CN108998403B - Bacillus subtilis recombinant strain and application thereof - Google Patents
Bacillus subtilis recombinant strain and application thereof Download PDFInfo
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- CN108998403B CN108998403B CN201811090744.1A CN201811090744A CN108998403B CN 108998403 B CN108998403 B CN 108998403B CN 201811090744 A CN201811090744 A CN 201811090744A CN 108998403 B CN108998403 B CN 108998403B
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- 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)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2457—Pullulanase (3.2.1.41)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01041—Pullulanase (3.2.1.41)
Abstract
The invention discloses a bacillus subtilis recombinant strain and application thereof, belonging to the field of genetic engineering and fermentation engineering. The invention adopts GRAS microorganism bacillus subtilis as a host, three bacillus licheniformis genomes as templates and polymerase chain reaction to amplify three promoters Pblma、PnpuAnd PαglyAnd connecting the three promoters with an encoding gene and a vector of archaea amylopullulanase SMApu to transform the host bacteria of the bacillus subtilis to obtain three strains of bacillus subtilis recombinant bacteria. The recombinant strain is cultured in a proper culture medium to produce the archaea starch pullulanase, so that the yield and the activity of the pullulanase can be improved.
Description
Technical Field
The invention relates to a bacillus subtilis recombinant strain and application thereof, in particular to a bacillus subtilis recombinant strain for expressing archaea amylopullulanase and application thereof, and belongs to the field of microbial genetic engineering and fermentation engineering.
Background
Amylopullulanase (amylopullulanase), also known as type 2pullulanase, is capable of catalyzing the hydrolysis of alpha-1, 6-glucosidic bonds and alpha-1, 4-glucosidic bonds in starch and pullulan; having (beta/alpha)7Catalytic structure in the catalytic reactionThe glutamic acid residue is taken as a nucleophilic group, and belongs to a glycoside hydrolase GH57 family. The starch pullulanase belonging to archaea such as Thermococcus, Pyrococcus, Staphylococcus, Caldiverga and Thermofilum has the optimal catalytic reaction temperature of 85-110 ℃, and has better application prospect in the fields of starch modification, maltodextrin production and the like. The archaea amylopullulanase SMApu related by the invention is derived from archaea Staphylothermus marinus F1.
The culture medium components required by the growth of archaea are complex, higher (or lower) temperature and stronger acidic (or alkaline) conditions are often required in the culture process, and strict anaerobic environment is also required in some cases, and toxic gases are released. In order to research the archaea amylopullulanase, the Escherichia coli which is a microorganism easy to culture is mainly adopted for gene recombination expression at present. However, Escherichia coli is a pathogenic bacterium for humans, and endotoxin is produced during growth, so that it is not suitable as a host bacterium for food enzymes. Bacillus subtilis does not produce endotoxin, is Generally Recognized As Safe (GRAS) bacteria, and has become an industrial production bacterium of various food enzymes. However, archaea have codon usage bias, which is manifested by the codons of some amino acids that archaea often use, which bacteria rarely use; this often results in either incorrect expression of the archaeal gene in the bacteria or too low an expression level. Archaebacteria amylopullulanase genes from the glycoside hydrolase GH57 family are also often difficult to heterologously express in bacillus subtilis due to the lack of suitable promoters and stable vectors.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a recombinant Bacillus subtilis strain.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the bacillus subtilis recombinant strain is characterized in that the recombinant strain contains PblmaPromoter or PnpuPromoter or PαglyA promoter, and an encoding gene of archaea amylopullulanase SMApu, PblmaThe nucleotide sequence of the promoter is shown as a sequence table SEQ ID NO.1, and the P isnpuThe nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO.2, and P is shownαglyThe nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO.3, and the nucleotide sequence of the coding gene of the archaebacteria amylopullulanase SMApu is shown in a sequence table SEQ ID NO. 4.
The construction method of the recombinant bacterium specifically comprises the following steps:
polymerase Chain Reaction (PCR) amplification of P using Bacillus licheniformis ATCC27811 (American type culture Collection ATCC deposit) genome as templateblmaThe promoter or P gene is amplified by Polymerase Chain Reaction (PCR) using Bacillus licheniformis DSM13 (deposited in DSMZ of Germany Collection of microorganisms and cells) as templatenpuThe promoter or P is amplified by Polymerase Chain Reaction (PCR) using Bacillus licheniformis B.licheniformis sATCC 9789 (American type culture Collection ATCC deposit) genome as templateαglyPromoter of PblmaPromoter or PnpuPromoter or PαglyThe promoter, the coding gene of the archaea amylopullulanase SMApu and an expression vector pUBERT 29 are connected and recombined to construct a recombinant plasmid, and then the recombinant plasmid is transformed into bacillus subtilis to obtain recombinant bacteria.
The expression vector plasmid pURBRT 29, the construction method of which has been disclosed in the paper "enzymatized synthesis of glycosylated mutant used in microbial engineering from Bacillus stearothermophilus expressed in Bacillus subtilis in journal of the Science of Food and Agriculture" in 2010.
The invention also protects the application of any one of the bacillus subtilis recombinant bacteria in preparing archaea pullulanase.
A method for producing archaea pullulanase by using the bacillus subtilis recombinant bacteria comprises the step of culturing any bacillus subtilis recombinant bacteria in a proper culture medium to obtain the pullulanase.
The bacillus subtilis recombinant archaebacteria pullulanase has high yield, and the archaebacteria pullulanase produced by the method has high temperature resistance and still has high activity at 100 ℃. The invention lays a foundation for the application of the archaea pullulanase in the food industry.
Drawings
FIG. 1 recombinant plasmid pUBERT-PblmaThe construction process diagram of (1);
FIG. 2 recombinant plasmid pUBERT-Pblma、pUBRT-Pnpu、pUBRT-PαglyPCR verification of (1);
FIG. 3 is a graph derived from pUBERT-PblmaSDS-PAGE electrophoresis images of various stages of purifying archaebacteria amylopullulanase in shake flask fermentation liquor of the bacillus subtilis recombinant bacteria.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
plasmid pUBERT 29: construction methods reference: choice, c.h., Kim, s.h., Jang, j.h., Park, j.t., Shim, j.h., Kim, y.w., Park, K.H (2010). enzymatized synthesis of glycosylated purified enzyme using macromolecular amplified in Bacillus subtilis in journal of the Science of Food and Agriculture,90, 1179-.
B, bacillus subtilis: most of the mutants are artificial mutants isolated from natural environments or known, such as Bacillus subtilis ISW1214(leuA8 metB5 hsrM1), B.subtilis 168, and the like. The bacillus subtilis host bacterium used in the present invention is b.
Bacillus licheniformis b. licheniformis DSM 13: DSMZ of German Collection of microorganisms and cells.
Bacillus licheniformis b.licheniformis ATCC 9789 and b.licheniformis ATCC 27811: american type culture Collection ATCC deposit.
Example 1 construction of recombinant Bacillus subtilis
(1) Promoter Pblma、PnpuAnd PαglyPreparation of
Genomic DNA (100ng, 2. mu.L) from B.licheniformis ATCC27811, B.licheniformis DSM13 and B.licheniformis ATCC 9789 were added as templates, respectively
Buffer solution (Mg)2+plus, 10. mu.L), sterile distilled water (31.5. mu.L), dNTP (2.5mM each, 4. mu.L),
a forward primer:
(F-BamHI,5 'ATTGGATCCTGAGGCTGTTTCTCGACCGGA-3'; 10. mu.M, 1. mu.L), and reverse primer:
(R-SalI,5′-CAAGTCGACCATATGGTTTCCCCCTTTTGG-3′；10μM，1μL)，
by using
HS DNA Polymerase (2.5U/. mu.L, 0.5. mu.L) catalyzes the Polymerase chain reaction (PCR, 30 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 20 s). Then, the AxyPrep PCR cleaning kit is used to remove other components in the reaction solution to obtain a purified promoter Pblma、PnpuAnd Pαgly。
(2) Cloning of archaea s.marinus F1 pullulanase SMApu gene
Using archaebacteria S.marinus F1 genomic DNA (100ng, 2. mu.L) as a template, dNTP (2.5mM each, 4. mu.L) was added,
buffer (Mg2+ plus, 10 μ L), forward primer:
(F-SalI,5 'TGAGTCGAACTTGGAAGTATTGGATAAGTAT-3'; 10. mu.M, 1. mu.L), reverse primer:
(R-XhoI,5 'TACACTCGAGTCTTGGATTTGGTAATAGTTT-3'; 10. mu.M, 1. mu.L) and sterilized distilled water (31.5. mu.L) were added thereto
HS DNA Polymerase (2.5U/. mu.L, 0.5. mu.L) catalyzes the Polymerase Chain Reaction (PCR) (30 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 120 s). Then, other components in the reaction solution are removed by using an AxyPrep PCR cleaning kit, and the purified encoding gene of the amylopullulanase is obtained.
(3) Construction of recombinant plasmid
DNA restriction enzyme QuickcutTMSalI (1. mu.L) and QuickcutTMXhoI (1. mu.L), Buffer solution 10 XQuickcut Green Buffer (1. mu.L), starch pullulanase encoding gene (0.2. mu.g), and sterilized distilled water supplemented to 10. mu.L, and incubating at 37 ℃ for 30 min; then, nucleic acid electrophoresis was performed, and the double-digested amylopullulanase gene was recovered by cutting with AxyPrep DNA gel recovery kit.
DNA restriction enzyme QuickcutTMBamHI (1. mu.L) and QuickcutTMSalI (1. mu.L), Buffer 10 XQuickcut Green Buffer (1. mu.L), promoter sequence (0.2. mu.g), and sterile distilled water supplemented to 10. mu.L, and incubated at 30 ℃ for 30 min; then, nucleic acid electrophoresis was performed, and the promoter sequence cleaved by double digestion was recovered by cutting with AxyPrep DNA gel recovery kit.
DNA restriction enzyme QuickcutTMBamHI (1. mu.L) and QuickcutTMXhoI (1. mu.L), Buffer 10 XQuickcut Green Buffer (1. mu.L), plasmid vector pUBERT 29 (0.2. mu.g), and sterile distilled water supplemented to 10. mu.L, and incubating at 30 ℃ for 30 min; then, alkaline phosphatase (30U/. mu.l, 1. mu.L) was added and incubated at 37 ℃ for 1 h; then, nucleic acid electrophoresis was performed, and the double-digested pUBERT 29 was recovered by cutting with AxyPrep DNA gel recovery kit.
Mixing the double-enzyme-digested starch pullulanase encoding gene (1.6 mu L), the double-enzyme-digested promoter sequence (1.6 mu L) and the double-enzyme-digested pUBERT 29(1.6 mu L) with the Ligation Mix (5 mu L) in the DNA Ligation kit, and preserving the temperature at 16 ℃ for 30 min; coli MC1061, coating LB Amp solid culture medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 15g/L, ampicillin 100. mu.g/L), culturing at 37 ℃ for 10-12h, picking up transformant, extracting recombinant plasmid and carrying out double enzyme digestion verification. After the restriction enzyme digestion, the DNA sequence of the recombinant plasmid is determined, and the positive clone contains the recombinant plasmid.
The plasmids constructed by three different promoters are respectively marked as recombinant plasmid pUBERT-Pblma、pUBRT-Pnpu、pUBRT-Pαgly。
(4) Preparation of competent cells
Taking out Bacillus subtilis ISW1214 glycerol bacterial liquid from a refrigerator at-80 ℃, streaking and coating the liquid on an LB solid culture medium after dissolution, placing the liquid in an incubator, and culturing the liquid overnight at 37 ℃. 3-4 single clones were picked with sterile toothpicks and placed in 5mL LB liquid medium, 37 ℃, 250rpm, shaking table overnight. Taking 1mL of bacillus subtilis liquid, putting the bacillus subtilis liquid into a 1.5mL centrifuge tube, centrifuging for 1min at 10000rpm, removing supernatant, adding 1mL of SPI culture medium to resuspend the precipitate, adding 9mL of SPI culture medium, and performing shake cultivation at 37 ℃ at 250 rpm; after 3 hours, 100 mu L of sample liquid is taken every half hour, diluted by 10 times by sterile water, the delta OD600 value is measured, when the delta OD600 value just tends to be stable, the culture is stopped, sterile glycerol is added to ensure that the concentration reaches 12.5 percent, and then the sterile split charging is carried out, and the sample liquid is used as the first culture bacteria and is stored in a refrigerator at the temperature of 80 ℃ below zero for standby. Before transformation, the first culture was taken out from a freezer at-80 ℃ and thawed at 37 ℃, and SPII medium was added, diluted 7.5 times, and placed in a 50mL sterilization tube. Shaking at 250rpm and 37 deg.C for 90min to obtain second culture.
(5) Transformation of competent cells:
respectively taking out 200-500 mu L of secondary culture bacteria, uniformly mixing the secondary culture bacteria with 20-50 mu L of each recombinant plasmid (10-100ng DNA) in a 1.5mL centrifuge tube, carrying out shake culture at 250rpm and 37 ℃ for 30min, adding 1-time volume of LB liquid culture medium, carrying out shake culture at 250rpm and 37 ℃ for 60min, then carrying out centrifugation at 13000rpm for 30s, removing 800 mu L of supernatant, carrying out heavy suspension on the precipitate, taking out, coating LB Kan solid culture medium (10 g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 15g/L of agar and 40 mu g/L of kanamycin) with a glass rod, placing the precipitate in an incubator, carrying out overnight culture at 37 ℃ to finally obtain three kinds of bacillus subtilis recombinant bacteria for expressing pullulanase genes, and respectivelyAs B.subtilis/Pblma/SMApu、B.subtilis/PnpuSMApu and Bαgly/SMApu。
Example 2 (comparative example) recombinant Bacillus subtilis B.subtilis/P43Construction of/SMApu
The construction method of the recombinant bacterium in this embodiment is completely the same as that of embodiment 1 except that the promoter is different. The promoter used in this example is a promoter P conventional in the art43By using
Forward primer F-BamHI, 5-
Reverse primer
R-SalI,5 '-GTCGACAAGCTTTCTGTTATTAATTCTTGTC-3' amplified from the B.subtilis 168 genome.
Example 3 (comparative example) recombinant Bacillus subtilis B.subtilis/PhagConstruction of/SMApu
The construction method of the recombinant bacterium in this embodiment is completely the same as that of embodiment 1 except that the promoter is different. The promoter used in this example is a promoter P conventional in the arthagBy using
The forward primer F-BamHI, 5-.
Example 4 fermentation of recombinant Bacillus subtilis and purification of enzyme
The three recombinant strains prepared in example 1 and the single colonies of the recombinant strains prepared in examples 2 and 3 were picked with sterile toothpicks and inoculated into medium A Kan broth (peptone 3.3%, yeast extract 2%, sodium chloride 0.74%, disodium hydrogen phosphate 0.8%, potassium dihydrogen phosphate 0.4%, casamino acid 2%, manganese chloride 0.06mM, kanamycin 80mg/L) and cultured at 37 ℃ and 250rpm for 30 hours.
Centrifuging the above five fermentation solutions for 20min at 7000g, resuspending the bacteria with 1/10 fermentation volume of lysis buffer (50M pH 7.4Tris-HCl,500mM NaCl,5mM imidazole), disrupting the cells (50mL) with ultrasonic waves (750W, 35% amplitude, 15min), centrifuging for 20min at 9000g to remove the disrupted cells, placing the obtained cell extract enzyme solution in 50mL centrifuge tube, treating in 70 deg.C water bath for 15min, passing the heat-treated enzyme solution through Ni ion-dextran affinity chromatography column, eluting with elution buffer (Tris-HCl 50M pH 7.4, sodium chloride 500mM, imidazole 500mM), loading into semipermeable membrane, dialyzing in buffer (Tris-HCl 50M pH 7.4) to remove sodium chloride and imidazole, and analyzing the purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), And monitoring the specific activity of the enzyme to finally obtain the purified archaea starch pullulanase.
The method for measuring the enzyme activity comprises the following steps: adding 150 mu L of 1% pullulan solution into 75 mu L of 200mmol/LpH5.0 acetic acid-sodium acetate buffer solution, uniformly mixing, setting the temperature of a dry bath to 100 ℃, preheating for 3min, then adding 75 mu L of archaea starch pullulanase SMApu, continuing to keep the temperature at 100 ℃ for 10min, immediately adding 900 mu L of 3, 5-dinitrosalicylic acid solution (DNS) to terminate the reaction, putting into a boiling water bath to boil for 5min, cooling to room temperature, and measuring the delta OD575 value by using an ultraviolet visible spectrophotometer. And (4) calculating the maltose concentration according to the maltose standard curve, and further calculating the enzyme activity according to the definition of the enzyme activity.
The results are shown in table 1:
as is clear from the table, when Bacillus subtilis is used the promoter P which is conventional in the art43Or PhagWhen the archaea pullulanase is expressed, the specific activity of the pullulanase is 19.6U/mg protein or 18.3U/mg protein, and the yield is 25.6 percent or 21.9 percent; while using the promoter P of the present inventionblma、PnpuOr PαglyThe fermentation result shows that the specific activity of the pullulanase produced by the three recombinant strains constructed by the invention is 31.9U/mg protein, 30.7U/mg protein or 31.2U/mThe yield of the g protein is respectively 30.8%, 29.9% or 30.1%, which is obviously improved compared with the recombinant bacteria using the conventional promoter.
Claims (3)
1. The bacillus subtilis recombinant strain is characterized in that the recombinant strain contains PblmaPromoter or PnpuPromoter or PαglyA promoter, and an encoding gene of archaea amylopullulanase SMApu, PblmaThe nucleotide sequence of the promoter is shown as SEQ ID NO.1 in the sequence table, and the PnpuThe nucleotide sequence of the promoter is shown as SEQ ID NO.2 in the sequence table, and the PαglyThe nucleotide sequence of the promoter is shown as SEQ ID NO.3 in the sequence table, and the nucleotide sequence of the archaebacteria amylopullulanase SMApu coding gene is shown as SEQ ID NO.4 in the sequence table.
2. The method for constructing a recombinant bacterium of bacillus subtilis according to claim 1, wherein the method for constructing the recombinant bacterium specifically comprises the following steps: amplification of P by polymerase chain reaction Using Bacillus licheniformis ATCC27811 genome as templateblmaPromoter, or P amplified by polymerase chain reaction using Bacillus licheniformis DSM13 genome as templatenpuPromoter, or P amplified by polymerase chain reaction using Bacillus licheniformis ATCC 9789 genome as templateαglyA promoter; adding the above-mentioned PblmaPromoter or PnpuPromoter or PαglyPromoter, and archaebacteria amylovoraAnd (3) carrying out recombination construction on the gene encoding the rucanase and an expression vector plasmid, and converting the gene encoding the rucanase into bacillus subtilis host bacteria to obtain recombinant bacteria.
3. The use of the recombinant Bacillus subtilis strain of claim 1 in the preparation of archaeal amylopullulanase.
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