CN102676399B - Ethyl carbamate (EC) catabolic enzyme generating bacterium for yellow wine - Google Patents
Ethyl carbamate (EC) catabolic enzyme generating bacterium for yellow wine Download PDFInfo
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Abstract
The invention relates to an ethyl carbamate (EC) catabolic enzyme generating bacterium for yellow wine, belonging to the fields of small enzyme strain generating bacteria related to food quality improvement and enzyme characteristic research. The classification designation of the strain is (Penicilliumvariabile)JN-A525, and the strain has been collected in China General Microbiological Culture Collection Center; and the collection number is CGMCC No.5763. The amino acid sequence of the strain in the amplified ITS area complete sequence is SEQ ID NO:1. The optimum pH value of the EC catabolic enzyme generated by the bacterium is relatively wide, ranging from 4.0 to 6.0; and the optimum temperature is 50 DEG C. The enzyme generated by the bacterium has relatively strong hydrolysis capacity for the EC under simulated yellow wine conditions (15% ethanol, 3% EC and pH 4.4); and the generated EC catabolic enzyme has the application potential in removing generated trace EC and MC in fermented foods, such as sake, yellow wine, rice wine and the like, and can be combined with acidic urease, thereby enhancing the quality and safety of many fermented foods.
Description
Technical field
A kind of urethanum (EC) degrading enzyme generation bacterium for yellow rice wine, relate to the evaluation of this bacterium and the fundamental characteristics of the enzyme that produces, and isolates specifically a strain and can produce EC degrading enzyme generation bacterium from the enteron aisle of mouse, by the ITS ordination, identifies its kind.The thick enzyme extract obtained is analyzed to the fundamental characteristics such as the specificity of its optimal pH, optimum temperuture, pH value and temperature stability scope, substrate and alcohol resistance, and investigated its Preliminary Applications effect in yellow rice wine.Belong to food quality and promote relevant little enzyme generation bacterium and enzyme characteristic research field.
Background technology
Urethanum (ethyl carbamate, referred to as EC) can cause lung tumor, lymphatic cancer, liver cancer, skin carcinoma etc.It is generally acknowledged that the main mechanism of carcinogenesis of EC is: approximately 0.1% urethanum is oxidized to N-hydroxyl-urethanum by Cytochrome P450, and the latter can induce Cu
2+the DNA damage of regulation and control, this damage is multiple is born on the residue of thymus pyrimidine (T) and cytosine(Cyt) (C), and then affects biological genetic material, the initiation canceration.
Both at home and abroad the research of urethanum in wine shown, the main path that in wine, urethanum forms is the urea approach, and the urethanum in China's yellow rice wine and Japanese sake more than 90% is to derive from reacting of contained urea and ethanol in wine:
The analysis of the formation approach by urethanum is known, and in leavened food (as bread, yogurt milk, cheese, soy sauce etc.) and alcoholic beverage (as grape wine, hard cider, Chinese rice wine and Japanese sake etc.), the formation of EC is mainly from the reaction of urea and ethanol.The method that reduces EC content in yellow rice wine can be divided into improvement and the enzymolysis process of traditional technology.At present, utilizing enzymolysis process to remove EC in yellow rice wine has become now and development trend in the future.Urea is the precursor substance that generates urethanum, utilizes acid urease to process yellow rice wine, can remove most of urea in yellow rice wine, thereby reduces the possibility that generates urethanum.The urethanum degrading enzyme can directly act on urethanum, effectively decomposes it for urea and ammonia, reduces the EC content produced in yellow rice wine.The discovery of a strain EC degrading enzyme producing bacterial strain and the relevant research to it in the present invention, for the better reduce with control of EC content in following yellow rice wine provides the means of supplementing out economy preferably.
Raising day by day along with standard of living, the domestic consumption to Wine rises increasingly, especially the alcoholic drinkss such as grape wine, rice wine, yellow rice wine, beer become the focus of people's consumption especially, and the EC contents level therefore effectively reduced in the alcohol drink is imperative.
Summary of the invention
The bacterial strain that the purpose of this invention is to provide a kind of EC of producing degrading enzyme, this EC degrading enzyme has active preferably under the working condition of the leavened foods such as pure mellow wine, yellow rice wine, rice wine; The invention provides the relevant zymologic property of the EC degrading enzyme that this bacterium and this bacterium produce and the result that this enzyme is applied in yellow rice wine.
Technical scheme of the present invention: a kind of urethanum EC degrading enzyme produces bacterium, be from the enteron aisle of mouse, to separate to obtain the bacterial strain that the EC degrading enzyme is produced in a strain, its Classification And Nomenclature be penicillium variable (
penicillium variabile) JN-A525, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.5763.
Described EC degrading enzyme produces bacterium, the amplification ITS district complete sequence of this bacterial strain, and its aminoacid sequence is SEQ ID NO:1.
The optimal pH of bacterial strain CGMCC enzyme that No.5763 produces is 4.0-6.0, and optimum temperuture is 50 ℃ of left and right; This enzyme is between 40-50 ℃, and between pH value 7-9, enzyme has stability preferably.
Bacterial strain CGMCC No.5763 of the present invention separates and obtains from the intestinal tissue of mouse and content.
The evaluation of bacterial strain:
(1) design Auele Specific Primer, primer title: ITS1, ITS4
(2) extract genomic dna.
(3) synthetic primer, amplification ITS district complete sequence, amplification is as shown in Figure 3.
(4) PCR product purification.
(5) PCR product order-checking.The amplification ITS district complete sequence of this bacterial strain, its aminoacid sequence is SEQ ID NO:1.
(6) phylogenetic tree: as shown in Figure 6.
The sequencing result that the EC lytic enzyme is produced to the rDNA ITS sequence of bacterium CGMCC No.5763 is compared by the Blast program on the NCBI website and Genbank amplifying nucleic acid database, find with
penicillium variabilerDNA ITS sequence similarity reach 97%, simultaneously the EC lytic enzyme produce bacterium CGMCC No.5763 with
penicillium variabileon the systematic evolution tree based on rDNA ITS sequence, in same branch, and the colonial morphology of the two, Mycelial morphological characteristic are identical, and physiological and biochemical property is basically identical, therefore identify this bacterial strain for be Penicillium (
penicillium) middle penicillium variable (
penicillium variabile) JN-A525.
This bacterial strain is on czapek's solution, and the bacterium colony quality is velvet-like, and densification is smooth, thinner; Edge mycelium yellow, slightly green; It is orange-yellow to reddish-brown that the bacterium colony reverse side is; Observing conidium in Electronic Speculum is oval or ellipse, and wall is smooth or slightly coarse.
The preparation of crude enzyme liquid and research
Slant medium: sucrose 30g/L, NaNO
33 g/L, K
2hPO
41 g/L, KCl 0.5 g/L, MgSO
40.5 g/L, FeSO
40.01 g/L, agar 15-20 g/L, pH nature;
Fermention medium: glucose 20g/L, peptone 10g/L, pH 6.0;
The preparation of spore suspension: with fresh, eugonic spore on agar slant under appropriate aseptic washing, then move into and be equipped with in the triangular flask of granulated glass sphere, on vibrator, fully vibration, make spore dispersed.
The spore suspension of this bacterium is inoculated in the Erlenmeyer flask that contains 70 mL fermented liquids with 3% inoculum size, is placed in constant temperature speed governing shaking table and carries out fermentation culture, rotating speed is 100 r/min, and temperature is 30 ℃.After fermentation culture 3d, fermented liquid is centrifugal, and the mycelium of gained utilizes ultrasonic disruption, 10000 *
g, 4 ℃, 20 min.The supernatant liquid of acquisition is placed in to 4 ℃ of preservations.Be 13.87g/L by the biomass in centrifugal acquisition fermented liquid, record total enzyme and live as 340.8U/L, thereby yield of enzyme that can the unit's of measuring thalline is 24.57u/g.
Enzyme activity determination method and thick enzyme zymologic property
(1) enzyme activity determination method:
1. principle: under certain condition, after the crude enzyme liquid of this bacterial strain and substrate (3% EC, pH4.4) reaction, after utilizing terminator, developer I, developer II to act on respectively.The depth of its color is directly proportional to the vigor of enzyme within the specific limits, therefore carry out colorimetric under the wavelength of 625nm, calculates enzyme activity.
2. enzyme reaction system: the crude enzyme liquid after dilution; Developer I: take 15g phenol and the 0.625g sodium nitroprusside is settled to 250mL with ultrapure water; Developer II: take 13.125g NaOH and 7.5mL NaClO is settled to 250mL with ultrapure water; Terminator: take the 10g trichoroacetic acid(TCA) and be settled to 100mL with ultrapure water.
3. NH
4 +the standardized solution preparation
1mL ammoniacal liquor is dissolved in substrate reactions liquid (3% EC, pH4.4) and is settled to 145mL, is made into the NH of 0.1mol/L
4 +solution, and, as mother liquor, be mixed with the NH of 0.1 mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L with identical substrate reactions liquid (3% EC, pH4.4)
4 +standardized solution.
4. the drafting of ammonium ion typical curve
Accurately pipette 1mL NH with transfer pipet
4 +normal gradients liquid is placed in respectively the 10mL colorimetric cylinder of serial number.37 ℃ of lower constant temperature insulation 30min, with inhaling the 1mL terminator in colorimetric cylinder, vibration mixes immediately.Add successively 1mL developer I and developer II, strong vibration, make it fully to mix again, reaction 20min.Ultrapure water is settled to 10mL, and colorimetric estimation OD value under 625nm, take the OD value as ordinate zou, NH
4 +gradient is the X-coordinate mapping, obtains typical curve.
5. the determination step that enzyme is lived
Get two 10mL colorimetric cylinders, add respectively 1mL substrate reactions liquid (3% EC, pH4.4) in two pipes, then add 1mL enzyme liquid in a colorimetric cylinder, add the 1mL ultrapure water in another colorimetric cylinder.Then, react 30min in 50 ℃ of constant water bath box after, respectively add the 1mL terminator in two pipes, the developer I and the 1mL developer II that add 1mL after mixing, strong concussion, continue after insulation 15 min, to take out in 50 ℃ of constant water bath box, with ultrapure water, is diluted to 10mL, 625nm place colorimetric, and record the OD value.
Formula is calculated in enzyme work:
Enzyme activity=Δ OD
625* n * k * 10/30
In formula: Δ OD
625: after enzyme reaction, sample determination and blank test optical density value is poor; N: enzyme activity determination liquid extension rate; K: the inverse of slope of standard curve; The 10:1mL sample liquid is diluted to the multiple of 10mL; 30: the time of enzyme reaction (min).
Enzyme activity definition: under normal pressure, 37 ℃, pH4.4 condition, per minute bottom exploded deposits yields 1 μ mol ammonia is an enzyme activity unit.
(2) thick enzyme zymologic property (seeing Fig. 4, Fig. 5)
Beneficial effect of the present invention: the present invention separates the bacterial strain obtain, and through being accredited as the penicillium variable bacterium, the EC degrading enzyme of its product is very stable between pH 7.0-9.0, but as pH higher than 10.0 or lower than 3.0 the time, the vigor of enzyme starts reduction rapidly until lose activity.The optimal pH that produces enzyme through this bacterium of test determination is 4.0-6.0, illustrates that its sphere of action is relatively wide.Temperature is in the scope of 40-50 ℃, and enzymic activity changes little, and enzyme is very stable, and when temperature, higher than 60 ℃, the activity of enzyme starts to reduce until inactivation rapidly.The optimum temperuture of producing enzyme through this bacterium of test determination is 50 ℃.And the enzyme that this bacterium is produced tested in the different wine precision, recording this enzyme has certain tolerance to the alcohol of 1%-15% concentration.Under simulation yellow rice wine (15% ethanol, 3% EC, pH4.4) condition, this enzyme has relatively strong Degradation to EC, and Urethylane (MC) is also had certain effect, but can not act on amino acids.Therefore this penicillium variable (
penicillium variabile)jN-A525 produces the EC degrading enzyme removal has produced in the leavened foods such as pure mellow wine, yellow rice wine, rice wine micro-EC and the application potential of MC.
The impact of solid-phase microextraction technical measurement enzyme liquid on Flavor in Rice Wine.
In yellow rice wine before and after head space-solid-phase microextraction method enzyme analysis processing, the content of EC, investigate the Preliminary Applications effect (in Table 1) that enzyme is removed EC in yellow rice wine.
The biological material specimens preservation: a kind of urethanum EC degrading enzyme produces bacterium, be from the enteron aisle of mouse, to separate to obtain the bacterial strain that the EC degrading enzyme is produced in a strain, its Classification And Nomenclature be penicillium variable (
penicillium variabile) JN-A525, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address: Beijing Institute of Microorganism, Academia Sinica, its deposit number is: CGMCC No.5763, preservation date on February 16th, 2012.
The accompanying drawing explanation
Fig. 1 ammonium ion concentration standard curve
.
The specificity that Fig. 2 bacterial strain produces the substrate of EC degrading enzyme.
Fig. 3 synthetic primer, amplification ITS district complete sequence, amplification.
The optimum temperuture of Fig. 4 enzyme reaction.
The optimal pH of Fig. 5 enzyme reaction.
Fig. 6 phylogenetic tree.
Embodiment
1, the separation of bacterial strain:
From mouse intestinal, one strain EC degrading enzyme produces bacterium.
Isolation medium used: 0.23% hydroxyethyl piperazine acetic acid, 0.1% vitamine mixture (30g/L choline hydrochloric acid, 10mg/L niacinamide, 2.5mg/L calcium pantothenate, 2.5mg/L thiamine hydrochloride, 1.25mg/L riboflavin, 0.75mg/L pyridoxal hydrochloric acid, 0.6mg/L para-amino benzoic acid, 0.5mg/L folic acid, 0.1mg/L vitamin H), 0.02% mineral mixture (0.25% urethanum, 0.2% ethanamide).
The evaluation of bacterial strain:
This bacterial strain is on czapek's solution, and the bacterium colony quality is velvet-like, and densification is smooth, thinner; Edge mycelium yellow, slightly green; It is orange-yellow to reddish-brown that the bacterium colony reverse side is; Observing conidium in Electronic Speculum is oval or ellipse, and wall is smooth or slightly coarse.
The sequencing result that the EC degrading enzyme is produced to the rDNA ITS sequence of bacterium JN-A525 is compared by the Blast program on the NCBI website and Genbank amplifying nucleic acid database, find with
penicillium variabilerDNA ITS sequence similarity reach 97%, simultaneously the EC lytic enzyme produce bacterium with
penicillium variabileon the systematic evolution tree based on rDNA ITS sequence, in same branch, and the colonial morphology of the two, Mycelial morphological characteristic are identical, and physiological and biochemical property is basically identical, therefore identify this bacterial strain for be Penicillium (
penicillium) middle penicillium variable (
penicillium variabile).
The preparation of crude enzyme liquid and research
Slant medium: sucrose 30g/L, NaNO
33 g/L, K
2hPO
41 g/L, KCl 0.5 g/L, MgSO
40.5 g/L, FeSO
40.01 g/L, agar 15-20 g/L, pH nature;
Fermention medium: glucose 20g/L, peptone 10g/L, pH 6.0;
The preparation of spore suspension: with fresh, eugonic spore on agar slant under appropriate aseptic washing, then move into and be equipped with in the triangular flask of granulated glass sphere, on vibrator, fully vibration, make spore dispersed.
The spore suspension of this bacterium is inoculated in the Erlenmeyer flask that contains 70 mL fermented liquids with 3% inoculum size, is placed in constant temperature speed governing shaking table and carries out fermentation culture, rotating speed is 100 r/min, and temperature is 30 ℃.After fermentation culture 3d, fermented liquid is centrifugal, and the mycelium obtained utilizes ultrasonic disruption, and 10000
* g, 4 ℃, 20 min.The supernatant liquid of acquisition is placed in to 4 ℃ of preservations.Be 13.87g/L by the biomass in centrifugal acquisition fermented liquid, record total enzyme and live as 340.8U/L, thereby yield of enzyme that can the unit's of recording thalline is 24.57u/g.
The EC degrading enzyme that this bacterium is produced carries out respectively the experiment of temperature, pH stability; The experiment of optimum temperuture and pH; Substrate specificity and the experiment of alcohol tolerance level.
The optimum temperuture of enzyme reaction and thermostability: use produced enzyme liquid respectively under differing temps (30-70 ℃) measure EC lytic enzyme enzyme work in simulation wine sample (alcoholic strength is 15%, EC content is 3%, pH4.4), in the time of 50 ℃, enzyme work reaches the highest.Between 40-50 ℃, enzyme lives in showing higher relative activity, and more than 55 ℃, enzyme inactivation alive is very fast.The thermostat water bath that enzyme liquid is placed in respectively to 40-75 ℃ is incubated, and in 1h, the sampling and measuring enzyme is alive simultaneously, and with the not enzyme contrast of heat treated, result draws: the EC degrading enzyme has stability preferably at 40-50 ℃.
The optimal pH of enzyme reaction and pH stability: measure under different pH values, the enzyme of enzyme liquid is lived, the optimum pH of enzyme effect as a result approximately 6.0.With the damping fluid of different pH values, by the mycelium dilution suitable multiple, 4 ℃ of insulation 1h, measure enzyme and live under standard conditions, and the highest enzyme work of take is 100%, calculates relative enzyme and lives, and between known this enzyme pH value 7-9, enzyme has stability preferably.
The specificity of EC degrading enzyme substrate: take respectively glycine, L-glutamic acid, Urethylane, urethanum, γ-aminobutyric acid is substrate, under the same terms, measures enzyme and lives.As shown in Figure 2, this enzyme has relatively strong hydrolysis to discharge the ability of ammonium ion to urethanum, and also can act on Urethylane, but, a little less than effect, some amino acid is not had to effect substantially.
Alcohol resistance: getting respectively ethanol content is 0,3%, 6%, 9%, 12%, 15%, 18%, 21%, 24%, 27%, and EC content 3%, in the substrate 1mL of the pH4.5 colorimetric cylinder good in mark, respectively adds 1mL enzyme liquid, and 50 ℃ of water-bath 30min measure the OD value.Experimental result is the work of EC lytic enzyme enzyme along with the increase of ethanol content reduces.
3, the impact of enzyme liquid on Flavor in Rice Wine
Adopt the impact of solid-phase microextraction technical measurement enzyme liquid on Flavor in Rice Wine.
Sample one is contrast, through GC-MS, detects, and check variety contains 36 kinds of flavour substancess, comprising 18 kinds of ester classes, 5 kinds of acids, 4 kinds of alcohols, 2 kinds of phenols, a kind of ketone, 2 kinds of alkyl compounds, a kind of furan compound.Sample two is yellow rice wine of processing through enzyme liquid, contains 38 kinds of flavour substancess, wherein phenols increased a kind of 2,6-di-tert-butyl-4-cresylol, alcohols has increased a kind of 2-methyl butanol, acids has increased a kind of amine DA-6 acid, has lacked a kind of furfuran compound.Sample three is yellow rice wine of processing through the enzyme liquid of deactivation, and containing 37 flavor substances, wherein many a kind of alcohols are 2,3-butanediol.All the other fundamental sum contrasts keep the same.This description of test enzyme liquid the flavour substances in yellow rice wine is not had to adsorption, through the rice wine flavor material of enzyme liquid effect, atomic little variation is arranged, substantially do not affect whole local flavor and the quality of yellow rice wine.
Enzyme is removed the Preliminary Applications effect of EC in yellow rice wine
With the EC in headspace solid-phase microextraction technology enrichment yellow rice wine, then carry out quantitative analysis with gas chromatography-mass spectrography.
Headspace solid-phase microextraction: draw the head space bottle that the wine sample 8ml processed is placed in 20mL, add 8 μ LPC inner mark solutions with microsyringe, then add 3.1gNaCl, screw bottle cap, insert extracting head, at 70 ℃ of lower 250r/min constant temperature, stir extraction 45min.
The makings chromatography-mass spectroscopy is used in conjunction detection: after extraction, extracting head is taken out, insert 250 ℃ of thermal desorption 5min of gas chromatographic sample introduction mouth, carrier gas is high-purity He, flow velocity 2mL/min; Splitless injecting samples.
The sample condition that yellow rice wine is processed by enzyme:
Contrast: by every mL yellow rice wine, add the yellow rice wine sample after 37 ℃ of 0.7mL ultrapure waters are processed 1h;
Sample one: by every mL yellow rice wine, add the yellow rice wine sample after 37 ℃ of 0.1U crude enzyme liquids are processed 1h;
Sample two: sample is processed to 1h once 100 ℃ of boiling water baths enzyme that goes out;
Sample three: by every mL yellow rice wine, add the yellow rice wine sample after 37 ℃ of 0.3U crude enzyme liquids are processed 1h;
Sample four: sample three is processed to 1h through 100 ℃ of boiling water baths enzyme that goes out;
Sample five: by every mL yellow rice wine, add the yellow rice wine sample after 37 ℃, 0.5U enzyme powder is processed 1h;
In each sample, the EC clearance is as shown in table 1.
Table 1 EC degrading enzyme is removed the Preliminary Applications effect of EC in yellow rice wine
| Sample | Contrast | Sample one | Sample two | Sample three | Sample four | Sample five |
| EC content (μ g/L) | 154.28 | 127.49 | 135.79 | 112.14 | 139.75 | 98.81 |
| Enzyme liquid absorption EC(%) | — | — | 11.98 | — | 9.05 | — |
| Enzymolysis is removed EC(%) | — | 6.11 | — | 19.22 | — | 35.95 |
| The total clearance of EC (%) | — | 18.09 | — | 28.27 | — | 35.95 |
Result shows: after the crude enzyme liquid that this bacterium produces is deducted it certain adsorption possessed, EC in yellow rice wine is still shown to obvious Degradation, and after enzymolysis in yellow rice wine the main volatile flavour substances substantially do not have the impact, while adding enzyme powder sample in yellow rice wine, enzymolysis is always removed EC and is led and can reach 35.95%, therefore the enzyme that this bacterium produces has larger potentiality on EC in removing yellow rice wine, for good basis has been set up in the application that the quality at yellow rice wine and fermented type alcoholic beverage promotes.
<210> SEQ ID NO:1
<211> 610
<212> DNA
<213 > penicillium variable (Penicillium variabile) JN-A525
<400> 1
aagacttccg taggtgaacc tgcggaagga tcattaccga gtgcgggttc caacgagccc 60
aacctcccac ccgtgtttac tgttaccgcg ttgcctcggc gggcccactg gggcctggcc 120
ccggtcgccg gggggcttct gcccccgggc ccgcgcccgc cgaagcgccc tggaaccctg 180
tctgaatagt gagtctgagt gggatattaa atcattaaaa ctttcaacaa cggatctctt 240
ggttccggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc 300
cgtgaatcat cgaatctttg aacgcacatt gcgccccctg gcattccggg gggcatgcct 360
gtccgagcgt catttctgcc ctccagcacg cctgggtgtt gggtgctgtc cccccgggga 420
cacgccccaa aagcagtggc ggcgccgcgt cgggtcctcg agcgtatggg gctctgtcac 480
ccgctcggga gggactcggt cggcgctggt cttccttctg gcgacccttc ggggctcgtc 540
tcctctggtt gacctcggat caggtaggac tacccgctga acttaagcat atcaataagc 600
aaaagaataa 610
Claims (2)
1. a urethanum EC degrading enzyme produces bacterium, its Classification And Nomenclature be penicillium variable (
penicillium variabile) JN-A525, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.5763.
2. EC degrading enzyme according to claim 1 produces bacterium, and it is characterized in that: the ITS region sequence of described bacterium is as shown in SEQ ID NO:1.
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| CN102994433B (en) * | 2012-12-27 | 2014-07-09 | 江南大学 | Acid producing klebsiella oxytoca for synthesizing acidity ethyl carbamate hydrolase and application thereof |
| CN103937714A (en) * | 2014-04-14 | 2014-07-23 | 江南大学 | Bacterial strain producing acid urease and EC degrading enzyme and application of bacterial strain |
| CN104266984B (en) * | 2014-10-11 | 2016-08-17 | 江南大学 | A kind of spectrophotometric method of quick detection ethyl carbamate content |
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN113337417B (en) * | 2021-03-30 | 2023-06-06 | 大连工业大学 | Agrobacterium capable of efficiently degrading ethyl carbamate and application thereof |
| 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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