Recombinant Escherichia coli for expressing cholyl glycine hydrolase and application of recombinant Escherichia coli
By introducing the optimized Bifidobacterium breve CGH gene into Escherichia coli, the problems of low CGH fermentation level and activity were solved, and efficient expression and high-activity CGH production were achieved.
Patent Information
- Application Number
- CN202510791604.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-09-09
AI Technical Summary
The fermentation level and activity of cholylglycine hydrolase (CGH) expressed by existing Escherichia coli are low, which cannot meet the current needs.
The CGH encoding gene from Bifidobacterium breve was introduced into Escherichia coli, and the preferred codons were optimized to construct recombinant Escherichia coli, and highly active CGH was expressed through fermentation.
High yield and high activity of CGH were achieved, with an expression yield of 135 mg/L and an enzyme activity of 1186 U/mg.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of bile ylglycine hydrolase production, in particular to a recombinant Escherichia coli expressing bile ylglycine hydrolase and an application thereof. Background Art
[0002] Choloylglycine hydrolase (CGH), also known as glycocholate hydrolase, is a member of the bile salt hydrolase (BSH) family. CGH plays a key role in bile acid metabolism, catalyzing the hydrolysis of cholylglycine (glycocholate). This enzyme plays a crucial role in maintaining bile acid homeostasis, lipid metabolism, and intestinal microbial ecology.
[0003] CGH plays a wide and profound role in human physiology. Within the intestinal microecological environment, it influences the acid-base balance by regulating bile acid composition and concentration. The production of free bile acids lowers intestinal pH. This acidic environment inhibits the growth of some harmful bacteria while promoting the proliferation of probiotics, thereby reshaping the intestinal microbiome and maintaining microecological homeostasis. In metabolic regulation, secondary bile acids produced by CGH, such as deoxycholic acid and lithocholic acid, act as ligands to activate the farnesoid X receptor (FXR) and pregnane X receptor (PXR). Activation of these nuclear receptors modulates the expression of a range of genes related to glucose and lipid metabolism and energy balance, significantly influencing physiological processes such as insulin sensitivity and lipid synthesis and degradation. Furthermore, in terms of immune regulation, free bile acids produced by hydrolysis enhance the expression of tight junction proteins between intestinal epithelial cells, strengthening the intestinal barrier function and preventing pathogen invasion. They also regulate the differentiation and activity of immune cells, participating in the regulation of inflammatory responses and playing a key role in maintaining intestinal immune homeostasis.
[0004] Currently, methods for producing CGH primarily include fermentation using wild microbial strains and fermentation using genetically engineered strains. Fermentation using wild microbial strains, such as Lactobacillus, Coprococcus, and Escherichia coli, offers the advantage of requiring less culturing of wild strains. However, it suffers from low enzyme activity. While some studies have employed strain mutagenesis to increase enzyme production, overall, these levels remain insufficient.
[0005] Genetic engineering methods primarily involve expressing CGH genes from other sources in E. coli. Several studies have also investigated targeted mutagenesis of CGH genes to enhance enzyme activity, thermostability, or alkaline resistance. While these methods can significantly improve CGH fermentation levels or alter certain enzyme properties, current CGH fermentation levels and enzyme activity still fall short of current needs. Research continues on the pursuit of highly efficient and highly active CGH expression. Summary of the Invention
[0006] In order to solve the technical problems of low fermentation level and low activity of cholylglycine hydrolase (CGH) expressed in existing Escherichia coli, the present invention provides a recombinant Escherichia coli expressing CGH and application thereof.
[0007] The specific technical solutions of the present invention are: In the first aspect, the present invention provides a recombinant Escherichia coli expressing bile ylglycine hydrolase, which carries a gene encoding bile ylglycine hydrolase. bsh The amino acid sequence of the bile ylglycine hydrolase is shown in SEQ ID No. 1.
[0008] The present invention is achieved by Bifidobacterium breve The gene encoding a CGH derived from Bifidobacterium breve was introduced into Escherichia coli to produce recombinant E. coli. After fermentation, highly active CGH was isolated from the recombinant E. coli. This approach overcomes the low activity of CGHs expressed in conventional E. coli. The amino acid sequence of the CGH derived from Bifidobacterium breve is shown in SEQ ID No. 1.
[0009] As the preferred embodiment of the above-mentioned recombinant Escherichia coli, bsh The nucleotide sequence of the gene is shown in SEQ ID No.2.
[0010] The coding gene of CGH derived from Bifidobacterium breve was optimized by preferred codons to obtain the nucleotide sequence shown in SEQ ID No. 2 bsh Gene. bsh The gene was introduced into recombinant Escherichia coli for fermentation expression, and the yield of expressed CGH reached 135 mg / L, and the activity of the prepared CGH reached 1186 U / mg.
[0011] As a preferred embodiment of the above-mentioned recombinant Escherichia coli, the recombinant Escherichia coli is Escherichia coli BL21 (DE3).
[0012] In a second aspect, the present invention provides a method for constructing a recombinant Escherichia coli expressing bile ylglycine hydrolase, comprising the following steps: Step (S.1), connecting the gene encoding the cholylglycine hydrolase having the amino acid sequence shown in SEQ ID No. 1 to an expression vector to construct a CGH expression vector; Step (S.2): transform the CGH expression vector into Escherichia coli BL21 (DE3) cells to obtain recombinant Escherichia coli, which is recorded as recombinant Escherichia coli BSH.
[0013] As a preferred embodiment of the above construction method, the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.
[0014] As a preferred embodiment of the above construction method, step (S.1) is: (a) Introduce EcoRI restriction enzyme sequence at the 5′-end of SEQ ID No.2, remove the stop codon at the 3′-end and introduce Xho I enzyme cleavage sequence, and then chemically synthesize the DNA nucleotide sequence; (b) The DNA nucleotide sequence obtained in step (a) and the expression vector pET-28a(+) were double-digested with EcoRI and XhoI, respectively, to obtain bsh Enzyme digestion products of gene fragments and expression vector pET-28a(+); (c) bsh The gene fragment was connected with the enzyme-digested product of the expression vector pET-28a(+), and then transformed into Escherichia coli DH5α competent cells to obtain the recombinant plasmid pET-28a-bsh.
[0015] As a preferred embodiment of the above construction method, the ligation is carried out using T4 DNA ligase.
[0016] As a preferred embodiment of the above construction method, the transformation adopts a heat shock method.
[0017] In a third aspect, the present invention provides the use of the above-mentioned recombinant Escherichia coli in the fermentation production of bileylglycine hydrolase.
[0018] Preferably, the application method comprises the following steps: inoculating the recombinant Escherichia coli and fermenting and culturing to obtain bile ylglycine hydrolase, wherein the fermentation and culturing temperature is 25-40°C.
[0019] More specifically, the application method comprises the following steps: After seed expansion, the recombinant E. coli BSH was inoculated into the expression medium containing 50 μg / mL kanamycin at an inoculum concentration of 4% to 6% by volume, and cultured at 35-37°C and 220-240 rpm for 4-6 h until the OD 600= 1.17-1.24, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5-0.75 mmol / L, and induce the culture at 25-28°C and 150-180 rpm for 10-14 h until OD 600 = 8.63-11.4, obtaining a culture medium containing E. coli BSH, which was then separated and purified to obtain CGH. The final concentration of the expression medium is: yeast extract powder 20-30 g / L, tryptone 10-14 g / L, glycerol 4-6 g / L, KH2PO4 2-3 g / L, K2HPO4 8-10 g / L, the solvent is deionized water, pH 6.8-7.0.
[0020] Furthermore, the recombinant E. coli seed expansion culture method is as follows: E. coli BSH cells frozen in 20% glycerol aqueous solution are streaked onto LB slant medium containing 50 μg / mL kanamycin and cultured at 37°C for 20-24 hours. 2-3 loops of cells from the slant culture are picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 35-37°C and 150-200 rpm for 12-14 hours until the OD reaches 0. 600 =3.54-3.76, obtaining seed solution for expanded culture; the LB liquid medium is composed of: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L NaCl, and deionized water as the solvent, with a pH of 7.0-7.2. The Escherichia coli BSH cells frozen in the 20% glycerol aqueous solution are cultured in LB liquid medium containing 50 μg / mL kanamycin at 37°C and 200 rpm for 12 hours, then mixed with an equal volume of 40% glycerol aqueous solution and stored in a -80°C freezer.
[0021] Furthermore, the method for isolating and purifying CGH is as follows: centrifuging the Escherichia coli BSH culture medium (preferably at 4°C, 8000×g for 5-10 min), collecting the bacteria and resuspending them in a binding buffer, disrupting the cells by ultrasonication, and then centrifuging again (preferably at 4°C, 8000×g for 5-10 min), collecting the supernatant, and purifying the supernatant by Ni-NTA column chromatography, dialyzing, concentrating, and freeze-drying to obtain CGH; the binding buffer composition is: 50 mmol / LNa2HPO4, 300 mmol / L NaCl, and the pH is adjusted to 8.0 with 1 mol / L NaOH; the volume of the binding buffer is 4-5 mL / g based on the wet weight of the bacteria.
[0022] Furthermore, the ultrasonic cell disruption conditions are: in an ice water bath, power 200 W, working for 3 s, interval 3 s, working 200 times.
[0023] Furthermore, the supernatant was purified by Ni-NTA column chromatography, dialyzed, concentrated, and freeze-dried. The specific method was as follows: the Ni-NTA column was rinsed with distilled water and pre-equilibrated with 3 to 4 column volumes of binding buffer. The cell lysis supernatant after centrifugation was injected at a flow rate of 1 to 2 mL / min. After the injection, the column was washed with 5 to 10 column volumes of washing buffer until the outflow of washing buffer A 280 ≈0, and then eluted with elution buffer until the A 280 ≈0, and all the eluate was collected; the eluate was placed in a dialysis bag (molecular weight cutoff of 15 kDa), and dialyzed with binding buffer to remove imidazole. The dialyzate was transferred to an ultrafiltration tube (molecular weight cutoff of 10 kDa) and centrifuged and concentrated. The concentrate was freeze-dried at -80°C and a vacuum of 50 Pa to obtain CGH freeze-dried enzyme powder; the washing buffer was the binding buffer supplemented with imidazole at a final concentration of 5 mmol / L; the elution buffer was the binding buffer supplemented with imidazole at a final concentration of 40 mmol / L.
[0024] Furthermore, the specific steps of preparing CGH by recombinant E. coli include: (1) Escherichia coli BSH cells preserved in 20% glycerol aqueous solution were inoculated into LB slant medium containing 50 μg / mL kanamycin and cultured at 37°C for 20-24 h to obtain slant cells; the final concentration of the LB slant medium was as follows: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L, the solvent was deionized water, pH 7.0-7.2, and sterilized by high-pressure steam at 121°C for 20 min; (2) Pick 2-3 loops of the slant bacteria from step (1) and inoculate them into LB liquid culture medium containing 50 μg / mL kanamycin. Incubate at 35-37°C and 150-200 r / min in a shaking incubator for 12-14 h until the OD reaches 600 =3.54~3.76 seed solution; (3) According to the inoculum volume fraction of 4% to 6%, the seed solution prepared in step (2) was inoculated into the expression medium containing 50 μg / mL kanamycin, and cultured in a shaking incubator at 35-37°C and 220-240 r / min for 4-6 h until the OD 600 =1.17~1.24, add IPTG with a final concentration of 0.5~0.75 mmol / L as an inducer, and culture in a shaking incubator at 25~28℃ and 150~180 r / min for 10~14 h to obtain the OD 600 =8.63~11.4 culture medium; (4) The culture medium of step (3) was centrifuged at 4°C and 8000 × g for 5-10 min to collect the cells, and 4-5 mL of binding buffer was added to each gram of wet cells to resuspend them. The cells were ultrasonically disrupted in an ice-water bath (power 200 W, working for 3 s, interval 3 s, working 200 times), and the cell disruption solution was centrifuged at 4°C and 8000 × g for 5-10 min, and the supernatant was collected; (5) After the Ni-NTA column is rinsed with distilled water, it is pre-equilibrated with 3 to 4 column volumes of binding buffer. The supernatant of the cell lysate after centrifugation in step (4) is injected at a flow rate of 1 to 2 mL / min. After the injection is completed, the column is washed with 5 to 10 column volumes of washing buffer until the outflow of washing buffer A 280 ≈0; then use elution buffer to elute to the A of the effluent 280 The eluate was collected and placed in a dialysis bag (molecular weight cutoff: 15 kDa) for dialysis to remove imidazole. The dialysate was concentrated by centrifugation in an ultrafiltration tube (molecular weight cutoff: 10 kDa). The concentrate was placed in a culture dish and freeze-dried to obtain lyophilized CGH powder.
[0025] Compared with the prior art, the present invention has the following technical effects: 1. The present invention introduces a gene encoding a CGH derived from Bifidobacterium breve into Escherichia coli to produce recombinant E. coli. After fermentation, highly active CGH is isolated from the recombinant E. coli. This solves the problem of low activity of CGH expressed in existing E. coli cultures. The amino acid sequence of the CGH derived from Bifidobacterium breve is shown in SEQ ID No. 1.
[0026] 2. The coding gene of CGH derived from Bifidobacterium breve was optimized with preferred codons to obtain the nucleotide sequence shown in SEQ ID No. 2 bsh Gene. bsh The gene was introduced into recombinant Escherichia coli for fermentation expression, and the yield of expressed CGH reached 135 mg / L, and the activity of the prepared CGH reached 1186 U / mg. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the map of the recombinant plasmid pET-28a-bsh.
[0028] Figure 2 The electrophoresis diagram of the recombinant plasmid pET-28a-bsh after double enzyme digestion, where M is: Marker; N is: bsh sequence.
[0029] Figure 3This is the SDS-PAGE electrophoresis diagram of E. coli GD strain expressing CGH, where M is Marker; 1 is the negative control of E. coli BL21 carrying pET-28a(+); and 2 is the cell lysate of E. coli BSH strain.
[0030] Figure 4 is the glycine concentration—A 570 Standard curve. DETAILED DESCRIPTION
[0031] The present invention will be further described below with reference to the following embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0032] In the embodiments of the present invention, the LB plate medium has a final concentration and composition and preparation method of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and deionized water as the solvent, pH 7.0. The LB plate medium is placed in a conical flask, sealed with 8 layers of gauze, sterilized by high-pressure steam at 121°C for 20 minutes, and poured into 9 cm diameter sterile Petri dishes before solidification, with 15-20 mL per dish. The LB slant medium has a final concentration and composition similar to the plate medium. 4 mL of LB slant medium is placed in a test tube, sealed with a silicone stopper, sterilized by high-pressure steam at 121°C for 20 minutes, and set aside to form a slant before solidification. The final concentration and composition of the LB liquid medium is similar to the LB plate medium, except that the agar is not present. 25 mL of LB liquid medium is placed in a 150 mL conical flask, sealed with 8 layers of gauze, and sterilized by high-pressure steam at 121°C for 20 minutes.
[0033] In the examples of the present invention, kanamycin was added to the LB slant medium, LB plate medium, LB liquid medium, and expression medium used to culture recombinant Escherichia coli before inoculation at a final concentration of 50 μg / mL. Kanamycin was added in the form of an aqueous solution at a concentration of 50 mg / mL.
[0034] In the embodiment of the present invention, the E. coli culture solution OD 600 , is the absorbance measured at a wavelength of 600 nm with the uninoculated culture medium as a reference; for example, the OD value of the undiluted culture medium is 600 If it is greater than 1.5, dilute it appropriately with deionized water and then multiply the absorbance by the dilution factor.
[0035] Example 1 Construction of recombinant Escherichia coli expressing CGH The recombinant E. coli expressing CGH of the present invention is constructed according to the following steps: (1) According to the NCBI database provided by Bifidobacterium breve ( Bifidobacterium breve ) of CGH gene sequence (Gene symbol: bsh ; Gene ID: 29241525), using GenSmart TM (https: / / www.genscript.com / tools / gensmart-codon-optimization) to optimize the preferred codons of E. coli. The codon-optimized DNA sequence is shown in SEQ ID No.2. bsh The CGH amino acid sequence encoded by the gene is shown in SEQ ID No. 1. Introduced upstream of the SEQ ID No. 2 sequence BamH I restriction enzyme site (GGATCC), downstream removal of the stop codon and introduction Xho I restriction enzyme site (CTCGAG), the DNA sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd.; (2) The DNA sequence synthesized in step (1) and the expression vector pET-28a(+) were respectively Eco RI and Xho I double enzyme digestion, the digestion products were recovered by electrophoresis and gel cutting, and the target gene fragment and the vector were connected with T4 DNA ligase to obtain the recombinant plasmid (see the attached plasmid map). Figure 1 The recombinant plasmid was transformed into E. coli DH5α competent cells, positive transformants were screened, and the plasmid was extracted for double enzyme digestion identification. The sequence size was consistent with the target gene size (954 bp). The agarose gel electrophoresis diagram is attached. Figure 2 The DNA of the band was recovered by tapping, and sequencing confirmed that the inserted sequence was correct. The recombinant plasmid was named pET-28a-bsh. (3) The recombinant plasmid pET-28a-bsh was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, cultured on LB plates containing 50 μg / mL kanamycin, and the positive transformant colonies were picked and transferred to LB slant culture medium to obtain the recombinant bsh coli, named E. coli BSH strain; (4) Pick three loops of E. coli BSH cells cultured on LB slant, inoculate into 50 mL of LB liquid medium, and culture at 37°C and 220 rpm for 4 h (OD 600 =0.793), IPTG was added at a final concentration of 0.5 mmol / L, and the culture was induced at 28°C and 150 rpm for 10 h until OD600 =2.78; (5) 20 mL of the culture medium prepared in step (4) was centrifuged at 4°C and 8000 × g for 10 min to collect the cells, and the cells were resuspended in 5 mL (1 / 4 of the original culture medium) of pH 6.0, 0.2 mol / L PB buffer. The cells were disrupted by ultrasonication in an ice-water bath (power 50 W, working for 3 s, interval 3 s, working 100 times), and centrifuged at 4°C and 8000 × g for 5 min. The supernatant was collected to obtain the cell lysate; (6) The cell lysate from step (5) was analyzed by SDS-PAGE protein electrophoresis (see Appendix Figure 3 ), the recombinant bacteria expressed a protein with a molecular weight of approximately 35 kDa, which was consistent with the molecular weight of CGH (35.13 kDa), indicating that the recombinant Escherichia coli expressing CGH was successfully constructed.
[0036] The restriction endonuclease BamH I and Xho I and T4 DNA ligase were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. The plasmid vector pET-28a, Escherichia coli DH5α strain, and BL21(DE3) strain were purchased from Beijing Qingke Biotechnology Co., Ltd. Plasmid miniprep kits and DNA gel recovery kits were purchased from Beijing Solaibao Technology Co., Ltd. Example 2 Enzyme activity of CGH expressed by E. coli BSH strain The CGH activity of E. coli BSH strain expressed in non-optimized expression medium was determined.
[0037] (1) 0.1 mL of Escherichia coli BSH culture solution frozen in 20% glycerol aqueous solution was inoculated into LB slant medium and cultured at 37°C for 24 h to obtain slant cells; (2) Pick 2 loops of cells from the slant in step (1) with an inoculation loop and inoculate them into 25 mL of LB liquid medium. Incubate in a shaker at 37°C and 150 rpm for 12 h to obtain the OD value. 600 =3.54 of seed solution; (3) According to the inoculum volume fraction of 4%, 2 mL of the seed solution prepared in step (2) was inoculated into 50 mL of expression medium and cultured in a shaking incubator at 37°C and 220 r / min for 4 h (OD 600 =1.17), add IPTG with a final concentration of 0.5 mmol / L as an inducer, and culture in a shaking incubator at 28°C and 150 r / min for 6-16 h. During this period, 4 mL of the sample was sampled every 2 h to measure the OD 600 The results of enzyme activities in the culture medium are shown in Table 1.
[0038] Table 1 OD of E. coli BSH strains at different expression culture times 600 and CGH vitality Induction culture time (h) <![CDATA[OD 600 ]]> CGH activity (U / mL) 6 3.42 70.4 8 6.53 98.6 10 7.61 131 12 8.57 137 14 864 142 16 8.35 125 From the data in Table 1, it can be seen that during the cultivation of E. coli BSH strain, after IPTG with a final concentration of 0.5 mmol / L was added to the culture medium for induction, the enzyme activity reached a high level after 10 h of induction. The enzyme activity reached the highest level (142 U / mL) after 14 h of induction. The enzyme activity began to decline after 16 h of induction. Therefore, the optimal induction time is 10-14 h.
[0039] The final concentration of the expression medium is as follows: yeast extract powder 20 g / L, trypsin 10 g / L, glycerol 4 g / L, KH2PO4 3 g / L, K2HPO4 10 g / L, the solvent is deionized water, pH 7.0. 50 mL of the expression medium is placed in a 250 mL Erlenmeyer flask, tied with 8 layers of gauze, and sterilized with high-pressure steam at 121°C for 20 min.
[0040] The activity determination method of the CGH is: (1) Preparation of glycine standard curve: Prepare glycine aqueous solutions with concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mmol / L using PB buffer at pH 6.0. Take 1 mL of glycine aqueous solution of different concentrations in a test tube, add 1 mL of ninhydrin colorimetric solution, mix thoroughly, and heat in boiling water for 15 min. After cooling with tap water, add 3 mL of 70% (v / v) ethanol aqueous solution and measure the absorbance (A) at a wavelength of 570 nm using a spectrophotometer. 570 ). With glycine concentration as the horizontal axis, A 570 As the vertical axis, plot the glycine concentration - A 570 Standard curve (attached Figure 4 ).
[0041] (2) Preparation of bacterial lysis solution: The culture medium of E. coli BSH strain was centrifuged at 4°C and 8000 × g for 5 min to collect the cells. An equal volume of PB buffer (pH 6.0) was added and the cells were repeatedly oscillated and suspended. The cells were disrupted by ultrasonication in an ice-water bath (power 50 W, 3 s operation, 3 s interval, 100 operations). The cell disruption solution was centrifuged at 4°C and 8000 × g for 5-10 min, and the supernatant was collected.
[0042] (3) Determination of CGH enzyme activity of samples: Take 0.1 mL of cell lysis supernatant diluted by appropriate multiples and add it to 1.8 mL of PB buffer (pH 6.0, 0.2 mol / L) and 0.1 mL of 200 mmol / L sodium glycocholate aqueous solution. After the mixed system is kept at 37°C for 30 min, 2 mL of 15% (v / v) trichloroacetic acid aqueous solution is added. After centrifugation at 4°C and 10,000 × g for 10 min, 1 mL of supernatant is mixed with 1 mL of ninhydrin colorimetric solution and placed in a boiling water bath for 15 min. After cooling with tap water, 3 mL of 70% (v / v) ethanol aqueous solution is added and the absorbance at a wavelength of 570 nm (A) is measured using a spectrophotometer. 570 ), by glycine concentration—A 570 The production of glycine (μmol) in the enzyme reaction system was calculated using the standard curve.
[0043] CGH enzyme activity definition: At pH 6.0 and 37°C, the amount of enzyme required to release 1 μmol of glycine from the substrate per minute is 1 activity unit (U).
[0044] The pH 6.0 PB buffer is a sodium hydrogen phosphate-potassium dihydrogen phosphate buffer, prepared by weighing 14.2 g of anhydrous Na2HPO4 and dissolving it in 1 L of distilled water to prepare a 0.2 mol / L Na2HPO4 stock solution; and weighing 13.6 g of anhydrous KH2PO4 and dissolving it in 1 L of distilled water to prepare a 0.2 mol / L KH2PO4 stock solution. The Na2HPO4 and KH2PO4 solutions are mixed in a volume ratio of 12.2:87.8 to obtain a pH 6.0, 0.2 mol / L PB buffer.
[0045] Example 3 Expression of CGH in E. coli BSH under optimal conditions Under the optimal conditions, the method for expressing CGH in E. coli BSH is as follows: (1) 0.1 mL of Escherichia coli BSH culture solution frozen in 20% glycerol aqueous solution was inoculated into LB slant medium and cultured at 37°C for 20 h to obtain slant cells; (2) Pick up 2 loops of cells from the slant in step (1) with an inoculation loop and inoculate them into 25 mL of LB liquid medium. Incubate in a shaker at 35°C and 200 rpm for 14 h to obtain the OD value. 600 =3.76 seed solution; (3) According to the inoculum volume fraction of 6%, 3 mL of the seed solution prepared in step (2) was inoculated into 50 mL of expression medium and cultured in a shaking incubator at 35°C and 240 r / min for 6 h (OD 600=1.24), IPTG was added as an inducer at a final concentration of 0.75 mmol / L, and the cells were cultured in a shaker at 25°C and 180 rpm for 14 h. The OD 600 = 10.7. The final concentration of the expression medium is as follows: yeast extract powder 30 g / L, tryptone 14 g / L, glycerol 6 g / L, KH2PO4 2 g / L, K2HPO4 8 g / L, the solvent is deionized water, pH 6.8. 50 mL of the expression medium is placed in a 250 mL Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by high-pressure steam at 121°C for 20 min. (4) According to the method described in Example 2, the CGH activity of the culture medium of Escherichia coli BSH cultured in this example was measured to be 176 U / mL.
[0046] Example 4 Isolation and Purification of CGH According to the method of Example 3, E. coli BSH cells were obtained by induction culture, and CGH was obtained by separation and purification according to the following steps: (1) Prepare OD according to the method of Example 3 600 =11.4, centrifuge 100 mL of culture medium at 8000 × g for 5 minutes at 4°C to collect the cells, yielding 2.14 g of wet cells. Add 10.7 mL of binding buffer (5 mL / g of wet cell weight) and disrupt the cells using ultrasonic waves in an ice-water bath (power 200 W, 3 s operation, 3 s interval, 200 cycles). Centrifuge the cell disruption solution at 8000 × g for 10 minutes at 4°C, and collect the supernatant to yield approximately 10 mL of cell lysate. (2) After washing with distilled water, the Ni-NTA column (1.6 cm × 2.5 cm) was pre-equilibrated with 4 column volumes of binding buffer. The entire cell lysate from step (1) was injected at a flow rate of 1 mL / min. After injection, the column was washed with wash buffer until the outflow of wash buffer A 280 ≈0, and then eluted with elution buffer until the A 280 ≈0, collect all eluates; (3) The eluate collected in step (2) was placed in a dialysis bag (molecular weight cutoff of 15 kDa) and dialyzed against binding buffer to remove imidazole. The dialyzate was concentrated in an ultrafiltration tube (molecular weight cutoff of 10 kDa), and the concentrate was freeze-dried in a culture dish at -80°C and a vacuum of 20 Pa to obtain 13.5 mg of CGH lyophilized powder. Thus, the fermentation yield of recombinant E. coli BSH expressing CGH was 135 mg / L.
[0047] According to the method described in Example 2, the enzyme activity of the CGH lyophilized powder prepared in this example was measured to be 1186 U / mg.
[0048] The binding buffer was prepared by adding 50 mmol / L Na2HPO4, 300 mmol / L NaCl, and adjusting the pH to 8.0 with 1 mol / L NaOH. The wash buffer was prepared by adding imidazole to a final concentration of 5 mmol / L in the binding buffer. The elution buffer was prepared by adding imidazole to a final concentration of 40 mmol / L in the binding buffer.
[0049] In the examples of the present invention, unless specific experimental methods are described, conventional molecular biology experimental methods are used, such as the methods described in Molecular Biology Experiment Guide (3rd edition, Science Press, 2015), edited by Wei Qun, or the product instructions provided by the kit manufacturer.
[0050] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.
[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.
Claims
1. A recombinant Escherichia coli expressing bile ylglycine hydrolase, characterized in that: It carries the gene encoding bileylglycine hydrolase bsh The amino acid sequence of the bile ylglycine hydrolase is shown in SEQ ID No.
1.
2. The recombinant Escherichia coli expressing bile ylglycine hydrolase according to claim 1, characterized in that: described bsh The nucleotide sequence of the gene is shown in SEQ ID No.
2.
3. The recombinant Escherichia coli expressing bile ylglycine hydrolase according to claim 1 or 2, characterized in that: The recombinant Escherichia coli is Escherichia coli BL21 (DE3).
4. The method for constructing a recombinant Escherichia coli according to any one of claims 1 to 3, wherein: The following steps are involved: Step (S.1), connecting the gene encoding the cholylglycine hydrolase having the amino acid sequence shown in SEQ ID No. 1 to an expression vector to construct a CGH expression vector; Step (S.2): transform the CGH expression vector into Escherichia coli BL21 (DE3) cells to obtain recombinant Escherichia coli.
5. The construction method according to claim 4, wherein: The nucleotide sequence of the coding gene is shown in SEQ ID No.
2.
6. The construction method according to claim 4 or 5, wherein: Step (S.1) is: (a) Introduce EcoRI restriction enzyme sequence at the 5′-end of SEQ ID No.2, remove the stop codon at the 3′-end and introduce Xho I enzyme cleavage sequence, and then chemically synthesize the DNA nucleotide sequence; (b) The DNA nucleotide sequence obtained in step (a) and the expression vector pET-28a(+) were double-digested with EcoR I and Xho I, respectively, to obtain bsh Enzyme digestion products of gene fragments and expression vector pET-28a(+); (c) bsh The gene fragment was connected with the enzyme-digested product of the expression vector pET-28a(+), and then transformed into Escherichia coli DH5α competent cells to obtain the recombinant plasmid pET-28a-bsh.
7. The construction method according to claim 6, wherein: The ligation was performed using T4 DNA ligase.
8. The construction method according to claim 4, wherein: The transformation was performed using a heat shock method.
9. Use of the recombinant Escherichia coli according to any one of claims 1 to 3, or the recombinant Escherichia coli constructed by the construction method according to any one of claims 4 to 8, in the fermentative production of cholylglycine hydrolase.
10. The use according to claim 9, characterized in that: The application method comprises the following steps: inoculating the recombinant Escherichia coli and fermenting and culturing to obtain bile ylglycine hydrolase, wherein the fermentation and culturing temperature is 25-40°C.