CN105524907A - Bile salt hydrolase mutant with substrate specificity improved - Google Patents

Bile salt hydrolase mutant with substrate specificity improved Download PDF

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CN105524907A
CN105524907A CN201610070608.0A CN201610070608A CN105524907A CN 105524907 A CN105524907 A CN 105524907A CN 201610070608 A CN201610070608 A CN 201610070608A CN 105524907 A CN105524907 A CN 105524907A
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bile salt
mutant
salt hydrolase
seqidno
genetic engineering
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CN105524907B (en
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刘松
堵国成
陈坚
毕洁
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Jiangnan University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • 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 a bile salt hydrolase mutant with substrate specificity improved, and belongs to the field of enzyme engineering. In a mode of site-specific mutagenesis, tyrosine (Tyr24), alanine (Ala58) and phenylalanine (Phe65) on of amino acid sites of lactobacillus salivarius derived bile salt hydrolase BSH1 in a substrate-binding pocket are mutated into alanine, glutamine and alanine, so that hydrophobic interaction and binding mode between enzyme molecules and a substrate are changed, and the substrate specificity of the bile salt hydrolase is improved. Zymoprotein, after transformation, is beneficial for reducing the production amount of bile acid in a conjugated bile salt hydrolysis reaction, capable of improving the bile salt tolerance of lactobacillus and is conducive to researches on the application of the bile salt hydrolase to regulate the composition and the flow rate of host bile.

Description

The bile salt hydrolase mutant that a kind of Substratspezifitaet improves
Technical field
The present invention relates to the bile salt hydrolase mutant that a kind of Substratspezifitaet improves, belong to enzyme engineering field.
Background technology
Milk-acid bacteria (LAB), as probiotic bacterium, is the industrial strain of a class in field widespread uses such as food, agricultural, medical science.In order to effectively play its prebiotic function, milk-acid bacteria needs tolerance from the various plysiochemical barrier of host gastrointestinal tract environment, and wherein, in enteron aisle bile is one of coercing of facing of milk-acid bacteria in conjunction with cholate.Bile salt hydrolase (BSH) in current known extensive existence and enteric microorganism born of the same parents has substrate specificity widely, almost can be hydrolyzed and allly in bile generate free cholic acid and amino acid in conjunction with cholate, remove the toxicity in conjunction with cholate, but too much cholic acid can cause new coercing again, therefore improve the Substratspezifitaet of BSH, can play and reduce cholaligenic object.
Meanwhile, bile acide, as a kind of semiochemicals, in biological cycle, take part in biochemical metabolism and the adjustment of body lipid, cholesterol and sugar, and therefore, researchist thinks that the composition of change bile acide and flow can as a kind of new direction for the treatment of metabolism syndrome.The Substratspezifitaet using genetic engineering means to improve bile salt hydrolase can not only improve the intestinal colonisation ability of milk-acid bacteria, also for using BSH to regulate host health situation to provide technology platform.
Summary of the invention
In order to solve the problem, the invention provides the bile salt hydrolase mutant that a kind of Substratspezifitaet improves, described sudden change carries out rite-directed mutagenesis to the bile salt hydrolase BSH1 internal amino acid deriving from saliva lactobacillus of aminoacid sequence as shown in SEQIDNO.1, thus improve the Substratspezifitaet of bile salt hydrolase.
Described sudden change is that the tyrosine of bile salt hydrolase the 24th is sported L-Ala, obtains mutant Y24A; Or be glutamine by the alanine mutation of 58, obtain mutant A58Q; Or the phenylalanine of 65 is sported L-Ala, obtain mutant F65A.
The aminoacid sequence of described bile salt hydrolase mutant Y24A, A58Q, F65A is respectively as SEQIDNO:2, SEQIDNO:3, SEQIDNO:4.
In one embodiment of the invention, the nucleotide sequence of described mutant is respectively as shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7.
Second object of the present invention is to provide the nucleotide fragments of described bile salt hydrolase mutant of encoding, the carrier containing described bile salt hydrolase mutant, and express described bile salt hydrolase mutant genetic engineering bacterium.
In one embodiment of the invention, described genetic engineering bacterium is intestinal bacteria, is transformation of E. coli after being connected with expression vector by the gene containing the described bile salt hydrolase mutant of coding.
In one embodiment of the invention, described genetic engineering bacterium is that pET-20b (+) is carrier, E.coliBL21 (DE3) is mutant described in host expresses.
3rd object of the present invention is to provide a kind of method of producing bile salt hydrolase, is the genetic engineering bacterium utilizing the coding nucleotide fragments of described bile salt hydrolase mutant, the carrier containing described bile salt hydrolase mutant or express described bile salt hydrolase mutant.
In one embodiment of the invention, described method collects thalline by after the genetic engineering bacterium inducing culture of the described bile salt hydrolase mutant of expression, and broken wall purifying obtains; Described broken wall purifying is after collecting thalline, washs centrifugal 2 times with the phosphate buffered saline buffer of 0.1M, pH6.0, and resuspended, ultrasonication 5min, collected by centrifugation supernatant liquor, the ultra-filtration membrane being 10kDa through nickel post affinity column chromatography and molecular weight cut-off carries out purified concentration, obtains single recombinant protein.
Described inducing culture cultivates seed liquor, is 1% be inoculated in TB liquid nutrient medium by inoculum size, 37 DEG C of cultivations; Thalline grows to OD 600during for 0.5-0.6, add IPTG induction, and culture temperature is dropped to 20 DEG C, cultivate 24h.
The present invention is claimed described bile salt hydrolase mutant also; or utilize and produce the method for bile salt hydrolase and produce the bile salt hydrolase that obtains in food, agricultural or the application prepared in medicine, especially preparing the application in probiotics, medicine expression vector or fodder additives.
4th object of the present invention is to provide a kind of method improving bile salt hydrolase BSH1 Substratspezifitaet, that the tyrosine of the bile salt hydrolase protein molecule inner 24th of aminoacid sequence as shown in SEQIDNO.1 is sported L-Ala, or be glutamine by the 58th alanine mutation, or the phenylalanine of the 65th is sported L-Ala.
Name about mutant:
Represent mutant, such as S32A with " parent amino acid site replace after amino acid ", refer to, on the basis of parent amino acid sequence, the amino acid S (Serine, Ser) of the 32nd is sported amino acid A (L-Ala, Ala).In the present invention, with the sequence shown in SEQIDNO.1 for parental array.
Beneficial effect of the present invention:
The present invention is by the mode of rite-directed mutagenesis, amino acid in bile salt hydrolase substrate binding pocket is suddenlyd change, change the hydrophobic interaction between enzyme molecule and substrate, and then affect the Binding Capacity mode of enzyme molecule, improve the Substratspezifitaet of bile salt hydrolase.Mutant F24A of the present invention only has hydrolytic activity to four kinds in six kinds of substrates, and reduces about 30% to the relative enzyme of GCDCA with TDCA is alive; And mutant protein A58Q and F65A only has hydrolytic activity to five kinds in six kinds of substrates, and A58Q reduces the specificity of more than 50%, F65A to GDCA to GDCA, GCDCA, TCA, TDCA is significantly improved.Substratspezifitaet and the specificity of mutant F24A, A58Q and F65A of the present invention are obtained for raising, for using BSH to regulate host health situation to provide technology platform, also for the industrial application of probiotic strain is laid a good foundation.
Accompanying drawing explanation
Fig. 1: bile salt hydrolase wild-type BSH1 compares with the substrate specificity of mutant protein; The mensuration of substrate specificity is by dividing
Not with six kinds in conjunction with cholate for substrate, measure and live than enzyme, soprano is 100%.
Embodiment
LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.0;
TB substratum: peptone 12g/L, yeast extract paste 24g/L, glycerine 8g/L, 17mmol/LKH 2pO 4, 72mmol/LK 2hPO 4.
Bile salt hydrolase enzyme activity determination method: get 10 μ L enzymes liquid 0.1M phosphate buffered saline buffer (pH6.0) and be diluted to 90 μ L, add 10 μ L to mix in conjunction with cholate (200mM), 30min is hatched in 37 DEG C, add isopyknic 15% (w/v) trichoroacetic acid(TCA) termination reaction, get 10 μ L supernatant liquors after centrifugal to mix with 190 μ L ninhydrin reagents, in 100 DEG C of reaction 15min, measure light absorption value in 570nm place after cooling, calculate according to glycine typical curve.Wherein, consisting of of ninhydrin reagent: 0.5mL1% (w/v) triketohydrindene hydrate (being dissolved in 0.5M, pH5.5 citrate buffer), 1.2mL glycerine, the citrate buffer solution of 0.2mL0.5M, pH5.5.
Embodiment 1: the structure of recombinant bacterium
The DNA carrying the recombinant plasmid pET21 of bile salt hydrolase BSH1 encoding gene (aminoacid sequence is as SEQIDNO.1) built with early stage is for template, and carry out pcr amplification according to KOD-Plus-Neo test kit specification sheets, primer sequence is:
F24A-F:5 '-TTAGATTTAGATTTTTCAGCTGGTGAGGAGGTAATCATT-3 ' (sequence is as SEQIDNO.8)
F24A-R:5 '-AATGATTACCTCCTCACCAGCTGAAAAATCTAAATCTAA-3 ' (sequence is as SEQIDNO.9)
A58Q-F:5 '-ATAGGTGTTGGAATTGTCCAGAATGATTATCCACTGTAT-3 ' (sequence is as SEQIDNO.10)
A58Q-R:5 '-ATACAGTGGATAATCATTCTGGACAATTCCAACACCTAT-3 ' (sequence is as SEQIDNO.11)
F65A-F:5 '-AATGATTATCCACTGTATGCTGATGCTATTAATGAGGAT-3 ' (sequence is as SEQIDNO.12)
F65A-R:5 '-ATCCTCATTAATAGCATCAGCATACAGTGGATAATCATT-3 ' (sequence is as SEQIDNO.13)
PCR primer is the competent cell that recombinant plasmid pET22Q58A is converted into cloning host E.coilJM109, on the LB flat board of penbritin, and the positive bacterium colony of picking.37 DEG C of incubator overnight extract plasmid after cultivating, the raw work order-checking qualification through Shanghai.Check order correct plasmid (containing aminoacid sequence respectively as SEQIDNO:2, SEQIDNO:3, SEQIDNO:4, nucleotide sequence is as the sequence of SEQIDNO:5, SEQIDNO:6, SEQIDNO:7), transformation of E. coli E.coilBL21 (DE3), obtains recombinant bacterium.
Embodiment 2: the abduction delivering of zymoprotein and activity identification in recombinant bacterium
To express the recombination bacillus coli of bile salt hydrolase mutant in embodiment 1, picking individual colonies is inoculated in LB liquid nutrient medium, 37 DEG C, 12h is cultivated under 200rpm, be transferred in TB substratum, inoculum size is 1%, in 37 DEG C, be cultured to thalline under 200rpm condition and grow to OD 600during for 0.5-0.6, add IPTG induction, IPTG concentration is 1mM, and culture temperature is dropped to 20 DEG C, cultivates 24h.
Embodiment 3: the preparation of mutant protein and the mensuration of Substratspezifitaet thereof
Collect the thalline after embodiment 2 fermentation, centrifugal 2 times are washed with 0.1M phosphate buffered saline buffer (pH6.0), and it is resuspended, ultrasonication 5min (working hour: intermittent time=2:4), collected by centrifugation supernatant liquor, the bile salt hydrolase mutant protein after the ultra-filtration membrane acquisition purified concentration that nickel post affinity column chromatography and molecular weight cut-off are 10kDa.
Compare bile salt hydrolase mutant F24A, A58Q, F65A and wild-type BSH1 (SEQIDNO:1) to the specificity of six kinds of substrates.Wherein six kinds of substrates be respectively sweet ammonia in conjunction with cholate (GCA), sweet ammonia deoxidation in conjunction with cholate (GDCA), the deoxidation of sweet ammonia goose in conjunction with cholate (GCDCA), ox sulphur in conjunction with cholate (TCA), the deoxidation of ox sulphur in conjunction with cholate (TDCA), the deoxidation of ox sulphur goose in conjunction with cholate (TCDCA).
The measurement result of Substratspezifitaet as shown in Figure 1.Compared with wild-type bile salt hydrolase BSH1, bile salt hydrolase mutant F24A only has hydrolytic activity to four kinds in six kinds of substrates, and reduces about 30% to the relative enzyme of GCDCA with TDCA is alive; And mutant protein A58Q and F65A only has hydrolytic activity to five kinds in six kinds of substrates, and A58Q reduces the specificity of more than 50%, F65A to GDCA to GDCA, GCDCA, TCA, TDCA is significantly improved.Above data show, Substratspezifitaet and the specificity of mutant F24A, A58Q and F65A of the present invention are obtained for raising.
In addition, contriver also suddenlys change to F130 site, and result display substrate specificity does not change, and still has hydrolytic activity to these six kinds of substrates.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. a bile salt hydrolase mutant for Substratspezifitaet raising, it is characterized in that, the aminoacid sequence of described mutant is as shown in SEQIDNO:2, SEQIDNO:3 or SEQIDNO:4.
2. mutant according to claim 1, is characterized in that, the nucleotide sequence of described mutant is as shown in SEQIDNO:5, SEQIDNO:6 or SEQIDNO:7.
3. the nucleotide fragments of bile salt hydrolase mutant described in coding claim 1.
4. the carrier containing bile salt hydrolase mutant described in claim 1.
5. express bile salt hydrolase mutant described in claim 1 genetic engineering bacterium.
6. genetic engineering bacterium according to claim 5, is characterized in that, described genetic engineering bacterium is intestinal bacteria.
7. genetic engineering bacterium according to claim 6, is characterized in that, described genetic engineering bacterium is that pET-20b (+) is carrier, E.coliBL21 (DE3) is mutant described in host expresses.
8. in a kind of method of producing bile salt hydrolase, it is characterized in that, is utilize the arbitrary described genetic engineering bacterium of the nucleotide fragments described in claim 3, carrier according to claim 4 or claim 5-7.
9. method according to claim 8, is characterized in that, described method collects thalline by after genetic engineering bacterium inducing culture, and broken wall purifying obtains; Described broken wall purifying is after collecting thalline, washs centrifugal 2 times with the phosphate buffered saline buffer of 0.1M, pH6.0, and resuspended, ultrasonication 5min, collected by centrifugation supernatant liquor, the ultra-filtration membrane being 10kDa through nickel post affinity column chromatography and molecular weight cut-off carries out purified concentration, obtains single recombinant protein.
10. the bile salt hydrolase that the arbitrary described bile salt hydrolase mutant of claim 1-2, or the arbitrary described method of claim 8-9 obtains is in food, agricultural or the application prepared in medicine.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005161A1 (en) * 1991-09-11 1993-03-18 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Lactobacilli having genetically modified bile salt hydrolytic activity, production and use thereof
CN102220408A (en) * 2011-05-10 2011-10-19 江南大学 Lactic acid bacteria for producing bile salt hydrolase as well as screening method and application thereof
CN103756989A (en) * 2013-11-11 2014-04-30 江南大学 Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof
CN103992991A (en) * 2013-11-11 2014-08-20 江南大学 Bile salt hydrolase (BSH) mutant and use thereof
WO2014145958A2 (en) * 2013-03-15 2014-09-18 Seres Health, Inc. Network-based microbial compositions and methods
CN104928206A (en) * 2015-04-29 2015-09-23 东北农业大学 Lactobacillus acidophilus with high cholate hydrolase activity and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005161A1 (en) * 1991-09-11 1993-03-18 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Lactobacilli having genetically modified bile salt hydrolytic activity, production and use thereof
CN102220408A (en) * 2011-05-10 2011-10-19 江南大学 Lactic acid bacteria for producing bile salt hydrolase as well as screening method and application thereof
WO2014145958A2 (en) * 2013-03-15 2014-09-18 Seres Health, Inc. Network-based microbial compositions and methods
CN103756989A (en) * 2013-11-11 2014-04-30 江南大学 Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof
CN103992991A (en) * 2013-11-11 2014-08-20 江南大学 Bile salt hydrolase (BSH) mutant and use thereof
CN104928206A (en) * 2015-04-29 2015-09-23 东北农业大学 Lactobacillus acidophilus with high cholate hydrolase activity and application thereof

Non-Patent Citations (1)

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Title
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