阅读说明：本技术 一种环糊精葡萄糖基转移酶突变体及其应用 (Cyclodextrin glucosyltransferase mutant and application thereof ) 是由 韩瑞枝 柴宝成 倪晔 于 2019-11-08 设计创作，主要内容包括：本发明公开了一种环糊精葡萄糖基转移酶突变体及其应用,属于酶工程和微生物工程技术领域。本发明CGTase突变体对长链糖基化染料木素的产物特异性高；利用本发明的CGTase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％。(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.)

1. A cyclodextrin glucosyltransferase mutant, characterized in that the cyclodextrin glucosyltransferase mutant is obtained by mutating the 156 th alanine and 174 th leucine of cyclodextrin glucosyltransferase whose starting amino acid sequence is shown in 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.

2. The cyclodextrin glucosyltransferase mutant according to claim 1, wherein the cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine and leucine 174 to proline of cyclodextrin glucosyltransferase having 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 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.

3. A gene encoding the cyclodextrin glucosyltransferase mutant according to claim 1 or 2.

4. A recombinant plasmid carrying the gene of claim 3.

5. The recombinant plasmid of claim 4, wherein the vector of the recombinant plasmid is a pET-20b (+) plasmid, a pET-22b (+) plasmid, or a pET-28a (+) plasmid.

6. A host cell carrying the gene of claim 3 or the recombinant plasmid of claim 4 or 5.

7. The host cell of claim 6, wherein the host cell is a bacterium or a fungus.

8. The method for producing a cyclodextrin glucosyltransferase mutant according to claim 1 or 2, which comprises inoculating the host cell according to claim 6 or 7 into a fermentation medium, and fermenting 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 or 2.

9. 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 or 2 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.

10. Use of the cyclodextrin glucosyltransferase mutant according to claim 1 or 2 or the gene according to claim 3 or the recombinant plasmid according to claim 4 or 5 or the host cell according to claim 6 or 7 or the method of claim 8 or the method according to claim 9 for producing long-chain glycosylated genistein.

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, MikamiB, Baik MY, Park CS, Park KH.2005.glycosylation of genistein in the international soluble formation: JAgricFood 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 dietary approach expression and flavor free solids. JAgric food chem 54: 3819-26). 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, JunHJ, Kim SY, Auh JH, Moon TW, Lee SJ, Park KH.2006.Water-soluble genistein uppers-regulated antioxidant genes expressing and collagen free radicals. JAgric Food Chem 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 research shows 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 genetic in soluble formation. 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) with high specificity to long-chain glycosylated genistein products, and EC 2.4.1.19.

[ 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 cyclodextrin glucosyltransferase mutant 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 in 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 cyclodextrin glucosyltransferase mutant is obtained by mutating alanine 156 to valine, leucine 174 to proline and alanine 166 to lysine of cyclodextrin glucosyltransferase with an original amino acid sequence shown in 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: enzyme extracting solution0.1mL of a buffer solution containing 0.9mL of a buffer solution prepared in advance with 50mM phosphate (pH 6.5) and having a concentration of 30 g.L was added^-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.

The enzyme activity of cyclodextrin glucosyltransferase is defined as that the enzyme amount required for producing α -cyclodextrin in an amount of 1. mu. mol/min under the condition is one enzyme activity unit.