CN112481280A - Method for preparing rare ginsenoside CK by gene combination transformation and application - Google Patents
Method for preparing rare ginsenoside CK by gene combination transformation and application Download PDFInfo
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Abstract
The invention discloses a method for preparing rare ginsenoside CK by gene combination transformation and application. The invention utilizes bacillus subtilis (B.subtilis)Bacillus subtilis str.168) And Bifidobacterium (Bifidobacterium breve689b) The glycosidase produced by the gene can convert ginsenoside Rc into rare ginsenoside CK in vitro with high efficiency.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a method for preparing rare ginsenoside CK by utilizing gene combination transformation and application, and particularly relates to a method for preparing rare ginsenoside CK by utilizing escherichia coli to co-express bacillus subtilis alpha-L-arabinofuranosidase and bifidobacterium beta-glucosidase to transform ginsenoside Rc.
Background
Ginsenosides are the most important active ingredients in ginseng, belong to tetracyclic triterpenoids, and can be classified into dammarane-type and oleanolic-type saponins according to the difference in the types and numbers of Glycosidic bonds (Glycosidic bonds) at the C-3, C-6 and C-20 positions on aglycone, wherein the dammarane-type saponins are further classified into protopanaxadiol-type saponins (PPD-type saponins mainly comprising Rb1, Rb2, Rc, Rd and the like) and protopanaxatriol-type saponins (PPT-type saponins including Re, Rg1 and the like). The main ginsenosides such as Rb1, Rb2, Rc, Rd, Re and Rg1, etc. and Rare ginsenosides such as Rg3, Rh1, Rh2, F1, F2, CK, C-O, CY and C-Mc, etc. are classified according to the content of ginsenosides in plants. To date, over 100 ginsenosides have been found in ginseng plants. However, Rb1, Rb2, Rc, Rd, Re and Rg1 account for more than 80% of total saponins, and these main ginsenoside molecules usually contain 3-4 glycosyl groups, and are difficult to be directly absorbed by human bodies due to the abundance of the glycosyl groups, so that the physiological activity of rare ginsenosides is generally lower than that of the glycosyl groups.
Among the currently known ginsenosides, rare ginsenosides such as F2, Rg3, Rh2, CK, etc. having low sugar group have high absorption rate in the human body. The rare ginsenoside has important application values in the aspects of resisting cancer, tumors and thrombi, improving immunity, resisting inflammation, resisting aging, resisting diabetes and anxiety, preventing and relieving senile dementia and the like. Researches show that the ginsenoside CK has the unique advantages that other ginsenosides can not be substituted in the aspects of resisting cell mutation, inhibiting tumor cell metastasis, inducing tumor cell apoptosis, reversing tumor cell drug resistance, resisting tumor-induced angiogenesis and the like. It can be used in combination with radiotherapy and chemotherapy to enhance the effect of radiotherapy and chemotherapy. In addition, the ginsenoside CK has antiallergic and antiinflammatory activities, and has neuroprotective, antidiabetic and antiaging effects. And the pharmacological activity of the ginsenoside CK has the characteristics of multiple targets, high activity and low toxicity.
In order to obtain high-activity rare ginsenoside with low glycosyl content, a large number of researches on the aspects of conversion by using intestinal bacteria, microorganisms, cloned enzymes and the like are reported. The research finds that the ginsenoside CK is protopanaxadiol type saponin and is the most main metabolite in human intestinal tracts after being taken. In addition, most protopanaxadiol-type saponins can be absorbed by human bodies only after being metabolized into CK, so that the ginsenoside CK protopanaxadiol-type saponins are real molecules with pharmacological effects in the bodies, and other diol-type ginsenosides are only prodrugs. Although ginsenoside CK plays an important pharmacological activity in the human body, since the content thereof is very rare in nature, it is very difficult to separate it from plants such as ginseng by the conventional method, which imposes a great limitation on the use of rare ginsenoside CK.
The major difference from the rare ginsenosides is the kind and position of glycosyl group according to the structure of the major ginsenosides. At present, the research on the preparation of rare ginsenoside mainly adopts a chemical hydrolysis method and a glycosidase biotransformation method. The well-known chemical hydrolysis method has poor selectivity, low yield, difficult purification and easy environmental pollution. The rare ginsenoside is prepared by converting the specific type of glycosidase generated by organisms, and has the advantages of good selectivity, high yield, few byproducts, no pollution, easiness in large-scale production and the like. Although the biotransformation method requires a complicated route, rare ginsenosides such as CK, which are difficult to obtain by chemical methods, can be obtained.
A great deal of research has been conducted on the production of rare ginsenosides by the conversion of glycosidases produced by organisms, and at present, the main focus has been on the utilization of screening strains or enzymes from microorganisms such as soil microorganisms and fungi to convert main ginsenosides, and the research and optimization of conversion conditions to achieve higher-level production of rare ginsenosides. Hong et al obtain fungi Monascus pilosus KMU103 by screening, and convert ginsenoside in Ginseng radix Rubri with the fungi Monascus pilosus KMU103 to obtain a series of rare ginsenosides, such as Rh1、Rh2、Rg3And the like. Cheng et al screened from soil to Caulobacter leidynia GP45, and found that Rb can be converted by beta-glucosidase in the soil1Is converted into CK. Liu et al found that ginsenoside enzyme (ginsenosidase) in Aspergillus niger belongs to one of glycosidases, and PPD type main ginsenoside Rb can be converted by different ways1、Rb2Conversion of Rc and Rd to C-O, F2C-Mc, CY and CK. Bae and the like adopt intestinal lactobacillus to convert PPD type ginsenoside to prepare ginsenoside CK, and the result shows that bifidobacterium B.minimu is appliedCo-fermentation of m KK-1 and B.choerinum KK-2 to transform Rb1The best effect is achieved, and the conversion rate can reach about 41%. Although there have been reports on the production of ginsenoside CK using the bioconversion method of ginsenoside Rb 1. But the conversion efficiency is low, and the production requirement cannot be met. Rc is one of the varieties with higher content in the main ginsenoside, and how to convert ginsenoside Rc into ginsenoside CK specifically and efficiently is still one of the problems to be solved urgently. There is no report on the research that the glycosidase produced by Bifidobacterium (Bifidobacterium breve 689b) and bacillus subtilis can convert ginsenoside Rc into rare ginsenoside CK in vitro with high efficiency. The method has the advantages of high specificity, low cost, mild reaction conditions, high selectivity and good safety, and is a feasible method for producing the rare ginsenoside CK.
Disclosure of Invention
The invention aims to overcome the technical defects and defects that the existing natural enzyme is difficult to obtain, the single enzyme activity is not high, the glucoside compounds such as ginsenoside and the like have high selectivity and are complex to operate, and the like, and provides a method for preparing rare ginsenoside CK by converting ginsenoside Rc by bacillus subtilis alpha-L-arabinofuranosidase and bifidobacterium beta-glucosidase with optimized colibacillus coexpression codons.
In order to achieve the above object, the technical solution provided by the present invention is:
the method for preparing the rare ginsenoside CK by utilizing gene combination transformation comprises the following steps:
(1) carrying out codon optimization on a gene BsAbfA of alpha-L-arabinofuranosidase from Bacillus subtilis str.168 to obtain a BsAbfA-op gene; carrying out codon optimization on a beta-glucosidase gene BbBgl2 from Bifidobacterium (Bifidobacterium breve 689b) to obtain a BbBgl2-op gene; and an Internal ribosomal-binding site (IRBS) sequence is inserted between the BsAbfA-op gene and the BbBgl2-op gene; wherein, the BsAbfA gene sequence is shown as SEQ ID NO.1, the BsAbfA-op gene sequence is shown as SEQ ID NO.2, the BbBgl2 gene sequence is shown as SEQ ID NO.3, the BbBgl2-op gene sequence is shown as SEQ ID NO.4, and the IRBS sequence is shown as SEQ ID NO. 5;
(2) connecting a gene sequence obtained by inserting an IRBS sequence between a BsAbfA-op gene and a BbBgl2-op gene to a position between BamH I and EcoR I enzyme cutting sites of a pET28a vector to obtain a recombinant expression vector containing the BsAbfA-op and BbBgl2-op genes, wherein the recombinant expression vector is named as pET-BsAbfA-BbBgl2-op, and the gene sequence of the recombinant expression vector pET-BsAbfA-BbBgl2-op is shown as SEQ ID NO. 6;
(3) transforming a recombinant expression vector pET-BsAbfA-BbBgl2-op into escherichia coli BL21, and preparing a recombinant strain containing BsAbfA-op and BbBgl2-op genes through kanamycin screening, PCR and sequencing detection;
(4) culturing the recombinant bacterium obtained in the step (3) at 37 ℃ until OD600 is 0.4-0.6, and inducing with 0.1-1.0mmol/L isopropyl-beta-D-thiogalactoside (IPTG) for 2-10 hours to obtain a recombinant bacterium liquid;
(5) dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 1-50g/L, and mixing the ginsenoside Rc substrate solution with IPTG-induced recombinant bacterial liquid according to volume ratio of 1: (1-10), reacting for 12-24 hours at the temperature of 30-50 ℃ and the pH of 4-6, inactivating in a water bath at the temperature of 70-80 ℃, preferably 80 ℃ for 10-20min, preferably 20min, centrifuging at room temperature (preferably 25 ℃) for 10-15min, preferably 12000rpm for 10-15min, and taking the supernatant to obtain the rare ginsenoside CK.
Preferably, in the step (2), the BsAbfA-op and BbBgl2-op genes are sequentially positioned at the downstream of the T7 promoter of the pET28a vector.
Preferably, in the step (4), the concentration of the fresh cells in the recombinant bacterium liquid is 10-40g/L, and preferably 20 g/L.
The invention also provides a recombinant expression vector for preparing the rare ginsenoside CK, and the gene sequence of the recombinant expression vector is shown in SEQ ID NO.6 and is named as pET-BsAbfA-BbBgl 2-op.
The invention also provides a recombinant bacterium for preparing the rare ginsenoside CK, which is prepared by transforming the recombinant expression vector pET-BsAbfA-BbBgl2-op of claim 4 into escherichia coli BL21, and then carrying out kanamycin screening, PCR and sequencing detection on the escherichia coli BL21 to obtain the recombinant bacterium containing BsAbfA-op and BbBgl2-op genes. The enzyme activity of the enzyme expressed by the recombinant bacteria is higher than that of a single component, and the enzyme property analysis shows that the optimum reaction pH of the co-expressed enzyme is 5.0, the optimum reaction temperature is 40 ℃, the co-expressed enzyme can keep higher activity within the range of pH5-7, the co-expressed enzyme has higher temperature stability within the range of 30-50 ℃, and the enzyme activity is maintained above 85%.
The invention is further illustrated below:
the alpha-L-arabinofuranosidase (BsAbfA) gene is from Bacillus subtilis (168), the beta-glucosidase (BbBgl2) gene is from Bifidobacterium (Bifidobacterium breve 689b), according to the preference of escherichia coli to codon, the structural characteristics of ginsenoside and enzyme-substrate combination simulation data (figure 1 and figure 2), the result shows that BsAbfA and ginsenoside Rc have good combination ability and good selectivity, the BsAbfA and BbBgl2 genes are subjected to codon optimization, the optimized gene sequences BsAbfA-op (SEQ ID N0.2) and BbBgl2-op (SEQ ID N0.4) are obtained by adopting a whole-gene synthesis method, an IRBS sequence is added between the two genes, a BamH I sequence and an EcoR I sequence are respectively added on the downstream of the gene sequences connected in series, and the BamH I sequence and an enzyme digestion site sequence are connected through enzyme digestion and a connecting site, and a ligase is obtained by adopting a whole-gene synthesis method, The method comprises the steps of converting Escherichia coli TOP10 by a calcium chloride method, extracting a recombinant plasmid (pET-BsAbfA-BbBgl2-op) after kanamycin screening, enzyme digestion and sequencing identification, converting Escherichia coli BL21 by the calcium chloride method, and obtaining Escherichia coli capable of efficiently expressing recombinase (beta-glucosidase and alpha-L-arabinofuranosidase) after kanamycin screening. Culturing the recombinant bacteria containing BsAbfA-op and BbBgl2-op genes at 37 ℃ until OD600 is 0.4-0.6, and inducing with isopropyl-beta-D-thiogalactoside (IPTG) of 0.1-1.0mmol/L for 2-10 hours to obtain recombinant bacteria liquid with high activity of two enzyme proteins. The two enzymes produced in the recombinant bacteria are expressed in the same bacteria through BsAbfA-op and BbBgl2-op genes after codon optimization, have higher enzyme activity and are higher than the enzyme activity expressed after co-expression of non-optimized genes (BsAbfA and BbBgl2) or single gene optimization (BsAbfA-op or BbBgl 2-op).
Both beta-glucosidase and alpha-L-arabinofuranosidase belong to glycosidases, and both glycosidases occur in large amounts in nature. However, the functions of beta-glucosidase or alpha-L-arabinofuranosidase from different sources, even from the same source and similar in structure, are significantly different, and the type of glycosidic bond of the substrate and the steric structure of the substrate influence the substrate-converting ability (Biotechnology Lett (2016)38: 1775-1780. Microbiology (2009),155,2739-2749. Process Biochemistry (2010),45, 1226-1235). Meanwhile, the activity of the enzyme and the substrate conversion ability are greatly influenced by the difference of the enzymatic reaction conditions. Therefore, it is difficult to predict the functions of some existing glycosidases based on their activities, and even though some glycosidases may have partial transforming ability in the original cells, their biological activities are significantly different when they are expressed heterologously in other microorganisms. On the one hand, the expression level of heterologous cells is influenced, and on the other hand, the activity of the enzyme expressed heterologously is not necessarily optimal and it is often difficult to perform its specific function.
In the research of producing specific ginsenoside by glycosidase conversion, it is found that few glycosidases are available for converting ginsenoside, and that more rare ones are available for selectively converting ginsenoside Rc to CK. Even a few glycosidases having ginsenoside converting activity are difficult to use due to low activity and the like. Before the present invention, no study on the transformation of ginsenoside Rc by exogenous BsAbfA gene and BbBgl2 was found. Screening and gene structure optimization prove that BsAbfA gene and BbBgl2 gene encoded protein after codon optimization have higher activity of converting ginsenoside Rc, and the BsAbfA-op and BbBgl2-op genes are co-expressed in escherichia coli cells to produce two enzymes, so that ginsenoside Rc can be efficiently converted into Rd, and finally the ginsenoside Rd is converted into rare ginsenoside CK, the recombinant bacteria containing BsAbfA-op and BbBgl2-op genes induced by IPTG react with the ginsenoside Rc for 12 hours, and the conversion efficiency of the Rc to generate the rare ginsenoside CK reaches more than 76%; reacting with ginsenoside Rc for 24 hr, and the conversion efficiency of Rc to rare ginsenoside CK reaches above 85%. The cells have the advantages of easy absorption and the like of the transformed product ginsenoside CK, have unique pharmacological action and important clinical medicinal value, and the method can improve the application value of the medicinal component containing the ginsenoside Rc in the medicinal market.
In conclusion, the invention utilizes the glycosidase produced by Bifidobacterium (Bifidobacterium breve 689b) and bacillus subtilis to convert ginsenoside Rc into rare ginsenoside CK in vitro with high efficiency. The method has the advantages of high specificity, low cost, mild reaction conditions, short conversion period, high catalytic efficiency, good selectivity, good stability, good safety, few byproducts, easy industrial production and the like, and is a feasible method for producing rare ginsenoside.
Drawings
FIG. 1 shows the docking results of ginsenoside Rc and BsAbfA protein molecules;
FIG. 2 shows the major sites of ginsenoside Rc binding to BsAbfA protein molecules;
FIG. 3 shows the results of polyacrylamide gel electrophoresis analysis of crude enzyme solution extracted from recombinant bacteria and purified enzyme under the conditions of example 1; in the figure, 1 represents the crude enzyme solution extracted, 2 and 3 represent BsAbfA and BbBgl2 enzymes separated and purified by nickel column affinity method; m is a protein molecular weight standard;
FIG. 4 shows the results of thin layer chromatography of recombinant bacteria transformed with ginsenoside Rc under the conditions of example 10 and example 14; in the figure, 0h represents that the recombinant bacteria react with the ginsenoside Rc for 0 hour, 12h represents that the recombinant bacteria react with the ginsenoside Rc for 12 hours, 24h represents that the recombinant bacteria react with the ginsenoside Rc for 24 hours, and S represents the ginsenosides Rc, Rd, F2 and CK reference;
FIG. 5 shows the results of High Performance Liquid Chromatography (HPLC) of recombinant bacteria transformed with ginsenoside Rc under the conditions of examples 10 and 14; in the figure, 12h shows that the recombinant bacteria reacted with ginsenoside Rc for 12 hours, 24h shows that the recombinant bacteria reacted with ginsenoside Rc for 24 hours, and S shows ginsenoside Rc, Rd, F2 and CK control.
Detailed Description
Example 1
1. Culturing the recombinant strain by using a Luria-Bertani (LB) liquid culture medium; the preparation method of the LB culture medium comprises the following steps: taking 10g of tryptone, 5g of yeast extract and 10g of NaCl, adding a proper amount of water for dissolving, adjusting the pH to 7.0 by using 5mol/L sodium hydroxide (NaOH) after solute is dissolved, then using deionized water for constant volume to 1L, and performing steam sterilization at 121 ℃ and high pressure for 20 min.
2. The specific method for fermenting and culturing the recombinant bacteria comprises the following steps: inoculating the recombinant bacteria to a solid LB culture medium containing 50 mu g/mL kanamycin, culturing overnight (8-10 hours) at 37 ℃, then selecting a single colony to be inoculated in an LB liquid culture medium containing 50 mu g/mL kanamycin, culturing at 37 ℃ until the absorbance OD600 is 0.4-0.6, adding 0.1-1mmol/L IPTG (isopropyl thiogalactoside) and inducing for 2-10 hours to obtain recombinant bacteria fermentation liquor.
3. Centrifuging the recombinant bacterium fermentation liquor at 4 ℃ and 8000rpm for 10min, removing supernatant, collecting thalli cells, dividing the collected cells into two parts, and adding a fresh liquid LB culture medium into one part until the cell concentration is 20g/L for later use. The other part of the cells was washed 2 times with PBS buffer (pH6.5); crushing for 30min on ice by using an ultrasonic cell crusher, wherein the crushing conditions are as follows: the power is 200W, the ultrasonic time is 2s, the interval time is 3s, the working frequency is 30 times, after the ultrasonic crushing is finished, the mixture is centrifuged at 12000rpm and 4 ℃ for 20min, and the supernatant is taken to obtain the crude enzyme solution. And (3) purifying the crude enzyme solution by using a nickel column, wherein the eluent is phosphate buffer solution with the pH value of 6.8, repeating the steps for a plurality of times, separating to obtain purified recombinant enzyme, and analyzing the molecular weight of the purified recombinant enzyme by using polyacrylamide gel electrophoresis (SDS-PAGE), wherein the molecular weight of BsAbfA-op and BbBgl2-op are respectively about 57kDa and 81kDa (shown in a figure 3).
FIG. 3 shows the results of polyacrylamide gel electrophoresis analysis of crude enzyme solution extracted from recombinant bacteria and purified enzyme under the conditions of example 1; in the figure, 1 represents the crude enzyme solution extracted, 2 and 3 represent BsAbfA and BbBgl2 enzymes separated and purified by nickel column affinity method; m is a protein molecular weight standard; compared with molecular weights of predicted proteins of BsAbfA (sequence numbers AL009126.3, 2938330 and 2939832 in GenBank database) and BbBgl2 (sequence numbers CP006715.1, 1716905 and 1719178 in GenBank database), the results show that the molecular weights of purified BsAbfA and BbBgl2 are consistent with theoretical values (57kDa and 81kDa), respectively.
4. Determination of recombinant enzyme Activity
The Folin-phenol method is used for determining the content of the separated and purified protein and then determining the activity of two recombinases, and the specific method for determining the activity of BsAbfA-op recombinases is as follows: the activity of the enzyme is determined by taking p-nitrobenzene-alpha-L-arabinofuranoside (pNPA) as a substrate and p-nitrophenol (pNP) as a product. 200. mu.L of the recombinant enzyme was added to 200. mu.L of pNPA (10mM), pH6.5, 55 ℃ and incubated for 2 hours, and then an equal volume of 200mM Na was added2CO3pNP was measured at 405 nm. 1 enzyme activity unit (U) was defined as 1. mu. mol pNP released in 1 min. The results showed that alpha-L-arabinofuranosidase KmThe value was 3.45X 10-3The mol/L, alpha-L-arabinofuranosidase has strong catalytic activity.
The specific method for measuring the activity of the BbBgl2-op recombinase comprises the following steps: the enzyme activity was determined using p-nitrophenyl-beta-D-glucoside (pNPG) as substrate and p-nitrophenol (pNP) as product. 200. mu.L of the recombinant enzyme was added to 200. mu.L of pNPG (10mM), pH6.5, 55 ℃ and incubated for 2 hours, followed by addition of an equal volume of 200mM Na2CO3pNP was measured at 405 nm. 1 enzyme activity unit (U) was defined as 1. mu. mol pNP released in 1 min. The results show that beta-glucosidase KmThe value is 1.48X 10-3The mol/L, beta-glucosidase has strong catalytic activity.
Example 2
Preparing a recombinant BL21 bacterial solution containing BsAbfA (SEQ ID N0.1) and BbBgl2(SEQ ID N0.3) genes (neither codon is optimized) and an Internal ribosomal-binding site (IRBS) sequence (SEQ ID N0.5) between the two genes by the culture method in example 1 to prepare a bacterial solution with a cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 3
Preparing a recombinant BL21 bacterial solution containing only BsAbfA (SEQ ID N0.1) gene (codon is not optimized) by the culture method in example 1 to prepare a bacterial solution with a cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 4
Preparing a recombinant BL21 bacterial solution only containing BbBgl2(SEQ ID N0.3) gene (codon is not optimized) by the culture method in the example 1 to prepare a bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 5
Preparing a recombinant BL21 bacterial solution only containing BsAbfA-op (SEQ ID N0.2) gene (codon optimization) by using the culture method in the example 1 to prepare a bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 6
Preparing a recombinant BL21 bacterial solution only containing BbBgl2-op (SEQ ID N0.4) gene (codon optimization) by using the culture method in the example 1 to prepare a bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 7
Recombinant BL21 bacterial fluid containing BsAbfA and BbBgl2 proteins was prepared by the culture method in example 3 and example 4, and bacterial fluid with cell concentration of 40g/L was prepared (mass ratio of two bacterial fluids is 1: 1); dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 8
Preparing a recombinant BL21 bacterial solution containing BsAbfA-op and BbBgl2-op proteins by the culture method in the embodiment 5 and the embodiment 6 to prepare a bacterial solution with the cell concentration of 40g/L (the mass ratio of the two bacterial solutions is 1: 1); dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 9
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:1, reacting at 40 deg.C for 12 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 10
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 12 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 11
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:3, reacting at 40 deg.C for 12 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 12
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:4, reacting at 40 deg.C for 12 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 13
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:1, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 14
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:2, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 15
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:3, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 16
Preparing a recombinant BL21 bacterial solution containing a pET-BsAbfA-BbBgl2-op vector by using the culture method in the embodiment 1, and preparing the bacterial solution with the cell concentration of 40 g/L; dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 40g/L, mixing the ginsenoside Rc solution with recombinant bacteria solution according to volume ratio of 1:4, reacting at 40 deg.C for 24 hr with pH of 5.0, inactivating in 80 deg.C water bath for 20min, centrifuging at 25 deg.C at 10000rpm for 10min, and oven drying the supernatant at 60 deg.C to obtain rare ginsenoside CK.
Example 17
HPLC determination method
The reaction solution of the induced bacteria and the substrate in the examples 2 to 17 and the reaction solution of the substrate without the bacteria solution were centrifuged at 8000rpm at 25 ℃ for 5min, the supernatant was dried at 60 ℃, and then dissolved by ultrasonic waves with an appropriate amount of methanol, respectively, and filtered through 0.45 μm filter membrane, and the volume was determined, and the content was measured by HPLC. The conversion rate of ginsenoside Rc was calculated according to the molar ratio of the reaction substrate to the product, and the calculation results are shown in Table 1.
The HPLC determination conditions are as follows: the chromatographic column is a Diamonsil C18 column (150 mm. times.4.6 mm, 5 μm); the mobile phase is acetonitrile-50 mmol/L dipotassium hydrogen phosphate/potassium dihydrogen phosphate 10: 90(V/V) (phosphoric acid is used for adjusting the pH value to 4.0); the flow rate is 1.0 mL/min; the column temperature was 30 ℃ and the detection wavelength was 203 nm.
In examples 10 and 14, the TLC results of ginsenoside Rc to CK are shown in fig. 4, 0h indicates that the recombinant bacterium reacts with ginsenoside Rc for 0 hour, 12h indicates that the recombinant bacterium reacts with ginsenoside Rc for 12 hours, 24h indicates that the recombinant bacterium reacts with ginsenoside Rc for 24 hours, and S indicates ginsenoside Rc, Rd, F2 and CK control; the results showed that ginsenoside Rc was mostly converted to F2 and CK after 12 hours of reaction, and that conversion of ginsenoside Rc to CK was further improved after 24 hours of reaction.
In examples 10 and 14, the HPLC results of conversion of ginsenoside Rc to CK are shown in fig. 5,12 h indicates that the recombinant bacterium reacted with ginsenoside Rc for 12 hours, 24h indicates that the recombinant bacterium reacted with ginsenoside Rc for 24 hours, and S indicates ginsenoside Rc, Rd, F2 and CK control; the result shows that after the reaction is carried out for 12 hours, the ginsenoside is mainly converted into ginsenoside Rd, F2 and CK, and after the reaction is carried out for 24 hours, the ginsenoside is mostly converted into CK, which indicates that the recombinant bacterium in the invention can lead the ginsenoside Rc to be finally converted into CK through Rd and F2 conversion pathways.
The results of the above examples show that the conversion rate of ginsenoside Rc into CK by the two genetically non-optimized recombinant bacteria used in the invention is only 32.4% (example 2); none of the recombinant bacteria containing the unoptimized or optimized single gene could transform ginsenoside Rc to CK (examples 3-6); the conversion rate of the recombinant bacterial liquid composition containing BsAbfA and BbBgl2 proteins to ginsenoside Rc is very low (39.6%, example 7); the conversion rate of ginsenoside Rc by the recombinant bacteria liquid combined bacteria liquid respectively containing BsAbfA-op and BbBgl2-op proteins is obviously improved (54.1 percent, example 8); the recombinant strain co-expressing codon-optimized BsAbfA-op and BbBgl2-op genes can convert ginsenoside Rc into rare ginsenoside CK, and the conversion rate can reach 70-85%. The method has the advantages of simple operation, low cost, high yield and environmental protection. The method can realize the industrial preparation of the rare ginsenoside CK, can meet the market requirements of the medicine and food industries, and has high market application value.
TABLE 1 analysis of conversion rate of recombinant bacteria to ginsenoside Rc to CK
| CK conversion (%) | |
| Example 2 | 32.4 |
| Example 3 | 0 |
| Example 4 | 0 |
| Example 5 | 0 |
| Example 6 | 0 |
| Example 7 | 39.6 |
| Example 8 | 54.1 |
| Example 9 | 73.2 |
| Example 10 | 76.5 |
| Example 11 | 71.4 |
| Example 12 | 70.3 |
| Example 13 | 82.4 |
| Example 14 | 85.3 |
| Example 15 | 84.4 |
| Example 16 | 80.2 |
Sequence listing
<110> Hunan engineering college
<120> method for preparing rare ginsenoside CK by gene combination transformation and application
<141> 2020-12-10
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1488
<212> DNA
<213> null
<400> 1
atgtctgaac atcaagcagt gattcaaaca gatatcgcaa aaggaaccat taacaaaaat 60
atatacggtc attttgctga gcatttagga agagggattt atgaggggat ctgggtcgga 120
acggactcag acatccccaa tatcaacggg atacgaaagg acgtgctgga ggcgctcaaa 180
cagctgcaca ttcctgtcct taggtggccg ggcgggtgtt ttgcggacga ataccattgg 240
gcaaacggtg tcggtgaccg taagacaatg ctgaacactc actggggcgg tacaattgaa 300
tcaaatgaat tcggaacgca tgaatttatg atgctttgcg agctgcttga atgcgagcca 360
tatatttgcg gcaatgtcgg aagcggaacc gttcaggaaa tgtcggagtg gattgagtat 420
atgacatttg aagaaggcac gccgatgtca gactggagaa agcaaaatgg aagagaagag 480
ccttggaagc tgaaatattt cggcgtgggc aatgaaaact ggggctgcgg cggcaacatg 540
catcccgaat actacgcaga tctgtaccgg cgttttcaga cttatgtccg caattacagc 600
gggaatgaca tttataaaat tgcaggcggc gcaaatgtgg atgattttaa ttggacggac 660
gtgcttatga aaaaagccgc tggcctgatg gacgggttga gtcttcatta ttacacgatt 720
ccgggggatt tctggaaggg caaaggatca gccacagaat tcacggaaga tgaatggttt 780
attacgatga aaaaagccaa atacatcgat gaattgattc aaaaacacgg cacgattatg 840
gaccggtacg atccggagca gcgggtcggg ctgattattg atgaatgggg cacgtggttt 900
gatcccgagc caggcacgaa tcccggtttc ttatatcagc aaaacaccat tcgtgatgca 960
ctggtggcgg cttctcattt ccacattttc catcagcatt gccgccgggt gcaaatggcc 1020
aacatcgccc aaacagtaaa tgttcttcaa gcgatgattt tgactgaggg agagcggatg 1080
cttttgacac cgacgtacca tgtattcaat atgtttaagg tgcaccagga cgcttctctt 1140
ttagccacag agacaatgtc tgccgactat gaatggaacg gtgaaacgct tccgcaaatc 1200
agcatttcag cgtcgaaaca agctgaaggc gatatcaata tcacaatttg caacatcgat 1260
caccaaaaca aagcagaggc ggaaatcgag ctgaggggcc tacacaaggc agcggaccat 1320
tccggagtca ttcttacggc agaaaaaatg aatgcgcata acacgtttga cgatcctcat 1380
catgtcaaac cggaatcctt cagacaatac acgctcagca aaaacaaact gaaagtaaaa 1440
ctcccgccaa tgtcagtcgt cttacttacg ctgcgtgctg attcttaa 1488
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atgaaaaaag cgcgaatgat tgtagacaaa gaatataaaa tcggtgaagt agataaacgg 60
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gatcaccctg aagcggatga agatggattt agaaaagatg tccagtcgct gatcaaagaa 180
ttacaggttc ccatcatccg ctatccgggc ggaaactttt tatccggata caactgggag 240
gacggtgtcg gaccagtcga aaaccgcccg agacggcttg acttggcatg gcaaacgaca 300
gaaaccaatg aagtgggaac aaatgaattt ttatcttggg caaaaaaggt gaacactgag 360
gtcaatatgg ccgtcaacct tggcacaaga ggcatagatg ccgcccgtaa tctcgttgaa 420
tattgcaacc atccgaaagg ctcttactgg agtgatttaa gaagatcgca tggctatgaa 480
cagccgtatg gcatcaaaac atggtgctta ggaaacgaaa tggatggacc atggcagatc 540
ggccacaaaa cagctgatga atacggacgg cttgccgcag agacagcaaa ggtcatgaag 600
tgggttgacc catcaattga actcgttgcc tgcggcagct caaacagcgg tatgccgacc 660
tttatcgatt gggaagcgaa ggtgcttgag catacgtatg agcatgtcga ctatatctct 720
cttcacactt actacggaaa ccgggataac aatctgccaa actacttggc acgttctatg 780
gatttggatc attttatcaa atcagtcgct gcgacctgtg actatgtaaa agcaaaaaca 840
cgcagcaaga aaactatcaa tctctctctg gatgaatgga acgtctggta ccactcaaat 900
gaggctgata aaaaagtcga gccgtggatc actgcgcgtc cgattttaga ggatatttac 960
aattttgaag atgccttatt agtcggctct ctgctcatta cgatgctgca gcacgcagac 1020
cgtgtgaaaa ttgcgtgtct tgcacagctt gttaatgtca tcgcgccgat catgacggaa 1080
aaaggcggag aagcatggag acagccgatt ttctatccat acatgcatgc ttctgtttac 1140
ggaaggggcg agtcactgaa accgcttatt tcttctccta agtacgattg ttctgatttc 1200
actgatgtgc catatgttga tgctgctgtt gtgtactctg aagaggaaga aacactcact 1260
atttttgcgg taaacaaggc tgaggatcag atggagacgg agatttcgct cagaggcttt 1320
gaatcctacc aaatcgcaga gcacatcgta cttgagcatc aggatatcaa agcaacaaac 1380
cagcataaca gaaaaaatgt cgttccgcat tccaacggat catcgtctgt cagcgaaaac 1440
ggcttaactg ctcatttcac gccgctttcc tggaatgtga tccgcctgaa aaaacagtca 1500
taa 1503
<210> 3
<211> 2274
<212> DNA
<213> null
<400> 3
atgagcgagt ccacctaccc gtccgtcaag gatctgaccc tggaagagaa ggcatccctc 60
acctccggcg gcgacgcctg gcatctgcag ggcgtggagt ccaaaggcat cccaggctac 120
atgatcactg acggtcctca cggtctaagg aagtccctgg cctccagcgc cggtgagacc 180
gaccttgacg actccgtgcc cgccacctgc ttcccgccgg ccgccggctt gtccagctct 240
tggaatcctg agctcattca caaggttggt gaggcgatgg ctgaggagtg cattcaggag 300
aaggtggctg tgattcttgg ccccggcgtc aatatcaagc gcaacccgct cggcggtcgc 360
tgcttcgaat actggtccga agacccgtac ttggccggtc atgaggccat cggcattgtt 420
gaaggcgtgc agtccaaggg cgtgggcacg tcgctcaagc acttcgccgc caacaatcag 480
gaaaccgacc gactgcgcgt cgatgcccgc atcagcccgc gcgccctgcg cgagatctac 540
ttcccggcct tcgaacacat cgtcaagaag gcccagccgt ggaccatcat gtgctcctac 600
aaccgcatca acggcgtgca ttccgcacaa aaccattggc tgctgaccga cgtgctgcgc 660
gacgaatggg gcttcgatgg catcgtcatg tccgactggg gcgccgatca tgaccgtggg 720
gcctccctga atgcaggcct gaatctggaa atgccgccga gctacaccga cgaccagatt 780
gtctacgccg tccgcgacgg cctaatcacc cccgcccagc tcgatcgtat ggctcagggc 840
atgattgact tggtaaacaa gacccgcgct gcaatgagta tcgataatta ccgtttcgat 900
gtggacgccc acgatgaagt cgcccatcag gccgctattg aatccattgt gatgctcaag 960
aacgatgatg cgattctgcc gctgaacgcc ggtccagtcg ccaatccgtc cgccacgccc 1020
cagaagatcg ccgttatcgg cgaattcgcc cgcaccccgc gctaccaggg cggcggctcc 1080
tcccacatca cccccaccaa gatgaccagc ttcctcgaca cgctcgccga gcgcggcatc 1140
aaggccgact tcgcccccgg cttcacgctc gatctggaac cggccgaccc ggccctcgaa 1200
tccgaagccg tggaaaccgc caagaacgcc gacgttgtcc tcatgttcct gggcctgccg 1260
gaagccgcgg aatccgaagg cttcgaccgc gacaccctcg acatgcccgc caagcagatc 1320
accctactcg aacaggtcgc cgccgcgaac cagaacgtcg tggtcgtact gtccaacggt 1380
tccgtgatca ccgtggcccc gtgggccaag aacgccaagg gcatcctcga atcctggttg 1440
ctcggccagt ccggtggccc ggcgctcgcc gatgtgatct tcggccaggt cagcccgtcc 1500
ggcaagctcg cccagtcgat tccgctggac atcaacgatg acccgagcat gctgaactgg 1560
ccgggcgagg aaggccacgt cgactacggc gagggcgtgt tcgtcggtta ccgctactac 1620
gacacctacg gcaaggctgt cgactacccg ttcggctacg gcctgagcta cgccacgttc 1680
gagatcaccg gtgtcgccgt tgccaagacc ggcgcgaaca ccgccaccgt gaatgccact 1740
gtgaccaaca cctccgatgt ggacgctgcc gaaaccgtgc aggtgtacgt tgtgccgggc 1800
aaggccgacg tggctcgccc gaagcacgag ctcaagggct tcaccaaggt gttcctcaag 1860
tccggtgagt ccaagaccgt gaccatcgac ctcgacgagc gcgcgttcgc ctactggtcc 1920
gaaaagtaca acgactggca cgtggaggcc ggcgaatatg ccatcgaagt gggcgtgagc 1980
tcccgcgaca ttgccgacac cgttgccgtg gccctcgatg gcgacggcaa gacccagccg 2040
ctcaccgaat ggtccaccta cggcgagtgg gaggccgatc cgttcggcgc caagatcgtg 2100
gccgccgtgg ccgccgccgg cgaggccggc gagctgccga agcttccgga taatgcgatg 2160
atgcgcatgt tcctcaactc catgcccatc aactcgctgc ccaccctgct gggcgaaggc 2220
ggcaagaaga tcgcccagtt catggttgac gagtacgcca agctgagcaa gtaa 2274
<210> 4
<211> 2274
<212> DNA
<213> null
<400> 4
atgagcgaaa gcacctatcc gagcgtgaag gacctgaccc tggaggaaaa agcgagcctg 60
accagcggtg gcgatgcgtg gcacctgcaa ggtgttgaga gcaagggcat cccgggttac 120
atgattaccg acggcccgca cggtctgcgt aaaagcctgg cgagcagcgc gggtgaaacc 180
gatctggacg atagcgttcc ggcgacctgc ttcccgccgg cggcgggtct gagcagcagc 240
tggaacccgg agctgatcca caaagttggc gaagcgatgg cggaggaatg catccaggaa 300
aaggtggcgg ttattctggg cccgggtgtt aacatcaaac gtaacccgct gggtggccgt 360
tgcttcgagt attggagcga agacccgtac ctggcgggtc atgaggcgat cggcattgtg 420
gaaggtgttc aaagcaaggg cgtgggtacc agcctgaaac actttgcggc gaacaaccag 480
gagaccgacc gtctgcgtgt tgatgcgcgt attagcccgc gtgcgctgcg tgagatctat 540
ttcccggcgt ttgaacacat tgtgaagaaa gcgcaaccgt ggaccattat gtgcagctac 600
aaccgtatca acggtgttca cagcgcgcag aaccactggc tgctgaccga cgtgctgcgt 660
gatgagtggg gcttcgacgg tatcgttatg agcgattggg gcgcggacca tgatcgtggt 720
gcgagcctga acgcgggcct gaacctggaa atgccgccaa gctataccga cgatcaaatc 780
gtgtacgcgg ttcgtgacgg tctgattacc ccggcgcagc tggaccgtat ggcgcaaggc 840
atgatcgatc tggtgaacaa aacccgtgcg gcgatgagca ttgacaacta tcgttttgac 900
gtggatgcgc atgatgaggt tgcgcaccag gcggcgatcg aaagcattgt tatgctgaag 960
aacgacgatg cgatcctgcc gctgaacgcg ggtccggtgg cgaacccgag cgcgaccccg 1020
caaaaaatcg cggttattgg cgagttcgcg cgtaccccgc gttaccaggg tggcggtagc 1080
agccacatca ccccgaccaa gatgaccagc tttctggaca ccctggcgga acgtggtatt 1140
aaagcggatt ttgcgccggg ttttaccctg gacctggagc cggcggaccc ggcgctggag 1200
agcgaagcgg tggaaaccgc gaagaacgcg gacgtggttc tgatgtttct gggtctgccg 1260
gaggcggcgg agagcgaagg ctttgaccgt gataccctgg atatgccggc gaaacagatc 1320
accctgctgg aacaagttgc ggcggcgaac cagaacgtgg ttgtggttct gagcaacggt 1380
agcgtgatca ccgttgcgcc gtgggcgaag aacgcgaaag gtattctgga gagctggctg 1440
ctgggtcaaa gcggcggtcc ggcgctggcg gacgtgattt tcggtcaagt tagcccgagc 1500
ggcaagctgg cgcagagcat cccgctggac attaacgacg atccgagcat gctgaactgg 1560
ccgggcgagg aaggtcacgt ggattatggc gagggtgtgt ttgttggtta tcgttactat 1620
gacacctacg gcaaggcggt tgattacccg ttcggctatg gtctgagcta cgcgaccttt 1680
gaaatcaccg gtgtggcggt tgcgaaaacc ggtgcgaaca ccgcgaccgt gaacgcgacc 1740
gttaccaaca ccagcgatgt ggatgcggcg gagaccgtgc aggtttacgt ggttccgggc 1800
aaggcggacg ttgcgcgtcc gaagcacgag ctgaaaggtt tcaccaaggt gtttctgaaa 1860
agcggcgaaa gcaagaccgt taccattgac ctggatgagc gtgcgttcgc gtattggagc 1920
gaaaaataca acgattggca cgtggaggcg ggtgaatatg cgatcgaagt gggcgttagc 1980
agccgtgaca ttgcggatac cgtggcggtt gcgctggatg gtgatggtaa aacccaaccg 2040
ctgaccgaat ggagcaccta cggcgagtgg gaagcggacc cgtttggcgc gaagatcgtt 2100
gcggcggttg cggcggcggg tgaagcgggt gaactgccga aactgccgga taacgcgatg 2160
atgcgtatgt tcctgaacag catgccgatc aacagcctgc cgaccctgct gggcgagggc 2220
ggtaagaaaa ttgcgcagtt tatggttgac gaatacgcga agctgagcaa ataa 2274
<210> 5
<211> 36
<212> DNA
<213> null
<400> 5
aataattttg tttaacttta agaaggagat atacat 36
<210> 6
<211> 9182
<212> DNA
<213> null
<400> 6
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccatgaaaaa agcgcgaatg attgtagaca aagaatataa aatcggtgaa 5220
gtagataaac ggatttatgg ctcgtttatc gaacatatgg gtcgtgcggt atatgaaggc 5280
atatacgagc ctgatcaccc tgaagcggat gaagatggat ttagaaaaga tgtccagtcg 5340
ctgatcaaag aattacaggt tcccatcatc cgctatccgg gcggaaactt tttatccgga 5400
tacaactggg aggacggtgt cggaccagtc gaaaaccgcc cgagacggct tgacttggca 5460
tggcaaacga cagaaaccaa tgaagtggga acaaatgaat ttttatcttg ggcaaaaaag 5520
gtgaacactg aggtcaatat ggccgtcaac cttggcacaa gaggcataga tgccgcccgt 5580
aatctcgttg aatattgcaa ccatccgaaa ggctcttact ggagtgattt aagaagatcg 5640
catggctatg aacagccgta tggcatcaaa acatggtgct taggaaacga aatggatgga 5700
ccatggcaga tcggccacaa aacagctgat gaatacggac ggcttgccgc agagacagca 5760
aaggtcatga agtgggttga cccatcaatt gaactcgttg cctgcggcag ctcaaacagc 5820
ggtatgccga cctttatcga ttgggaagcg aaggtgcttg agcatacgta tgagcatgtc 5880
gactatatct ctcttcacac ttactacgga aaccgggata acaatctgcc aaactacttg 5940
gcacgttcta tggatttgga tcattttatc aaatcagtcg ctgcgacctg tgactatgta 6000
aaagcaaaaa cacgcagcaa gaaaactatc aatctctctc tggatgaatg gaacgtctgg 6060
taccactcaa atgaggctga taaaaaagtc gagccgtgga tcactgcgcg tccgatttta 6120
gaggatattt acaattttga agatgcctta ttagtcggct ctctgctcat tacgatgctg 6180
cagcacgcag accgtgtgaa aattgcgtgt cttgcacagc ttgttaatgt catcgcgccg 6240
atcatgacgg aaaaaggcgg agaagcatgg agacagccga ttttctatcc atacatgcat 6300
gcttctgttt acggaagggg cgagtcactg aaaccgctta tttcttctcc taagtacgat 6360
tgttctgatt tcactgatgt gccatatgtt gatgctgctg ttgtgtactc tgaagaggaa 6420
gaaacactca ctatttttgc ggtaaacaag gctgaggatc agatggagac ggagatttcg 6480
ctcagaggct ttgaatccta ccaaatcgca gagcacatcg tacttgagca tcaggatatc 6540
aaagcaacaa accagcataa cagaaaaaat gtcgttccgc attccaacgg atcatcgtct 6600
gtcagcgaaa acggcttaac tgctcatttc acgccgcttt cctggaatgt gatccgcctg 6660
aaaaaacagt cataaaataa ttttgtttaa ctttaagaag gagatataca tatgagcgaa 6720
agcacctatc cgagcgtgaa ggacctgacc ctggaggaaa aagcgagcct gaccagcggt 6780
ggcgatgcgt ggcacctgca aggtgttgag agcaagggca tcccgggtta catgattacc 6840
gacggcccgc acggtctgcg taaaagcctg gcgagcagcg cgggtgaaac cgatctggac 6900
gatagcgttc cggcgacctg cttcccgccg gcggcgggtc tgagcagcag ctggaacccg 6960
gagctgatcc acaaagttgg cgaagcgatg gcggaggaat gcatccagga aaaggtggcg 7020
gttattctgg gcccgggtgt taacatcaaa cgtaacccgc tgggtggccg ttgcttcgag 7080
tattggagcg aagacccgta cctggcgggt catgaggcga tcggcattgt ggaaggtgtt 7140
caaagcaagg gcgtgggtac cagcctgaaa cactttgcgg cgaacaacca ggagaccgac 7200
cgtctgcgtg ttgatgcgcg tattagcccg cgtgcgctgc gtgagatcta tttcccggcg 7260
tttgaacaca ttgtgaagaa agcgcaaccg tggaccatta tgtgcagcta caaccgtatc 7320
aacggtgttc acagcgcgca gaaccactgg ctgctgaccg acgtgctgcg tgatgagtgg 7380
ggcttcgacg gtatcgttat gagcgattgg ggcgcggacc atgatcgtgg tgcgagcctg 7440
aacgcgggcc tgaacctgga aatgccgcca agctataccg acgatcaaat cgtgtacgcg 7500
gttcgtgacg gtctgattac cccggcgcag ctggaccgta tggcgcaagg catgatcgat 7560
ctggtgaaca aaacccgtgc ggcgatgagc attgacaact atcgttttga cgtggatgcg 7620
catgatgagg ttgcgcacca ggcggcgatc gaaagcattg ttatgctgaa gaacgacgat 7680
gcgatcctgc cgctgaacgc gggtccggtg gcgaacccga gcgcgacccc gcaaaaaatc 7740
gcggttattg gcgagttcgc gcgtaccccg cgttaccagg gtggcggtag cagccacatc 7800
accccgacca agatgaccag ctttctggac accctggcgg aacgtggtat taaagcggat 7860
tttgcgccgg gttttaccct ggacctggag ccggcggacc cggcgctgga gagcgaagcg 7920
gtggaaaccg cgaagaacgc ggacgtggtt ctgatgtttc tgggtctgcc ggaggcggcg 7980
gagagcgaag gctttgaccg tgataccctg gatatgccgg cgaaacagat caccctgctg 8040
gaacaagttg cggcggcgaa ccagaacgtg gttgtggttc tgagcaacgg tagcgtgatc 8100
accgttgcgc cgtgggcgaa gaacgcgaaa ggtattctgg agagctggct gctgggtcaa 8160
agcggcggtc cggcgctggc ggacgtgatt ttcggtcaag ttagcccgag cggcaagctg 8220
gcgcagagca tcccgctgga cattaacgac gatccgagca tgctgaactg gccgggcgag 8280
gaaggtcacg tggattatgg cgagggtgtg tttgttggtt atcgttacta tgacacctac 8340
ggcaaggcgg ttgattaccc gttcggctat ggtctgagct acgcgacctt tgaaatcacc 8400
ggtgtggcgg ttgcgaaaac cggtgcgaac accgcgaccg tgaacgcgac cgttaccaac 8460
accagcgatg tggatgcggc ggagaccgtg caggtttacg tggttccggg caaggcggac 8520
gttgcgcgtc cgaagcacga gctgaaaggt ttcaccaagg tgtttctgaa aagcggcgaa 8580
agcaagaccg ttaccattga cctggatgag cgtgcgttcg cgtattggag cgaaaaatac 8640
aacgattggc acgtggaggc gggtgaatat gcgatcgaag tgggcgttag cagccgtgac 8700
attgcggata ccgtggcggt tgcgctggat ggtgatggta aaacccaacc gctgaccgaa 8760
tggagcacct acggcgagtg ggaagcggac ccgtttggcg cgaagatcgt tgcggcggtt 8820
gcggcggcgg gtgaagcggg tgaactgccg aaactgccgg ataacgcgat gatgcgtatg 8880
ttcctgaaca gcatgccgat caacagcctg ccgaccctgc tgggcgaggg cggtaagaaa 8940
attgcgcagt ttatggttga cgaatacgcg aagctgagca aataagaatt cgagctccgt 9000
cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 9060
caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc 9120
ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg 9180
at 9182
Claims (8)
1. A method for preparing rare ginsenoside CK by utilizing gene combination transformation is characterized by comprising the following steps:
(1) carrying out codon optimization on a gene BsAbfA of alpha-L-arabinofuranosidase from Bacillus subtilis str.168 to obtain a BsAbfA-op gene; carrying out codon optimization on a beta-glucosidase gene BbBgl2 from Bifidobacterium (Bifidobacterium breve 689b) to obtain a BbBgl2-op gene; an IRBS sequence is inserted between the BsAbfA-op gene and the BbBgl2-op gene; wherein, the BsAbfA gene sequence is shown as SEQ ID NO.1, the BsAbfA-op gene sequence is shown as SEQ ID NO.2, the BbBgl2 gene sequence is shown as SEQ ID NO.3, the BbBgl2-op gene sequence is shown as SEQ ID NO.4, and the IRBS sequence is shown as SEQ ID NO. 5;
(2) connecting a gene sequence obtained by inserting an IRBS sequence between a BsAbfA-op gene and a BbBgl2-op gene to a position between BamH I and EcoR I enzyme cutting sites of a pET28a vector to obtain a recombinant expression vector containing the BsAbfA-op and BbBgl2-op genes, wherein the recombinant expression vector is named as pET-BsAbfA-BbBgl2-op, and the gene sequence of the recombinant expression vector pET-BsAbfA-BbBgl2-op is shown as SEQ ID NO. 6;
(3) transforming a recombinant expression vector pET-BsAbfA-BbBgl2-op into escherichia coli BL21, and preparing a recombinant strain containing BsAbfA-op and BbBgl2-op genes through kanamycin screening, PCR and sequencing detection;
(4) culturing the recombinant bacterium obtained in the step (3) at 37 ℃ until OD600 is 0.4-0.6, inducing with 0.1-1.0mmol/L IPTG for 2-10 hours, centrifuging, and adding fresh LB culture medium to prepare recombinant bacterium liquid;
(5) dissolving ginsenoside Rc in methanol, mixing with acetic acid-sodium acetate buffer solution with pH of 4.0-6.0 to prepare ginsenoside Rc substrate solution with concentration of 1-50g/L, mixing the ginsenoside Rc substrate solution with IPTG induced recombinant bacteria solution according to volume ratio of 1 (1-10), reacting at pH of 4-6 and 30-50 deg.C for 12-24 hr, inactivating in 70-80 deg.C water bath for 10-20min, centrifuging at room temperature for 10-15min, and collecting supernatant to obtain rare ginsenoside CK.
2. The method for preparing rare ginsenoside CK using gene combination transformation as claimed in claim 1, wherein in the step (2), BsAbfA-op and BbBgl2-op genes are sequentially located at the downstream of T7 promoter of pET28a vector.
3. The method for preparing a rare ginsenoside CK using gene combination transformation as claimed in claim 1, wherein in the step (4), the fresh cell concentration in the recombinant bacterium solution is 10-40 g/L.
4. A recombinant expression vector is characterized in that the gene sequence of the recombinant expression vector is shown in SEQ ID NO.6 and is named as pET-BsAbfA-BbBgl 2-op.
5. The use of the recombinant expression vector of claim 4 in the preparation of rare ginsenoside CK.
6. A recombinant bacterium is characterized in that the recombinant bacterium is a recombinant bacterium containing BsAbfA-op and BbBgl2-op genes, which is prepared by transforming the recombinant expression vector pET-BsAbfA-BbBgl2-op of claim 4 into escherichia coli BL21 and then carrying out kanamycin screening, PCR and sequencing detection.
7. The recombinant strain as claimed in claim 6, wherein the enzyme activity of the enzyme expressed by the recombinant strain is maintained to be more than 85% under the conditions of pH5-7 and 30-50 ℃.
8. The use of the recombinant bacterium of claim 6 in the preparation of rare ginsenoside CK.
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| CN114395563A (en) * | 2022-02-11 | 2022-04-26 | 湖南工程学院 | PgABCG11 gene for regulating JA-Ile transport in ginseng cell and encoding protein and application thereof |
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| KIM T.H.等: "Enhanced Production of β-D-glycosidase and α-L-arabinofuranosidase in Recombinant Escherichia coli in Fed-batch Culture for the Biotransformation of Ginseng Leaf Extract to Ginsenoside Compound K", 《BIOTECHNOLOGY AND BIOPROCESS ENGINEERING》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410321A (en) * | 2020-11-26 | 2021-02-26 | 昆明理工大学 | Beta-glucosidase Ttbgl3 and application thereof |
| CN112410321B (en) * | 2020-11-26 | 2022-01-28 | 昆明理工大学 | Beta-glucosidase Ttbgl3 and application thereof |
| CN114395563A (en) * | 2022-02-11 | 2022-04-26 | 湖南工程学院 | PgABCG11 gene for regulating JA-Ile transport in ginseng cell and encoding protein and application thereof |
| CN114395563B (en) * | 2022-02-11 | 2023-06-16 | 湖南工程学院 | PgABCG11 gene for regulating and controlling JA-Ile transport in ginseng cells, and encoding protein and application thereof |
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