CN117821348A - Recombinant bacillus subtilis for extracellular secretion expression of kappa-carrageenan - Google Patents
Recombinant bacillus subtilis for extracellular secretion expression of kappa-carrageenan Download PDFInfo
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- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical compound O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 title claims abstract description 42
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- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
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- 150000004676 glycans Chemical class 0.000 description 1
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- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention discloses a recombinant bacillus subtilis for extracellular secretion expression of kappa-carrageenan enzyme, belonging to the technical fields of genetic engineering and enzyme engineering. The invention uses food-grade bacillus subtilis as an expression host and plasmid pP43NMK as an expression vector to realize the heterologous expression of kappa-carrageenan enzyme. The invention successfully realizes the extracellular secretion expression of the kappa-carrageenan enzyme by removing the signal peptide of the kappa-carrageenan enzyme and optimizing the fermentation process, and the enzyme activity can reach 458.94U/mL which is higher than most of the kappa-carrageenan enzyme reported at present, and the higher extracellular secretion expression level is beneficial to the later mass production, separation and purification. The kappa-carrageenan enzyme obtained by fermenting the recombinant bacterium can be used for efficiently producing functional kappa-carrageenan oligosaccharides through hydrolyzing kappa-carrageenan green, and a new path is provided for efficiently preparing the carrageenan oligosaccharides.
Description
Technical Field
The invention relates to recombinant bacillus subtilis for extracellular secretion expression of kappa-carrageenan enzyme, belonging to the technical fields of genetic engineering and enzyme engineering.
Background
Carrageenan (Carrageenan) is used as a natural marine sulfated polysaccharide with a unique structure, and is formed by connecting D-galactose through alternating alpha-1, 3 and beta-1, 4 glycosidic bonds, and the application of the Carrageenan is greatly limited because the Carrageenan has high viscosity and is not easy to be absorbed by human bodies. The carrageenan oligosaccharide (Carrageenan oligosaccharide) is a degradation product of carrageenan, and the research shows that the activity of the carrageenan oligosaccharide is obviously improved compared with that of the carrageenan, and the physical property of the carrageenan oligosaccharide is obviously improved, and the molecular weight of the carrageenan oligosaccharide is greatly reduced compared with that of the carrageenan, so that the solubility, the antioxidant, antiviral, immunoregulatory and other biological activities of the carrageenan are improved, and the application range of the carrageenan is widened to a certain extent. At present, the methods for preparing carrageenan oligosaccharides mainly comprise a physical method, a chemical method and an enzymatic method.
The enzymatic method for preparing the carrageenan oligosaccharide has the advantages of mild condition, strong reaction specificity, less environmental pollution and the like, and gradually replaces the traditional method for preparing the carrageenan oligosaccharide. However, the direct fermentation of strains isolated from nature to produce carrageenase has the problems of low yield, complex culture conditions, unstable enzyme production, high enzyme purification cost and the like. Therefore, the gene engineering method is adopted to carry out heterologous expression on the carrageenase genes separated from the nature so as to improve the expression quantity of carrageenase, and has important significance for large-scale preparation of carrageenase and industrialization of carrageenase oligosaccharides. Carrageenase (carrageenase) can be classified into kappa-carrageenase, iota-carrageenase and lambda-carrageenase according to the substrate specificity. In practical applications, kappa-carrageenan (CgkA enzyme) is used in a large number, but the preparation of kappa-carrageenan still faces two problems: 1) The recombinant kappa-carrageenan enzyme is secreted and expressed in the form of intracellular enzyme, and the preparation of crude enzyme is obtained by further crushing and centrifuging, so that the production cost is increased and the efficiency is reduced in actual production; 2) Most of the existing researches adopt escherichia coli for heterologous expression, and the escherichia coli expression system has the problems of low expression quantity, endotoxin, inclusion body formation, target protein misfolding and the like in industrial application, so that the escherichia coli expression system is not beneficial to the application of enzymes in the fields of foods, medicines and the like. In the prior art, patent CN101784660A discloses that kappa-carrageenan is expressed in bacillus subtilis, but the activity and the expression quantity are still low. Therefore, there is a need to develop expression systems capable of efficiently exocrine expression of kappa-carrageenan enzymes.
Disclosure of Invention
In order to solve the problems, the invention provides a recombinant bacillus subtilis for efficiently secreting and expressing kappa-carrageenan enzyme extracellularly, a safer food-grade expression system is established, and heterologous extracellular secretion expression of the kappa-carrageenan enzyme is realized.
The first object of the present invention is to provide a recombinant bacillus subtilis expressing a kappa-carrageenan enzyme having an amino acid sequence as shown in SEQ ID NO. 1.
In one embodiment, the recombinant bacillus subtilis hosts bacillus subtilis Bacillus subtilis WB 600.
In one embodiment, the kappa-carrageenan enzyme with the amino acid sequence shown in SEQ ID NO.1 is obtained by deleting the signal peptide sequence of the wild-type kappa-carrageenan enzyme derived from Zobellia galactanivorans; the amino acid sequence of the Zobellia galactanivorans-derived wild-type kappa-carrageenan enzyme is shown as SEQ ID NO.5, and the nucleotide sequence of the signal peptide of the wild-type kappa-carrageenan enzyme is shown as SEQ ID NO. 4.
In one embodiment, the nucleotide sequence of the gene for kappa-carrageenan enzyme is shown in SEQ ID NO. 2.
In one embodiment, the vector used for the expression is a plasmid that can autonomously replicate in the recombinant bacillus subtilis, including but not limited to plasmid pP43NMK.
In one embodiment, the nucleotide sequence of the plasmid pP43NMK is shown in SEQ ID NO. 3.
A second object of the present invention is to provide a process for producing kappa-carrageenan enzyme comprising the step of fermentation using the recombinant Bacillus subtilis described above.
In one embodiment, the fermentation is to perform activation culture on the recombinant bacillus subtilis in a seed culture medium to obtain a seed solution, and then transfer the seed solution into a fermentation culture medium according to an inoculum size of 1% -5% (v/v) for fermentation culture.
In one embodiment, the fermentation is carried out by activating and culturing the recombinant bacillus subtilis in a seed culture medium to obtain a seed solution, and transferring the seed solution into a 250mL conical flask filled with 50mL fermentation culture medium at an inoculum size of 1% -5% (v/v).
In one embodiment, the seed fluid inoculum size is preferably 4% (v/v).
In one embodiment, the composition of the seed medium comprises: 2-8 g/L yeast powder, 5-15 g/L tryptone and 5-15 g/L NaCl.
In one embodiment, the pH of the seed medium is from 6.5 to 7.5.
In one embodiment, the fermentation mediumThe composition comprises: 5-10 g/L tryptone, 25-35 g/L yeast powder, K 2 HPO 4 15～20g/L，KH 2 PO 4 1-3 g/L and 5-10 g/L of glycerol.
In one embodiment, the fermentation medium has a pH of 7 to 8.
In one embodiment, the conditions of the fermentation culture are 20 to 37℃and 180 to 280r/min. Preferably at 25℃and 180-280 r/min.
In one embodiment, the fermentation culture is performed for a period of time ranging from 12 to 96 hours.
It is a third object of the present invention to provide the use of said recombinant bacillus subtilis or said method in the food field.
In one embodiment, the use comprises the use of the recombinant bacillus subtilis or the method in the preparation of kappa-carrageenan oligosaccharides.
The invention has the beneficial effects that:
the invention discovers that the wild type kappa-carrageenan enzyme (Zobellia galactanivorans source) can not realize heterologous extracellular secretion expression in bacillus subtilis, and the extracellular activity of the wild type kappa-carrageenan enzyme can not be detected.
Drawings
FIG. 1 is a construction flow of a recombinant plasmid of kappa-carrageenan; wherein F2 and R2 are target gene amplification primers, and the short line represents a homology arm;
FIG. 2 is a graph showing the effect of fermentation temperature and time on kappa-carrageenan shake flask fermentation;
FIG. 3 is an SDS-PAGE protein electrophoresis of kappa-carrageenan;
FIG. 4 is an HPLC analysis of the preparation of the enzymatic hydrolysate using recombinant kappa-carrageenan.
The specific embodiment is as follows:
the present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The following examples relate to the methods for enzyme activity detection as follows:
the enzyme activity of kappa-carrageenase is detected by using a 3, 5-dinitrosalicylic acid method (DNS method for short). According to the following conditions and steps: 0.1mL of the enzyme solution was added to 0.9mL of the substrate (substrate kappa-carrageenan was prepared to have a final concentration of 0.5% (w/v) with Tris-HCl (pH 7.5,50 mM)), reacted at 40℃for 15min, 1mL of DNS was added to stop the reaction, boiled in boiling water for 5min, immediately placed in ice water, 4mL of distilled water was added, and the absorbance was measured at 520 nm. The enzyme activity was calculated from the D-galactose standard curve. The enzyme solution inactivated at high temperature was used as a blank.
One enzyme activity unit (U) defines: under the above reaction conditions, the amount of enzyme required for 1. Mu.g of reducing sugar (in terms of D-galactose) was produced in 1 min.
The analytical methods for kappa-carrageenan enzymatic hydrolysis products are described in the examples below:
the enzymatic hydrolysate was analyzed by High Performance Liquid Chromatography (HPLC), specific analysis conditions: an inverted C18 column is adopted; a detector: an ultraviolet detector; mobile phase: phase A10 mM ammonium acetate (pH 4.5); phase B acetonitrile. Gradient elution is carried out for 0-60min,80-60% of phase A and 20-40% of phase B; ultraviolet wavelength 254nm; column temperature 40 ℃; the flow rate is 0.5mL/min; the sample injection amount was 10. Mu.L.
EXAMPLE 1 construction of Bacillus subtilis secretory expression System
The construction of the expression vector CgkA/pP43NMK is shown in FIG. 1. The primers used for amplifying the pP43NMK vector are F1/R1 in Table 1, and the primers used for amplifying the target gene CgkA are F2/R2 in Table 1.
TABLE 1 construction of the Bacillus subtilis expression System
The PCR system is as follows: max (Max)Master Mix 25. Mu.L, forward primer (10M) 2. Mu.L, reverse primer (10M) 2. Mu.L, template DNA 1. Mu.L, ddH was added 2 O to 50. Mu.L. The PCR amplification conditions were: pre-denaturation at 98℃for 3min; further 30 cycles (98 ℃ C. 10s,60 ℃ C. 15s,68 ℃ C. 1 min); finally, the temperature is kept at 68 ℃ for 10min.
The primers in Table 1 were used to amplify the linearized vector pP43NMK obtained by the above PCR system and the target gene with homology arms at both ends were ligated by homologous recombination. The homologous recombination reaction system is as follows: 0.03pmol of vector, 0.06pmol of target gene, 4. Mu.L of 5 XCE II Buffer, 2. Mu.L of Exnase II, and ddH were added 2 O to 20. Mu.L, and reacted at 37℃for 30min. The ligation product was transformed into Escherichia coli JM, and an LB plate with ampicillin was applied, and single colonies were picked for sequencing and colony PCR verification, and recombinant plasmid CgkA/pP43NMK containing the kappa-carrageenan enzyme gene was obtained by extraction. The recombinant plasmid was transferred into an expression host B.subtilis WB600 to obtain a recombinant genetically engineered bacterium B.subtilis WB600 (CgkA/pP 43 NMK).
Example 2 fermentative production of recombinant kappa-Carrageenan enzyme
LB medium: yeast powder 5g/L, tryptone 10g/L, naCl 10g/L, pH 7.0.
TB medium: tryptone 6g/L, yeast powder 30g/L, K 2 HPO 4 16.432g/L，KH 2 PO 4 2.314g/L, 5g/L glycerol and pH 7.5.
(1) Activating and culturing host bacteria: recombinant genetically engineered bacterium B.sublis WB600 (CgkA/pP 43 NMK) obtained according to the method described in example 1 was streaked on LB solid medium, placed in a 37℃incubator overnight for cultivation, and positive single colonies were picked up and inoculated in 250mL Erlenmeyer flasks containing 50mL LB liquid medium. Kanamycin was added at a final concentration of 5 μg/mL prior to inoculation. The Erlenmeyer flask was placed in a 200r/min rotary shaker and incubated at 37℃for 14h.
(2) Optimizing fermentation culture conditions: the seed solution was transferred to a 250mL Erlenmeyer flask containing 50mL of fermentation medium at an inoculum size of 4% (v/v). Shaking culture was performed in a shaker at 20℃and 25℃and 30℃and 37℃for 12 to 96 hours (rotation speed 200 r/min), respectively, and kanamycin was added at a final concentration of 5. Mu.g/mL before inoculation. After fermentation, the fermentation broth is centrifuged for 15min at 4 ℃ and 10000rpm, and the supernatant is collected to obtain crude enzyme liquid. As a result of measuring the enzyme activity of the crude enzyme solution, the extracellular enzyme activity gradually increased with the increase of the fermentation time, as shown in FIG. 2. The extracellular enzyme activity can reach 458.89 (U/mL) after shaking culture for 24 hours at 25 ℃ and 200 r/min.
EXAMPLE 3 isolation and purification of recombinant kappa-Carrageenan
The nickel column was equilibrated with 5 column volumes of Binding buffer (50 mM Tris-HCl,500mM NaCl,pH 7.5); after the fermentation crude enzyme solution is subjected to membrane passing treatment by a 0.45 mu m water system membrane, loading is started, and the flow rate is 1mL/min; after the nickel column was equilibrated with Binding buffer, part of the hetero-protein was washed off with Wash buffer (50 mM Tris-HCl,500mM NaCl,20mM imidazole, pH 7.5), followed by gradient Elution of the target protein with an Elutation buffer (50 mM Tris-HCl,500mM NaCl,500mM imidazole, pH 7.5). The collected target protein was dialyzed in a dialysate (50 mM Tris-HCl, pH 7.5) for 24 hours to obtain pure enzyme. Finally, the identification is carried out by means of SDS-PAGE protein electrophoresis and enzyme activity measurement. SDS-PAGE of proteins shows the results of FIG. 3.
EXAMPLE 4 use of recombinant kappa-Carrageenan
And (3) carrying out enzymolysis on the kappa-carrageenan by using recombinant kappa-carrageenan enzyme to degrade the kappa-carrageenan into a series of kappa-carrageenan oligosaccharides.
0.1mL of pure enzyme and 0.9mL of substrate with the concentration of 0.5% (w/v) are reacted for 3 hours at the temperature of 40 ℃ by using the inactivated enzyme liquid instead of the pure enzyme as a control group, and the rotating speed of a water bath shaking table is 200-300 rpm. Taking 0.5mL of enzymolysis product, sequentially adding 0.2mL of 0.2M derivatization reagent AEC (3-amino-9-ethylcarbazole) dissolved in methanol and 25 mu L of 0.5M NaBH 3 CN (sodium cyanoborohydride) and 50 μl acetic acid. The mixture was kept in a 70℃water bath for 1 hour, and 0.1mL of 1M NaOH was added to terminate the reaction. Finally, 1mL of chloroform was added for extraction. The supernatant was centrifuged at 8000rpm for 5min and analyzed by HPLC through a 0.22 μm organic membrane. As a result, as shown in FIG. 4, the enzymatic products of reaction 3h were mainly kappa-carrageenan disaccharide (DP 2), tetrasaccharide (DP 4) and octasaccharide (DP 8). And DP4 was the highest, 62.16% conversion, followed by 15.49% DP2 sugar conversion, with the lowest DP8 conversion of 7.15%. The conversion rate of the whole reaction system reaches 84.80 percent.
Comparative example 1
The nucleotide sequence of the target gene self signal peptide shown in SEQ ID NO.4 is added after the first start codon of the gene sequence shown in SEQ ID NO. 2. The amino acid sequence of the gene code added with the signal peptide code sequence is shown as SEQ ID NO. 5. Through gene synthesis, the recombinant strain B.subtilis WB600 (Signal-CgkA/pP 43 NMK) is obtained by constructing the recombinant strain into a plasmid pP43NMK and transforming the recombinant strain into a host B.subtilis WB600 and performing sequencing verification.
The recombinant strain B.subtilis WB600 (Signal-CgkA/pP 43 NMK) was fermented to verify its ability to express kappa-carrageenan enzyme extracellularly, and the fermentation method was the same as in example 2, and the fermentation result showed that no extracellular enzyme activity was detected, i.e., extracellular enzyme activity was 0U/mL.
Claims (10)
1. The recombinant bacillus subtilis is characterized in that the recombinant bacillus subtilis expresses kappa-carrageenan enzyme with an amino acid sequence shown as SEQ ID NO. 1.
2. The recombinant bacillus subtilis according to claim 1, wherein the recombinant bacillus subtilis hosts bacillus subtilis Bacillus subtilis WB 600.
3. The recombinant bacillus subtilis according to claim 1, wherein the expression uses plasmid pP43NMK as an expression vector, and the nucleotide sequence of the expression vector is shown in SEQ ID No. 3.
4. A process for producing kappa-carrageenan enzyme comprising the step of fermentation using the recombinant bacillus subtilis of any one of claims 1-3.
5. The method of claim 4, wherein the fermenting comprises: activating and culturing the recombinant bacillus subtilis in a seed culture medium to obtain a seed solution, and transferring the seed solution into a fermentation culture medium according to the inoculum size of 1% -5% (v/v) for fermentation culture.
6. The method of claim 5, wherein the composition of the seed medium comprises: 2-8 g/L yeast powder, 5-15 g/L tryptone and 5-15 g/L NaCl.
7. The method of claim 5, wherein the composition of the fermentation medium comprises: 5-10 g/L tryptone, 25-35 g/L yeast powder, K 2 HPO 4 15～20g/L，KH 2 PO 4 1-3 g/L and 5-10 g/L of glycerol.
8. The method according to claim 5, wherein the fermentation culture temperature is 20 to 37 ℃.
9. Use of the recombinant bacillus subtilis according to any one of claims 1 to 3, or the method according to any one of claims 4 to 8 in the food field.
10. The use according to claim 9, characterized in that the use comprises the use of the recombinant bacillus subtilis or the method in the preparation of kappa-carrageenan oligosaccharides.
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