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WO2021088352A1 - 一种生产长链糖基化染料木素的方法 - Google Patents

一种生产长链糖基化染料木素的方法 Download PDF

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WO2021088352A1
WO2021088352A1 PCT/CN2020/091254 CN2020091254W WO2021088352A1 WO 2021088352 A1 WO2021088352 A1 WO 2021088352A1 CN 2020091254 W CN2020091254 W CN 2020091254W WO 2021088352 A1 WO2021088352 A1 WO 2021088352A1
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genistein
seq
cyclodextrin glucosyltransferase
alanine
mutant
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PCT/CN2020/091254
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French (fr)
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韩瑞枝
倪晔
柴宝成
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江南大学
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Priority claimed from CN201911084700.2A external-priority patent/CN110804597B/zh
Priority claimed from CN201911084769.5A external-priority patent/CN110734946B/zh
Application filed by 江南大学 filed Critical 江南大学
Priority to US17/123,226 priority Critical patent/US11203743B2/en
Publication of WO2021088352A1 publication Critical patent/WO2021088352A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin

Definitions

  • the invention relates to a method for producing long-chain glycosylated genistein, which belongs to the technical fields of enzyme engineering and fermentation engineering.
  • Genistein also known as 5,7,4-trihydroxyisoflavone, genistein or genistein, is widely distributed in nature, mainly in soybeans, mung beans, alfalfa, oats, barley, black Wheat, wheat, corn and other cereal plants are non-estrogen compounds with weak estrogen-like effects, and have extremely high application prospects in the fields of medicine and health care.
  • genistein products are mainly focused on the prevention and treatment of cardiovascular disease, female menopausal syndrome, breast hyperplasia, breast cancer, prostate cancer, etc., as a preventive agent for tumor cells, it has the characteristics of not killing normal cells, so It is highly valued by the medical circles of various countries.
  • due to the strong hydrophobicity of genistein its bioavailability is low, and it is difficult to achieve the purpose of clinical treatment of diseases, which greatly limits its application in the fields of medicine and health care. Therefore, how to improve the solubility of genistein in aqueous solution has become the focus of attention at home and abroad.
  • glycosylation of genistein does not affect its physiological and biochemical functions (see references for details: Chung MJ, Kang AY, Lee KM, Oh E, Jun HJ, Kim SY, Auh JH, Moon TW, Lee SJ,Park KH.2006.Water-soluble genistin glycoside isoflavones up-regulate antioxidant metallothionein expression and scavenge free radicals.J Agric Food Chem 54:3819-26).
  • glycosylated genistein can be hydrolyzed in the body into glucose and genistein that can be absorbed by the human body, which is safer (see references: Chung MJ, Kang AY, Lee KM, Oh E ,Jun HJ,Kim SY,Auh JH,Moon TW,Lee SJ,Park KH.2006.Water-soluble genistin glycoside isoflavones up-regulate antioxidant metallothionein expression and scavenge free radicals.J Agric FoodChem 54:38. Therefore, genistein can be glycosylated to increase its water solubility.
  • Cyclodextrin glycosyltransferase (CGTase or CGTase for short, EC2.4.1.19) is a common enzyme that can catalyze glycosylation reactions. This enzyme can be used to glycosylate genistein. On this basis, studies have shown that the longer the sugar chain connected to glycosylated genistein, the better the water solubility (see references: Li D, Roh SA, Shim JH, Mikami B, Baik MY, Park CS, Park KH.2005.Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification.J Agric Food Chem 53:6516-24). Therefore, the production of large amounts of long-chain glycosylated genistein is essential to improve its application value.
  • the existing cyclodextrin glucosyltransferase has low synthesis efficiency of long-chain glycosyl genistein and high synthesis efficiency of short-chain glycosyl genistein, which greatly limits the use of cyclodextrin glucosyltransferase. Yield of synthetic long-chain glycosylated genistein. Therefore, it is urgent to find a method for producing long-chain glycosylated genistein with high yield.
  • the technical problem to be solved by the present invention is to provide a method for producing long-chain glycosylated genistein with high yield.
  • the present invention provides a cyclodextrin glucosyltransferase mutant, which is obtained by modifying the amino acid sequence of the cyclodextrin glucosyltransferase as shown in SEQ ID NO.1. Mutation of the 156th alanine and the 174th leucine of Glucosyltransferase;
  • the cyclodextrin glucosyltransferase mutant is obtained by changing the starting amino acid sequence of the 156th alanine, the 174th leucine and the 174th leucine of the cyclodextrin glucosyltransferase shown in SEQ ID NO.1. Alanine at position 166 was mutated.
  • the cyclodextrin glucosyltransferase mutant is obtained by changing the starting amino acid sequence of the cyclodextrin glucosyltransferase shown in SEQ ID NO.1 to the 156th alanine of the cyclodextrin glucosyltransferase. Mutation to valine and mutation of leucine at position 174 to proline, named A156V/L174P;
  • the cyclodextrin glucosyltransferase mutant is made by mutating the 156th alanine of the cyclodextrin glucosyltransferase whose starting amino acid sequence is shown in SEQ ID NO.1 to valine, and the 174th alanine.
  • Leucine at position mutated to proline and alanine at position 166 to tyrosine named A156V/L174P/A166Y;
  • the cyclodextrin glucosyltransferase mutant is made by mutating the 156th alanine of the cyclodextrin glucosyltransferase whose starting amino acid sequence is shown in SEQ ID NO.1 to valine, and the 174th alanine. Leucine was mutated to proline and the 166th alanine was mutated to valine, named A156V/L174P/A166V;
  • the cyclodextrin glucosyltransferase mutant is made by mutating the 156th alanine of the cyclodextrin glucosyltransferase whose starting amino acid sequence is shown in SEQ ID NO.1 to valine, and the 174th alanine. Leucine was mutated to proline and the 166th alanine was mutated to glycine, named A156V/L174P/A166G;
  • the cyclodextrin glucosyltransferase mutant is made by mutating the 156th alanine of the cyclodextrin glucosyltransferase whose starting amino acid sequence is shown in SEQ ID NO.1 to valine, and the 174th alanine. Leucine was mutated to proline and the 166th alanine was mutated to lysine, named A156V/L174P/A166K.
  • the amino acid sequence of the cyclodextrin glucosyltransferase mutant is SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO.7.
  • nucleotide sequence encoding the cyclodextrin glucosyltransferase is shown in SEQ ID No.2.
  • the present invention also provides a gene encoding the aforementioned cyclodextrin glucosyltransferase mutant.
  • the invention also provides a recombinant plasmid carrying the above-mentioned gene.
  • the vector of the recombinant plasmid is pET-20b(+) plasmid, pET-22b(+) plasmid or pET-28a(+) plasmid.
  • the vector of the recombinant plasmid is pET-20b(+) plasmid.
  • the present invention also provides a host cell carrying the above-mentioned gene or the above-mentioned recombinant plasmid.
  • the host cell is bacteria or fungi.
  • the host cell is Escherichia coli.
  • the present invention also provides a method for preparing the above-mentioned cyclodextrin glucosyltransferase mutant.
  • the method includes inoculating the above-mentioned host cell into a fermentation medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to obtain a fermentation supernatant The fermentation supernatant is separated to obtain the above-mentioned cyclodextrin glucosyltransferase mutant.
  • the present invention also provides a method for producing long-chain glycosylated genistein.
  • the method is to contain maltodextrin, genistein and cyclodextrin glycosyltransferase (CGTase or CGTase for short).
  • the reaction system of EC 2.4.1.19) is reacted at a pH of 4-8, a temperature of 30-60°C, and a rotation speed of 120-180rpm to obtain a reaction solution; the reaction solution is separated to obtain a long-chain glycosylated dye Lignin;
  • the cyclodextrin glucosyltransferase is an amino acid sequence such as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.
  • the long-chain glycosylated genistein refers to tetra-glycosylated genistein, penta-glycosylated genistein, and/or hexa-glycosylated genistein.
  • the method includes dissolving genistein in dimethyl sulfoxide to prepare a genistein solution; dissolving maltodextrin in buffer A to prepare a maltodextrin solution; Dissolve cyclodextrin glucosyltransferase in buffer B to prepare an enzyme solution; mix the genistein solution, maltodextrin solution and enzyme solution to obtain a reaction system; set the reaction system at a pH of 4-8 and a temperature of 30 The reaction is carried out under the conditions of -60°C and a rotating speed of 120-180 rpm to obtain a reaction liquid; the reaction liquid is separated to obtain a long-chain glycosylated genistein.
  • the pH of the reaction is 4 or 8, and the temperature is 45-50°C.
  • the cyclodextrin glucosyltransferase is a cyclodextrin glucosyltransferase whose amino acid sequence is shown in SEQ ID NO.4.
  • the buffer A is PBS buffer, citrate buffer or sodium acetate buffer.
  • the buffer B is PBS buffer, citrate buffer or sodium acetate buffer.
  • the concentration of the buffer A is 25-75 mmol/L.
  • the concentration of the buffer B is 25-75 mmol/L.
  • the concentration of the genistein solution is 5-15 g/L.
  • the concentration of the maltodextrin solution is 20-60 g/L.
  • the concentration of the enzyme solution is 10-20 U/L.
  • the volume ratio of the genistein solution, maltodextrin solution and enzyme solution is 2 ⁇ 4:4 ⁇ 6:1 ⁇ 3.
  • the reaction time is 20-24h.
  • the present invention also provides the use of the above-mentioned cyclodextrin glucosyltransferase mutant or the above-mentioned gene or the above-mentioned recombinant plasmid or the above-mentioned host cell or the above-mentioned preparation method or the above-mentioned method in the production of long-chain glycosylated genistein.
  • the present invention provides cyclodextrin glucosyltransferase mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P with high specificity to the product of long-chain glycosylated genistein /A166G and A156V/L174P/A166K; using the cyclodextrin glucosyltransferase mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G and A156V/L174P/A166K of the present invention, respectively
  • the yield of long-chain glycosylated genistein produced with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase and maltodext
  • the present invention provides a high-yield method for producing long-chain glycosylated genistein.
  • Using this method to produce long-chain glycosylated genistein can improve the long-chain glycosylated genistein in the reaction solution.
  • the content and the ratio of the content of long-chain glycosylated genistein in the reaction solution to the total glycosylated genistein content in the reaction solution increasing the content of long-chain glycosylated genistein in the reaction solution to 10.3g/L ,
  • the ratio of the content of long-chain glycosylated genistein in the reaction liquid to the total content of glycosylated genistein in the reaction liquid is increased to 70%.
  • Figure 1 The effect of reaction temperature on the ratio of the molar content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution to the molar content of the total glycosylated genistein in the reaction solution.
  • Figure 2 The effect of reaction pH on the ratio of the molar content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution to the molar content of the total glycosylated genistein in the reaction solution.
  • E. coli JM109 and E. coli BL21 (DE3) involved in the following examples were purchased from Beina Biotech, and the pET-20b(+) plasmid was purchased from Novagen. (The above-mentioned strains of Escherichia coli BL21 (DE3) can be purchased, and no preservation for patent procedures is required)
  • LB liquid medium yeast powder 5.0g ⁇ L -1 , tryptone 10.0g ⁇ L -1 , NaCl 10.0g ⁇ L-1, ampicillin 100 ⁇ g ⁇ L -1 .
  • LB solid medium yeast powder 5.0g ⁇ L -1 , tryptone 10.0g ⁇ L -1 , NaCl 10.0g ⁇ L -1 , agar powder 15g ⁇ L -1 , ampicillin 100 ⁇ g ⁇ L -1 .
  • Cyclodextrin glucosyltransferase enzyme activity determination method take 0.1mL enzyme solution, add 0.9mL pre-prepared with 50mM phosphate buffer (pH 6.5) concentration of 30g ⁇ L -1 soluble starch solution, in 40 After reacting at °C for 10 minutes, add 1.0mL 1.0M hydrochloric acid to stop the reaction, then add 1.0mL of 0.1mM methyl orange prepared with 50mM phosphate buffer, incubate at 16°C for 20min, and measure the absorbance at 505nm.
  • cyclodextrin glucosyltransferase enzyme activity Under these conditions, the amount of enzyme required to generate 1 ⁇ mol ⁇ -cyclodextrin per minute is one unit of enzyme activity.
  • Example 1 Preparation and expression of different cyclodextrin glucosyltransferases
  • the plasmid was sequenced to obtain the correctly sequenced recombinant plasmid pET20b-CGT; the correctly sequenced recombinant plasmid pET20b-CG was transformed into Escherichia coli BL21( DE3), the recombinant E. coli pET20b-CGT/E.coli BL21 is obtained.
  • the obtained recombinant plasmid pET20b-CGT was used as a template for site-directed mutation, and mutants A156V/L174P (amino acid sequence shown in SEQ ID NO. 3), A156V/L174P/A166Y (amino acid sequence shown in SEQ ID 4), A156V/L174P/A166V (amino acid sequence is shown in SEQ ID NO.5), A156V/L174P/A166G (amino acid sequence is shown in SEQ ID NO.6), A156V/L174P/A166K (amino acid The sequence is shown in SEQ ID NO. 7), A156S, A156L and L174M;
  • the primers used for mutant A156V are as follows:
  • Reverse primer 5'-GTTATCATACAG AAC ACCATT-3' (SEQ ID No. 9);
  • the primers used for mutation L174P are as follows:
  • Reverse primer 5'-GTTGTGATGGAA CGG GCCAGC-3' (SEQ ID No. 11);
  • the primers used for mutation A166Y are as follows:
  • Reverse primer 5'-GTCATTCGAGTA GTA ACCCAGCAG-3' (SEQ ID No. 13);
  • the primers used for mutant A166V are as follows:
  • Reverse primer 5'-GTCATTCGAGTA AAC ACCCAGCAG-3' (SEQ ID No. 15);
  • the primers used for mutant A166G are as follows:
  • Reverse primer 5'-GTCATTCGAGTA ACC ACCCAGCAG-3' (SEQ ID No. 17);
  • the primers used for mutation A166K are as follows:
  • Reverse primer 5'-GTCATTCGAGTA TTT ACCCAGCAG-3' (SEQ ID No. 19);
  • the primers used for mutant A156S are as follows:
  • Reverse primer 5'-GTTATCATACAG AGA ACCATT-3' (SEQ ID No. 21);
  • the primers used for mutation A156L are as follows:
  • Reverse primer 5'-GTTATCATACAG CAG ACCATTG-3' (SEQ ID No.23);
  • the primers used for mutation L174M are as follows:
  • Reverse primer 5'-GTTGTGATGGAA CAT GCCAGC-3' (SEQ ID No. 25),
  • PCR reaction systems are: 5 ⁇ PrimeSTAR Buffer (Mg 2+ Plus) 5 ⁇ L, 2.5mM dNTPs 4 ⁇ L, 10 ⁇ M forward primer 1 ⁇ L, 10 ⁇ M reverse primer 1 ⁇ L, template DNA 1 ⁇ L, 2.5U/ ⁇ L PrimeSTAR Taq HS 0.5 ⁇ L, add Double distilled water to 50 ⁇ L;
  • the PCR product amplification conditions were: 98°C pre-denaturation for 3min; then 98°C for 10s, 57°C for 15s, 72°C for 6min, 30 cycles; finally 72°C for 10min.
  • the PCR amplified product was detected by 1% agarose gel electrophoresis. After the detection, 0.5 ⁇ L of methylated template digestion enzyme (Dpn I) was added to 10 ⁇ L of the amplified product. Under the conditions of reaction for 1.5h, the amplified product after Dpn I treatment was transformed into E. coli JM109, the transformed product was spread on LB solid medium, cultured at 37°C for 8h, the transformant was picked on the LB solid medium and connected to LB Cultured in liquid medium, cultured at 37°C for 10 hours and then extracted the plasmid.
  • Dpn I methylated template digestion enzyme
  • the plasmid was sequenced to obtain the correct sequenced encoding mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G , A156V/L174P/A166K, A156S, A156L and L174M gene; Transform the correctly sequenced recombinant plasmid into Escherichia coli BL21(DE3) to obtain the coding mutant A156V/L174P, A156V/L174P/A166Y , A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M.
  • E. coli pET20b-CGT/E.coli BL21 and coding mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and Recombinant Escherichia coli with L174M gene was spread on LB solid medium and cultured at 37°C for 8-10 hours to obtain a single colony; a single colony was picked into the LB liquid medium and cultured at 37°C for 12-14 hours to obtain a seed solution; The seed solution was connected to the LB liquid medium according to the inoculum amount of 4% (v/v).
  • the flow rate is 1mL/min
  • the detection wavelength is 280nm
  • the pure enzymes of mutants A156V/L174P, A156V/L174P/A166Y, A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M were obtained and freeze-dried for later use.
  • Example 2 Product specificity of different cyclodextrin glucosyltransferases to different glycosylated genistein
  • the genistein (purchased from Sigma) was dissolved in dimethyl sulfoxide (DMSO) to prepare a genistein solution with a final concentration of 7.5g/L; maltodextrin (purchased from Shanghai Shenggong Biological Engineering Co., Ltd.) ) was dissolved in PBS buffer (50mM, pH 6.5) to prepare a maltodextrin solution with a final concentration of 40g/L; the lyophilized mutants A156V/L174P, A156V/L174P/A166Y, The pure enzymes of A156V/L174P/A166V, A156V/L174P/A166G, A156V/L174P/A166K, A156S, A156L and L174M are dissolved in PBS buffer (50mM, pH 6.5) to prepare a final concentration of 15U/L of CGTase enzyme solution ; Take 300 ⁇ L of genistein solution, 500 ⁇ L of maltodextrin solution and 200
  • the short-chain glycosylated genistein is monoglycosylated genistein, diglycosylated genistein, and triglycosylated genistein.
  • long-chain glycosylated genistein is a mixture of tetra-glycosylated genistein, penta-glycosylated genistein, and hexa-glycosylated genistein
  • Molar content calculates the ratio (%) of the molar content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution to the molar content of the total glycosylated genistein in the reaction solution and in the reaction solution
  • the content of short-chain glycosylated genistein and long-chain glycosylated genistein are shown in Table 1-2; among them, the short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution were detected by HPLC.
  • the content of chain glycosylated genistein accounts for the proportion (%) of the total glycosylated genistein content in the reaction solution.
  • the yield of long-chain glycosylated genistein produced by mutant A156V/L174P with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase.
  • maltodextrin as the glycosyl donor and using genistein as the glycosyl acceptor to produce long-chain glycosylated genistein increased by 62.5%;
  • the yield of long-chain glycosylated genistein produced by mutant A156V/L174P/A166Y with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase.
  • maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor to produce long-chain glycosylated genistein increased by 165%;
  • the yield of long-chain glycosylated genistein produced by mutant A156V/L174P/A166V with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase.
  • Maltodextrin is used as the glycosyl donor, and the production of long-chain glycosylated genistein with genistein as the glycosyl acceptor has increased by 112.5%;
  • the yield of long-chain glycosylated genistein produced by mutant A156V/L174P/A166G with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase.
  • maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor to produce long-chain glycosylated genistein, the yield increased by 37.5%;
  • the yield of long-chain glycosylated genistein produced by mutant A156V/L174P/A166K with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor is higher than that of wild-type cyclodextrin glucosyltransferase.
  • the yield of long-chain glycosylated genistein produced by maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor increased by 59.4%.
  • Example 3 The effect of reaction temperature on the yield of long-chain glycosylated genistein
  • Example 2 On the basis of Example 2, the mutant A156V/L174P/A166Y with the highest specificity for the long-chain glycosylation dye product was selected, and the reaction temperature was replaced with 30°C, 35°C, 40°C, 45°C, 50°C, respectively. °C, 55°C, 60°C.
  • the short-chain glycosylated genistein in the reaction solution was detected by HPLC (here the short-chain glycosylated genistein is monoglycosylated genistein, diglycosylated genistein, and triglycosylated genistein).
  • Genistein and long-chain glycosylated genistein (where long-chain glycosylated genistein is tetraglycosylated genistein, pentaglycosylated genistein, and hexaglycosylated genistein
  • the molar content of the mixture calculate the molar content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution to the ratio of the molar content of the total glycosylated genistein in the reaction solution (% ) And the content (g/L) of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution.
  • the test results are shown in Table 4 and Figure 1.
  • the mutant A156V/L174P/A166Y is used to produce long-chain glycosylated genistein with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor.
  • the yield of sulphate is the highest, which can reach 10.2 ⁇ 10.4g/L, which is 21.4 ⁇ 23.8% higher than when the temperature is 40°C.
  • the temperature should be controlled at 45-50°C.
  • Example 2 On the basis of Example 2, the mutant A156V/L174P/A166Y with the highest specificity for long-chain glycosylation dye products was selected, the reaction temperature was controlled to 50°C, and the reaction pH was replaced with 4, 5, and 6 respectively. , 7, 8.
  • the short-chain glycosylated genistein in the reaction solution was detected by HPLC (here the short-chain glycosylated genistein is monoglycosylated genistein, diglycosylated genistein, and triglycosylated genistein).
  • Genistein and long-chain glycosylated genistein (where long-chain glycosylated genistein is tetraglycosylated genistein, pentaglycosylated genistein, and hexaglycosylated genistein
  • the molar content of the mixture calculate the molar content of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution to the ratio of the molar content of the total glycosylated genistein in the reaction solution (% ) And the content (g/L) of short-chain glycosylated genistein and long-chain glycosylated genistein in the reaction solution.
  • the test results are shown in Table 5 and Figure 2.
  • the mutant A156V/L174P/A166Y is used to produce long-chain glycosylated genistein with maltodextrin as the glycosyl donor and genistein as the glycosyl acceptor.
  • the yield is the highest, reaching 10.2 ⁇ 10.3g/L, which is about 20% higher than when the pH is 6.5.
  • the pH should be controlled to 4.

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Abstract

提供了一种生产长链糖基化染料木素的方法，该方法可提高反应液中长链糖基化染料木素的含量以及比例，将反应液中长链糖基化染料木素的含量提高至10.3g/L，将反应液中长链糖基化染料木素的含量占反应液中总糖基化染料木素含量的比例提高至70％。还提供了生产长链糖基化染料木素的环糊精葡萄糖基转移酶突变体及其在生产长链糖基化染料木素中的应用。

Description

一种生产长链糖基化染料木素的方法 技术领域
本发明涉及一种生产长链糖基化染料木素的方法，属于酶工程以及发酵工程技术领域。
背景技术
染料木素(Genistein)，又称5,7,4-三羟基异黄酮、染料木黄酮或金雀异黄素，广泛分布于自然界中，主要存在于大豆、绿豆、苜蓿、燕麦、大麦、黑麦、小麦、玉米等谷类植物中，是一种具有弱雌激素样作用的非雌激素类化合物，在医药以及保健等领域均具有极高的应用前景。
目前，染料木素类产品主要集中于预防以及治疗心血管疾病、女性更年期综合症、乳腺增生、乳腺癌、前列腺癌等方面，其作为肿瘤细胞的预防剂，具有不杀伤正常细胞的特点，因此受到各国医药界的高度重视。然而，由于染料木素具有很强疏水性，导致其生物利用度低，难以达到临床治疗疾病的目的，这大大限制了其在医药以及保健等领域中的应用。因此，如何提高染料木素在水溶液中的溶解度，成为目前国内外关注的焦点。
有研究表明，二葡萄糖基染料木素和三葡萄糖基染料木素在水中的溶解度分别是染料木素的3700倍和44000倍(具体可见参考文献：Li D,Roh SA,Shim JH,Mikami B,Baik MY,Park CS,Park KH.2005.Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification.J Agric Food Chem 53:6516-24)。也有研究表明，将染料木素进行糖基化并不影响其生理生化功能(具体可见参考文献：Chung MJ,Kang AY,Lee KM,Oh E,Jun HJ,Kim SY,Auh JH,Moon TW,Lee SJ,Park KH.2006.Water-soluble genistin glycoside isoflavones up-regulate antioxidant metallothionein expression and scavenge free radicals.J Agric Food Chem 54:3819-26)。并且，有研究表明，糖基化染料木素在体内内会水解为可被人体吸收的葡萄糖与染料木素，安全性较高(具体可见参考文献：Chung MJ,Kang AY,Lee KM,Oh E,Jun HJ,Kim SY,Auh JH,Moon TW,Lee SJ,Park KH.2006.Water-soluble genistin glycoside isoflavones up-regulate antioxidant metallothionein expression and scavenge free radicals.J Agric Food Chem 54:3819-26)。因此，可通过将染料木素进行糖基化以提高其水溶性。
环糊精葡萄糖基转移酶(Cyclodextrin glycosyltransferase，简称CGTase或CGT酶，EC2.4.1.19)是常见的可催化糖基化反应的酶，可使用此酶对染料木素进行糖基化。在此基础上，研究表明，糖基化染料木素所连糖链越长，水溶性越好(具体可见参考文献：Li D,Roh SA,Shim JH,Mikami B,Baik MY,Park CS,Park KH.2005.Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification.J Agric Food Chem  53:6516-24)。因此，生产大量的长链糖基化染料木素对提高其应用价值至关重要。
但是，现有的环糊精葡萄糖基转移酶合成长链糖基染料木素的效率较低，合成短链糖基染料木素的效率较高，这大大限制了利用环糊精葡萄糖基转移酶合成长链糖基化染料木素的产量。因此，急需找到一种产量高的生产长链糖基化染料木素的方法。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种产量高的生产长链糖基化染料木素的方法。
[技术方案]
为解决上述技术问题，本发明提供了一种环糊精葡萄糖基转移酶突变体，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸以及第174位亮氨酸进行突变得到的；
或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸、第174位亮氨酸以及第166位丙氨酸进行突变得到的。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸以及第174位亮氨酸突变为脯氨酸得到的，命名为A156V/L174P；
或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为酪氨酸得到的，命名为A156V/L174P/A166Y；
或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为缬氨酸得到的，命名为A156V/L174P/A166V；
或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为甘氨酸得到的，命名为A156V/L174P/A166G；
或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为赖氨酸得到的，命名为A156V/L174P/A166K。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶突变体的氨基酸序列为SEQ ID  NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7。
在本发明的一种实施方式中，编码所述环糊精葡萄糖基转移酶的核苷酸序列如SEQ ID No.2所示。
本发明还提供了编码上述环糊精葡萄糖基转移酶突变体的基因。
本发明还提供了携带上述基因的重组质粒。
在本发明的一种实施方式中，所述重组质粒的载体为pET-20b(+)质粒、pET-22b(+)质粒或pET-28a(+)质粒。
在本发明的一种实施方式中，所述重组质粒的载体为pET-20b(+)质粒。
本发明还提供了携带上述基因或上述重组质粒的宿主细胞。
在本发明的一种实施方式中，所述宿主细胞为细菌或真菌。
在本发明的一种实施方式中，所述宿主细胞为大肠杆菌。
本发明还提供了上述环糊精葡萄糖基转移酶突变体的制备方法，所述方法为将上述宿主细胞接种至发酵培养基中进行发酵，获得发酵液；将发酵液进行离心，获得发酵上清液；将发酵上清液进行分离，获得上述环糊精葡萄糖基转移酶突变体。
本发明还提供了一种生产长链糖基化染料木素的方法，所述方法为将含有麦芽糊精、染料木素和环糊精葡萄糖基转移酶(Cyclodextrin glycosyltransferase，简称CGTase或CGT酶，EC 2.4.1.19)的反应体系于pH为4～8、温度为30～60℃、转速为120～180rpm的条件下进行反应，得到反应液；将反应液进行分离，得到长链糖基化染料木素；所述环糊精葡萄糖基转移酶为氨基酸序列如SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7所示的环糊精葡萄糖基转移酶中的一种或一种以上。所述长链糖基化染料木素是指四糖基化染料木素、五糖基化染料木素和/或六糖基化染料木素。
在本发明的一种实施方式中，所述方法为将染料木素溶解于二甲基亚砜中配制成染料木素溶液；将麦芽糊精溶解于缓冲液A中配制成麦芽糊精溶液；将环糊精葡萄糖基转移酶溶解于缓冲液B中配制成酶液；将染料木素溶液、麦芽糊精溶液和酶液混合得到反应体系；将反应体系于pH为4～8、温度为30～60℃、转速为120～180rpm的条件下进行反应，得到反应液；将反应液进行分离，得到长链糖基化染料木素。
在本发明的一种实施方式中，所述反应的pH为4或8、温度为45～50℃。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶为氨基酸序列如SEQ ID NO.4所示的环糊精葡萄糖基转移酶。
在本发明的一种实施方式中，所述缓冲液A为PBS缓冲液、柠檬酸缓冲液或乙酸钠缓冲 液。
在本发明的一种实施方式中，所述缓冲液B为PBS缓冲液、柠檬酸缓冲液或乙酸钠缓冲液。
在本发明的一种实施方式中，所述缓冲液A的浓度为25～75mmol/L。
在本发明的一种实施方式中，所述缓冲液B的浓度为25～75mmol/L。
在本发明的一种实施方式中，所述染料木素溶液的浓度为5～15g/L。
在本发明的一种实施方式中，所述麦芽糊精溶液的浓度为20～60g/L。
在本发明的一种实施方式中，所述酶液的浓度为10～20U/L。
在本发明的一种实施方式中，所述染料木素溶液、麦芽糊精溶液和酶液的体积比为2～4:4～6:1～3。
在本发明的一种实施方式中，所述反应的时间为20～24h。
本发明还提供了上述环糊精葡萄糖基转移酶突变体或上述基因或上述重组质粒或上述宿主细胞或上述制备方法或上述方法在生产长链糖基化染料木素中的应用。
[有益效果]
(1)本发明提供了对长链糖基化染料木素的产物特异性高的环糊精葡萄糖基转移酶突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G和A156V/L174P/A166K；利用本发明的环糊精葡萄糖基转移酶突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G和A156V/L174P/A166K分别以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量分别提高了62.5％、165％、112.5％、112.5％和59.4％。
(2)本发明提供了一种产量高的生产长链糖基化染料木素的方法，利用此方法生产长链糖基化染料木素可提高反应液中长链糖基化染料木素的含量以及反应液中长链糖基化染料木素的含量占反应液中总糖基化染料木素含量的比例，将反应液中长链糖基化染料木素的含量提高至10.3g/L，将反应液中长链糖基化染料木素的含量占反应液中总糖基化染料木素含量的比例提高至70％。
附图说明
图1：反应温度对反应液中短链糖基化染料木素以及长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例的影响。
图2：反应pH对反应液中短链糖基化染料木素以及长链糖基化染料木素的摩尔含量占反 应液中总糖基化染料木素摩尔含量的比例的影响。
具体实施方式
下面结合具体实施例，对本发明进行进一步的阐述。
下述实施例中涉及的大肠杆菌JM109以及大肠杆菌E.coli BL21(DE3)购自北纳生物，pET-20b(+)质粒购自Novagen公司。(上述菌株大肠杆菌E.coli BL21(DE3)可以购买得到，不需要进行用于专利程序的保藏)
下述实施例中涉及的培养基如下：
LB液体培养基：酵母粉5.0g·L -1、胰蛋白胨10.0g·L -1、NaCl 10.0g·L-1、氨苄青霉素100μg·L -1。
LB固体培养基：酵母粉5.0g·L -1、胰蛋白胨10.0g·L -1、NaCl 10.0g·L -1、琼脂粉15g·L -1、氨苄青霉素100μg·L -1。
下述实施例中涉及的检测方法如下：
环糊精葡萄糖基转移酶酶活测定方法：取酶液0.1mL，加入装有0.9mL预先用50mM磷酸缓冲液(pH 6.5)配制的浓度为30g·L -1的可溶性淀粉溶液中，在40℃下反应10min后，加入1.0mL 1.0M的盐酸停止反应，再加入1.0mL用50mM磷酸缓冲液配制的0.1mM甲基橙，在16℃下保温20min，在505nm下测定吸光度。
环糊精葡萄糖基转移酶酶活的定义：在该条件下每分钟生成1μmolα-环糊精所需酶量为一个酶活力单位。
实施例1：不同环糊精葡萄糖基转移酶的制备及表达
具体步骤如下：
化学合成编码氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的基因(基因的核苷酸序列如SEQ ID NO.2所示)；将获得的基因与pET-20b(+)质粒经双酶切(Nco I和Xho I)后进行连接，转化大肠杆菌JM109，转化产物涂布于LB固体培养基，于37℃培养8h，在LB固体培养基上挑取转化子，接入LB液体培养基培养，于37℃培养10h后提取质粒，将此质粒进行序列测定，获得测序正确的重组质粒pET20b-CGT；将测序正确的重组质粒pET20b-CG转化大肠杆菌E.coli BL21(DE3)，即获得重组大肠杆菌pET20b-CGT/E.coli BL21。
利用全质粒PCR技术，以获得的重组质粒pET20b-CGT为模板进行定点突变，获得突变体A156V/L174P(氨基酸序列如SEQ ID NO.3所示)、A156V/L174P/A166Y(氨基酸序列如SEQ ID NO.4所示)、A156V/L174P/A166V(氨基酸序列如SEQ ID NO.5所示)、A156V/L174P/A166G(氨基酸序列如SEQ ID NO.6所示)、A156V/L174P/A166K(氨基酸序 列如SEQ ID NO.7所示)、A156S、A156L以及L174M；
其中，突变A156V所用引物如下：
正向引物：5’-GCAGAAAATGGT GTTCTGTAT-3’(SEQ ID No.8)；
反向引物：5’-GTTATCATACAG AACACCATT-3’(SEQ ID No.9)；
突变L174P所用引物如下：
正向引物：5'-GACACCGCTGGC CCGTTCCAT-3'(SEQ ID No.10)；
反向引物：5'-GTTGTGATGGAA CGGGCCAGC-3'(SEQ ID No.11)；
突变A166Y所用引物如下：
正向引物：5'-TCACTGCTGGGT TACTACTCGAAT-3'(SEQ ID No.12)；
反向引物：5'-GTCATTCGAGTA GTAACCCAGCAG-3'(SEQ ID No.13)；
突变A166V所用引物如下：
正向引物：5'-TCACTGCTGGGT GTTTACTCGAAT-3'(SEQ ID No.14)；
反向引物：5'-GTCATTCGAGTA AACACCCAGCAG-3'(SEQ ID No.15)；
突变A166G所用引物如下：
正向引物：5'-TCACTGCTGGGT GGTTACTCGAAT-3'(SEQ ID No.16)；
反向引物：5'-GTCATTCGAGTA ACCACCCAGCAG-3'(SEQ ID No.17)；
突变A166K所用引物如下：
正向引物：5'-TCACTGCTGGGT AAATACTCGAAT-3'(SEQ ID No.18)；
反向引物：5'-GTCATTCGAGTA TTTACCCAGCAG-3'(SEQ ID No.19)；
突变A156S所用引物如下：
正向引物：5'-GCAGAAAATGGT TCTCTGTAT-3'(SEQ ID No.20)；
反向引物：5'-GTTATCATACAG AGAACCATT-3'(SEQ ID No.21)；
突变A156L所用引物如下：
正向引物：5'-GCAGAAAATGGT CTGCTGTAT-3'(SEQ ID No.22)；
反向引物：5'-GTTATCATACAG CAGACCATTG-3'(SEQ ID No.23)；
突变L174M所用引物如下：
正向引物：5'-GACACCGCTGGC ATGTTCCAT-3'(SEQ ID No.24)；
反向引物：5'-GTTGTGATGGAA CATGCCAGC-3'(SEQ ID No.25)，
PCR反应体系均为：5×PrimeSTAR Buffer(Mg 2+Plus)5μL，2.5mM dNTPs 4μL，10μM正向引物1μL，10μM反向引物1μL，模板DNA 1μL，2.5U/μL PrimeSTAR Taq HS 0.5μL， 加入双蒸水至50μL；
PCR产物扩增条件均为：98℃预变性3min；随后进行98℃10s，57℃15s，72℃6min，30个循环；最后72℃保温10min。
PCR扩增产物用1％琼脂糖凝胶电泳进行检测，检测结束后，向10μL扩增产物中加入0.5μL甲基化模板消化酶(Dpn I)，枪头吹吸进行混匀，于37℃条件下反应1.5h，将Dpn I处理后的扩增产物转化大肠杆菌JM109，转化产物涂布于LB固体培养基，于37℃培养8h，在LB固体培养基上挑取转化子，接入LB液体培养基培养，于37℃培养10h后提取质粒，将此质粒进行序列测定，获得测序正确的含有编码突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K、A156S、A156L以及L174M的基因的重组质粒；将测序正确的重组质粒转化大肠杆菌E.coli BL21(DE3)，即获得含有编码突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K、A156S、A156L以及L174M的基因的重组大肠杆菌。
将获得的重组大肠杆菌pET20b-CGT/E.coli BL21以及编码突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K、A156S、A156L以及L174M的基因的重组大肠杆菌涂布于LB固体培养基，于37℃培养8～10h，获得单菌落；挑取单菌落接入LB液体培养基，于37℃培养12～14h，获得种子液；将种子液按照4％(v/v)的接种量接入LB液体培养基，于30℃、120rpm培养至OD 600＝0.6后，在发酵液中加入终浓度为0.01mM的IPTG，于25℃、120rpm继续诱导培养90h，得到发酵液；将发酵液于4℃、1000rpm离心20min后，收集发酵上清液；在发酵上清液中加入70％固体硫酸铵盐析过夜，4℃、10000rpm离心20min，取沉淀物用适量含20mM磷酸钠、0.5M氯化钠、20mM咪唑、pH 7.4的缓冲液A溶解，并在缓冲液A中透析过夜后，通过0.22μm膜过滤后制成上样样品；Ni亲和柱用缓冲液A平衡后，将上样样品吸入Ni柱，使之完全吸附后，分别用缓冲液A、含20～480mM咪唑的缓冲液A、含480mM咪唑的缓冲液A的洗脱，流速1mL/min，检测波长为280nm，分部收集含环糊精葡萄糖基转移酶酶活的洗脱液；活力组分在50mM磷酸钠缓冲液(pH＝6)中透析过夜后，分别得到突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K、A156S、A156L以及L174M的纯酶，并冻干备用。
实施例2：不同环糊精葡萄糖基转移酶对不同糖基化染料木素的产物特异性
具体步骤如下：
将染料木素(购自Sigma公司)溶解于二甲基亚砜(DMSO)中配制成终浓度为7.5g/L的染料木素溶液；将麦芽糊精(购自上海生工生物工程有限公司)溶解于PBS缓冲液(50mM，pH 6.5)中配制成终浓度为40g/L的麦芽糊精溶液；分别将实施例1获得的冻干后的突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K、A156S、A156L以及L174M的纯酶溶解于PBS缓冲液(50mM，pH 6.5)中配制成终浓度为15U/L的CGTase酶液；分别取300μL染料木素溶液，500μL麦芽糊精溶液和200μL CGTase酶液混合于2mL的带盖小管内，放于40℃、120rpm摇床缓慢振荡20～24h，得到反应液。
通过HPLC检测反应液中短链糖基化染料木素(此处短链糖基化染料木素为一糖基化染料木素、二糖基化染料木素和三糖基化染料木素的混合物)和长链糖基化染料木素(此处长链糖基化染料木素为四糖基化染料木素、五糖基化染料木素和六糖基化染料木素的混合物)的摩尔含量，并计算反应液中短链糖基化染料木素和长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例(％)以及反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)，检测结果见表1-2；其中，HPLC检测反应液中短链糖基化染料木素以及长链糖基化染料木素的含量占反应液中总糖基化染料木素含量的比例(％)的方法为：反应液通过0.22μm滤膜过滤，使用Amethyst C18-H柱(4.6×250mm,Sepax,America)检测(具体检测条件见表3)；其中，长链糖基化染料木素的含量＝六糖基化染料木素的摩尔含量×六糖基化染料木素的分子量+五糖基化染料木素的摩尔含量×五糖基化染料木素的分子量+四糖基化染料木素的摩尔含量×四糖基化染料木素的分子量，短链糖基化染料木素的含量＝三糖基化染料木素的摩尔含量×三糖基化染料木素的分子量+二糖基化染料木素的摩尔含量×二糖基化染料木素的分子量+一糖基化染料木素的摩尔含量×一糖基化染料木素的分子量。
由表1-2可知，仅有突变体A156V/L174P、A156V/L174P/A166Y、A156V/L174P/A166V、A156V/L174P/A166G、A156V/L174P/A166K对长链糖基化染料木素的产物特异性高较野生型有了明显的提高；
其中，利用突变体A156V/L174P以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量提高了62.5％；
利用突变体A156V/L174P/A166Y以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量提高了165％；
利用突变体A156V/L174P/A166V以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量提高了112.5％；
利用突变体A156V/L174P/A166G以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量提高了37.5％；
利用突变体A156V/L174P/A166K以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量较利用野生型环糊精葡萄糖基转移酶以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量提高了59.4％。
表1不同环糊精葡萄糖基转移酶反应得到的反应液中短链糖基化染料木素以及长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例(％)
组别 长链基染料木素 短链糖基化染料木素
野生型 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
表2不同环糊精葡萄糖基转移酶反应得到的反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)
组别 长链基染料木素 短链糖基化染料木素
野生型 3.2 11.4
A156S 1.2 8.83
A156L 0.7 7.64
L174M 1.0 8.87
A156V/L174P 5.2 9.7
A156V/L174P/A166V 6.8 9.5
A156V/L174P/A166G 4.4 10.9
A156V/L174P/A166K 5.1 10.1
A156V/L174P/A166Y 8.5 8.0
表3 HPLC检测反应液中短链糖基化染料木素以及长链糖基化染料木素的含量的条件
Figure PCTCN2020091254-appb-000001
实施例3：反应温度对长链糖基化染料木素产量的影响
具体步骤如下：
在实施例2的基础上，选择对长链糖基化染料产物特异性最高的突变体A156V/L174P/A166Y，并且，将反应温度分别替换为30℃、35℃、40℃、45℃、50℃、55℃、60℃。
参照实施例2，通过HPLC检测反应液中短链糖基化染料木素(此处短链糖基化染料木素为一糖基化染料木素、二糖基化染料木素和三糖基化染料木素的混合物)和长链糖基化染料木素(此处长链糖基化染料木素为四糖基化染料木素、五糖基化染料木素和六糖基化染料木素的混合物)的摩尔含量，并计算反应液中短链糖基化染料木素和长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例(％)以及反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)，检测结果见表4和图1。
由表4可知，当温度为45～50℃时，利用突变体A156V/L174P/A166Y以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量最高，可达10.2～10.4g/L，较温度为40℃时提高了21.4～23.8％。
由图1可知，温度越高，利用突变体A156V/L174P/A166Y反应获得的反应液中长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例越高，可达49％以上， 较温度为40℃时提高了21％以上。
可见，利用突变体A156V/L174P/A166Y以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素时，应控制温度为45～50℃。
表4不同温度下反应得到的反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)
温度 长链基染料木素 短链糖基化染料木素
30℃ 6.8 8.9
35℃ 7.6 8.5
40℃ 8.4 8.0
45℃ 10.1 6.8
50℃ 10.0 6.6
55℃ 8.9 5.6
60℃ 5.7 3.6
实施例4：反应pH对长链糖基化染料木素产量的影响
具体步骤如下：
在实施例2的基础上，选择对长链糖基化染料产物特异性最高的突变体A156V/L174P/A166Y，将反应温度控制为50℃，并且，将反应pH分别替换为4、5、6、7、8。
参照实施例2，通过HPLC检测反应液中短链糖基化染料木素(此处短链糖基化染料木素为一糖基化染料木素、二糖基化染料木素和三糖基化染料木素的混合物)和长链糖基化染料木素(此处长链糖基化染料木素为四糖基化染料木素、五糖基化染料木素和六糖基化染料木素的混合物)的摩尔含量，并计算反应液中短链糖基化染料木素和长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例(％)以及反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)，检测结果见表5和图2。
由表5可知，当pH为4或8时，利用突变体A156V/L174P/A166Y以麦芽糊精作为糖基供体，以染料木素作为糖基受体生产长链糖基化染料木素的产量最高，可达10.2～10.3g/L，较pH为6.5时提高了20％左右。
由图2可知，当pH为4时，利用突变体A156V/L174P/A166Y反应获得的反应液中长链糖基化染料木素的摩尔含量占反应液中总糖基化染料木素摩尔含量的比例最高，可达70％，较pH为6.5时提高了30％。
可见，利用突变体A156V/L174P/A166Y以麦芽糊精作为糖基供体，以染料木素作为糖基 受体生产长链糖基化染料木素时，应控制pH为4。
表5不同pH下反应得到的反应液中短链糖基化染料木素以及长链糖基化染料木素的含量(g/L)
pH 长链基染料木素 短链糖基化染料木素
4 10.2 2.6
5 7.2 8.8
6 8.5 8.0
7 9.7 5.5
8 10.3 3.6
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (19)

  1. 一种环糊精葡萄糖基转移酶突变体，其特征在于，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸以及第174位亮氨酸进行突变得到的；
    或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸、第174位亮氨酸以及第166位丙氨酸进行突变得到的。
  2. 如权利要求1所述的一种环糊精葡萄糖基转移酶突变体，其特征在于，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸以及第174位亮氨酸突变为脯氨酸得到的；
    或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为酪氨酸得到的；
    或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为缬氨酸得到的；
    或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为甘氨酸得到的；
    或者，所述环糊精葡萄糖基转移酶突变体是通过将出发氨基酸序列如SEQ ID NO.1所示的环糊精葡萄糖基转移酶的第156位丙氨酸突变为缬氨酸、第174位亮氨酸突变为脯氨酸以及第166位丙氨酸突变为赖氨酸得到的。
  3. 如权利要求1或2所述的一种环糊精葡萄糖基转移酶突变体，其特征在于，所述环糊精葡萄糖基转移酶突变体的氨基酸序列为SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7。
  4. 编码权利要求1-3任一项所述环糊精葡萄糖基转移酶突变体的基因。
  5. 携带权利要求4所述基因的重组质粒。
  6. 如权利要求5所述的重组质粒，其特征在于，所述重组质粒的载体为pET-20b(+)质粒、pET-22b(+)质粒或pET-28a(+)质粒。
  7. 携带权利要求4所述基因或权利要求5或6所述重组质粒的宿主细胞。
  8. 如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为细菌或真菌。
  9. 权利要求1-3任一项所述环糊精葡萄糖基转移酶突变体的制备方法，其特征在于，所述 方法为将权利要求7或8所述的宿主细胞接种至发酵培养基中进行发酵，获得发酵液；将发酵液进行离心，获得发酵上清液；将发酵上清液进行分离，获得权利要求1-3任一项所述的环糊精葡萄糖基转移酶突变体。
  10. 一种生产长链糖基化染料木素的方法，其特征在于，所述方法为将含有麦芽糊精、染料木素和环糊精葡萄糖基转移酶的反应体系于pH为4～8、温度为30～60℃、转速为120～180rpm的条件下进行反应，得到反应液；将反应液进行分离，得到长链糖基化染料木素；所述环糊精葡萄糖基转移酶为氨基酸序列如SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7所示的环糊精葡萄糖基转移酶中的一种或一种以上。
  11. 如权利要求10所述的一种生产长链糖基化染料木素的方法，其特征在于，所述方法为将染料木素溶解于二甲基亚砜中配制成染料木素溶液；将麦芽糊精溶解于缓冲液A中配制成麦芽糊精溶液；将环糊精葡萄糖基转移酶溶解于缓冲液B中配制成酶液；将染料木素溶液、麦芽糊精溶液和酶液混合得到反应体系；将反应体系于pH为4～8、温度为30～60℃、转速为120～180rpm的条件下进行反应，得到反应液；将反应液进行分离，得到长链糖基化染料木素。
  12. 如权利要求10或11所述的一种生产长链糖基化染料木素的方法，其特征在于，所述反应的pH为4或8、温度为45～50℃。
  13. 如权利要求10-12任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述环糊精葡萄糖基转移酶为氨基酸序列如SEQ ID NO.4所示的环糊精葡萄糖基转移酶。
  14. 如权利要求11-13任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述缓冲液A为PBS缓冲液、柠檬酸缓冲液或乙酸钠缓冲液。
  15. 如权利要求11-14任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述缓冲液B为PBS缓冲液、柠檬酸缓冲液或乙酸钠缓冲液。
  16. 如权利要求11-15任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述染料木素溶液的浓度为5～15g/L。
  17. 如权利要求11-16任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述酶液的浓度为10～20U/L。
  18. 如权利要求11-17任一项所述的一种生产长链糖基化染料木素的方法，其特征在于，所述染料木素溶液、麦芽糊精溶液和酶液的体积比为2～4:4～6:1～3。
  19. 权利要求1-3任一项所述的环糊精葡萄糖基转移酶突变体或权利要求4所述的基因或权利要求5或6所述的重组质粒或权利要求7或8所述的宿主细胞或权利要求9所述的制备方法或权利要求10-18任一项所述的方法在生产长链糖基化染料木素中的应用。
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