CN105420210A - Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability - Google Patents
Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability Download PDFInfo
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- CN105420210A CN105420210A CN201510971638.4A CN201510971638A CN105420210A CN 105420210 A CN105420210 A CN 105420210A CN 201510971638 A CN201510971638 A CN 201510971638A CN 105420210 A CN105420210 A CN 105420210A
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- ethyl carbamate
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- carbamate hydrolase
- thermostability
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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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- 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
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/12—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation
- C12H1/14—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation with non-precipitating compounds, e.g. sulfiting; Sequestration, e.g. with chelate-producing compounds
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Abstract
The invention discloses ethyl carbamate hydrolytic enzyme mutants capable of improving the thermostability, and belongs to the technical field of gene engineering and enzyme engineering. By means of a molecular technical means, the ethyl carbamate hydrolytic enzyme mutants capable of improving the thermostability are obtained, and the half-life periods of the ethyl carbamate hydrolytic enzyme mutants Q328C and Q328V are increased by 7.46 times and 1.96 times respectively compared with a native enzyme. The mutants Q328C, Q328R and Q328 V all have better tolerance at 30 DEG C or above than the native enzyme. In addition, the tolerance to ethyl alcohol and the tolerance to acid of the mutant Q328C are improved.
Description
Technical field
The present invention relates to the ethyl carbamate hydrolase mutant that thermostability improves, belong to genetically engineered and technical field of enzyme engineering.
Background technology
Urethanum (Ethylcarbamate, EC) is leavened food (as soy sauce, fermented bean curd, pickles) and alcoholic beverage (grape wine, yellow rice wine, white wine) producing, generate in storage to human body, there is potential carinogenicity and genotoxic a kind of microscale harmful substance.Because the generting machanism of EC is complicated, stable in properties, once generate very difficult physics or chemical process removal, does not also have a kind of effective ways can eliminating EC in different fermentations product at present.The EC that applying biological enzyme process is removed in finished product is a kind of safer effective means.Urethane lytic enzyme (Urethanase, UH) can be degraded EC, generates harmless ethanol and carbonic acid gas, has potential industrial application value.Wild ethyl carbamate hydrolase less stable in the present invention, the transformation period at 40 DEG C is 3.67min.Thermally-stabilised difference is unfavorable for ethyl carbamate hydrolase application in the industry, therefore improves ethyl carbamate hydrolase stability by molecular modification and seems particularly important.
Summary of the invention
The problem to be solved in the present invention is to provide the ethyl carbamate hydrolase mutant that thermostability improves, and its relative parent's ethyl carbamate hydrolase has a point to suddenly change.
The aminoacid sequence of described ethyl carbamate hydrolase parent is as shown in SEQIDNO.1.On this basis, the glutamine of 328 is sported halfcystine, arginine or α-amino-isovaleric acid.
The present invention also provides a kind of preparation method of described ethyl carbamate hydrolase mutant, and concrete steps are as follows:
1) design the multiplex primer of rite-directed mutagenesis, carry out full plasmid amplification with the carrier carrying ethyl carbamate hydrolase encoding gene for template, obtain the recombinant vectors containing sudden change ethyl carbamate hydrolase;
2) recombinant vectors transformation of E. coli BL21 after suddenling change, abduction delivering, purifying obtains ethyl carbamate hydrolase mutant.
Ethyl carbamate hydrolase mutant thermostability provided by the invention all increases, wherein, the transformation period of mutant Q328C, Q328V and Q328R at 40 DEG C brings up to 27.4min, 7.18min and 3.99min respectively by the 3.67min contrasting (before sudden change).Mutant Q328C, Q328R, Q328V have tolerance more better than protoenzyme more than 30 DEG C.Simultaneous mutation body Q328C also increases to the tolerance of ethanol and sour tolerance.Ethyl carbamate hydrolase mutant can be used for the urethanum of degrading in food and fermented beverage wine better.
Accompanying drawing explanation
Fig. 1: temperature is on the impact of wild enzyme and mutant enzyme activity
Embodiment
The preparation of embodiment 1 ethyl carbamate hydrolase mutant
By analyzing the structure of ethyl carbamate hydrolase, we select to suddenly change to its amino acid of the 328th, design corresponding rite-directed mutagenesis primer (table 1).With recombinant plasmid pET20b-UH for masterplate, adopt PCR enzyme, utilize mutant primer to increase to recombinant plasmid pET20b-UH.Fragment after amplification utilized glue to reclaim test kit and carry out recovery purifying.Starch phosphorylase is adopted to carry out phosphorylation to fragment fragment after purifying.By the fragment after phosphorylation, utilize ligase enzyme to connect, in transformation of E. coli BL21 (DE3) competence, screening positive transformant, extracts plasmid, cuts after qualification, deliver to Shanghai biotechnology company limited sequence verification through enzyme.Intestinal bacteria recombinant bacterial strain containing order-checking correct plasmid is inoculated in LB substratum, in 37 DEG C, incubated overnight under 220r/min condition.By seed liquor by 3% inoculum size be transferred in TB substratum, in 37 DEG C, be cultured to OD under 220r/min condition
600be 0.6, adding ITPG to final concentration is 0.1mmol/L, 25 DEG C, cultivate 18h under 220r/min condition.
By recombinant bacterium bacterium liquid 4 DEG C, centrifugal 15min collects thalline under 9000r/min.Thalline 2 times is washed and resuspended thalline, ultrasonication with the phosphate buffer soln of 20mmol/L, pH7.0.By the bacterium liquid after fragmentation in 4 DEG C, centrifugal 20min collect supernatant liquor under 9000r/min condition.First use buffer A (phosphate buffered saline buffer containing 20mmol/L, pH7.4 of 250mmol/LNaCl, 20mmol/L imidazoles) balance nickel post during purifying, flow velocity is 1mL/min.Then loading, after buffer A balance nickel post, with buffer B (phosphate buffer soln containing 20mmol/L, pH7.4 of 250mmol/LNaCl, 500mmol/L imidazoles) gradient elution albumen.The elution fraction that collecting has enzyme to live carries out desalting treatment.
Table 1 ethyl carbamate hydrolase mutant primer sequence
Embodiment 2 enzyme activity determination method
Get 1mL enzyme liquid and 1mL ultrapure water (contrast), respectively add the EC solution of 1mL3%, react 15min in 37 DEG C of constant water bath box after, add 1mL10% trichoroacetic acid(TCA) termination reaction.1mL developer I (15g phenol and 0.625g sodium nitroprusside are settled to 250mL) and 1mL developer II (13.125gNaOH and 7.5mL clorox is settled to 250mL) is added after reaction terminating, in 37 DEG C of water baths, 20min is incubated after mixing, reaction terminates rear ultrapure water and is settled to 10mL, measures the absorbancy under 625nm.Enzyme is lived definition: normal pressure, 37 DEG C, under pH7.0 condition, 1min decomposes EC, and to produce enzyme amount needed for 1 μm of ol ammonia be an enzyme activity unit (U).
Embodiment 3 is suddenlyd change and is improved ethyl carbamate hydrolase thermostability and transformation period
By the wild enzyme after purifying and mutant thereof at different temperatures (20 DEG C ~ 65 DEG C, 5 DEG C, interval) carry out enzymatic reaction, measure its optimal reactive temperature.The optimal reactive temperature of wild enzyme and mutant is 30 DEG C, but mutant Q328C, Q328R, Q328V more than 30 DEG C than the good stability of wild enzyme.After 37 DEG C of 1 time of insulation, the residual enzyme of Q328C, Q328R, Q328V and UH is lived and is respectively 76.9%, 60.8%, 75.8% and 51.9%, improves 24.0%, 8.9% and 23.9% respectively than protoenzyme.At 40 DEG C, the residual enzyme of said mutation body is lived and is improve 31.7%, 19.7% and 28.9% (Fig. 1) than protoenzyme respectively.
Be incubated at wild enzyme after purifying and mutant thereof are placed in 40 DEG C, sampling and measuring enzyme is lived at regular intervals.By formula lnC
t=lnC
0+ kt, t
1/2=ln2/k calculates the transformation period of enzyme.Wherein, C
0that initial enzyme is lived, C
tenzyme corresponding when be the time being t is lived.Mutant Q328C, Q328V and Q328R transformation period at 40 DEG C brings up to 27.4min, 7.18min and 3.99min respectively by the 3.67min contrasting (before sudden change).7.46 times of wild enzyme, 1.96 times and 1.1 times respectively.Visible two kinds of mutant have raising, particularly Q328C in various degree to obtain to improve significantly.
Embodiment 4 is suddenlyd change and is improved ethyl carbamate hydrolase acid resistance and resistance to alcohol repellency
Wild UH enzyme after purifying and mutant thereof are carried out enzymatic reaction under different pH (4.5 ~ 7.0) conditions, and the enzyme measured under different pH value is lived.Wherein and Q328C the enzyme increase rate of living is comparatively obvious in acid condition: under pH6.0 condition, relative enzyme is lived and is improve 11%, and under pH4.5 condition, relative enzyme work also improves 11%.
Is reacted under the ethanol condition that wild UH enzyme after purifying and mutant thereof are 0%, 2%, 5%, 10%, 15% in concentration respectively, the enzyme measured under different ethanol concentration is lived.Under the ethanol condition of 2%, the residual enzyme work of wild enzyme UH is 55.2%, and the residual enzyme work of mutant enzyme Q328C is 70.2%, improves 25%.Under 5% ethanol condition, the residual enzyme work of wild enzyme is the residual enzyme work of 26.9%, Q328C is 40.2%, improves 13%.
As can be seen here, mutant Q328C also increases to the tolerance of ethanol and sour tolerance.
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 (6)
1. the ethyl carbamate hydrolase mutant of thermostability raising, it is characterized in that, relative to the ethyl carbamate hydrolase with aminoacid sequence shown in SEQIDNO.1, the glutamine of the 328th sports halfcystine, arginine or α-amino-isovaleric acid.
2. the nucleotide sequence of coding mutant according to claim 1.
3. carry carrier or the reconstitution cell of nucleotide sequence described in claim 2.
4. prepare a method for mutant described in claim 1, it is characterized in that, comprise the steps:
1) design the multiplex primer of rite-directed mutagenesis, carry out full plasmid amplification with the carrier carrying ethyl carbamate hydrolase encoding gene for template, obtain the recombinant vectors containing sudden change ethyl carbamate hydrolase;
2) recombinant vectors transformation of E. coli BL21 after suddenling change, abduction delivering, purifying obtains ethyl carbamate hydrolase mutant.
5. the application in the urethanum of ethyl carbamate hydrolase mutant described in claim 1 in degraded food and fermented beverage wine.
6. carrier described in claim 3 or reconstitution cell are in the application in the urethanum in food and fermented beverage wine of degrading.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342209A (en) * | 2020-11-26 | 2021-02-09 | 浙江大学 | Ethyl carbamate hydrolase mutant and application thereof |
| CN113774048A (en) * | 2021-10-15 | 2021-12-10 | 四川轻化工大学 | Ethyl carbamate hydrolase mutant and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714378A (en) * | 1995-03-31 | 1998-02-03 | The United States Of America As Represented By The Secretary Of The Navy | Pseudomonas chlororaphis microorganism polyurethane degrading enzyme obtained therefrom and method of using enzyme |
| CN103451216A (en) * | 2013-09-10 | 2013-12-18 | 江南大学 | Ethyl carbamate hydrolase gene, protein coded thereby and application |
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2015
- 2015-12-21 CN CN201510971638.4A patent/CN105420210B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714378A (en) * | 1995-03-31 | 1998-02-03 | The United States Of America As Represented By The Secretary Of The Navy | Pseudomonas chlororaphis microorganism polyurethane degrading enzyme obtained therefrom and method of using enzyme |
| CN103451216A (en) * | 2013-09-10 | 2013-12-18 | 江南大学 | Ethyl carbamate hydrolase gene, protein coded thereby and application |
Non-Patent Citations (1)
| Title |
|---|
| 吕思熠等: "氨基甲酸乙酯水解酶在枯草芽孢杆菌中的表达及发酵优化", 《过程工程学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342209A (en) * | 2020-11-26 | 2021-02-09 | 浙江大学 | Ethyl carbamate hydrolase mutant and application thereof |
| CN112342209B (en) * | 2020-11-26 | 2022-06-14 | 浙江大学 | Ethyl carbamate hydrolase mutant and application thereof |
| CN113774048A (en) * | 2021-10-15 | 2021-12-10 | 四川轻化工大学 | Ethyl carbamate hydrolase mutant and preparation method and application thereof |
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| CN105420210B (en) | 2018-08-07 |
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