A cyclodextrin glucosyltransferase mutant, base sequence, construction method and steviol glycoside conversion method

Abstract

The present application relates to cyclodextrin glucosyltransferase technical field, disclose a kind of cyclodextrin glucosyltransferase mutant, mutant is the 189th, the 255th mutation to protein (a) of wild-type cyclodextrin glucosyltransferase of amino acid sequence such as SEQ ID NO.1;Or mutant is the amino acid sequence of protein (a) is formed by the substitution, deletion or addition of one or more amino acid residues, with at least 90% of the homology of the amino acid sequence of protein (a), and the amino acid of corresponding protein (a) 189th, 255th site is same with protein (a), and the protein (b) with same or similar function of protein (a) is same or similar function.The mutant of the present application converts steviol glycoside into glucosyl steviol glycoside, the content of StevG1, RebAG1, StevG2 in product increases, and the taste and overall quality of glucosyl steviol glycoside are improved.

Description

Technical Field

[0001] This invention relates to the field of cyclodextrin glucosyltransferase technology, specifically to a cyclodextrin glucosyltransferase mutant, its base sequence, construction method, and steviol glycoside conversion method. Background Technology

[0002] Glucosylstevioside is a food additive obtained by extracting steviol glycosides from stevia leaves using an enzymatic method, followed by evaporation, concentration, and spray drying. In the national standard GB2760-2014, glucosylstevioside belongs to the flavoring and fragrance category. Compared to traditional steviol glycosides, it eliminates the natural bitterness, resulting in a better taste, purer sweetness, and a wider range of applications. Research has found that increasing the content of short-chain glucosylstevioside helps improve its taste and quality.

[0003] Currently, most steviol glycosides are converted to glucosyl steviol glycosides using cyclodextrin glucosyltransferases. However, most existing cyclodextrin glucosyltransferases lack substrate and product specificity, resulting in glucosyl steviol glycosides that are mixtures of various substances. The "degree of glucosylation" is difficult to control (the number of glucose groups linked at C13 and C19 positions significantly affects the product's taste). For example, compounds with relatively good taste, such as Rebaudioside D (RebD) and Rebaudioside M (RebM), have relatively low degrees of glucosylation. The lack of cyclodextrin glucosyltransferases specifically designed for steviol glycoside modification limits the improvement of glucosyl steviol glycoside quality, hindering a significant improvement in overall product quality.

[0004] Chinese patent application 202010253100.0, entitled "A mutant strain producing high-yield cyclodextrin glucosyltransferase and its application", discloses a mutant strain, Pichia pastoris CGT-2, which produces cyclodextrin glucosyltransferase with an enzyme activity as high as 60.4 Ul / mL, which can improve the conversion efficiency of substrates, but no reports have been found on the improvement of product quality. Summary of the Invention

[0005] In view of the current lack of cyclodextrin glucosyltransferases specifically for the conversion of steviol glycosides in the market, the purpose of this invention is to provide a cyclodextrin glucosyltransferase mutant, which, when applied to the enzymatic conversion process of steviol glycosides, results in a product with a high content of short-chain glucosyl steviol glycosides, thus improving taste and quality.

[0006] This invention provides the following technical solution: A cyclodextrin glucosyltransferase mutant, The mutant is a protein (a) obtained by mutating tyrosine to phenylalanine at position 189 and glycine to alanine at position 255 of the wild-type cyclodextrin glucosyltransferase as shown in SEQ ID NO.1. Alternatively, the mutant is a protein (b) formed by the substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of protein (a), having at least 90% homology with the amino acid sequence of protein (a), and having the same amino acids at positions 189 and 255 of protein (a) as protein (a), and having the same or similar functions as protein (a).

[0007] The amino acid sequence of the wild-type cyclodextrin glucosyltransferase CGTase-13 used in this invention is shown in SEQ ID NO. 1. This wild-type transferase is preferably derived from *Alkalihalobacillus oshimensis*, as reported in patent application CN202111292313.5. The mutant CGTase-13 was obtained by site-directed mutagenesis of amino acids 189 and 255 of this transferase. Y189F / G255A The amino acid sequence is shown in SEQ ID NO.2. When applied to the conversion of steviol glycosides to glucosyl steviol glycosides, the resulting product has a high content of short-chain glucosyl steviol glycosides and a good taste, showing great potential for industrial application.

[0008] The amino acid sequence SEQ ID NO.1 is as follows: Met Gly Val Thr Asn Lys Val Asn Tyr Ser Lys Asp Val Ile Tyr Gln IleVal Thr Asp Arg Phe Ser Asp Gly Asn Thr Ala Asn Asn Pro Thr Gly Thr Ile PheSer Gln Asn Cys Ser Asp Leu His Lys Tyr Cys Gly Gly Asp Trp Gln Gly Ile IleAsn Lys Met Asn Asp Gly Tyr Leu Thr Asp Leu Gly Ile Thr Ala Leu Trp Ile SerGln Pro Val Glu Asn Val Tyr Ala Leu His Pro Ser Gly Tyr Thr Ser Tyr His GlyTyr Trp Ala Arg Asp Tyr Lys Lys Thr Asn Pro Tyr Phe Gly Asn Phe Thr Asp PheAsp Arg Leu Val Ser Thr Ala His Ser Lys Gly Ile Lys Ile Ile Met Asp Phe ThrPro Asn His Ser Ser Pro Ala Leu Glu Thr Asn Pro Asn Tyr Val Glu Asn Gly AlaLeu Tyr Asn Asn Gly Ala Met Leu Gly Asn Tyr Ser Asn Asp Arg Asn Lys Leu PheHis His Asn Gly Gly Thr Asp Phe Ser Ser Tyr Glu Asp Ser Ile Tyr Arg Asn LeuTyr Asp Leu Ala Asp Tyr Asp Leu Asn Asn Lys Val Ile Asp Gln Tyr Leu Lys GluSer Ile Lys Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg Val Asp Ala Val LysHis Met Ser Glu Gly Trp Gln Thr Ser Leu Met Ser Asp Ile Tyr Ser His Lys ProVal Phe Thr Phe Gly Glu Trp PheLeu Gly Ser Gly Glu Val Asp Pro Gln Asn HisHis Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Gln Phe Gly Gln Thr IleArg Ser Val Leu Lys Asp Arg Thr Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile LysSer Thr Glu Lys Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe Ile Asp Asn HisAsp Met Ser Arg Phe Ser Val Gly Ser Ser Ser Asn Arg Gln Thr Asp Ile Ala LeuAla Val Leu Leu Thr Ser Arg Gly Val Pro Thr Ile Tyr Tyr Gly Thr Glu Gln TyrLeu Thr Gly Gly Asn Asp Pro Asp Asn Arg Lys Pro Met Lys Thr Phe Asp Arg SerThr Asn Ser Tyr Lys Ile Thr Ser Lys Leu Ala Ser Leu Arg Gln Arg Asn Ser AlaLeu Gly Tyr Gly Asn Thr Thr Glu Arg Trp Ile Asn Ser Asp Val Tyr Ile Tyr GluArg Lys Phe Gly Asn Ser Ile Val Leu Thr Ala Val Asn Ser Ser Asn Arg Asn GlnThr Ile Ser Asn Leu Asn Thr Ser Leu Pro Gln Gly Asn Tyr Thr Asp Glu Leu GlnGln Leu Leu Asp Gly Asn Ala Ile Thr Val Asn Ala Asn Gly Ser Ala Asn Ser PheGln Leu Arg Ala Asn Ser Val Ala Val Trp Gln Val Thr Lys Glu Ser Thr Ser ProLeu Ile Gly His Val Gly Pro Met Met Gly Lys Thr Gly AsnThr Val Thr Val Ser Gly Glu Gly Phe Gly Asp Lys Lys Gly Ser Val Leu Phe Gly Ser Thr Ser Ala Glu Ile Val Thr Trp Ser Asn Thr Glu Ile Gln Val Lys Val Pro Ser Val Thr Ala Gly His Tyr Asn Leu Ser Val Val Asn Ala Thr Asn Ala Lys Ser Pro Ala Tyr Glu Lys Phe Glu Val Leu Ser Gly Asn Gln Val Ser Val Arg Phe Ala Val Asn Asn Ala Thr Thr Asn Ser Gly Thr Asn Val Tyr Ile Val Gly Asn Val Ser Glu Leu Gly Asn Trp Asp Pro Asn Lys Ala Ile Gly Pro Met Phe Asn Gln Val Met Tyr Lys Tyr Pro Thr Trp Tyr Tyr Asp Ile Ser Val Pro Ala Gly Lys Asn Leu Glu Tyr Lys Tyr Ile Lys Lys Asp Gln Asn Gly Asn Val Ile Trp Gln Ser Gly Asn Asn Arg Thr Tyr Thr Ser Pro Ala Thr Gly Thr Asp Thr Val Ile Thr Asn Trp.

[0009] Amino acid sequence SEQ ID NO. 2 is as follows: Met Gly Val Thr Asn Lys Val Asn Tyr Ser Lys Asp Val Ile Tyr Gln IleVal Thr Asp Arg Phe Ser Asp Gly Asn Thr Ala Asn Asn Pro Thr Gly Thr Ile PheSer Gln Asn Cys Ser Asp Leu His Lys Tyr Cys Gly Gly Asp Trp Gln Gly Ile IleAsn Lys Met Asn Asp Gly Tyr Leu Thr Asp Leu Gly Ile Thr Ala Leu Trp Ile SerGln Pro Val Glu Asn Val Tyr Ala Leu His Pro Ser Gly Tyr Thr Ser Tyr His GlyTyr Trp Ala Arg Asp Tyr Lys Lys Thr Asn Pro Tyr Phe Gly Asn Phe Thr Asp PheAsp Arg Leu Val Ser Thr Ala His Ser Lys Gly Ile Lys Ile Ile Met Asp Phe ThrPro Asn His Ser Ser Pro Ala Leu Glu Thr Asn Pro Asn Tyr Val Glu Asn Gly AlaLeu Tyr Asn Asn Gly Ala Met Leu Gly Asn Tyr Ser Asn Asp Arg Asn Lys Leu PheHis His Asn Gly Gly Thr Asp Phe Ser Ser Tyr Glu Asp Ser Ile Tyr Arg Asn LeuPhe Asp Leu Ala Asp Tyr Asp Leu Asn Asn Lys Val Ile Asp Gln Tyr Leu Lys GluSer Ile Lys Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg Val Asp Ala Val LysHis Met Ser Glu Gly Trp Gln Thr Ser Leu Met Ser Asp Ile Tyr Ser His Lys ProVal Phe Thr Phe Gly Glu Trp PheLeu Ala Ser Gly Glu Val Asp Pro Gln Asn HisHis Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Gln Phe Gly Gln Thr IleArg Ser Val Leu Lys Asp Arg Thr Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile LysSer Thr Glu Lys Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe Ile Asp Asn HisAsp Met Ser Arg Phe Ser Val Gly Ser Ser Ser Asn Arg Gln Thr Asp Ile Ala LeuAla Val Leu Leu Thr Ser Arg Gly Val Pro Thr Ile Tyr Tyr Gly Thr Glu Gln TyrLeu Thr Gly Gly Asn Asp Pro Asp Asn Arg Lys Pro Met Lys Thr Phe Asp Arg SerThr Asn Ser Tyr Lys Ile Thr Ser Lys Leu Ala Ser Leu Arg Gln Arg Asn Ser AlaLeu Gly Tyr Gly Asn Thr Thr Glu Arg Trp Ile Asn Ser Asp Val Tyr Ile Tyr GluArg Lys Phe Gly Asn Ser Ile Val Leu Thr Ala Val Asn Ser Ser Asn Arg Asn GlnThr Ile Ser Asn Leu Asn Thr Ser Leu Pro Gln Gly Asn Tyr Thr Asp Glu Leu GlnGln Leu Leu Asp Gly Asn Ala Ile Thr Val Asn Ala Asn Gly Ser Ala Asn Ser PheGln Leu Arg Ala Asn Ser Val Ala Val Trp Gln Val Thr Lys Glu Ser Thr Ser ProLeu Ile Gly His Val Gly Pro Met Met Gly Lys Thr Gly AsnThr Val Thr Val SerGly Glu Gly Phe Gly Asp Lys Lys Gly Ser Val Leu Phe Gly Ser Thr Ser Ala GluIle Val Thr Trp Ser Asn Thr Glu Ile Gln Val Lys Val Pro Ser Val Thr Ala GlyHis Tyr Asn Leu Ser Val Val Asn Ala Thr Asn Ala Lys Ser Pro Ala Tyr Glu LysPhe Glu Val Leu Ser Gly Asn Gln Val Ser Val Arg Phe Ala Val Asn Asn Ala ThrThr Asn Ser Gly Thr Asn Val Tyr Ile Val Gly Asn Val Ser Glu Leu Gly Asn TrpAsp Pro Asn Lys Ala Ile Gly Pro Met Phe Asn Gln Val Met Tyr Lys Tyr Pro ThrTrp Tyr Tyr Asp Ile Ser Val Pro Ala Gly Lys Asn Leu Glu Tyr Lys Tyr Ile LysLys Asp Gln Asn Gly Asn Val Ile Trp Gln Ser Gly Asn Asn Arg Thr Tyr Thr SerPro Ala Thr Gly Thr Asp Thr Val Ile Thr Asn Trp.

[0010] The present invention also provides the base sequence encoding the above-mentioned cyclodextrin glucosyltransferase mutant.

[0011] The present invention also provides an expression vector containing the above-mentioned base sequence.

[0012] Preferably, the expression vector is a pGEX plasmid, a pHY300 plasmid, a pPIC3K plasmid, a pPIC9K plasmid, a pHY300PLK plasmid, a pET plasmid, or a Duet plasmid. Preferably, the pET plasmid is pET-15, pET-19, pET-20, pET-24, pET-28, or pET-32; preferably, the Duet plasmid is pRSFDuet-1 or pACYCDuet-1; and preferably, the pGEX plasmid is pGEX-4T-2 or pGEX-6P.

[0013] The present invention also provides a host cell containing the above-described base sequence or the above-described expression vector.

[0014] Preferably, the host cell is a bacterial or fungal cell. More preferably, the host cell is a recombinant prokaryotic cell or a eukaryotic cell; the prokaryotic cell is a Gram-negative or Gram-positive bacterium.

[0015] As a preferred embodiment of the present invention, the host cell is Escherichia coli BL21(DE3) cell or Pichia pastoris cell.

[0016] The present invention also provides a method for constructing the above-mentioned cyclodextrin glucosyltransferase mutant, wherein an expression vector containing the base sequence encoding the above-mentioned cyclodextrin glucosyltransferase mutant is transferred into a host cell by electroporation or chemical transformation.

[0017] As a preferred embodiment of the method of the present invention, the method includes the following specific steps: An expression vector containing the base sequence encoding the amino acid sequence SEQ ID NO.1 is subjected to site-directed mutagenesis using a first primer pair that mutates at position 189, transferred to a cloning host for amplification, the expression vector is extracted and sequenced, and expression vectors with mutations at the mutation site but not at non-target mutation sites are screened. Then, a second primer pair that mutates at position 255 is subjected to site-directed mutagenesis, transferred to a cloning host for amplification, the expression vector is extracted and sequenced, and expression vectors with mutations at the mutation site but not at non-target mutation sites are screened. The vector is then transferred to host cells for induction of expression, and the enzyme solution is collected. When the host cell is Escherichia coli BL21(DE3), the forward primer sequence of the first primer pair is shown in SEQ ID NO.3, the reverse primer sequence is shown in SEQ ID NO.4, the forward primer sequence of the second primer pair is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6. When the host cell is Pichia pastoris cell, the first primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.7, and the reaction primer sequence is shown in SEQ ID NO.8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.10. The second primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.11, and the reaction primer sequence is shown in SEQ ID NO.8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.12.

[0018] A method for converting steviol glycosides to glucosyl steviol glycosides includes the following steps: using a solution of soluble starch and steviol glycosides as a substrate, adding the above-mentioned cyclodextrin glucosyltransferase mutant, and reacting by shaking and heating.

[0019] Using the cyclodextrin glucosyltransferase mutant of the present invention to convert steviol glycosides, the content of StevG1, RebAG1, and StevG2 in the prepared glucosyl steviol glycoside product is increased compared with the wild-type transferase, the astringency and off-flavors of the product are reduced, the sweetness is increased, and the taste and overall quality of the product are improved.

[0020] As a preferred embodiment of the method of the present invention The concentration of soluble starch is 15–30 g / L, the concentration of steviol glycosides is 15–30 g / L, and the concentration of cyclodextrin glucosyltransferase mutant is 15–30 mg / L; the steviol glycosides used can be selected from steviol glycosides, rebaudioside A, or a mixture thereof; the reaction temperature is 35–50℃, and the reaction time is 18–30 h.

[0021] The beneficial effects of this invention are as follows: When the mutant Y189F / G255A obtained in this invention converts steviol glycosides into glucosyl steviol glycosides, the content of StevG1, RebAG1, and StevG2 in glucosyl steviol glycosides increases, the astringency of glucosyl steviol glycosides is significantly reduced, while off-flavors are reduced and sweetness increases, thus improving the taste and overall quality of glucosyl steviol glycosides. Attached Figure Description

[0022] Figure 1 This is a sequencing peak diagram at the mutation site (left: E. coli host; right: Pichia pastoris host).

[0023] Figure 2 These are SDS-PAGE images of the enzyme before and after mutation (top: E. coli host, purified by nickel column; bottom: Pichia pastoris host, extracellular enzyme solution).

[0024] Figure 3 This is a comparison of the peak areas of short-chain glucosylstevioside before and after the mutation.

[0025] Figure 4 It is a mesh diagram comparing the taste before and after the mutation. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below.

[0027] Unless otherwise specified, all raw materials used in this invention are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0028] This invention provides a cyclodextrin glucosyltransferase mutant, which is the product of mutations at positions 189 and 255 of the wild-type cyclodextrin glucosyltransferase shown in SEQ ID NO.1. This wild-type cyclodextrin glucosyltransferase CGTase-13 is derived from *Alkalihalobacillus oshimensis*, with accession number CGMCC No. 23164, specifically as described in Chinese patent application CN202111292313.5. During the conversion of steviol glycosides to glucosylsteviosides, the content of StevG1, RebAG1, and StevG2 in the product increases, astringency is significantly reduced, off-flavors are somewhat reduced, and the flavor and overall quality are improved.

[0029] In some embodiments provided by this invention, the mutant is a protein (a) obtained by mutating tyrosine to phenylalanine at position 189 and glycine to alanine at position 255 of the wild-type cyclodextrin glucosyltransferase as shown in SEQ ID NO.1, namely mutant CGTase-13. Y189F / G255A The amino acid sequence is shown in SEQ ID NO.2.

[0030] In some embodiments provided by the present invention, the mutant is a protein (b) formed by the substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of protein (a), having at least 90% homology with the amino acid sequence of protein (a), and having the same amino acids at positions 189 and 255 of protein (a) as protein (a), and having the same or similar functions as protein (a).

[0031] The present invention also provides the base sequence encoding the above-mentioned cyclodextrin glucosyltransferase mutant.

[0032] The present invention also provides an expression vector containing the above-mentioned base sequence.

[0033] In some embodiments provided by the present invention, the expression vector is a pGEX plasmid, a pHY300 plasmid, a pPIC3K plasmid, a pPIC9K plasmid, a pHY300PLK plasmid, a pET plasmid, or a Duet plasmid; preferably, the pET plasmid is pET-15, pET-19, pET-20, pET-24, pET-28, or pET-32; preferably, the Duet plasmid is pRSFDuet-1 or pACYCDuet-1; preferably, the pGEX plasmid is pGEX-4T-2 or pGEX-6P.

[0034] The present invention also provides a host cell containing the above-described base sequence or the above-described expression vector.

[0035] In some embodiments provided by this invention, the host cell is a bacterial or fungal cell. The host cell is a recombinant prokaryotic cell or a eukaryotic cell; the prokaryotic cell is a Gram-negative or Gram-positive bacterium; preferably, the host cell is *Escherichia coli* BL21(DE3) competent cells or *Pichia pastoris* cells.

[0036] The present invention also provides a method for constructing the above-mentioned cyclodextrin glucosyltransferase mutant, comprising transferring an expression vector containing the base sequence encoding the above-mentioned cyclodextrin glucosyltransferase mutant into host cells for expression by electroporation or chemical transformation.

[0037] In some embodiments provided by this invention, it can be constructed using the following specific methods: The expression vector containing the base sequence encoding the amino acid sequence SEQ ID NO.1 was site-directedly mutated using the first primer pair that mutated at position 189, transferred to the cloning host for amplification, and the expression vectors in the host cells were extracted and sequenced. Expression vectors with mutations at the mutation site but not at non-target mutation sites were screened. Then, the expression vectors were site-directedly mutated using the second primer pair that mutated at position 255, transferred to the cloning host for amplification, and the expression vectors in the host cells were extracted and sequenced. Expression vectors with mutations at the mutation site but not at non-target mutation sites were screened. The expression vectors were then transferred to the expression host to induce expression, and the enzyme solution was collected. When the expression host is Escherichia coli BL21(DE3), the forward primer sequence of the first primer pair is shown in SEQ ID NO.3, the reverse primer sequence is shown in SEQ ID NO.4, the forward primer sequence of the second primer pair is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6. When the expression host is Pichia pastoris, the first primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.7, and the reaction primer sequence is shown in SEQ ID NO.8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.10. The second primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.11, and the reaction primer sequence is shown in SEQ ID NO.8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.12.

[0038] In some embodiments provided by this invention, a large number of mutant CGTase-13 Y189F / G255AThe cyclodextrin glucosyltransferase mutant can be obtained through fermentation culture, specifically by inoculating the expression host carrying the above-mentioned base sequence or expression vector into the fermentation medium for fermentation. After fermentation, the fermentation broth is collected and centrifuged. After centrifugation, the cyclodextrin glucosyltransferase mutant is isolated from the supernatant obtained by centrifugation or from the bacterial cells.

[0039] This invention also provides a method for converting steviol glycosides into glucosyl steviol glycosides, comprising the following steps: Using a solution of soluble starch and steviol glycosides as a substrate, the above-mentioned cyclodextrin glucosyltransferase mutant was added, and the reaction was carried out under shaking and heating. Preferably, the concentration of soluble starch was 15-30 g / L, the concentration of steviol glycosides was 15-30 g / L, and the concentration of cyclodextrin glucosyltransferase mutant was 15-30 mg / L; preferably, the steviol glycosides were steviol glycoside, rebaudioside A, or a mixture thereof; preferably, the reaction temperature was 35-50°C, and the reaction time was 18-30 h.

[0040] The following specific implementation examples further illustrate this point. Example 1 Construct a mutant CGTase-13 using Escherichia coli BL21(DE3) as the expression host. Y189F / G255A Expression vector (1) CGTase-13 Y189F Construction of Escherichia coli expression vector Using a recombinant plasmid containing the base sequence encoding the amino acid sequence SEQ ID NO.1 as a template plasmid, the site-directed mutant sequence of CGTase was amplified by PCR using the Fast Mutagenesis System from Total Gold and the designed corresponding mutation primer pairs. The specific operation steps are as follows: ① Mutant primer pair (first primer pair) The forward primer 13#-E-Y189-F (SEQ ID NO.3) is: 5'-CATTTATCGCAACCTGTTTGATCTGGCGG-3', The reverse primer 13#-E-Y189F-R (SEQ ID NO.4) is: 5'-AACAGGTTGCGATAAAATGCTATCTTCATA-3'.

[0041] ②The reaction system is as follows: Table 1 PCR reaction system Plasmid (pET-28a-CGTase-13) 1 μl (concentration 10 ng / μl) Forward Primer 1μl Reverse Primer 1μl 2*TransStart FastPfu Fly PCR SuperMix 25μl Nuclease-free Water 22μl ③PCR conditions are as follows: Table 2 PCR conditions ④ Digestion of PCR products: Add 1 μl of DMT enzyme to the PCR products, mix well, and incubate at 37°C for 1 h.

[0042] ⑤ Transformation: i) Add 2-5 μl of DMT enzyme digestion product to 50 μl of DMT competent cells (add the ligation product when the competent cells are just thawed), gently mix, and incubate on ice for 20-30 min; ii) Heat shock at 42℃ for 45 seconds, then immediately ice bath for 2 minutes; iii) Add 250 μl of SOC or LB medium equilibrated to room temperature, and incubate at 200 rpm and 37°C for 1 h; iv) Preheat the plates with suitable resistance in a 37°C incubator (using kanamycin resistance, working concentration 50 ng / μl, add 500 μl of 10 mg / ml stock solution to 100 ml of culture medium); v) Streak the bacterial culture on a plate and incubate overnight at 37°C.

[0043] ⑥ Picking bacteria: On the second day, observe the overnight plates and find that colonies have grown on all of them. Pick 5 single clones from each plate and place them in 1ml LB (kanamycin, 2ml tube) and culture at 37℃ and 220rpm. Extract plasmids and send them for sequencing to confirm that the obtained plasmids have mutated at the target mutation site but have not mutated at non-target mutation sites.

[0044] (2) CGTase-13 Y189F / G255A Construction of Escherichia coli expression vector pET-28a-CGTase-13 Y189F The plasmid was used as the template plasmid. Using the Fast Mutagenesis System (FMS) single-point mutagenesis kit, the site-directed mutant sequence of CGTase was amplified by PCR using the designed corresponding mutation primer pair. The specific mutation primer pair (second primer pair) was as follows: The forward primer 13#-E-G255A-F (SEQ ID NO.5) is: 5'-TGGCGAGCGGCGAAGTGGATCCGCAGAACCATCATT-3', The reverse primer 13#-E-G255A-R (SEQ ID NO.6) is: 5'-TGCGGATCCACTTCGCCGCTCGCCAGAAACATTCG-3' The remaining steps are the same as above, and the results are attached. Figure 1 As shown.

[0045] Example 2 The mutant was expressed and purified by transforming the mutant plasmid into the expression host, Escherichia coli BL21(DE3) competent cells, following the same transformation steps as above.

[0046] Protein-induced expression: (1) Strains and conditions ① Two proteins need to be induced to express (CGTase-13 and CGTase-13) Y189F / G255A ): Escherichia coli BL21(DE3) competent cells containing the corresponding plasmid were seeded into 4 mL of LB medium with 50 μg / mL kanamycin and cultured overnight at 30°C and 220 rpm.

[0047] ② Transfer all the overnight cultured bacterial cultures to 400 mL LB + 50 μg / mL kanamycin medium and incubate at 30 °C and 220 rpm for 4 h.

[0048] ③ Add IPTG to a final concentration of 0.1 mM and incubate at 16°C and 220 rpm for 20 h.

[0049] (2) Ultrasonic cell disruption ① After inducing protein expression, the bacterial culture was centrifuged at 10000×g for 5 min at room temperature, and the cells were collected.

[0050] ② Add 30 mL of 20 mM PBS (containing 20 mM imidazole) to resuspend the cells and place them in an ice-water bath.

[0051] ③ Use a 6mm diameter amplitude rod, 20% intensity, and ultrasonically break the mixture for 15 minutes with a 2s pause and a 3s interval; ④ Centrifuge at 22000rpm for 20 minutes at 4℃, filter the supernatant through a 0.22μm filter membrane, and store at 4℃ for later use.

[0052] (3) Protein purification ① The supernatant after ultrasonic disruption was filtered through a 0.22μm filter membrane and then loaded into the AKTA system.

[0053] 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.

[0054] ③ Replace with 40mM imidazole PBS buffer and continue washing. After an absorption peak appears, the UV absorbance value decreases to the initial level.

[0055] ④ Replace with 250mM imidazole PBS buffer, elute, and start collecting the eluent when the UV absorbance index rises, 2 mL per tube, 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, 2 mL per tube, until the UV absorption level flattens out.

[0056] (4) SDS-PAGE ① Take 20 μL of the above protein purification collection solution, add it to 10 μL of 6× protein loading buffer, mix well, and incubate at 95℃ for 10 min.

[0057] ② Take two pieces of pre-prepared 12% polyacrylamide gel and put them into the electrophoresis tank.

[0058] ③ Load the samples in sequence, and the results are shown in the attached figure. Figure 2 As shown.

[0059] Example 3 Methods for converting steviol glycosides to glucosyl steviol glycosides (1) Prepare a solution of 20 g / L soluble starch + 20 g / L steviol glycoside (Haotian SG90) with water as substrate, bring the volume to 100% (v / v), and boil in a water bath for 10 min.

[0060] (2) After the substrate has cooled, add 20 mg / L enzyme solution, which is derived from the purified protein in Example 2; use 50 mM pH 7.0 PBS instead of enzyme solution as a control.

[0061] (3) Place the centrifuge tube in a 3L Erlenmeyer flask, place the Erlenmeyer flask in a 40℃ shaker, react at 220rpm for 22h, and then centrifuge at the maximum speed for 2min at room temperature.

[0062] (4) Take 100 μL of supernatant, add 900 μL of water, filter with a 0.22 μm filter membrane, and then perform the determination according to the HPLC procedure for the determination of glucosyl steviol glycosides in standard GB2760-2014.

[0063] The content of the produced short-chain glucosylsteviosides was expressed by the peak area corresponding to them in high-performance liquid chromatography (HPLC), and the results are shown in the attached figure. Figure 3 As shown, compared with the unmutated product, the content of StevG1, RebAG1 and StevG2 increased.

[0064] Example 4 Constructing a mutant CGTase-13 using Pichia pastoris cells as the expression host. Y189F / G255A expression carrier (1) CGTase-13 Y189F Construction of Pichia pastoris expression vector 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 FastMultiSite Mutagenesis System and the designed corresponding mutation primer pairs. The specific operation steps are as follows: ① Mutant primer pair (first primer pair) Segment 1 (First Group): The forward primer 13#-P-Y189F-F (SEQ ID NO.7) is: 5'-TATTTGATTTGGCTGATTATGACTTGAACAATAAAAG-3', The reverse primer PR (SEQ ID NO.8) is: 5'-AGCAGCACGCCATAGTGACTGGCG-3' Segment 2 (Second Group): The forward primer PF (SEQ ID NO.9) is: 5'-CGCCAGTCACTATGGCGTGCTGCT-3', The reverse primer 13#-P-Y189F-R (SEQ ID NO.10) is: 5'-AAGTCATAATCAGCCAAATCAAATAAGTTCCTATAA-3'.

[0065] ②The reaction system is as follows: Table 3 PCR reaction system Plasmid(pPIC9K-CGTase-13) 1 μl (concentration 10 ng / μl) Forward Primer 1μl Reverse Primer 1μl 2*TransStart FastPfu Fly PCR SuperMix 25μl Nuclease-free Water 22μl ③PCR conditions are as follows: Table 4 PCR conditions ④ Electrophoresis detection: Take 3-5 μl of PCR product and detect it by 1% agarose gel electrophoresis.

[0066] ⑤ Digestion of PCR products: Add 1 μl of DMT enzyme to the PCR products, mix well, and incubate at 37°C for 1 h.

[0067] ⑥ Purified fragments: Perform agarose gel electrophoresis, then cut the gel and recover the fragments using an Omega gel extraction kit, as follows: i) Add 600 μl of Bingding buffer (XP2) to a 1.5 ml tube containing the adhesive strip, ensuring the strip is completely submerged. Incubate in a 65°C water bath until the adhesive strip is completely melted. ii) Place the DNA mini column into a standard 2ml collection tube, add 700μl of the mixture into the centrifuge column, centrifuge at 10000×g for 15s, and wash the membrane once with 500μl of Bingding buffer (XP2). iii) Add 700 μl of SPW wash buffer, centrifuge at 10000×g for 15 s, and repeat once; iv) 10000×g air-to-air separation for 2 min; v) Dry in a 65℃ metal bath for 2 minutes; vi) Elute with 30 μl of deionized water, and repeat once.

[0068] Test concentrations: Fragment 1: 5.2 ng / μl; Fragment 2: 28.4 ng / μl.

[0069] ⑦ Mutant fragment assembly: Table 5 Fragment Assembly System 2*Assembly Mix 5μl Segment 1 4μl Segment 2 1μl Mix well and maintain at 50°C for 15 minutes. After the reaction is complete, place in an ice bath for a few seconds before using for conversion.

[0070] ⑧ Transformation: The steps are the same as above (the mutant expressed in the Pichia pastoris host was constructed using ampicillin sodium resistance, working concentration 100 μg / ml).

[0071] 9. Bacterial selection: The steps are the same as above. Extract plasmids and send them for sequencing to determine if the obtained plasmids have mutated at the target mutation site but not at non-target mutation sites.

[0072] (2) CGTase-13 Y189F / G255A Construction of Pichia pastoris expression vector pPIC9K-CGTase-13 Y189F The plasmid was used as the template plasmid. Using the FastMultiSite Mutagenesis System, the site-directed mutant sequence of CGTase was amplified by PCR using the designed corresponding mutation primer pairs. The specific operation steps are as follows: Mutant primer pair (second primer pair) Segment 1 (First Group): The forward primer 13#-P-G255A-F (SEQ ID NO.11) is: 5'-TGGCTTCTGGTGAAGTTGATCCACAAAATCATCATT-3', The reverse primer PR (SEQ ID NO.8) is: 5'-AGCAGCACGCCATAGTGACTGGCG-3' Segment 2 (Second Group): The forward primer PF (SEQ ID NO.9) is: 5'-CGCCAGTCACTATGGCGTGCTGCT-3', The reverse primer 13#-P-G255A-R (SEQ ID NO.12) is: 5'-TGTGGATCAACTTCACCAGAAGCCAAAAACCATTCA-3' The remaining steps are the same as above, and the results are attached. Figure 1 As shown.

[0073] Example 5: Expression of the mutant (Pichia pastoris) Plasmids carrying wild-type enzyme base sequences and those carrying mutant base sequences were digested with SacI to recover linearized DNA fragments. These fragments were then transformed into Pichia pastoris strain GS115 (purchased from Weidi Biotechnology) via chemical transformation. The transformed cells were plated on MD plates (13.4 g / L yeast basic nitrogen source (YNB); 0.4 mg / L biotin; 20 g / L glucose) and cultured at 28°C for 3 days. Transformants were obtained and screened using YPD-G418 and MM / MD plates. PCR verification confirmed the presence of recombinant strains (Mut+ phenotype). The recombinant strains were inoculated into 25 ml of BMGY medium (250 ml shake flask) and incubated at 28-30°C and 250 rpm until OD600 = 2-6. Cells were centrifuged at 1500-3000 g for 5 min at room temperature, the supernatant was removed, and the cells were resuspended in BMMY medium at OD600 = 1.0 for induction of expression (approximately 100-200 ml). Add the above culture to a 1L shake flask, cover with 4 layers of sterile gauze, and place in a shaker to continue growth. Add methanol every 24 hours to a final concentration of 1%, and induce culture for 96 hours.

[0074] Centrifuge, collect the extracellular enzyme solution, and perform SDS-PAGE analysis. The results are shown in the attached figure. Figure 2 As shown.

[0075] Example 6 Sensory Experiment (1) Concentration of crude enzyme solution The enzyme solution obtained in Example 5 was concentrated using a Millipore ultrafiltration centrifuge tube (15 ml / 10 kDa) (100-fold concentration), and the buffer was exchanged (>1000-fold) to remove culture medium components, so that the enzyme was in ultrapure water to avoid introducing impurities in subsequent reactions.

[0076] (2) Transglycosylation reaction Table 6 Glycosylation Reaction System (3) Sensory sample preparation The crude glucosyl steviol glycoside powder was obtained by centrifugation, filtration, and freeze-drying. It was then formulated to a sweetness level of 5 degrees sucrose for sensory evaluation. The results are as follows: Figure 4 As shown in Table 7.

[0077] Table 7 Summary of Sensory Experiment Results Sweetness 5.9 6.0 Sweetening speed 5.2 5.0 Aftertaste 2.7 2.7 bitterness 1.2 1.2 astringent 2.5 2.0 Mixed flavors 2.1 2.0 Overall preferences 74.9 77.8 from Figure 4 As can be seen from Table 7, the mutant CGTase-13 was used. Y189F / G255A The prepared glucosylsteviosides showed increased sweetness, significantly reduced astringency, and some off-flavors, resulting in improved overall palatability. The taste and quality of the obtained glucosylsteviosides were significantly better than before the mutation.

[0078] 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. A cyclodextrin glucosyltransferase mutant, characterized in that, The mutant is a protein obtained by mutating tyrosine to phenylalanine at position 189 and glycine to alanine at position 255 of the wild-type cyclodextrin glucosyltransferase, as shown in SEQ ID NO.

1.

2. Encoding the base sequence of the cyclodextrin glucosyltransferase mutant as described in claim 1.

3. An expression vector containing the base sequence as described in claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vectors are pGEX plasmid, pHY300 plasmid, pPIC3K plasmid, pPIC9K plasmid, pHY300PLK plasmid, pET plasmid, or Duet plasmid.

5. A host cell containing the base sequence as described in claim 2 or the expression vector as described in claim 3 or 4.

6. The host cell according to claim 5, characterized in that, The host cell is a bacterial or fungal cell.

7. The host cell according to claim 6, characterized in that, The host cell is Escherichia coli. BL21(DE3) Cells or Pichia pastoris cells.

8. A method for constructing a cyclodextrin glucosyltransferase mutant as described in claim 1, characterized in that, The expression vector containing the base sequence encoding the cyclodextrin glucosyltransferase mutant as described in claim 1 is transferred into host cells for expression by electroporation or chemical transformation.

9. The construction method according to claim 8, characterized in that, Includes the following steps: The expression vector containing the base sequence encoding the amino acid sequence SEQ ID NO.1 was site-directedly mutated using the first primer pair that mutated at position 189, and then transferred to an E. coli cloning host for amplification. The expression vector was extracted and sequenced. Expression vectors with mutations at the mutation site but not at non-target mutation sites were screened. Then, the expression vector was site-directedly mutated using the second primer pair that mutated at position 255, and then transferred to an E. coli cloning host for amplification. The expression vector was extracted and sequenced. Expression vectors with mutations at the mutation site but not at non-target mutation sites were screened. Then, the expression vector was transferred to the expression host for induction of expression, and the enzyme solution was collected. When the expression host is Escherichia coli BL21(DE3) At that time, the forward primer sequence of the first primer pair is shown in SEQ ID NO.3, the reverse primer sequence is shown in SEQ ID NO.4, the forward primer sequence of the second primer pair is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; When the expression host is Pichia pastoris, the first primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.7, and the reaction primer sequence is shown in SEQ ID NO.

8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.

10. The second primer pair consists of two sets. The forward primer sequence of the first set is shown in SEQ ID NO.11, and the reaction primer sequence is shown in SEQ ID NO.

8. The forward primer sequence of the second set is shown in SEQ ID NO.9, and the reaction primer sequence is shown in SEQ ID NO.

12.

10. A method for converting steviol glycosides into glucosyl steviol glycosides, characterized in that, Includes the following steps: Using a solution of soluble starch and steviol glycosides as a substrate, the cyclodextrin glucosyltransferase mutant as described in claim 1 was added, and the reaction was carried out by shaking and heating.

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