CN106755003A - The recombination bacillus coli that a kind of bile salt hydrolase enzyme activity is improved - Google Patents
The recombination bacillus coli that a kind of bile salt hydrolase enzyme activity is improved Download PDFInfo
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- CN106755003A CN106755003A CN201611153861.9A CN201611153861A CN106755003A CN 106755003 A CN106755003 A CN 106755003A CN 201611153861 A CN201611153861 A CN 201611153861A CN 106755003 A CN106755003 A CN 106755003A
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- bile salt
- salt hydrolase
- enzyme activity
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- bacillus coli
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- C—CHEMISTRY; METALLURGY
- 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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01024—Choloylglycine hydrolase (3.5.1.24), i.e. bile salt hydrolase
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Abstract
The invention discloses the recombination bacillus coli that a kind of bile salt hydrolase enzyme activity is improved, belong to bioengineering field.The present invention will carry out plasma mutagenesis and the heterogenous expression in Escherichia coli from the bile salt hydrolase gene of Bifidobacterium ATCC 27673, induced by IPTG, the relative enzyme activity of the bile salt hydrolase that recombination bacillus coli is produced is improved to 123.2%, 23.2% is improve compared with the control, induction 32h enzyme activity improves 21.6%, and the method is simple to operate, possess the potentiality of commercial Application.
Description
Technical field
The present invention relates to the recombination bacillus coli that a kind of bile salt hydrolase enzyme activity is improved, belong to bioengineering field.
Background technology
Serum cholesterol concentration is too high to be to cause the key factor of the angiocardiopathies such as coronary heart disease, in humans and animals body just
Level cholate is mainly and synthesize in liver by cholesterol, in the position of carbon 24 by steroid nucleus and a glycine or taurine by acyl
Amine key is connected.With reference to cholate storage in gall-bladder, by bile duct secretions to small intestine, the seizure meals courage of this combination cholate instinct
Sterol and fat, enterocyte is easier to be absorbed into blood to pass through them, while about 95% cholate enters
Hepato-enteric circulation, the cholate of 650mg can avoid being absorbed by enterocyte, therefore, largely it is present in intestines and stomach with reference to cholate.Grind
Study carefully and show, some probiotics can by producing bile salt hydrolase (bile salt hydrolase, BSH), decompose taurine or
The cholate that glycine is combined forms free amino acid and free cholic acid, and these probiotics combine cholate by this effect to reduce
Toxic action to itself.
Bile salt hydrolase (BSH) is, by a kind of endocellular enzyme of bsh gene codes, to be widely present in enteric microorganism, is
The main composition of degraded bile --- the enzyme needed for the conjugation cholic acid first step.Cholate hydrolase can be by conjugation cholic acid hydrolysis
Into taurine or glycine and free cholic acid, the latter can be made further degraded by other enteric microorganism in enteron aisle.Cholate
The physiological action of hydrolase is embodied in two aspects：First, influence host fat metabolic process, reduce fat digest and assimilate and
Reduce cholesterol levels etc.；2nd, the degraded of bile reduces toxicity of the bile to enteric microorganism, improves microbe survival
Intestinal environment；The amino acid and free fatty produced by degraded can also provide nutriment for microorganism.
Because bile salt hydrolase is typically derived from the microorganisms such as lactic acid bacteria, its growing environment and ferment strength are not suitable for greatly
Technical scale metaplasia is produced.On the other hand, most bile salt hydrolase expression quantity of report are low at present, easily form inclusion body.Therefore, sieve
Select it is a kind of can enzyme activity it is higher and can high efficient expression bile salt hydrolase gene for industrialized production bile salt hydrolase, and use
Bile salt hydrolase regulation living organism health status is significant.
The content of the invention
In order to overcome above mentioned problem, first purpose of the invention to be to provide the bile salt hydrolase base that a kind of enzyme activity is improved
Cause, the gene order is as shown in SEQ ID NO.1.
Second object of the present invention is to provide the cell line for expressing the bile salt hydrolase gene.
Third object of the present invention is to provide a kind of recombination bacillus coli, and the recombination bacillus coli is with pET-28a
(+) is carrier, with e. coli bl21 (DE3) as host, expresses gene shown in SEQ ID NO.1.
Fourth object of the present invention is to provide a kind of construction method of the recombination bacillus coli that enzyme activity is improved.
In one embodiment of the invention, methods described is by the base containing the coding bile salt hydrolase mutant
Converted into Escherichia coli after because being connected with expression vector.
In one embodiment of the invention, the recombination bacillus coli be pET-28b (+) for carrier, with E.coli
BL21 (DE3) is gene shown in host expresses SEQ ID NO.1.
5th purpose of the invention is to provide a kind of method for producing bile salt hydrolase, is by the recombination bacillus coli
It is seeded in TB culture mediums, 37 DEG C of 12~52h. of culture
In one embodiment of the invention, the inoculum concentration is by volume 1%.
In one embodiment of the invention, the thalline is to OD600For 0.8~1.0 when, add IPTG induction 28~
36h。
In one embodiment of the invention, the IPTG concentration is 0.1~0.15mol/L.
In one embodiment of the invention, also the zymotic fluid after Fiber differentiation is centrifuged for methods described, collects thalline,
And broken wall purifying obtains pure enzyme.
In one embodiment of the invention, the broken wall purifying is, by 6000~9000rpm of zymotic fluid centrifugations, to collect
After thalline, with the phosphate buffer washing thalline 2~3 times of 0.1M, pH 6~7,3~10min of ultrasonication is collected by centrifugation
Clear liquid, is purified by nickel post affinity column chromatography.
The present invention also provide the bile salt hydrolase gene food, health products, pharmaceutical product production field application.
Beneficial effects of the present invention：The present invention filters out a kind of gene of the high yield bile salt hydrolase in Bifidobacterium source,
The heterogenous expression of bile salt hydrolase gene is realized in Escherichia coli.The bile salt hydrolase production method that the present invention is provided passes through
IPTG is induced, and the relative enzyme activity of bile salt hydrolase is improved to 123.2%, and 23.2% is improve compared with the control, induces 32h enzymes
Work improves 21.6%, and the method is simple to operate, the potentiality for possessing commercial Application.
Brief description of the drawings
Fig. 1 is recombinant bacterium intracellular enzyme activity under different IPTG induction times.
Specific embodiment
LB culture mediums：Tryptone 10g/L, dusty yeast 5g/L, NaCl 10g/L, pH 7.0；
TB culture mediums：Peptone 12g/L, yeast extract 24g/L, glycerine 8g/L, 17mmol/L KH2PO4, 72mmol/L
K2HPO4。
Bile salt hydrolase activity identification method：Be added dropwise 5 μ L recipient bacteriums bacterium solutions to not Han You 0.01% Pig cholate (w/v) LB
On solid plate (containing 100 μ g/mL ampicillins and 24 μ g/mL IPTG), by the way that white or transparent precipitation circle inspection can be produced
Test the activity of bile salt hydrolase.
Bile salt hydrolase enzyme activity determination method：Take 10 μ L enzyme liquid 0.1M phosphate buffers (pH 6.0) and be diluted to 90 μ
L, adds 10 μ L combinations cholate (200mM) mixing, and 30min is incubated in 37 DEG C, adds 15% isometric (w/v) trichloroacetic acid end
Only react, 10 μ L of supernatant liquid are taken after centrifugation and is mixed with 190 μ L ninhydrin reagents, 15min is reacted in 100 DEG C, cool down after 570nm
Place determines light absorption value, is calculated according to glycine standard curve.Wherein, the composition of ninhydrin reagent is：(the w/ of 0.5mL 1%
V) ninhydrin (being dissolved in 0.5M, pH5.5 citrate buffer), 1.2mL glycerine, the citric acid of 0.2mL 0.5M, pH5.5 delays
Fliud flushing.
In specific embodiment enzyme activity is determined from ox sulphur deoxidation combination cholate.
The structure of the recombination bacillus coli of embodiment 1
With plasmid pET-28a (+) as carrier, (NCBI is logged in the bile salt hydrolase genes of synthesis Bifidobacterium ATCC 27673
Number be CP007522.1) construction recombination plasmid pET-28a (+)-bsh, conversion to E.coil JM109 competent cell, picking
Positive bacterium colony.Plasmid is extracted after 37 DEG C of incubator overnight cultures, through sequence verification, correct plasmid pET-28a (+)-bsh will be sequenced
Conversion to Escherichia coli E.coil BL21 (DE3), obtain recombination bacillus coli E.coil BL21 (DE3) pET-28a (+)-
bsh。
The screening of the bile salt hydrolase gene of embodiment 2
(1) it is seeded to containing 0.5% (mass fraction) half as starting strain with Bifidobacterium ATCC 27672 (being purchased from ATCC)
In the MRS culture mediums of cystine, 37 DEG C of Anaerobic culturels to logarithmic phase, the bacteria suspension for taking 10 μ L is added dropwise the load glass after the cooling that sterilizes
On piece, carry out plasma mutagenesis with ARTP mutation breedings instrument (think of radically reform biotechnology), mutation time be respectively 0s, 5s, 10s,
15s、20s、40s、60s。
(2) bacteria suspension after mutagenesis is transferred in the sterile tube equipped with 1mL physiological saline, then by Sample Dilution extremely
10-1~10-3Three concentration, take dilution coat screening and culturing medium (containing 0.1g/L Pig cholates LB solid plates) in, be placed in
24~48h of Anaerobic culturel in 37 DEG C of constant incubators, each treatment is in triplicate.
(3) 0.5% half Guang ammonia is contained to small order picking there is the single bacterium colony of transparent circle to be seeded to greatly by transparent circle
In 96 orifice plates of the MRS culture mediums of acid, 24~48h of Anaerobic culturel in 37 DEG C of constant incubators, centrifugation, collects thalline uses physiology
Salt water washing is simultaneously diluted to identical OD values, determines full cell enzyme activity.
(4) picking enzyme activity highest bacterial strain (being named as Bifidobacterium BH06) is collected by centrifugation by the method culture of step (3)
Thalline, extracts genome.Bsh genes with Genbank accession number 530821.1 expand Bifidobacterium as template design primer
The bsh genes of BH06, gel nucleic acid electrophoresis verify and are sequenced, and obtain the gene as shown in SEQ ID NO.1.
The structure of the recombination bacillus coli of embodiment 3
With plasmid pET-28a (+) as carrier, construction recombination plasmid pET-28a (+)-bsh06, conversion to E.coil
The competent cell of JM109, picking positive bacterium colony.Plasmid is extracted after 37 DEG C of incubator overnight cultures, through sequence verification, will be sequenced just
True plasmid pET-28a (+)-bsh06 is converted to Escherichia coli E.coil BL21 (DE3), obtains recombination bacillus coli E.coil
BL21(DE3)pET-28a(+)-bsh06。
The induced expression and enzyme activity determination of zymoprotein in the recombinant bacterium of embodiment 3
(1) will express bile salt hydrolase recombination bacillus coli E.coil BL21 (DE3) pET-28a (+)-bsh and
E.coil BL21 (DE3) pET-28a (+)-bsh06 picking individual colonies are inoculated into LB fluid nutrient mediums respectively, 37 DEG C,
12h is cultivated under 200rpm, is transferred in TB culture mediums, inoculum concentration is 1%, cultivate long to thalline under the conditions of 37 DEG C, 200rpm
To OD600For 0.8 when, add IPTG inductions, IPTG concentration is 0.1M, and cultivation temperature dropped into 20 DEG C, cultivates 24h.According to courage
Salt hydrolysis enzymatic characterization method, detects recipient bacterium and occurs in the LB solid plates containing 0.01% Pig cholate (w/v)
Bright precipitation circle, shows that the bile salt hydrolase mutant of recipient bacterium expression is active.
(2) it is control with E.coil BL21 (DE3) pET-28a (+)-bsh, two recombinant bacteriums is distinguished into picking individual colonies
It is inoculated into LB fluid nutrient mediums, 37 DEG C, 12h is cultivated under 200rpm, be transferred in TB culture mediums, inoculum concentration is 1%, in 37
DEG C, cultivate to thalline under the conditions of 200rpm and grow to OD600For 0.8 when, add IPTG induction, IPTG concentration be 0.15M, and will training
Foster temperature drops to 20 DEG C, cultivates 24h.By 6000~9000rpm of zymotic fluid centrifugations, with the phosphate buffer of 0.1M, pH 6~7
Washing thalline 2~3 times, and ultrasonication 5min, are collected by centrifugation supernatant and determine intracellular enzyme activity.Result shows, control strain born of the same parents
The intracellular enzyme activity of interior enzyme activity 112.29U/mL, recombinant bacterium E.coil BL21 (DE3) pET-28a (+)-bsh06 bile salt hydrolases is carried
It is high by 14.6%.
Influence of the different induction times of embodiment 4 to enzyme activity
Recombination bacillus coli E.coil BL21 (DE3) pET-28a (+)-bsh06 for expressing bile salt hydrolase is selected into list
Colony inoculation 37 DEG C, 12h is cultivated under 200rpm in LB fluid nutrient mediums, is transferred in TB culture mediums, and inoculum concentration is 1%, in
37 DEG C, cultivate to thalline under the conditions of 200rpm and grow to OD600For 0.8 when, add IPTG induction, IPTG concentration be 0.1M, and will training
Foster temperature drops to 20 DEG C, induces 16~36h.E.coil BL21 (DE3) pET-28a (+)-bsh enzyme activity measured with embodiment 3
It is control, determines the relative enzyme activity of E.coil BL21 (DE3) pET-28a (+)-bsh06 intracellulars under different induction times, as a result
As shown in Figure 1.Under 0.1M IPTG inductions, enzyme activity improves more than 10.3%, wherein relative enzyme activity is after induction 28h
123.2%, 23.2% is improve compared with the control, induction 32h enzyme activity improves 21.6%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
SEQUENCE LISTING
<110>Cao Shuhua
<120>The recombination bacillus coli that a kind of bile salt hydrolase enzyme activity is improved
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 942
<212> DNA
<213>Artificial sequence
<400> 1
atgtgcaccg ctgttcgttt cgacgacggt cagaacaaca tgtacttcgg tcgtaacctg 60
gactggtctg aagactacgg tgaaaaaatc gttttcgctc cgcacgacta ccactacgct 120
ccggctttca acgctgaaga caaaaaccac ccggttatcg gtatcggtat catcgttgaa 180
gacaccccgc tgtacttcga ctgcatgaac gacgctggtc tggctgttgc tggtctgaac 240
ttcgctaaat actgcaaata cgctaccgaa gctgttaact tcaccaccaa cgttgctgct 300
tacgaattcc cgctgtgggt tacccgtaac ttcacctctg ttgacgacgt tcaggaagct 360
ctgaaaaacg ttaccatcgt tggtaaaccg atcaacgacc gtttcccggt tgctaccctg 420
cactggatca tcgctgacaa cacccgttct atcgttgttg aatgcaccga agacggtatg 480
cacgtttacg acgacgacgt tgacgttctg accaaccagc cgccgttccc gcagcagatc 540
gaacacctgg acaactacgc ttacgtttct ccgcgtaccg gtaaatctgt taaatggggt 600
tcttctgaac tggaaaccaa ccaggactct aactcttctc agggtctgcc gggtggttac 660
ggttctatgg ctcgtttcgt tcgtgctgct tacaacaaca cccactaccc gacccagtct 720
ggtgaaaacg ctaacgttaa ccgtctgttc aaaaccctgt ctaccgctgc tgttatcgaa 780
ggtaccgcta tctctgctaa cggtgaattc gaaaaaaccc tgttctctga ctgctactct 840
accgctaccc agaccgttta cctgaaaaaa tacgacgaca tggctgttca ctcttacgct 900
gttaaagact tcgacgcttc ttctaaccag ctgcagtcta aa 942
Claims (10)
1. the bile salt hydrolase gene that a kind of enzyme activity is improved, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.1.
2. the cell line of bile salt hydrolase gene described in claim 1 is expressed.
3. a kind of recombination bacillus coli, it is characterised in that with pET-28a (+) as carrier, with e. coli bl21 (DE3) as place
It is main, gene shown in expression SEQ ID NO.1.
4. the construction method of the recombination bacillus coli that a kind of enzyme activity is improved, it is characterised in that methods described is will to be weighed containing coding
Profit requires that the gene of the bile salt hydrolase mutant described in 1 is converted into Escherichia coli after being connected with expression vector.
5. method according to claim 4, it is characterised in that the recombination bacillus coli be pET-28b (+) be carrier,
With E.coli BL21 (DE3) for gene shown in host expresses SEQ ID NO.1.
6. a kind of method for producing bile salt hydrolase, it is characterised in that be seeded to the recombination bacillus coli described in claim 3
In TB culture mediums, 37 DEG C of 12~52h of culture.
7. method according to claim 6, it is characterised in that the inoculum concentration is by volume 1%.
8. method according to claim 6, it is characterised in that the thalline culture to OD600For 0.8~1.0 when, add
IPTG is induced.
9. method according to claim 6, it is characterised in that methods described also by the zymotic fluid centrifugation after Fiber differentiation,
Collects thalline, and broken wall purifying obtains pure enzyme.
10. bile salt hydrolase gene described in claim 1 food, health products, pharmaceutical product production field application.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11291693B2 (en) | 2015-06-25 | 2022-04-05 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002130A (en) * | 2015-07-30 | 2015-10-28 | 江南大学 | Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002130A (en) * | 2015-07-30 | 2015-10-28 | 江南大学 | Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 无: "NCBI Reference Sequence: WP_004217908.1", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11291693B2 (en) | 2015-06-25 | 2022-04-05 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
| US11896627B2 (en) | 2015-06-25 | 2024-02-13 | Synlogic Operating Company, Inc. | Bacteria engineered to treat metabolic diseases |
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Application publication date: 20170531 |