain glucose steviol glycoside at the C-19 position is a glycosylated product in which one or two glucose groups are attached to the C-19 region of rebaudioside A.

9. A method for improving the utilization rate of maltose by α-cyclodextrin glucosyltransferase, characterized in that, The α-cyclodextrin glucosyltransferase with the amino acid sequence shown in SEQ ID NO.1 has a mutation at position 100, 183, or 328; Among them, the tyrosine at position 100 is mutated to isoleucine, and the phenylalanine at position 183 is mutated to leucine. Alternatively, the tyrosine at position 100 may be mutated to isoleucine, and the histidine at position 328 may be mutated to leucine. Alternatively, phenylalanine at position 183 may mutate into leucine, and histidine at position 328 may mutate into leucine.

10. The use of the α-cyclodextrin glucosyltransferase mutant of any one of claims 1 to 2, the polynucleotide of claim 3, the plasmid of claim 4, the cell of claim 5, or the Escherichia coli genetically engineered bacterium of claim 6 in the food, pharmaceutical, or biological fields.

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