CN110804597B - Cyclodextrin glucosyltransferase mutant and application thereof - Google Patents
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Abstract
The invention discloses a cyclodextrin glucosyltransferase mutant and application thereof, belonging to the technical field of enzyme engineering and microbial engineering. The CGTase mutant has high product specificity on long-chain glycosylation genistein; the yields of the long-chain glycosylated genistein produced by using the CGTase mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G and A156V/L174P/A166K of the invention respectively take maltodextrin as glycosyl donors, and the yields of the long-chain glycosylated genistein produced by using genistein as glycosyl acceptor are respectively improved by 62.5%, 165%, 112.5% and 59.4% compared with the yields produced by using wild-type cyclodextrin glucosyltransferase and taking maltodextrin as glycosyl donor.
Description
Technical Field
The invention relates to a cyclodextrin glucosyltransferase mutant and application thereof, belonging to the technical field of enzyme engineering and microbial engineering.
Background
Genistein (also known as genistein, etc.) is considered as a soybean isoflavone substance with the highest activity and function, and is often present in leguminous plants such as sophora fruit, subprostrate sophora root, etc. in the form of its glucoside derivative, genistin (also known as 4',5, 7-trihydroxyisoflavone-7-glucoside).
Genistein has a wide range of pharmacological effects in human and animal cells, mainly expressed as: (1) has effects in preventing cancer (breast cancer and prostate cancer). Genistein has effects of estrogen-like hormone and anti-hormone, and can inhibit activity of related enzyme in synthesis process of tumor cell, inhibit tumor angiogenesis in formation process of tumor cell, and delay or prevent tumor from becoming cancer cell. (2) Can be used for preventing cardiovascular diseases. Genistein can stimulate lipoprotein receptor with low density to produce positive regulation effect, promote cholesterol removal, inhibit platelet aggregation, and prevent and treat atherosclerosis. (3) Can be used for preventing postmenopausal diseases. Genistein is a typical phytoestrogen, and has estrogenic activity capable of relieving female climacteric syndrome and preventing postmenopausal diseases. (4) Has anti-osteoporosis effect. The estrogen activity of the genistein can activate an estrogen receptor and improve the activity of osteoblasts; in addition, the bone mineral can also increase the bone mineral density, inhibit the bone mass loss and have better improvement effect on the osteoporosis. Therefore, the genistein has extremely high application prospect in the field of medicine.
However, genistein has strong hydrophobicity, is almost insoluble in water, and has poor solubility in general organic solvents, which greatly limits the medicinal effect of genistein as oral medicines and intravenous injection medicines, and limits the application of genistein in the field of medicine. Therefore, how to improve the solubility of genistein in aqueous solution is the focus of attention at home and abroad.
At present, studies show that the solubility of diglucose-based genistein and triglucose-based genistein in water is 3700 times and 44000 times that of genistein (see specifically references: Li D, Roh SA, Shim JH, Mikami B, Baik MY, Park CS, Park KH.2005.glycosylation of genistein in soluble formation of cyclic carbohydrates by biological modification. JAgric Food Chem53: 6516-24). It has also been shown that glycosylation of genistein does not affect its physiological and biochemical functions (see in particular references: Chung MJ, KangAY, Lee KM, Oh E, Jun HJ, Kim SY, Auh JH, Moon TW, Lee SJ, Park KH.2006.Water-soluble genetic present in carbohydrate isoflavone ups up-relative important dietary approach expression and flavor free. Moreover, it has been found that glycosylated genistein is hydrolyzed in vivo into glucose and genistein, which can be absorbed by human body, and is highly safe (see references: Chung MJ, KangAY, Lee KM, Oh E, Jun HJ, Kim SY, Auh JH, Moon TW, Lee SJ, Park KH.2006.Water-soluble genistein blood sugar up-regulated antioxidant expression syndrome 54: 3819-26). Thus, the water solubility of genistein can be increased by glycosylating it.
Cyclodextrin glucosyltransferase (CGTase or CGT enzyme for short, EC2.4.1.19) is a common enzyme that catalyzes glycosylation reactions and can be used to glycosylate lignin. However, the existing cyclodextrin glucosyltransferase has low efficiency of synthesizing long-chain glycosyl genistein and high efficiency of synthesizing short-chain glycosyl genistein, and studies show that the longer the sugar chain connected with the glycosylated genistein, the better the water solubility (see the references: Li D, Roh SA, Shim JH, Mikami B, Baik MY, Park CS, Park KH.2005.glycosylation of the genetic in soluble use complex of cyclic carbohydrates by amino modification. J agricultural Food Chem53: 6516-24). Therefore, it is urgently needed to find a cyclodextrin glucosyltransferase with high specificity to a long-chain glycosylated genistein product so as to realize large-scale industrial production of the long-chain glycosylated genistein early.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing Cyclodextrin glucosyltransferase (CGTase or CGT enzyme for short, EC2.4.1.19) with high specificity to long-chain glycosylated genistein products.
[ solution ]
In order to solve the technical problems, the invention provides a cyclodextrin glucosyltransferase mutant, which is obtained by mutating 156 th alanine and 174 th leucine of cyclodextrin glucosyltransferase with the starting amino acid sequence shown as SEQ ID NO. 1;
or the mutant of the cyclodextrin glucosyltransferase is obtained by mutating the 156 th alanine, 174 th leucine and 166 th alanine of the cyclodextrin glucosyltransferase with the starting amino acid sequence shown as SEQ ID NO. 1.
In one embodiment of the invention, the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine and leucine 174 to proline of cyclodextrin glucosyltransferase with the starting amino acid sequence shown in SEQ ID NO.1, and is named as A156V/L174P;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to tyrosine of cyclodextrin glucosyltransferase with an original amino acid sequence shown in SEQ ID NO.1 and is named as A156V/L174P/A166Y;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to valine of cyclodextrin glucosyltransferase with an original amino acid sequence shown in SEQ ID NO.1, and is named as A156V/L174P/A166V;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to glycine of cyclodextrin glucosyltransferase with an original amino acid sequence shown in SEQ ID NO.1 and is named as A156V/L174P/A166G;
or the mutant of the cyclodextrin glucosyltransferase is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to lysine of the cyclodextrin glucosyltransferase with the starting amino acid sequence shown as SEQ ID NO.1 and is named as A156V/L174P/A166K.
In one embodiment of the invention, the nucleotide sequence encoding the cyclodextrin glucosyltransferase is set forth in SEQ ID No. 2.
The invention also provides a gene for coding the cyclodextrin glucosyltransferase mutant.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment of the present invention, the vector of the recombinant plasmid is pET-20b (+) plasmid, pET-22b (+) plasmid or pET-28a (+) plasmid.
In one embodiment of the present invention, the vector of the recombinant plasmid is pET-20b (+) plasmid.
The invention also provides a host cell carrying the gene or the recombinant plasmid.
In one embodiment of the invention, the host cell is a bacterium or a fungus.
In one embodiment of the invention, the host cell is E.coli.
The invention also provides a preparation method of the cyclodextrin glucosyltransferase mutant, which comprises the steps of inoculating the host cell into a fermentation culture medium for fermentation to obtain fermentation liquor; centrifuging the fermentation liquor to obtain fermentation supernatant; separating the fermentation supernatant to obtain the cyclodextrin glucosyltransferase mutant.
The invention also provides a method for producing long-chain glycosylation genistein, which comprises the steps of taking maltodextrin as a glycosyl donor, taking genistein as a glycosyl acceptor, and adding the cyclodextrin glucosyltransferase mutant into a reaction system containing maltodextrin and genistein for reaction to obtain a reaction solution; separating the reaction liquid to obtain the long-chain glycosylated genistein. The long-chain glycosylated genistein refers to tetra-glycosylated genistein, penta-glycosylated genistein and/or hexa-glycosylated genistein.
The invention also provides the application of the cyclodextrin glucosyltransferase mutant or the gene or the recombinant plasmid or the host cell or the preparation method or the method for producing the long-chain glycosylated genistein in producing the long-chain glycosylated genistein.
[ advantageous effects ]
The cyclodextrin glucosyltransferase mutant has high product specificity on long-chain glycosylated genistein; the yield of the long-chain glycosylated genistein produced by using the cyclodextrin glucosyltransferase mutant A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G and A156V/L174P/A166K respectively takes maltodextrin as a glycosyl donor, and the yield of the long-chain glycosylated genistein produced by using the genistein as the glycosyl acceptor is respectively improved by 62.5%, 165%, 112.5% and 59.4% compared with the yield produced by using the wild-type cyclodextrin glucosyltransferase and taking the maltodextrin as the glycosyl donor.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Coli JM109 and E.coli BL21(DE3) referred to in the examples below were obtained from North Nay organisms and pET-20b (+) plasmid was obtained from Novagen. (the above-mentioned strain E.coli BL21(DE3) is commercially available and does not require preservation for patent procedures)
The media involved in the following examples are as follows:
LB liquid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1NaCl 10.0. mu.g.L-1, ampicillin 100. mu.g.L-1。
LB solid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-115 g.L agar powder-1Ampicillin 100. mu.g.L-1。
The detection methods referred to in the following examples are as follows:
the enzyme activity determination method of cyclodextrin glucosyltransferase comprises the following steps: 0.1mL of the enzyme solution was added to a solution containing 0.9mL of a solution prepared in advance with 50mM phosphate buffer (pH 6.5) and having a concentration of 30 g.L-1After reacting at 40 ℃ for 10 minutes, 1.0mL of 1.0M hydrochloric acid was added to stop the reaction, 1.0mL of 0.1mM methyl orange prepared with 50mM phosphate buffer was added, the mixture was incubated at 16 ℃ for 20 minutes, and the absorbance was measured at 505 nm.
Definition of cyclodextrin glucosyltransferase enzyme activity: under the condition, the enzyme amount required for generating 1 mu mol of alpha-cyclodextrin per minute is one enzyme activity unit.
Example 1: preparation and expression of different cyclodextrin glucosyltransferases
The method comprises the following specific steps:
chemically synthesizing a gene (the nucleotide sequence of the gene is shown as SEQ ID NO. 2) of cyclodextrin glucosyltransferase with the amino acid sequence shown as SEQ ID NO. 1; connecting the obtained gene with pET-20b (+) plasmid after double enzyme digestion (Nco I and Xho I), transforming Escherichia coli JM109, coating the transformed product on LB solid culture medium, culturing at 37 ℃ for 8h, selecting a transformant on the LB solid culture medium, inoculating into LB liquid culture medium for culturing, culturing at 37 ℃ for 10h, extracting the plasmid, performing sequence determination on the plasmid, and obtaining a recombinant plasmid pET20b-CGT with correct sequencing; and (3) transforming the recombinant plasmid pET20b-CG with correct sequencing into Escherichia coli E.coli BL21(DE3) to obtain the recombinant Escherichia coli pET20b-CGT/E.coli BL 21.
Carrying out site-directed mutagenesis by using the obtained recombinant plasmid pET20b-CGT as a template by using a whole plasmid PCR technology to obtain a mutant A156V/L174P (the amino acid sequence is shown as SEQ ID NO. 3), A156V/L174P/A166Y (the amino acid sequence is shown as SEQ ID NO. 4), A156V/L174P/A166V (the amino acid sequence is shown as SEQ ID NO. 5), A156V/L174P/A166G (the amino acid sequence is shown as SEQ ID NO. 6), A156V/L174P/A166K (the amino acid sequence is shown as SEQ ID NO. 7), A156S, A156L and L174M;
the primers used for the mutation A156V are as follows:
a forward primer: 5' -GCAGAAAATGGTGTTCTGTAT-3’(SEQ ID No.8)；
Reverse primer: 5' -GTTATCATACAGAACACCATT-3’(SEQ ID No.9)；
The primers used for mutation L174P were as follows:
a forward primer: 5' -GACACCGCTGGCCCGTTCCAT-3'(SEQ ID No.10)；
Reverse primer: 5' -GTTGTGATGGAACGGGCCAGC-3'(SEQ ID No.11)；
The primers used for mutation a166Y were as follows:
a forward primer: 5' -TCACTGCTGGGTTACTACTCGAAT-3'(SEQ ID No.12)；
Reverse guideAn object: 5' -GTCATTCGAGTAGTAACCCAGCAG-3'(SEQ ID No.13)；
The primers used for mutation a166V were as follows:
a forward primer: 5' -TCACTGCTGGGTGTTTACTCGAAT-3'(SEQ ID No.14)；
Reverse primer: 5' -GTCATTCGAGTAAACACCCAGCAG-3'(SEQ ID No.15)；
The primers used for mutation a166G were as follows:
a forward primer: 5' -TCACTGCTGGGTGGTTACTCGAAT-3'(SEQ ID No.16)；
Reverse primer: 5' -GTCATTCGAGTAACCACCCAGCAG-3'(SEQ ID No.17)；
The primers used for mutation a166K were as follows:
a forward primer: 5' -TCACTGCTGGGTAAATACTCGAAT-3'(SEQ ID No.18)；
Reverse primer: 5' -GTCATTCGAGTATTTACCCAGCAG-3'(SEQ ID No.19)；
The primers used for mutation a156S were as follows:
a forward primer: 5' -GCAGAAAATGGTTCTCTGTAT-3'(SEQ ID No.20)；
Reverse primer: 5' -GTTATCATACAGAGAACCATT-3'(SEQ ID No.21)；
The primers used for mutation a156L were as follows:
a forward primer: 5' -GCAGAAAATGGTCTGCTGTAT-3'(SEQ ID No.22)；
Reverse primer: 5' -GTTATCATACAGCAGACCATTG-3'(SEQ ID No.23)；
The primers used for mutation L174M were as follows:
a forward primer: 5' -GACACCGCTGGCATGTTCCAT-3'(SEQ ID No.24)；
Reverse primer: 5' -GTTGTGATGGAACATGCCAGC-3'(SEQ ID No.25)，
The PCR reaction systems are as follows: 5 × PrimeSTAR Buffer (Mg)2+Plus) 5. mu.L, 4. mu.L of 2.5mM dNTPs, 1. mu.L of 10. mu.M forward primer, 1. mu.L of 10. mu.M reverse primer, 1. mu.L of template DNA, 0.5. mu.L of 2.5U/. mu.LPrimeSTAR Taq HS, and double distilled water was added to 50. mu.L;
the PCR product amplification conditions were all: pre-denaturation at 98 ℃ for 3 min; then carrying out 30 cycles of 10s at 98 ℃, 15s at 57 ℃ and 6min at 72 ℃; finally, keeping the temperature at 72 ℃ for 10 min.
Detecting the PCR amplification product by using 1% agarose gel electrophoresis, after the detection is finished, adding 0.5 mu L of methylated template digestive enzyme (Dpn I) into 10 mu L of the amplification product, blowing and sucking a gun head for uniformly mixing, reacting for 1.5h at 37 ℃, converting the amplification product treated by the Dpn I into escherichia coli JM109, coating the conversion product on an LB solid culture medium, culturing for 8h at 37 ℃, picking up a transformant on the LB solid culture medium, inoculating the transformant into an LB liquid culture medium for culturing, extracting a plasmid after culturing for 10h at 37 ℃, and carrying out sequence determination on the plasmid to obtain a recombinant plasmid which correctly sequences and contains genes encoding mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L P/A166K, A S, A39156 and L M; coli BL21(DE3) was transformed with the recombinant plasmid with the correct sequencing to obtain recombinant E.coli containing the genes encoding mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M.
Coating the obtained recombinant Escherichia coli pET20b-CGT/E.coli BL21 and recombinant Escherichia coli LB of genes encoding mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M on a solid culture medium, and culturing at 37 ℃ for 8-10 h to obtain a single colony; selecting a single colony, inoculating the single colony into an LB liquid culture medium, and culturing at 37 ℃ for 12-14 h to obtain a seed solution; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 4% (v/v), culturing at 30 deg.C and 120rpm to OD600After the concentration is 0.6, adding IPTG with the final concentration of 0.01mM into the fermentation liquor, and continuously carrying out induction culture for 90 hours at 25 ℃ and 120rpm to obtain the fermentation liquor; centrifuging the fermentation liquor at 4 deg.C and 1000rpm for 20min, and collecting the fermentation supernatant; adding 70% solid ammonium sulfate into the fermentation supernatant, salting out overnight, centrifuging at 4 deg.C and 10000rpm for 20min, dissolving the precipitate with appropriate amount of buffer solution A containing 20mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole and pH 7.4, dialyzing in buffer solution A overnight, and filtering with 0.22 μ M membrane to obtain sample; after the Ni affinity column is equilibrated with buffer solution A, the sample is loadedSucking Ni column, adsorbing completely, eluting with buffer solution A, buffer solution A containing 20-480 mM imidazole and buffer solution A containing 480mM imidazole respectively at flow rate of 1mL/min and detection wavelength of 280nm, and collecting eluate containing cyclodextrin glucosyltransferase activity; after the active components were dialyzed overnight in 50mM sodium phosphate buffer (pH 6), pure enzymes of mutants a156V/L174P, a156V/L174P/a166Y, a156V/L174P/a166V, a156V/L174P/a166G, a156V/L174P/a166K, a156S, a156L, and L174M were obtained, respectively, and lyophilized for use.
Example 2: product specificity of different Cyclodextrin glucosyltransferases for Long-chain glycosylated Genistein
The method comprises the following specific steps:
dissolving genistein (purchased from Sigma) in dimethyl sulfoxide (DMSO) to prepare a genistein solution with a final concentration of 7.5 g/L; dissolving maltodextrin (purchased from Shanghai Biotech engineering Co., Ltd.) in PBS buffer (50mM, pH 6.5) to obtain maltodextrin solution with final concentration of 40 g/L; pure enzymes of the freeze-dried mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M obtained in example 1 are dissolved in PBS buffer (50mM, pH 6.5) to prepare CGTase enzyme solution with the final concentration of 15U/L; respectively taking 300 mu L of genistein solution, 500 mu L of maltodextrin solution and 200 mu L of CGTase enzyme solution, mixing the obtained mixture in a 2mL small tube with a cover, and slowly oscillating the mixture in a shaker at 40 ℃ and 120rpm for 20-24 hours to obtain reaction liquid.
Detecting the molar contents of short-chain glycosylated genistein (here, the short-chain glycosylated genistein is a mixture of mono-glycosylated genistein, di-glycosylated genistein and tri-glycosylated genistein) and long-chain glycosylated genistein (here, the long-chain glycosylated genistein is a mixture of tetra-glycosylated genistein, penta-glycosylated genistein and hexa-glycosylated genistein) in the reaction solution by HPLC, and calculating the proportion (%) of the molar contents of the short-chain glycosylated genistein and the long-chain glycosylated genistein in the reaction solution to the molar content of the total glycosylated genistein in the reaction solution and the contents (g/L) of the short-chain glycosylated genistein and the long-chain glycosylated genistein in the reaction solution, wherein the detection results are shown in tables 1-2; wherein, the method for detecting the proportion (%) of the content of the short-chain glycosylated genistein and the long-chain glycosylated genistein in the reaction solution in the total content of the glycosylated genistein in the reaction solution by HPLC comprises the following steps: the reaction solution was filtered through a 0.22 μm filter and detected using an Ethylst C18-H column (4.6X 250mm, Sepax, America) (specific detection conditions are shown in Table 3); wherein the content of the long-chain glycosylated genistein is equal to the molar content of the hexa-glycosylated genistein x the molecular weight of the hexa-glycosylated genistein + the molar content of the penta-glycosylated genistein x the molecular weight of the penta-glycosylated genistein + the molar content of the tetrasaccharide-genistein x the molecular weight of the tetrasaccharide-genistein, and the content of the short-chain glycosylated genistein is equal to the molar content of the trisaccharide-genistein x the molecular weight of the trisaccharide-genistein + the molar content of the diglycosylated genistein x the molecular weight of the diglycosylated genistein + the molar content of the monoglycosylated genistein x the molecular weight of the monoglycosylated genistein.
As can be seen from tables 1-2, only mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G and A156V/L174P/A166K have obviously improved product specificity on long-chain glycosylated genistein compared with the wild type;
wherein, the yield of the long-chain glycosylated genistein produced by using the mutant A156V/L174P and the genistein as the glycosyl donor is improved by 62.5 percent compared with the yield of the long-chain glycosylated genistein produced by using the wild-type cyclodextrin glucosyltransferase and the genistein as the glycosyl acceptor, wherein the maltodextrin is used as the glycosyl donor;
the mutant A156V/L174P/A166Y takes maltodextrin as glycosyl donor, and the yield of the long-chain glycosylated genistein produced by taking genistein as glycosyl acceptor is improved by 165 percent compared with the yield of the long-chain glycosylated genistein produced by taking wild-type cyclodextrin glucosyltransferase and maltodextrin as glycosyl donor and genistein as glycosyl acceptor;
the mutant A156V/L174P/A166V takes maltodextrin as glycosyl donor, and the yield of the long-chain glycosylated genistein produced by taking genistein as glycosyl acceptor is improved by 112.5 percent compared with the yield of the long-chain glycosylated genistein produced by taking wild-type cyclodextrin glucosyltransferase and maltodextrin as glycosyl donor and genistein as glycosyl acceptor;
the mutant A156V/L174P/A166G takes maltodextrin as glycosyl donor, and the yield of the long-chain glycosylated genistein produced by taking genistein as glycosyl acceptor is improved by 37.5 percent compared with the yield of the long-chain glycosylated genistein produced by taking wild-type cyclodextrin glucosyltransferase and maltodextrin as glycosyl donor and genistein as glycosyl acceptor;
the mutant A156V/L174P/A166K takes maltodextrin as glycosyl donor, and the yield of the long-chain glycosylated genistein produced by taking genistein as glycosyl acceptor is increased by 59.4 percent compared with the yield of the long-chain glycosylated genistein produced by taking wild-type cyclodextrin glucosyltransferase and maltodextrin as glycosyl donor and genistein as glycosyl acceptor.
TABLE 1 molar ratio (%)% of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution obtained by the reaction with different cyclodextrin glycosyltransferases to the total glycosylated genistein in the reaction solution
| Group of | Long chain radical genistein | Short chain glycosylated genistein |
| Wild type | 15 | 85 |
| A156S | 7 | 93 |
| A156L | 5 | 95 |
| L174M | 6 | 94 |
| A156V/L174P | 25 | 75 |
| A156V/L174P/A166V | 29 | 71 |
| A156V/L174P/A166G | 20 | 80 |
| A156V/L174P/A166K | 24 | 76 |
| A156V/L174P/A166Y | 40 | 60 |
TABLE 2 content (g/L) of short-chain glycosylated genistein and long-chain glycosylated genistein in reaction solution obtained by reaction of different cyclodextrin glucosyltransferases
TABLE 3 conditions for HPLC determination of the content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> cyclodextrin glucosyltransferase mutant and application thereof
<160> 25
<170> PatentIn version 3.3
<210> 1
<211> 687
<212> PRT
<213> Artificial sequence
<400> 1
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Ala Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Ala Tyr Ser Asn Asp Thr Ala Gly Leu Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 2
<211> 2061
<212> DNA
<213> Artificial sequence
<400> 2
tcaccggaca cctcagtgga caataaagtt aacttcagca ccgatgttat ctaccagatc 60
gtcacggacc gttttgcgga tggtgaccgc accaacaatc cggcaggcga tgctttcagc 120
ggtgaccgtt ctaatctgaa actgtatttt ggcggtgatt ggcagggcat tatcgataaa 180
attaacgacg gttacctgac cggcatgggt gtgacggcgc tgtggatcag ccaaccggtg 240
gaaaacatca cctcagttat caaatactcg ggcgtcaaca atacgtctta tcatggttac 300
tgggcccgtg attttaaaca gaccaacgac gcgtttggcg atttcgccga ctttcaaaat 360
ctgattgata ccgcacatgc tcacaacatt aaagtggtta tcgatttcgc cccgaaccac 420
acctctccgg cagatcgcga caatccgggc tttgcagaaa atggtgctct gtatgataac 480
ggctcactgc tgggtgcata ctcgaatgac accgctggcc tgttccatca caacggcggt 540
acggatttta gtaccattga agacggtatc tataaaaatc tgtacgatct ggctgacatc 600
aaccataaca ataacgcgat ggatgcctat ttcaaatcag caattgacct gtggctgggc 660
atgggtgttg atggcatccg ctttgacgcg gtcaaacaca tgccgttcgg ttggcagaaa 720
tcgtttgtga gcagcattta tggcggtgat cacccggttt ttaccttcgg cgaatggtat 780
ctgggtgctg atcagacgga tggcgacaat atcaaatttg cgaacgaatc tggtatgaat 840
ctgctggatt ttgaatatgc acaagaagtc cgtgaagtgt ttcgcgataa aacggaaacc 900
atgaaagacc tgtacgaagt gctggcctca accgaatcgc agtatgatta cattaataac 960
atggtgacct tcatcgacaa tcacgatatg gaccgttttc aggttgcggg ctcaggtacg 1020
cgcgccaccg aacaagcgct ggcactgacg ctgacctcgc gtggcgttcc ggcgatttat 1080
tacggcaccg aacagtatat gacgggcgat ggtgacccga ataaccgcgc catgatgacg 1140
agtttcaata ccggcaccac ggcatataaa gtgattcaag cactggctcc gctgcgtaaa 1200
tccaacccgg caatcgccta cggcaccacc accgaacgtt gggtgaataa cgatgttctg 1260
attatcgaac gcaaatttgg tagttccgcg gccctggtcg ccattaatcg caactcatcg 1320
gcagcttatc cgatcagtgg tctgctgagc agcctgccag cgggcaccta ctccgatgtg 1380
ctgaatggcc tgctgaatgg taacagcatt accgtgggct ctggcggtgc ggttacgaac 1440
tttaccctgg cagcgggcgg caccgcagtt tggcagtata cggctccgga aaccagcccg 1500
gcgatcggta atgtcggtcc gacgatgggc caaccgggta acattgtgac gatcgatggt 1560
cgtggtttcg gcggtacggc tggcaccgtg tactttggta cgaccgcggt caccggcagt 1620
ggtattgtgt cctgggaaga tacgcagatt aaagcggtca tcccgaaagt ggcagctggc 1680
aaaaccggtg tcagcgtgaa aacgagttcc ggcaccgcca gtaatacgtt caaatccttt 1740
aacgttctga ccggtgatca ggttacggtc cgctttctgg tcaaccaagc gaataccaac 1800
tatggcacga atgtttacct ggtcggcaac gcggccgaac tgggttcctg ggacccgaat 1860
aaagccattg gtccgatgta taaccaggtt atcgcaaaat acccgagctg gtattacgat 1920
gtgagcgttc cggcgggcac caaactggac ttcaaattca ttaaaaaagg cggtggcacg 1980
gtgacctggg aaggtggcgg taaccatacc tacacgaccc cggcgagcgg cgttggcacg 2040
gtgacggtgg attggcaaaa t 2061
<210> 3
<211> 687
<212> PRT
<213> Artificial sequence
<400> 3
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Val Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Ala Tyr Ser Asn Asp Thr Ala Gly Pro Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 4
<211> 687
<212> PRT
<213> Artificial sequence
<400> 4
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Val Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Tyr Tyr Ser Asn Asp Thr Ala Gly Pro Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 5
<211> 687
<212> PRT
<213> Artificial sequence
<400> 5
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Val Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Val Tyr Ser Asn Asp Thr Ala Gly Pro Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 6
<211> 687
<212> PRT
<213> Artificial sequence
<400> 6
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Val Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Gly Tyr Ser Asn Asp Thr Ala Gly Pro Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 7
<211> 687
<212> PRT
<213> Artificial sequence
<400> 7
Ser Pro Asp Thr Ser Val Asp Asn Lys Val Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Val Thr Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn
20 25 30
Asn Pro Ala Gly Asp Ala Phe Ser Gly Asp Arg Ser Asn Leu Lys Leu
35 40 45
Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Leu Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Thr Ser Val Ile Lys Tyr Ser Gly Val Asn Asn Thr Ser
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Gln Thr Asn Asp Ala Phe
100 105 110
Gly Asp Phe Ala Asp Phe Gln Asn Leu Ile Asp Thr Ala His Ala His
115 120 125
Asn Ile Lys Val Val Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Asp Arg Asp Asn Pro Gly Phe Ala Glu Asn Gly Val Leu Tyr Asp Asn
145 150 155 160
Gly Ser Leu Leu Gly Lys Tyr Ser Asn Asp Thr Ala Gly Pro Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Ile Glu Asp Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Ile Asn His Asn Asn Asn Ala Met Asp
195 200 205
Ala Tyr Phe Lys Ser Ala Ile Asp Leu Trp Leu Gly Met Gly Val Asp
210 215 220
Gly Ile Arg Phe Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Val Ser Ser Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe
245 250 255
Gly Glu Trp Tyr Leu Gly Ala Asp Gln Thr Asp Gly Asp Asn Ile Lys
260 265 270
Phe Ala Asn Glu Ser Gly Met Asn Leu Leu Asp Phe Glu Tyr Ala Gln
275 280 285
Glu Val Arg Glu Val Phe Arg Asp Lys Thr Glu Thr Met Lys Asp Leu
290 295 300
Tyr Glu Val Leu Ala Ser Thr Glu Ser Gln Tyr Asp Tyr Ile Asn Asn
305 310 315 320
Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Gln Val Ala
325 330 335
Gly Ser Gly Thr Arg Ala Thr Glu Gln Ala Leu Ala Leu Thr Leu Thr
340 345 350
Ser Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr
355 360 365
Gly Asp Gly Asp Pro Asn Asn Arg Ala Met Met Thr Ser Phe Asn Thr
370 375 380
Gly Thr Thr Ala Tyr Lys Val Ile Gln Ala Leu Ala Pro Leu Arg Lys
385 390 395 400
Ser Asn Pro Ala Ile Ala Tyr Gly Thr Thr Thr Glu Arg Trp Val Asn
405 410 415
Asn Asp Val Leu Ile Ile Glu Arg Lys Phe Gly Ser Ser Ala Ala Leu
420 425 430
Val Ala Ile Asn Arg Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Leu
435 440 445
Leu Ser Ser Leu Pro Ala Gly Thr Tyr Ser Asp Val Leu Asn Gly Leu
450 455 460
Leu Asn Gly Asn Ser Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn
465 470 475 480
Phe Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro
485 490 495
Glu Thr Ser Pro Ala Ile Gly Asn Val Gly Pro Thr Met Gly Gln Pro
500 505 510
Gly Asn Ile Val Thr Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly
515 520 525
Thr Val Tyr Phe Gly Thr Thr Ala Val Thr Gly Ser Gly Ile Val Ser
530 535 540
Trp Glu Asp Thr Gln Ile Lys Ala Val Ile Pro Lys Val Ala Ala Gly
545 550 555 560
Lys Thr Gly Val Ser Val Lys Thr Ser Ser Gly Thr Ala Ser Asn Thr
565 570 575
Phe Lys Ser Phe Asn Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe
580 585 590
Leu Val Asn Gln Ala Asn Thr Asn Tyr Gly Thr Asn Val Tyr Leu Val
595 600 605
Gly Asn Ala Ala Glu Leu Gly Ser Trp Asp Pro Asn Lys Ala Ile Gly
610 615 620
Pro Met Tyr Asn Gln Val Ile Ala Lys Tyr Pro Ser Trp Tyr Tyr Asp
625 630 635 640
Val Ser Val Pro Ala Gly Thr Lys Leu Asp Phe Lys Phe Ile Lys Lys
645 650 655
Gly Gly Gly Thr Val Thr Trp Glu Gly Gly Gly Asn His Thr Tyr Thr
660 665 670
Thr Pro Ala Ser Gly Val Gly Thr Val Thr Val Asp Trp Gln Asn
675 680 685
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence
<400> 8
gcagaaaatg gtgttctgta t 21
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence
<400> 9
gttatcatac agaacaccat t 21
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence
<400> 10
gacaccgctg gcccgttcca t 21
<210> 11
<211> 21
<212> DNA
<213> Artificial sequence
<400> 11
gttgtgatgg aacgggccag c 21
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence
<400> 12
tcactgctgg gttactactc gaat 24
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence
<400> 13
gtcattcgag tagtaaccca gcag 24
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence
<400> 14
tcactgctgg gtgtttactc gaat 24
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence
<400> 15
gtcattcgag taaacaccca gcag 24
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence
<400> 16
tcactgctgg gtggttactc gaat 24
<210> 17
<211> 24
<212> DNA
<213> Artificial sequence
<400> 17
gtcattcgag taaccaccca gcag 24
<210> 18
<211> 24
<212> DNA
<213> Artificial sequence
<400> 18
tcactgctgg gtaaatactc gaat 24
<210> 19
<211> 24
<212> DNA
<213> Artificial sequence
<400> 19
gtcattcgag tatttaccca gcag 24
<210> 20
<211> 21
<212> DNA
<213> Artificial sequence
<400> 20
gcagaaaatg gttctctgta t 21
<210> 21
<211> 21
<212> DNA
<213> Artificial sequence
<400> 21
gttatcatac agagaaccat t 21
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence
<400> 22
gcagaaaatg gtctgctgta t 21
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence
<400> 23
gttatcatac agcagaccat tg 22
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence
<400> 24
gacaccgctg gcatgttcca t 21
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence
<400> 25
gttgtgatgg aacatgccag c 21
Claims (9)
1. A cyclodextrin glucosyltransferase mutant is characterized in that the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine and leucine 174 to proline of cyclodextrin glucosyltransferase with an original amino acid sequence shown as SEQ ID NO. 1;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to tyrosine of cyclodextrin glucosyltransferase with the starting amino acid sequence shown in SEQ ID No. 1;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to valine of cyclodextrin glucosyltransferase with the starting amino acid sequence shown in SEQ ID No. 1;
or the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to glycine of cyclodextrin glucosyltransferase with the starting amino acid sequence shown in SEQ ID No. 1;
or the mutant of the cyclodextrin glucosyltransferase is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to lysine of the cyclodextrin glucosyltransferase with the starting amino acid sequence shown as SEQ ID No. 1.
2. A gene encoding the cyclodextrin glucosyltransferase mutant of claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. The recombinant plasmid of claim 3, wherein the vector of the recombinant plasmid is a pET-20b (+) plasmid, a pET-22b (+) plasmid, or a pET-28a (+) plasmid.
5. A host cell carrying the gene of claim 4 or the recombinant plasmid of claim 3 or 4.
6. The host cell of claim 5, wherein the host cell is a bacterium or a fungus.
7. The method for producing a cyclodextrin glucosyltransferase mutant according to claim 1, which comprises inoculating the host cell according to claim 5 or 6 into a fermentation medium, and fermenting the inoculated host cell to obtain a fermentation broth; centrifuging the fermentation liquor to obtain fermentation supernatant; separating the fermentation supernatant to obtain the cyclodextrin glucosyltransferase mutant of claim 1.
8. A method for producing long-chain glycosylated genistein, which comprises using maltodextrin as glycosyl donor and genistein as glycosyl acceptor, and adding the cyclodextrin glucosyltransferase mutant of claim 1 into a reaction system containing maltodextrin and genistein to perform reaction to obtain a reaction solution; separating the reaction liquid to obtain the long-chain glycosylated genistein.
9. Use of the cyclodextrin glucosyltransferase mutant according to claim 1 or the gene according to claim 2 or the recombinant plasmid according to claim 3 or 4 or the host cell according to claim 5 or 6 or the method according to claim 7 or the method according to claim 8 for producing long-chain glycosylated genistein.
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| PCT/CN2020/091254 WO2021088352A1 (en) | 2019-11-08 | 2020-05-20 | Method for producing long-chain glycosylated genistein |
| US17/123,226 US11203743B2 (en) | 2019-11-08 | 2020-12-16 | Method for producing long-chain glycosylated genistein |
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| US11203743B2 (en) | 2019-11-08 | 2021-12-21 | Jiangnan University | Method for producing long-chain glycosylated genistein |
| WO2021088352A1 (en) * | 2019-11-08 | 2021-05-14 | 江南大学 | Method for producing long-chain glycosylated genistein |
| CN111394328B (en) * | 2020-04-16 | 2022-02-15 | 江南大学 | Cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof |
| CN112410315B (en) * | 2020-11-30 | 2022-04-15 | 江南大学 | Method for producing long-chain glycosylated sophoricoside |
| CN113817704B (en) * | 2021-10-22 | 2023-07-18 | 江南大学 | Cyclodextrin glucosyltransferase with improved organic solvent tolerance and preparation method thereof |
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| CN108486080A (en) * | 2018-04-04 | 2018-09-04 | 江南大学 | A kind of cyclodextrin glycosyltransferase and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486080A (en) * | 2018-04-04 | 2018-09-04 | 江南大学 | A kind of cyclodextrin glycosyltransferase and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity;Han RZ等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20200131;第86卷(第7期);第e00004-20号 * |
| Position 228 in Paenibacillus macerans cyclodextrin cyclodextrin glycosyltransferase is critical for 2-O-d-glucopyranosyl-l-ascorbicacid synthesis;Chen S等;《JOURNAL OF BIOTECHNOLOGY》;20170410;第247卷;第18-24页 * |
| 登录号：JX412224.1;Liu H等;《GenBank》;20121129;第1-2064位 * |
| 登录号：P04830;Wu L等;《UniProtKB》;20140226;第1-714位 * |
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