阅读说明：本技术 一种重组β-葡萄糖苷酶CB-3B及其在生产人参皂苷Rg3中的应用 (Recombinant β -glucosidase CB-3B and application thereof in production of ginsenoside Rg3 ) 是由 周义发 原野 胡晨星 孙琳 张梦珊 台桂花 于 2019-11-15 设计创作，主要内容包括：本发明公开了一种重组β-葡萄糖苷酶CB-3B及其在生产人参皂苷Rg3中的应用。重组β-葡萄糖苷酶CB-3B的氨基酸序列如SEQ ID NO：2或SEQ ID NO：4所示。实验证明,蛋白质CB-3B为β-葡萄糖苷酶,能够水解结合于末端非还原性的β-D-葡萄糖键,同时释放出β-D-葡萄糖和相应的配基。利用蛋白质CB-3B和糖苷水解酶水解人参皂苷Rb1,可以生产人参皂苷Rg3。本发明具有重要应用价值。(The invention discloses a recombinant β -glucosidase CB-3B and application thereof in producing ginsenoside Rg 3. the amino acid sequence of the recombinant β -glucosidase CB-3B is shown as SEQ ID NO.2 or SEQ ID NO. 4. experiments prove that the protein CB-3B is β -glucosidase, can hydrolyze β -D-glucose bond bound to non-reducing end, and simultaneously release β -D-glucose and corresponding aglycone, the ginsenoside Rb1 is hydrolyzed by the protein CB-3B and glycoside hydrolase, and the ginsenoside Rg3 can be produced.)

1. Protein CB-3B, which is a1) or a2) or a3) or a4) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) the amino acid sequence is SEQ ID NO: 4;

a4) the protein with β -glucosidase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by a1) or a2) or a 3).

2. A nucleic acid molecule encoding the protein CB-3B according to claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as b1) or b2) or b3) or b 4):

b1) the coding region SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) the nucleotide sequence is SEQ ID NO: 3, a DNA molecule shown in seq id no;

b4) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in B1) or B2) or B3), derived from a fiber bacterium and encoding the protein CB-3B of claim 1;

b5) a DNA molecule derived from Cellulosium and encoding the protein CB-3B as claimed in claim 1, which hybridizes under stringent conditions with a nucleotide sequence defined in B1) or B2) or B3).

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3.

5. The use of the protein CB-3B as claimed in claim 1 as c1) or c2) or c3) or c4) or c5) or c 6):

c1) application in producing ginsenoside Rg 3;

c2) application in preparing products for producing ginsenoside Rg 3;

c3) use as β -glucosidase;

c4) the application in preparing products with β -glucosidase function;

c5) application in hydrolyzing ginsenoside Rb 1;

c6) application in preparing products for hydrolyzing ginsenoside Rb1 is provided.

6. The use of the nucleic acid molecule of claim 2 or 3 as c1) or c2) or c3) or c4) or c5) or c 6):

c1) application in producing ginsenoside Rg 3;

c2) application in preparing products for producing ginsenoside Rg 3;

c3) use as β -glucosidase;

c4) the application in preparing products with β -glucosidase function;

c5) application in hydrolyzing ginsenoside Rb 1;

c6) application in preparing products for hydrolyzing ginsenoside Rb1 is provided.

7. Use according to claim 5 or 6, characterized in that: in the c1) or c2), the production of the ginsenoside Rg3 uses ginsenoside Rb1 as a substrate.

8. A method for producing ginsenoside Rg3, comprising the steps of (a 1): hydrolyzing ginsenoside Rb1 with the protein CB-3B and glycoside hydrolase according to claim 1.

9. The method of claim 8, wherein: the glycoside hydrolase is glycoside hydrolase BglPC 28;

the glycoside hydrolase BglPC28 is d1) or d2) or d3) as follows:

d1) the amino acid sequence is SEQ ID NO: 5;

d2) in SEQ ID NO: 5, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

d3) the protein with the glycoside hydrolase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by d1) or d 2).

10. The method of claim 8 or 9, wherein: the method further comprises step (a 2); the step (a2) is as follows: after completion of step (a1), purification was performed.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to recombinant β -glucosidase CB-3B and application thereof in production of ginsenoside Rg 3.

Background

The ginsenoside Rg3 has good pharmacological activities such as anti-tumor and anti-inflammatory activities, and has high potential application value. However, the content of the ginsenoside Rg3 in ginseng is low, and the industrial preparation of the ginsenoside Rg3 is difficult to extract from ginseng, so that the application of the ginsenoside Rg3 is limited.

The content of ginsenoside Rb1 in total saponins of Ginseng radix is up to 15%, and the content in total saponins of radix Panacis Quinquefolii and Notoginseng radix is up to 25% and 30%. Ginsenoside Rb1 and ginsenoside Rg3 have the same aglycone structure except that the number of glycosyl groups attached to the aglycone C20 position is different. Therefore, the ginsenoside Rg3 can be obtained by hydrolyzing the glucosyl group at C20 position in the molecule of ginsenoside Rb 1. Wherein, the specificity of acid hydrolysis is poor, the byproducts are many, the yield is low, and the separation and purification of the product are difficult. The biotransformation has the advantages of mild conditions, good specificity, high yield, little pollution and the like, and is an ideal method for preparing the ginsenoside Rg 3.

Disclosure of Invention

The invention aims to prepare the ginsenoside Rg 3.

The invention firstly protects a protein CB-3B which can be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) the amino acid sequence is SEQ ID NO: 4;

a4) the protein with β -glucosidase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by a1) or a2) or a 3).

Wherein, SEQ ID NO: 2 may consist of 765 amino acid residues; SEQ ID NO: 4 may consist of 785 amino acid residues.

To facilitate purification of the protein in a1), the protein of SEQ ID NO: 2 to the amino terminus or carboxy terminus of the protein shown in table 1.

TABLE 1 sequence of tags

The protein according to a4), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a4) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a4) above can be obtained by converting the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and/or is missense mutated by one or more base pairs, and/or is obtained by linking the coding sequence of the tag shown in table 1 above at its 5 'end and/or 3' end.

Nucleic acid molecules which code for the protein CB-3B also belong to the scope of protection of the invention.

The nucleic acid molecule encoding the protein CB-3B can be a DNA molecule shown as B1) or B2) or B3) or B4) as follows:

b1) the coding region SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) the nucleotide sequence is SEQ ID NO: 3, a DNA molecule shown in seq id no;

b4) a DNA molecule which has 75% or more than 75% identity with the nucleotide sequence defined by B1) or B2) or B3), is derived from a fiber bacterium and encodes the protein CB-3B;

b5) a DNA molecule which is derived from a fiber bacterium and codes for the protein CB-3B and hybridizes with the nucleotide sequence defined by B1) or B2) or B3) under strict conditions.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 2301 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2; SEQ ID NO: 3 consists of 2361 nucleotides, SEQ ID NO: 3 encodes the nucleotide sequence of SEQ id no: 4.

The nucleotide sequence encoding the protein CB-3B of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of said protein CB-3B isolated in accordance with the present invention as long as they encode said protein CB-3B are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 2 or SEQ ID NO: 4, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the sequence of nucleotides of the protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines which contain nucleic acid molecules which code for the protein CB-3B also belong to the scope of protection of the invention.

The recombinant vector containing the nucleic acid molecule for coding the protein CB-3B can be obtained by inserting SEQ ID NO: 1 in the sequence listing.

The expression vector may be a pET-28a vector.

The recombinant vector containing the nucleic acid molecule for coding the protein CB-3B can be specifically a recombinant plasmid pET-28a/CB-3B obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes NdeI and BamHI of a pET-28a vector with a DNA molecule with the nucleotide sequence shown in SEQ ID No. 1.

The recombinant microorganism containing the nucleic acid molecule encoding the protein CB-3B may be a recombinant bacterium obtained by introducing any of the above-mentioned recombinant vectors containing the nucleic acid molecule encoding the protein CB-3B into a starting microorganism.

The recombinant microorganism containing the nucleic acid molecule for encoding the protein CB-3B can be specifically a recombinant bacterium obtained by introducing the recombinant plasmid pET-28a/CB-3B into an original microorganism.

The starting microorganism may be Escherichia coli.

The Escherichia coli can be specifically Escherichia coli BL21(DE 3).

The invention also protects the application of any one of the proteins CB-3B, which can be c1), c2), c3), c4), c5) or c 6):

c1) application in producing ginsenoside Rg 3;

c2) application in preparing products for producing ginsenoside Rg 3;

c3) use as β -glucosidase;

c4) the application in preparing products with β -glucosidase function;

c5) application in hydrolyzing ginsenoside Rb 1;

c6) application in preparing products for hydrolyzing ginsenoside Rb1 is provided.

The invention also protects the application of the nucleic acid molecule for coding the protein CB-3B, which can be c1), c2), c3), c4), c5) or c 6):

c1) application in producing ginsenoside Rg 3;

c2) application in preparing products for producing ginsenoside Rg 3;

c3) use as β -glucosidase;

c4) the application in preparing products with β -glucosidase function;

c5) application in hydrolyzing ginsenoside Rb 1;

c6) application in preparing products for hydrolyzing ginsenoside Rb1 is provided.

In the c1) or c2), the production of the ginsenoside Rg3 uses ginsenoside Rb1 as a substrate. Specifically, the ginsenoside Rb1 can be used as a substrate, and a substance containing total saponins of ginsenoside Rb1 can be used as a substrate. The total saponin containing ginsenoside Rb1 can be protopanaxadiol type total saponin, radix Panacis Quinquefolii, Notoginseng radix, etc.

In the above, β -glucosidase means an enzyme capable of hydrolyzing a bond of β -D-glucose bound to a terminal non-reducing group while releasing β -D-glucose and a corresponding ligand β -glucosidase activity is defined as that 1U of enzyme is required for β -glucosidase to catalyze the production of 1. mu. mol of PNP from p-Nitrophenyl- β -D-glucopyranoside (p-Nitrophenyl β -D-glucopyranoside, PNPG) at 37 ℃ per minute.

The invention also provides a method for producing ginsenoside Rg3, which can comprise the following steps (a 1): the ginsenoside Rb1 is hydrolyzed by using any one of the protein CB-3B and glycoside hydrolase.

In the step (a1), the hydrolysis system may comprise ginsenoside Rb1, buffer, any one of the proteins CB-3B and glycoside hydrolase. The buffer solution may be Na₂HPO₄-NaH₂PO₄And (4) a buffer solution. The Na is₂HPO₄-NaH₂PO₄The buffer solution may specifically be pH6.0, 100mM Na₂HPO₄-NaH₂PO₄And (4) a buffer solution. In one embodiment of the invention, the hydrolysis system is 5L, and comprises 250g of ginsenoside Rb1, 500mL of ginsenoside Rb1 with pH of 6.0 and 100mM Na₂HPO₄-NaH₂PO₄The kit comprises a buffer solution, 75kU recombinant β -glucosidase CB-3B, 25kU recombinant glucosidase BglPC28 and water.

In the step (a1), the hydrolysis parameter may be 150-200rpm (e.g., 150-170rpm, 170-200rpm, 150rpm, 170rpm or 200rpm) for 4-6h (e.g., 4-5h, 5-6h, 4h, 5h or 6h) of stirring.

In any of the above processes, the glycoside hydrolase may be glycoside hydrolase BglPC 28; the glycoside hydrolase BglPC28 may be d1) or d2) or d3) as follows:

d1) the amino acid sequence is SEQ ID NO: 5;

d2) in SEQ ID NO: 5, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

d3) the protein with the glycoside hydrolase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by d1) or d 2).

The glycoside hydrolase BglPC28 may specifically be a recombinant glycosidase BglPC28(Chang-Hao Cui, et al, Characterisation of a Ginsenoside-transduction β -glucopyranosase from Paenibacillus communis and Its Application for Enhanced Production of minor Ginsenoside F2.10.1371/journel. bone 0085727).

The method of any of the above further comprising the step (a 2); the step (a2) is as follows: after completion of step (a1), purification was performed. The purification may be a silica gel column purification.

In one embodiment of the present invention, the purification steps are specifically as follows:

(1-1) after completion of step (a1), centrifuging and collecting the precipitate.

(1-2) after completion of the step (1-1), the precipitate was washed with an aqueous ethanol solution, and the supernatant was collected.

(1-3) after the step (1-2) is completed, concentrating;

(1-4) after completion of the step (1-3), removing ethanol.

(1-5) drying after completing the step (1-4).

In the step (1-1), the centrifugation parameter may be 4000rpm centrifugation for 15 min.

In the step (1-2), "washing the precipitate with an ethanol aqueous solution, and collecting the supernatant" may specifically be performed by re-dissolving the precipitate in a 95% (v/v) ethanol aqueous solution, stirring at 70 ℃ for 2h, centrifuging at 4000rpm for 15min, and collecting the supernatant 1 and the precipitate 1; adding 95% (v/v) ethanol water solution into the precipitate 1 for resuspension, fully stirring, centrifuging again, and collecting the supernatant 2. And combining the supernatant 1 and the supernatant 2 to obtain a supernatant combined solution.

In the step (1-3), "concentration" may be achieved by rotary evaporation at 50 ℃ using a rotary evaporator.

In the step (1-4), "removing ethanol" may be carried out by filtration.

In the step (1-5), the drying may be vacuum drying, freeze drying, spray drying or atmospheric drying.

Experiments prove that the protein CB-3B is β -glucosidase, can be hydrolyzed and combined with β -D-glucose bond with non-reducing tail end, and simultaneously releases β -D-glucose and corresponding aglycone, the ginsenoside Rb1 is hydrolyzed by the protein CB-3B and the glycoside hydrolase, and the ginsenoside Rg3 can be produced.

Drawings

FIG. 1 is a SDS-PAGE result of total protein solution before IPTG induction, total protein solution after IPTG induction and purified recombinant β -glucosidase CB-3B.

FIG. 2 shows the effect of pH on the activity and stability of recombinant β -glucosidase CB-3B enzyme, wherein ■ is pH2.0-6.0, 50mM NaAc-HAc buffer, □ is pH2.0-6.0, 50mM NaAc-HAc buffer tolerance, ● is pH6.0-8.0, 50mM Na₂HPO₄-NaH₂PO₄A buffer solution, ○ pH6.0-8.0, 50mM Na₂HPO₄-NaH₂PO₄The buffer solution is tolerant, wherein ▲ is pH8.0-11.0, 50mM Glycine-NaOH buffer solution, △ is pH8.0-11.0, 50mM Glycine-NaOH buffer solution.

FIG. 3 shows the effect of temperature on the activity of recombinant β -glucosidase CB-3B enzyme.

FIG. 4 is a graph showing the effect of temperature on the stability of recombinant β -glucosidase CB-3B.

FIG. 5 shows the result of TLC detection of recombinant β -glucosidase CB-3B converted ginsenoside.

FIG. 6 shows the results of HPLC detection in example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Cellulosimicrobium sp.21 is preserved in China general microbiological culture Collection center (CGMCC, address No.3 of Xilu No.1 Beijing, Chaoyang, Beijing) in 2013 at 5.14.M, and the preservation number is CGMCC No. 7587.

β -definition of enzyme activity of glucosidase enzyme β -glucosidase enzyme amount required for catalyzing p-Nitrophenyl- β -D-glucopyranoside (p-Nitrophenyl β -D-glucopyranoside, PNPG) to generate 1 μmol PNP per minute at 37 ℃ is 1U.

In the following examples, the PNP standard curve is: y is 45.3x (R)²＝0.9996，y：OD_405nmAnd x: μ mol). The preparation method of the PNP standard curve comprises the following steps: (1) accurately weighing 1.39mg of p-nitrophenol (PNP), dissolving with a proper amount of deionized water, and then diluting to 10mL with deionized water to obtain a PNP mother solution with the concentration of 1 mM; (2) taking 0 μ L, 40 μ L, 80 μ L, 120 μ L, 160 μ L, 200 μ L or 240 μ L PNP mother liquor, adding 200 μ L Na with concentration of 0.5M₂CO₃Adding deionized water to the water solution to a constant volume of 1000 μ L, and mixing to obtain a solution to be detected; (3) adding 200 mu L of solution to be detected into a 96-well plate, and detecting a light absorption value under 405nm wavelength by using an enzyme-labeling instrument; (4) and drawing a PNP standard curve by taking the concentration of PNP in the liquid to be detected as an abscissa and the corresponding light absorption value as an ordinate.