CN102676399A - 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 yellow rice wine produces bacterium with urethanum (EC) degrading enzyme, relates to the evaluation of this bacterium and the fundamental characteristics of the enzyme that produces, and from the enteron aisle of mouse, isolates a strain specifically and can produce EC degrading enzyme generation bacterium, identifies its kind through the ITS ordination.The thick enzyme that extract to obtain is analyzed the fundamental characteristics such as specificity and alcohol resistance of its ph optimum, optimum temperuture, pH value and temperature stability scope, substrate, 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 abbreviates EC as) can cause lung tumor, lymphatic cancer, liver cancer, skin carcinoma etc.It is generally acknowledged that the main mechanism of carcinogenesis of EC is: about 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 pilosity is born on the residue of thymus pyrimidine (T) and cytosine(Cyt) (C), and then the biological genetic material of influence, causes canceration.
Research to urethanum in the wine both at home and abroad shows that the main path that urethanum forms in the wine is the urea approach, and the urethanum in China's yellow rice wine and the japanese sake more than 90% is to derive from contained urea and alcoholic acid reaction in the wine:
The analysis of the formation approach through urethanum can know that the formation of EC is mainly reacted from urea and alcoholic acid in leavened food (like bread, yogurt milk, cheese, soy sauce etc.) and the beverage (like wine, hard cider, Chinese rice wine and japanese sake etc.).The method that reduces EC content in the yellow rice wine can be divided into the improvement and the enzymolysis process of traditional technology.At present, utilize enzymolysis process to remove that EC has become now and development trend in the future in the yellow rice wine.Urea is the precursor substance that generates urethanum, utilizes acid urease to handle yellow rice wine, can remove most of urea in the yellow rice wine, thereby reduces the possibility that generates urethanum.The urethanum degrading enzyme then can directly act on urethanum, effectively decomposes it and is urea and ammonia, reduces the EC content that has produced in the yellow rice wine.Among the present invention a strain EC degrading enzyme produce the discovery of bacterial strain with to its relevant research, better minimizing and control provide the means of supplementing out economy preferably for EC content in the following yellow rice wine.
Raising day by day along with standard of living; Domestic consumption to Wine rises day by day; Especially alcoholic drinkss such as wine, rice wine, yellow rice wine, beer become the focus of people's consumption especially, and the EC contents level that therefore effectively reduces 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 have active preferably under the working condition of leavened foods such as pure mellow wine, yellow rice wine, rice wine; The present invention provides the relevant zymologic property of the EC degrading enzyme that this bacterium and this bacterium produces and the result that this enzyme is used in yellow rice wine.
Technical scheme of the present invention: a kind of urethanum EC degrading enzyme produces bacterium, is 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 called after penicillium variable (
Penicillium variabile) JN-A525, being preserved in China Committee for Culture Collection of Microorganisms 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 ph optimum of bacterial strain CGMCC enzyme that No.5763 produces is 4.0-6.0, and optimum temperuture is about 50 ℃; This enzyme is between 40-50 ℃, and between the pH value 7-9, enzyme has stability preferably.
Bacterial strain CGMCC No.5763 of the present invention separates to obtain from intestinal tissue of mouse and content.
The evaluation of bacterial strain:
(1) design specific primers, 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 the rDNA ITS sequence of bacterium CGMCC No.5763 is compared through the Blast program on the NCBI website and Genbank amplifying nucleic acid DB, find and
Penicillium variabileRDNA ITS sequence similarity reach 97%, simultaneously the EC lytic enzyme produce bacterium CGMCC No.5763 with
Penicillium variabileOn systematic evolution tree, be in same branch, and the colonial morphology of the two, mycelia morphological specificity are identical based on rDNA ITS sequence, the physiological and biochemical property 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, and is thinner; The edge mycelium is yellow, has a little green; It is orange-yellow to reddish-brown that the bacterium colony reverse side is; In Electronic Speculum, observing conidium is oval or ellipse, and wall is smooth or coarse slightly.
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: wash fresh on the agar slant, eugonic spore with an amount of sterilized water, move into then and be equipped with in the triangular flask of granulated glass sphere, fully vibration makes the spore homodisperse on vibrator.
The spore suspension of this bacterium is inoculated in the Erlenmeyer flask that contains 70 mL fermented liquids with 3% inoculum size, places constant temperature speed governing shaking table to carry out fermentation culture, rotating speed is 100 r/min, and temperature is 30 ℃.Behind the fermentation culture 3d, fermented liquid is centrifugal, and the mycelium of gained utilizes ultrasonic disruption, 10000 *
g, 4 ℃, 20 min.The supernatant liquid that obtains is placed 4 ℃ of preservations.Biomass through in the centrifugal acquisition fermented liquid is 13.87g/L, record total enzyme and live and be 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, the crude enzyme liquid of this bacterial strain and substrate (3% EC, pH4.4) after the reaction, utilize terminator, developer I, developer II to act on respectively after.The depth of its color is directly proportional with the vigor of enzyme within the specific limits, so under the wavelength of 625nm, carry out colorimetric, calculates enzyme activity.
2. enzyme reaction system: the crude enzyme liquid after the dilution; Developer I: take by weighing 15g phenol and the 0.625g sodium nitroprusside is settled to 250mL with ultrapure water; Developer II: take by weighing 13.125g NaOH and 7.5mL NaClO is settled to 250mL with ultrapure water; Terminator: take by weighing the 10g trichoroacetic acid(TCA) and be settled to 100mL with ultrapure water.
3. NH
4 +Standard solution preparation
1mL ammoniacal liquor is dissolved in substrate reactions liquid, and (3% EC pH4.4) and be settled to 145mL, is made into the NH of 0.1mol/L
4 +Solution, and as mother liquor, (3% EC pH4.4) is 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
4 +Standardized solution.
4. the drafting of ammonium ion typical curve
Accurately pipette 1mL NH with transfer pipet
4 +Normal gradients liquid places the 10mL tube comparison tubes of serial number respectively.37 ℃ of following constant temperature insulation 30min are immediately with inhaling the 1mL terminator in tube comparison tubes, the vibration mixing.Add 1mL developer I and developer II more successively, strong vibration makes it abundant mixing, reaction 20min.Ultrapure water is settled to 10mL, and colorimetric estimation OD value under 625nm is an ordinate zou with the OD value, NH
4 +Gradient is the X-coordinate mapping, obtains typical curve.
5. the enzyme determination step of living
Get two 10mL tube comparison tubess, (3% EC pH4.4), adds 1mL enzyme liquid again in a tube comparison tubes, in another tube comparison tubes, add the 1mL ultrapure water in two pipes, to add 1mL substrate reactions liquid respectively.Then, in 50 ℃ of constant water bath box, behind the reaction 30min, in two pipes, respectively add the 1mL terminator; The developer I and the 1mL developer II that add 1mL behind the mixing; Strong concussion continues in 50 ℃ of constant water bath box, to take out behind insulation 15 min, is diluted to 10mL with ultrapure water; 625nm place colorimetric, and record OD value.
Formula is calculated in enzyme work:
Enzyme activity=Δ OD
625* n * k * 10/30
In the formula: Δ OD
625: sample determination and blank test OD value is poor after the enzyme reaction; 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: PM bottom exploded deposits yields 1 μ mol ammonia is an enzyme activity unit under normal pressure, 37 ℃, pH4.4 condition.
(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 is higher than 10.0 or when being lower than 3.0, the vigor of enzyme begins reduction rapidly up to losing activity as pH.The ph optimum that produces enzyme through this bacterium of test determination is 4.0-6.0, and the relative broad of its reach is described.Temperature is in 40-50 ℃ scope, and enzymic activity changes little, and enzyme is very stable, and when temperature is higher than 60 ℃, the activity of enzyme begins to reduce up to inactivation rapidly.The optimum temperuture of producing enzyme through this bacterium of test determination is 50 ℃.And the enzyme that this bacterium is produced makes an experiment in the different wine precision, and 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 stronger Degradation relatively to EC, and Urethylane (MC) is also had certain effect, but can not act on amino acids.So this penicillium variable (
Penicillium variabile)JN-A525 produces the EC degrading enzyme has removal has produced in leavened foods such as pure mellow wine, yellow rice wine, rice wine micro-EC and the application potential of MC.
SPME-gas chromatography mass spectrometry technical measurement enzyme liquid is to the influence of flavour substances in the yellow rice wine.
The content of EC in the yellow rice wine before and after head space-SPME-gas chromatography mass spectrometry method enzyme analysis is handled is investigated enzyme is removed EC in yellow rice wine Preliminary Applications effect (seeing table 1).
The biological material specimens preservation: a kind of urethanum EC degrading enzyme produces bacterium, is 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 called after penicillium variable (
Penicillium variabile) JN-A525, be preserved in China Committee for Culture Collection of Microorganisms 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.
Description of drawings
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 ph optimum of Fig. 5 enzyme reaction.
Fig. 6 phylogenetic tree.
Embodiment
1, the separation of bacterial strain:
One strain EC degrading enzyme produces bacterium from mouse intestinal.
Used isolation medium: 0.23% hydroxyethyl piperazine acetate, 0.1% vitamine mixture (30g/L choline hydrochloric acid, 10mg/L vitamin PP, 2.5mg/L VA, 2.5mg/L thiamine hydrochloride, 1.25mg/L vitamin G, 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, and is thinner; The edge mycelium is yellow, has a little green; It is orange-yellow to reddish-brown that the bacterium colony reverse side is; In Electronic Speculum, observing conidium is oval or ellipse, and wall is smooth or coarse slightly.
The sequencing result that the EC degrading enzyme is produced the rDNA ITS sequence of bacterium JN-A525 is compared through the Blast program on the NCBI website and Genbank amplifying nucleic acid DB, find and
Penicillium variabileRDNA ITS sequence similarity reach 97%, simultaneously the EC lytic enzyme produce bacterium with
Penicillium variabileOn systematic evolution tree, be in same branch, and the colonial morphology of the two, mycelia morphological specificity are identical based on rDNA ITS sequence, the physiological and biochemical property 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: wash fresh on the agar slant, eugonic spore with an amount of sterilized water, move into then and be equipped with in the triangular flask of granulated glass sphere, fully vibration makes the spore homodisperse on vibrator.
The spore suspension of this bacterium is inoculated in the Erlenmeyer flask that contains 70 mL fermented liquids with 3% inoculum size, places constant temperature speed governing shaking table to carry out fermentation culture, rotating speed is 100 r/min, and temperature is 30 ℃.Behind the fermentation culture 3d, fermented liquid is centrifugal, and the mycelium that obtains utilizes ultrasonic disruption, and 10000
* g, 4 ℃, 20 min.The supernatant liquid that obtains is placed 4 ℃ of preservations.Biomass through in the centrifugal acquisition fermented liquid is 13.87g/L, record total enzyme and live and be 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 the experiment of temperature, pH stability respectively; 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 the enzyme liquid that produced respectively under differing temps (30-70 ℃) measure EC lytic enzyme enzyme work in the simulation wine appearance (alcoholic strength is 15%, EC content is 3%, pH4.4), enzyme work reaches the highest in the time of 50 ℃.Between 40-50 ℃, enzyme lives in showing has higher relative vigor, and more than 55 ℃, enzyme inactivation alive is very fast.Place 40-75 ℃ thermostat water bath to be incubated respectively enzyme liquid, the sampling and measuring enzyme is alive simultaneously in 1h, and with the not enzyme contrast of heat treated, the result draws: the EC degrading enzyme has stability preferably at 40-50 ℃.
The ph optimum of enzyme reaction and pH stability: measure under the different pH values, the enzyme of enzyme liquid is lived, the optimum pH of enzyme effect as a result about 6.0.Damping fluid with different pH values dilutes suitable multiple with thalline, and 4 ℃ of insulation 1h measure enzyme and live under the standard conditions, be 100% with the highest enzyme work, calculate relative enzyme and live, and can know that enzyme has stability preferably between this enzyme pH value 7-9.
The specificity of EC degrading enzyme substrate: be substrate with glycocoll, L-glutamic acid, Urethylane, urethanum, γ-An Jidingsuan respectively, under the same terms, measure enzyme and live.Can know that by Fig. 2 this enzyme has stronger relatively hydrolysis to discharge the ability of ammonium ion to urethanum, and also can act on Urethylane, but a little less than the effect, some amino acid is not then had effect basically.
Alcohol resistance: getting ethanol content respectively is 0,3%, 6%, 9%, 12%, 15%, 18%, 21%, 24%, 27%, and EC content 3%, the substrate 1mL of pH4.5 are in the good tube comparison tubes of mark, and each 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, enzyme liquid is to the influence of flavour substances in the yellow rice wine
Adopt of the influence of SPME-gas chromatography mass spectrometry technical measurement enzyme liquid to flavour substances in the yellow rice wine.
Appearance one is contrast, detects through GC-MS, 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.Appearance two is yellow rice wine of handling through enzyme liquid, contains 38 kinds of flavour substancess, and wherein phenols has increased a kind ofly 2, and 6-di-tert-butyl-4-cresylol, alcohols have increased a kind of 2-methyl butanol, and acids has increased a kind of amine DA-6 acid, has lacked a kind of furfuran compound.Appearance three is yellow rice wine of handling through the enzyme liquid of deactivation, contains flavour substances in 37, wherein many a kind of alcohols promptly 2,3-butyleneglycol.All the other fundamental sum contrasts keep the same.This description of test enzyme liquid the flavour substances in the yellow rice wine is not had adsorption, through the rice wine flavor material of enzyme liquid effect atomic little variation is arranged, do not influence the whole local flavor and the quality of yellow rice wine basically.
Enzyme is removed the Preliminary Applications effect of EC in yellow rice wine
EC with in the headspace solid-phase microextraction technology enrichment yellow rice wine carries out quantitative analysis with gas chromatography-mass spectrography again.
Headspace solid-phase microextraction: draw the head space bottle that the wine appearance 8ml that handled places 20mL, add 8 μ LPC inner mark solutions with microsyringe, add 3.1gNaCl again, the bottle cap of screwing inserts extracting head, stirs extraction 45min at 70 ℃ of following 250r/min constant temperature.
The logotype of makings chromatography-mass spectroscopy detects: after extraction finishes extracting head is taken out, insert 250 ℃ of thermal desorption 5min of gc injection port, carrier gas is high-purity He, flow velocity 2mL/min; Split stream sampling not.
The sample condition that yellow rice wine is handled by enzyme:
Contrast: add the yellow rice wine sample after 37 ℃ of 0.7mL ultrapure waters are handled 1h by every mL yellow rice wine;
Appearance one: add the yellow rice wine sample after 37 ℃ of 0.1U crude enzyme liquids are handled 1h by every mL yellow rice wine;
Appearance two: appearance is handled 1h once 100 ℃ of boiling water baths enzyme that goes out;
Appearance three: add the yellow rice wine sample after 37 ℃ of 0.3U crude enzyme liquids are handled 1h by every mL yellow rice wine;
Appearance four: appearance three is handled 1h through 100 ℃ of boiling water baths enzyme that goes out;
Appearance five: add the yellow rice wine sample after 37 ℃ in 0.5U enzyme powder is handled 1h by every mL yellow rice wine;
The EC clearance is as shown in table 1 in each sample.
Table 1 EC degrading enzyme is removed the Preliminary Applications effect of EC in yellow rice wine
| Sample | Contrast | Appearance one | Appearance two | Appearance three | Appearance four | Appearance 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 |
The result shows: after it certain adsorption that possesses of crude enzyme liquid deduction that this bacterium produced; EC in the yellow rice wine is still shown tangible Degradation; And behind the enzymolysis in the yellow rice wine main volatile flavour substances do not have influence basically, when in yellow rice wine, adding enzyme powder sample, enzymolysis is always removed EC and is led and can reach 35.95%; Therefore the enzyme that this bacterium produced has bigger potentiality on the 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 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 called after penicillium variable (
Penicillium variabile) JN-A525, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, its deposit number is: CGMCC No.5763.
2. EC degrading enzyme according to claim 1 produces bacterium, the amplification ITS district complete sequence of this bacterial strain, and its aminoacid sequence is SEQ ID NO:1.
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| CN102994433A (en) * | 2012-12-27 | 2013-03-27 | 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 |
| CN104266984A (en) * | 2014-10-11 | 2015-01-07 | 江南大学 | Spectrophotometric method for quickly detecting content of ethyl carbamate |
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN113337417A (en) * | 2021-03-30 | 2021-09-03 | 大连工业大学 | Agrobacterium capable of efficiently degrading ethyl carbamate and application thereof |
| CN114807102A (en) * | 2022-05-16 | 2022-07-29 | 安徽工程大学 | Ethanol-resistant amidase, gene, expression vector, engineering bacterium, preparation method and application |
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2012
- 2012-04-26 CN CN 201210125030 patent/CN102676399B/en not_active Expired - Fee Related
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994433A (en) * | 2012-12-27 | 2013-03-27 | 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 |
| CN104266984A (en) * | 2014-10-11 | 2015-01-07 | 江南大学 | Spectrophotometric method for quickly detecting content of ethyl carbamate |
| 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 |
| CN113337417A (en) * | 2021-03-30 | 2021-09-03 | 大连工业大学 | Agrobacterium capable of efficiently degrading ethyl carbamate 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 |
| CN114807102A (en) * | 2022-05-16 | 2022-07-29 | 安徽工程大学 | Ethanol-resistant amidase, gene, expression vector, engineering bacterium, preparation method and application |
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