CN104818265B - A kind of bifunctional enzyme of resistance to ethanol and its application of degrade urea and urethanes - Google Patents
A kind of bifunctional enzyme of resistance to ethanol and its application of degrade urea and urethanes Download PDFInfo
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
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- C12H1/003—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages by a biochemical process
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- 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)
- C12Y305/01005—Urease (3.5.1.5)
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- 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)
- C12Y305/01075—Urethanase (3.5.1.75)
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Abstract
The invention discloses the present invention relates to a kind of bifunctional enzyme of resistance to ethanol and its application of degrade urea and urethanes, belong to technical field of bioengineering.The enzyme comes from bacillus licheniformis (Bacillus licheniformis 9945A) urase, is resistant to efficient degradation urethanes while high concentration ethanol.The present invention utilizes escherichia expression system BL21 (DE3)/pRSFDuet 1, it is successfully realized the high efficient expression of the urease gene with EC hydrolysis activities, enzyme activity reaches 2.93U/ml during using EC as substrate, and enzyme activity reaches 5.81U/ml during using urea as substrate.Recombinase 37 DEG C of reservation 4h in 20% ethanol, can still retain its more than 50% activity during using EC as substrate.Recombinase prepared by the present invention is the elimination for realizing EC and urea in yellow rice wine, realizes that EC hydrolase industrialized productions are laid a good foundation, and has huge economy and social benefit.
Description
Technical field
The present invention relates to a kind of bifunctional enzyme of resistance to ethanol and its application of degrade urea and urethanes, belong to biology
Field of engineering technology.
Background technology
Urethanes (Ethyl Carbamate or Urethane, abbreviation EC), be it is a kind of have genetoxic and compared with
The material (can cause lung neoplasm, lymph cancer, liver cancer, cutaneum carcinoma etc.) of strong carcinogenicity, is widely present in a variety of fermented foods (such as
Soy sauce, vinegar, pickles) and alcoholic beverage (such as yellow rice wine, white wine, grape wine, sake, brandy) in.Human intake's ammonia
Base Ethyl formate mainly passes through No alcoholic beverages and food.Urethanes has turned into one of influence human health not
Negligible factor.The urethanes for taking effective ways to eliminate in fermented food or beverage is extremely urgent.
The EC eliminated in fermented food mainly has two methods：
When reduction or eliminate EC in zymotic fluid precursor substance (mainly urea) content, this is to lead in the world at present
Way.Urase can break down urea into ammonia and CO2, EC generations can be effectively reduced, utilize acid urea in process of production
EC contents are the most commonly used methods in enzyme control finished wine.The acid urease for wine of the production such as the U.S., Japan at present has been achieved with
Commercialization.Because urea is not unique precursor substance for forming EC, therefore this method is difficult to thoroughly elimination EC formation.
Another kind is direct degraded urethanes.EC hydrolases (Urethanase, abbreviation UH) can drop EC
Solve as ethanol, ammonia and carbon dioxide, so as to the EC that effectively degrades.Due to more than EC Forming Mechanisms and more complicated, simultaneously
The mechanism for suppressing a variety of formation EC is relatively difficult, it is difficult to EC precursor substances are thoroughly eradicated, so as to be difficult to thoroughly eliminate EC formation,
And EC formed after because its structure is highly stable, it is difficult to eradicate, therefore application biological enzyme is removed and formed in finished product
EC, it is a kind of ideal minimizing technology.
For at present, EC hydrolases that document report has obtained, parsed only two of amino acid sequence：One
It is that Japanese Akutsu-Shigeno Y have parsed an acyl ammonia enzyme in 2006 from Rhodococcus equi TB-60, but
It only has very faint degradation for EC, is defined as that EC hydrolases are a bit forced, at the same its alcohol resistance nor
It is often poor, therefore may not apply in the fermented foods such as yellow rice wine；The second is obtained from Lysinibacillus fusiformis
One amide transferase, but its alcohol resistance is poor, does not have under high ethano concentration in any activity, pH4.5 environment also without work
Property, therefore also it is difficult to the application in yellow rice wine.Because UH gene order is not yet effectively parsed, only by traditional side
Method purifies production UH enzymes cost height from wild mushroom and production capacity is limited, it is difficult to large-scale commercial application.
The urase that is obtained of the present invention its still have in high concentration ethanol (20%v/v) efficient EC hydrolytic enzyme activities (with
2 times of enzyme activity when enzyme activity when urea is substrate is only using EC as substrate) so that it has huge application in yellow rice wine
Value, while the industrialized production for resolving to EC hydrolases of its gene order is laid a good foundation.
Urease gene sequence is made up of structural gene and its auxiliary gene, and structural gene compiles urase albumen, its auxiliary gene
Supplementary structure gene expression is played, the albumen of expression of structural gene is modified (for example transmit metal to catalytic active center
Ion) so as to formed maturation urase.The urase monomer structure of separate sources is different.Urase wherein from bacterium is usual
Polymer is formed by three different subunit A, B, C, its catalytic active center is located at C subunits.Urea obtained in the present invention
Enzyme is made up of three structural gene ureA ureB ureC and 5 auxiliary gene ureE ureF ureG ureD ureH, and it is urged
Change activated centre and be located at C subunits, final ripe protein is the tripolymer of tri- subunit compositions of A, B and C.Single expression owns
Gene, express expressing protein assembled in vitro or simultaneously the activity expression that all genes are possible to obtain the urase.
The content of the invention
First purpose of the present invention is to provide one kind and is resistant to high concentration ethanol efficient degradation urethanes simultaneously
With the bifunctional enzyme and its gene order of urea.The nucleotide sequence such as SEQ ID NO:Shown in 1 or it is coding SEQ ID
NO:The amino acid sequence of protein shown in 2.
Structural subunits UreA, UreB, UreC of the bifunctional enzyme amino acid sequence：(a) respectively such as SEQ ID
NO.2, SEQ ID NO.3, shown in SEQ ID NO.4；(b) it is to pass through substitution, missing on the basis of amino acid sequence in (a)
Or addition one or several amino acid obtain, and encode with ethyl carbamate hydrolase and urease activity by (a)
Derivative protein, such as in C-terminal or N-terminal addition or missing one or several amino acid residues, addition fusion tag etc., although
Modified in form but do not change the enzyme activity of albumen.
The bifunctional enzyme, in one embodiment of the invention, there is SEQ ID NO:Nucleotides sequence shown in 1
Row.
The sequence, it is by the way that purifying is come from into Bacillus in one embodiment of the invention
The laser desorption ionization flight time mass spectrum (SALDI- that licheniformis 9945A EC hydrolases are strengthened by surface
TOF-MS peptide fragment accurate molecular weight identification) is carried out, searches for the albumen that Mascot standard protein databases are matched, according to
Bacillus licheniformis 9945A genomic informations in ncbi database and obtain.
The bifunctional enzyme, in one embodiment of the invention, Bacillus can be derived from
licheniformis 9945A、Bacillus licheniformis 12759、Bacillus licheniformis G-1、
Any one in Bacillus licheniformis S16, Bacillus licheniformis BL-09 etc..
Second object of the present invention is to provide a kind of method for urethanes of degrading, and is to utilize to derive from lichens bud
The difunctional urase of spore bacillus is come urethanes of degrading.
The difunctional urase contains one of following characteristics：(a) Structural subunits UreA, UreB, UreC amino acid sequence
Respectively as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4；(b) amino acid sequence of Structural subunits is in (1)
By substituting, lacking or add what one or several amino acid obtained on the basis of middle amino acid sequence, and coding has ammonia
The protein as derived from (a) of base Ethyl formate hydrolase and urease activity；(c) there is SEQ ID NO:Nucleotides shown in 1
Sequence.
The difunctional urase, it is by from bacillus licheniformis genome in one embodiment of the invention
Clone obtains urease gene, obtained from then urease gene is expressed in Host Strains.
The expression, in one embodiment of the present invention, it is：Obtain SEQ ID NO:Nucleotide sequence shown in 1, will
The nucleotide sequence, which is connected on expression plasmid carrier, builds recombinant expression plasmid, is obtained after recombinant expression plasmid conversion Host Strains
Obtain recombinant bacterium.
The Host Strains can be Escherichia coli, bacillus subtilis, huge bud in one embodiment of the invention
Any one in spore bacillus, bacillus licheniformis, Lactococcus lactis or saccharomycete, wherein it is preferred that e. coli bl21 DE3.
Described recombinant plasmid, can be commercially available plasmid, clay, bacteriophage in one embodiment of the invention
Deng through Protocols in Molecular Biology means by the urease gene sequence connect into and it is built-up, wherein it is preferred that pRSFDuet-1.
The expression, in one embodiment of the present invention, it is specifically：(1) nucleotide sequence such as SEQ ID NO.1 are obtained
Shown urease gene ureBL；(2) target gene ureBL is connected to carrier pRSFDuet-1 and obtains recombinant plasmid
pRSFDuet-ureBL；(3) recombinant plasmid that step 2 obtains is imported in e. coli bl21 DE3 and obtains recombination bacillus coli
BL21DE3 (pRSFDuet-ureBL), urease gene are expressed by T7 promoter regulations in recombinant bacterium；(4) recombinant bacterium is utilized
BL21DE3 (pRSFDuet-ureBL) fermenting and producing has the urase of EC hydrolysis activities.
The fermentation of the recombinant bacterium, in one embodiment of the invention, Ke Yishi：By E.coli BL21 (DE3)/
The culture of pRSFDuet-ureBL genetic engineering bacteriums is to certain cell concentration (such as OD600=0.6-0.8), addition derivant (such as IPTG
Ultimate density is 1mM), EC urase efficiently drops in culture certain time (such as 10-30h), the resistance to ethanol of high efficient expression.
Urethanes and urea side of the urase in the fermented foods such as yellow rice wine are eliminated is also claimed in the present invention
The application in face.
Beneficial effects of the present invention：Find that there is degraded urea and efficient degradation urethanes and resistance to second simultaneously first
The urase of alcohol, the enzyme can be used for the urethanes for reducing alcoholic beverage.The enzyme is expressed in Escherichia coli, with EC
For substrate when enzyme activity reach 2.93U/ml, enzyme activity reaches 5.81U/m during using urea as substrate.When this urase is using EC as substrate simultaneously
With high alcohol resistance, more than 50% activity can be still kept after 37 DEG C of insulation 4h in 20% ethanol.
Brief description of the drawings
Fig. 1：UreBL PCR amplification electrophoresis patterns, wherein M is DNAMarker (10,000bp, Takara company), and 1 is
UreBL pcr amplification product；
Fig. 2：Plasmid pRSFDuet-ureBL BamH I and Xho I double digestion electrophoresis patterns, wherein, M DNAMarker
(10000bp, Takara company)；1 be pRSFDuet-ureBL through BamH I and Xho I double digestion samples；
Fig. 3：Expression plasmid pRSFDuet-ureBL structure schematic diagram；
Fig. 4：The SDS-PAGE protein electrophoresises of recombinant bacterium E.coli BL21 (DE3)/pRSFDuet-ureBL expression products
Figure；Wherein 1 is breaking-wall cell supernatant after recombinant bacterium induction, and 2 be the precipitation after breaking-wall cell high speed centrifugation after recombinant bacterium induces,
3 be breaking-wall cell supernatant after the recombinant bacterium BL21 (DE3) without ureBL genes/pRSFDuet-1 inductions, and 4 be without ureBL
Breaking-wall cell precipitates after the recombinant bacterium BL21 (DE3) of gene/pRSFDuet-1 inductions.
Fig. 5：Alcohol resistance collection of illustrative plates when the restructuring urase of engineering strain expression is using EC as substrate.
Embodiment
Materials and methods
LB culture mediums：(film solid media separately adds 1.5% agar by dusty yeast 5g/L, peptone 10g/L, sodium chloride 10g/L
Powder)
TB culture mediums：Following component is dissolved in 0.9L water：Peptone 12g, yeast extract 24g, glycerine 4ml.Respectively
Autoclaving after component dissolving.60 DEG C are cooled to, then adds the 0.17mmol/L KH of 100ml sterilizings2PO4, 0.72mmol/
LK2HPO4Solution.
Used restriction enzyme is purchased from Fermentas companies, and glue reclaim kit is purchased from TAKARA companies, specifically
Reaction
Condition and application method refer to its specification.Genome extraction agent box is purchased from Omega companies, is said using with reference to it
Bright book.Following commercialization plasmid and Escherichia coli are used for Gene clone and expression：
PRSFDuet-1 (Novagen, the U.S.)
E.coli BL21 (DE3) (Novagen, the U.S.)
Ethyl carbamate hydrolase and urase enzyme activity determination method：Two 10mL colorimetric cylinders are taken, are separately added into 1mL enzyme liquids
With the enzyme liquid of 1mL inactivations.Then 1mL 3%EC (or urea) substrate solution is separately added into two pipes (with 20mM pH's 4.5
Citrate buffer solution is prepared), after reacting 30min in 37 DEG C of constant water bath box, in two each addition 1mL terminators (10% 3 of pipe
Monoxone), (15g phenol and 0.625g sodium nitroprussides are settled to addition 1mL developer I with ultra-pure water after mixing
It is strong to shake 250mL) with 1mL developers II (13.125g NaOH and 7.5mLNaClO are settled to 250mL with ultra-pure water), after
Continue and taken out after being incubated 20min in 37 DEG C of constant water bath box, 10mL is diluted to ultra-pure water, 625nm places colorimetric, measure OD values,
Calculate enzyme activity (standard curve is drawn using ammonium chloride).
Ethyl carbamate hydrolase and the definition of urase enzyme-activity unit：In normal pressure, 20mM pH4.5 citric acid-sodium citrates
Under the conditions of buffer solution and 37 DEG C, degraded EC (or urea) per minute generates 1 μm of ol NH4 +Required enzyme amount is an enzyme activity
Unit.
Embodiment 1：Encode resistance to ethanol efficient degradation EC urease genes ureBL clone
According to bacterial genomes extracts kit (OMEGA) operation instruction extraction bacillus licheniformis (Bacillus
Licheniformis 9945A) genomic DNA.Urease gene sequence (the GenBank announced according to NCBI:CP005965.1,
GI：521287266-521287273), designing forward primer F and reverse primer R, (sequence is respectively such as SEQ ID NO.5, SEQ ID
Shown in NO.6), forward primer adds BamH I restriction enzyme sites and its protection base, reverse primer plus Xho I restriction enzyme sites and
Its protection base is as follows：
Forward primer F：5'-CGCGGATCCGATGCAACTATTACCGCGTGAAGTAG-3'
Reverse primer R：5'-CCGCTCGAGTTAAATCCAAAGGTTAAATAAACCC-3'
Using bacillus licheniformis genomic DNA as template, above-mentioned specific primer is primer, to ureBL gene (nucleotides
Sequence is as shown in SEQ ID NO.1) expanded.Amplification system：According to PrimerSTAR HS DNA Polymerase
(TAKARA) kit specification prepares 50 μ l reaction systems.Amplification condition is：98 DEG C of 30s, 1 circulation, 98 DEG C of 10s, 54 DEG C
10s, 72 DEG C of 5min 48s, 30 circulations, 72 DEG C of 10min, mono- circulation.PCR primer is entered using 0.8% agarose gel electrophoresis
Row gel extraction, reclaims about 5.8kb DNA fragmentation (as shown in Figure 1), and recovery method is said with reference to glue reclaim kit (TAKARA)
Bright book is carried out.Recovery product serves Hai Sheng works biotech firm measure nucleotide sequence.
Embodiment 2：The structure of expression vector and expression system
The PCR primer that the method for embodiment 1 is obtained is through BamH I and Xho I double digestions, after gel extraction, and through BamH I
Connected with the pRSFDuet-1 plasmids of Xho I double digestions, be transferred to E.coli BL21DE3 competent cells, be coated on containing 50 μ
On the LB solid plates of g/ml kanamycins, 37 DEG C of culture 12h, with forward primer F and positive gram of reverse primer R, PCR identification
It is grand.Bacterium colony PCR is identified in correct LB fluid nutrient mediums of the positive colony access containing 50 μ g/ml kanamycins and cultivated, is carried
Plasmid is taken, carries out plasmid double digestion checking.Verify that correct plasmid (as shown in Figure 2) is sent to through BamH I and Xho I double digestions
Shanghai Sheng Gong biotech firms are sequenced.Thus obtain recombinating heterogenous expression system BL21 (DE3)/pRSFDuet-ureBL of urase.
Embodiment 3：The recombination expression of resistance to ethanol efficient degradation urethanes urase
Picking genetic engineering bacterium BL21 (DE3)/pRSFDuet-ureBL single bacterium colonies, it is inoculated in 25mL, containing 50 μ g/mL cards
In the LB fluid nutrient mediums of that mycin, 37 DEG C of concussion and cultivates are stayed overnight.It is forwarded to by 1% inoculum concentration containing 50 μ g/mL cards within second day
In the TB culture mediums of that mycin, cultivate to the dense OD of bacterium600When=0.6, add IPTG to final concentration of 1mmol/L and induce, 30 DEG C of trainings
10h is supported, thalline is collected by centrifugation, cell is resuspended with 20mM pH4.5 citric acids-citrate buffer solution, passes through ultrasonication, centrifugation
Supernatant is taken, determines enzyme activity, and expressing quantity is detected by SDS-PAGE.As can be known from Fig. 4, with being not connected with purposeful band
Empty carrier swimming lane is compared, and swimming lane is at 62kDa after induction and above and below 14KDa has with predicting target stripe of the same size, it was demonstrated that
Recombinant protein realizes expression in this expression system, and enzyme activity is：Enzyme activity reaches 2.93U/ml during using EC as substrate, with urea
For substrate when enzyme activity reach 5.81U/ml.
Example 4：Alcohol resistance when resistance to ethanol efficient degradation urethanes urase is using EC as substrate
The urase crude enzyme liquid that BL21 (DE3)/pRSFDuet-ureBL fermentations are obtained, is placed in 20% ethanol, 37 DEG C of guarantors
Temperature some hours, per a sample measure enzyme activity is taken every other hour, according to ethyl carbamate hydrolase described in materials and methods
Enzyme activity determination method, using without 20% Ethanol Treatment enzyme liquid activity as 100%, measurement result is as shown in Figure 5：
Enzyme liquid in 20% ethanol 37 DEG C insulation 4h after can still keep its 50% and the above activity.Thus say
It is bright, there is high alcohol resistance, the elimination for ethyl carbamate in yellow wine has huge when this urase is using EC as substrate
Application value.
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, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (6)
1. application of the difunctional urase of resistance to ethanol of degraded urea and urethanes in terms of urethanes of degrading, institute
Stating urase is：(1) as the amino acid sequence structure as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively
Subunit UreA, UreB and UreC are formed；Or (2) such as SEQ ID NO:Nucleotide sequence shown in 1.
2. the difunctional urase of resistance to ethanol of degraded urea and urethanes urethanes in alcoholic beverage of degrading
Using the urase is：(1) as amino acid sequence respectively as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4
Structural subunits UreA, UreB and UreC composition；Or (2) such as SEQ ID NO:Nucleotide sequence shown in 1.
A kind of 3. method for urethanes of degrading, it is characterised in that methods described is to utilize to derive from bacillus licheniformis
Urase be come urethanes of degrading, the urase：(1) by amino acid sequence respectively such as SEQ ID NO.2, SEQ ID
Structural subunits UreA, UreB and UreC composition shown in NO.3 and SEQ ID NO.4；Or (2) such as SEQ ID NO:Core shown in 1
Nucleotide sequence.
4. according to the method for claim 3, it is characterised in that the urase is by from bacillus licheniformis genome
Clone obtains urease gene, obtained from then urease gene is expressed in Host Strains.
5. according to the method for claim 4, it is characterised in that the Host Strains are Escherichia coli, bacillus subtilis, huge
Any one in Bacterium anthracoides, bacillus licheniformis, Lactococcus lactis or saccharomycete.
6. according to the method for claim 4, it is characterised in that the expression is：(1) nucleotide sequence such as SEQ ID are obtained
Urease gene ureBL shown in NO.1；(2) target gene ureBL is connected to carrier pRSFDuet-1 and obtains recombinant plasmid
pRSFDuet-ureBL；(3) the recombinant plasmid pRSFDuet-ureBL of acquisition is imported in e. coli bl21 (DE3) and weighed
Group e. coli bl21 (DE3)/pRSFDuet-ureBL, urease gene are expressed by T7 promoter regulations in recombinant bacterium；(4) it is sharp
There is the urase of ethyl carbamate hydrolase vigor with recombinant bacterium BL21 (DE3)/pRSFDuet-ureBL fermenting and producings.
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| CN105462998B (en) * | 2015-12-23 | 2019-06-07 | 江南大学 | A kind of difunctional acid urease structural gene and its expression and application |
| CN106011118B (en) * | 2016-06-07 | 2019-09-17 | 江南大学 | A kind of Fe3+ dependent form food-grade acid urase and its application in yellow rice wine |
| CN105820992A (en) * | 2016-06-07 | 2016-08-03 | 江南大学 | Lactic acid bacteria capable of efficiently expressing Fe3+ dependent-form food grade acid urease |
| CN105950527B (en) * | 2016-06-07 | 2019-09-17 | 江南大学 | A kind of bacillus subtilis of high efficient expression Fe3+ dependent form food-grade acid urase |
| CN106282071B (en) * | 2016-10-26 | 2019-07-23 | 江南大学 | The bacillus amyloliquefaciens of one plant of degradation urethanes and urea |
| CN107502574B (en) * | 2017-09-13 | 2019-10-08 | 江南大学 | The bacillus licheniformis of one plant of degradation urethanes and its precursor |
| CN110055240A (en) * | 2019-04-25 | 2019-07-26 | 江南大学 | A kind of urase recombinase of degradable low concentration urethanes |
| CN114807102B (en) * | 2022-05-16 | 2023-08-04 | 安徽工程大学 | Ethanol-resistant amidase, gene, expression vector, engineering bacterium, preparation method and application |
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