WO2015035730A1

WO2015035730A1 - Ethyl carbamate hydrolase gene and protein encoded thereby and use thereof

Info

Publication number: WO2015035730A1
Application number: PCT/CN2013/090631
Authority: WO
Inventors: 陈坚; 方芳; 李京京; 刘庆涛; 堵国成
Original assignee: 江南大学
Priority date: 2013-09-10
Filing date: 2013-12-27
Publication date: 2015-03-19
Also published as: CN103451216B; CN103451216A

Abstract

Disclosed are an ethyl carbamate hydrolase gene UH and the protein encoded thereby and the use thereof, wherein the gene has the nucleotide sequence as shown by SEQ ID NO.1 and the protein encoded thereby has the amino acid sequence as shown by SEQ ID NO.2. A genetic engineering strain is constructed by using the provided UH gene, and a recombinantly expressed enzyme is obtained therefrom with an enzyme activity of 83.5 U/g of wet bacterial cells.

Description

A urethane hydrolase gene and its encoded protein and application

Technical field

The invention discloses a urethane hydrolase gene and a protein encoded thereby, in particular a urethane hydrolase gene derived from Bacillus lycopene and a protein encoded thereby.

Background technique

Urethane (English name: urethane or ethyl Carbamate (EC), also known as urethane, urethane, is an intermediate for medicines, pesticides, and fragrances, or a cosolvent for the production of sleeping pills, sedatives, injections, and colorants for printing and dyeing industries. It can also be used in biochemical research. In addition, urethane itself can be used as a medicine, has anti-cancer properties, and is used for the treatment of multiple myeloma and chronic leukemia.

Nettleship in the 1940s Experiments have shown that urethane has a carcinogenic effect. Mainly can cause lung cancer, lymphoma, liver cancer, skin cancer and so on. As a by-product, urethane is widely found in fermented foods (such as bread, yogurt, cheese, soy sauce, etc.) and alcoholic beverages (such as wine, rice wine, cider, and Japanese sake). The body's intake of urethane is mainly through the consumption of alcoholic beverages and food. Urethane has become a factor that can not be ignored influencing human health.

Current governments in the fermented food EC The content has been introduced to the corresponding limit standard. Ethyl urethane is also widely found in liquor, wine, soy sauce, etc. produced in China, which not only restricts the export of related products in China, but also affects the health of ordinary consumers. . Therefore, it is extremely urgent to take effective measures to control or eliminate urethane in fermented foods or beverages.

In the 1990s, Japanese scientists screened strains that could degrade urethane, such as Bacillus. Licheniformis, Citrobacter sp., Micrococcus species, etc., capable of degrading urethane to ethanol, ammonia And carbon dioxide, turning carcinogens into non-toxic organic molecules, effectively solves the problem of food safety. However, the amount of enzyme produced by wild bacteria is small, the purification step of protein is cumbersome, and some are non-safe strains, etc., and have not been widely used in the food industry. This makes it important to excavate genes with urethane hydrolase activity and to construct efficient exogenous expression systems. By genetic engineering, direct cloning and expression of the desired urethane hydrolase, changing the amount of enzymes produced by wild bacteria, etc., provided a new idea and laid the foundation for the industrial application of urethane hydrolase.

Summary of the invention

The technical problem to be solved by the present invention is to provide a urethane hydrolase gene having a nucleotide sequence of SEQ ID NO: 1 Shown or is the nucleotide sequence encoding the protein of SEQ ID NO: 2.

The present invention performs N-terminal sequencing of the urethane hydrolase purified from Bacillus lysine, and the N-terminal 12 amino acid residues are aligned with the NCBI database, and the encoding of the urethane is hydrolyzed by designing specific primers. The gene is amplified by the enzyme and a recombinant vector is constructed to transform the host cell, and the gene is expressed in E. coli. Therefore, the object of the present invention is to provide a urethane hydrolase gene, a recombinant vector containing the same, a recombinant genetic engineering strain transformed by the recombinant vector, and the use thereof in preparing a recombinant urethane hydrolase .

The specific technical scheme adopted by the present invention is to pass the N-terminal 12 amino acid residues of the urethane hydrolase which has been sequenced and NCBI. Database alignment, designing specific primers based on the alignment results to amplify the target gene and ligating the cloning vector, and sequencing the nucleic acid sequence, such as SEQ ID NO. 1 in the sequence listing. Shown in which the coding sequence (CDS) is terminated from the 1st base of DNA to the 1419th, ATG is the transcription start codon, TAA The transcriptional stop codon is obtained by sequence translation as shown in SEQ ID NO. 2 of the Sequence Listing. Of course, as known to those skilled in the art, the urethane hydrolase gene of the present invention may also be encoded by the SEQ in the sequence listing. The other nucleotide sequence of the protein consisting of the amino acid sequence shown by ID NO. 2; and the urethane hydrolase of the present invention is not limited to having SEQ ID NO. 2 in the sequence listing. The sequence of the amino acid composition shown may also be a protein derived from SEQ ID NO: 2, which has a substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 2 and one or more amino acid residues, such as at the C terminus and / or N One or several amino acid residues are added or deleted at the end, a fusion tag or the like is added, although the form is modified without changing the enzyme activity of the protein.

The urethane hydrolase gene can be obtained from Bacillus lysine (Lysinibacillus) Fusiformis), such as Lysinibacillus fusiformis SCO2.

Contains SEQ ID NO.1 The gene expression vector of the indicated nucleotide sequence is also in the scope of protection of the present invention, which is successfully constructed by ligating the cloned target gene sequence into various gene expression vectors by conventional biotechnological means, and the vector used may be Commercially available plasmids, cosmids, phage, and the like, of which the expression vector pET20b(+) is preferred.

Another problem to be solved by the present invention is to construct an engineered strain containing a recombinant gene expression vector, which is to transform the recombinant vector into Escherichia coli, and obtain the recombinant genetic engineering bacteria for induction culture, and the culture solution is separated to obtain recombinant carbamate. The bacterial cell of the ethyl ester hydrolase; specifically, the expression vector containing the urethane hydrolase gene is transformed into the corresponding expression host to construct an exogenous expression system. If the above expression vector pET20b(+)-UH will be constructed The E. coli BL21(DE3) competent cells were transformed into E. coli BL21(DE3)/pET20b(+)-UH exogenous expression system.

Another technical problem solved by the present invention is to provide a method for preparing a recombinant urethane hydrolase, which is prepared by transforming a recombinant expression vector into an expression host to construct an expression system, and culturing and expressing under certain conditions to prepare a recombinant urethane hydrolysis. Enzyme. Wherein, the expression host may be a plant, an animal or a bacterium, and the preferred expression host is Escherichia coli, such as the genetically engineered bacteria obtained as described above: E. coli BL21(DE3)/pET20b(+)-UH. The better method is to use E. coli BL2 (DE3)/pET20b(+)-UH The genetically engineered bacteria are cultured to a certain bacterial concentration (such as OD600=0.35-0.8), and an inducer (such as IPTG final concentration of 0.05-1 mM) is added for a certain period of time (such as 10-30). h), efficient expression of recombinant urethane hydrolase.

DRAWINGS

Figure 1 is a PCR amplification electropherogram of UH of the present invention, wherein 1 is a PCR amplification product of UH; 2 is DNA Marker (2000 bp, Takara company)

Figure 2 is a double-cut analysis map of Nde I and BamH I of plasmid pET20b(+)-UH of the present invention, wherein DNA Marker (5000 bp, Takara); 2 pET20b(+)-UH was digested by Nde I and BamH I.

Figure 3 is a genetically engineered strain of the present invention E. coli BL21(DE3)/pET20b(+)-UH SDS-PAGE protein electrophoresis map of the expressed product, wherein 1 is a genetically engineered strain E.coli BL21(DE3)/pET20b(+)-UH non-inducing product; It is the protein molecular weight standard (Biyuntian Company); 3 is the product of genetic engineering bacteria E. coli BL21(DE3)/pET20b(+)-UH induced by IPTG for 15 hours.

detailed description

The invention is further illustrated by the following examples.

Materials and methods in the following examples:

The molecular cloning technique used is referred to J. Sambrook et al., Guide to Molecular Cloning.

The restriction enzymes and gel recovery kits used were purchased from Fermentas The company, the specific reaction conditions and methods of use refer to its instructions. The genomic extraction kit was purchased from Omega and used with reference to its instructions.

The following commercial plasmids and E. coli are used for gene cloning and expression.

pMD19-T (Takara, China)

pET20b(+) (Novagen, USA)

E. coli JM 109 (Novagen, United States)

E.coli BL21 (DE3) (Novagen, United States)

Example 1: Cloning of the UH gene from the B. lycopene genome

A single colony of Bacillus lysine bacillus activated by streaking on a plate was inoculated into LB liquid medium and cultured for about 15 h. In the logarithmic growth phase, the cells are collected. The genomic DNA is extracted by reference to the genomic extraction kit step. Design specific primers based on protein sequence alignment results, including forward primer 1 and reverse primer 2, forward primer 1 Add Nde I, reverse primer 2 plus BamH I, as follows:

Forward primer 1:5'-GGA ATT CCA TAT GAT GCG GAC ATT GCT GTA CGT-3'

Reverse Primer 2: 5'-CGG GAT CCT TAG ATA TTA GCA AAA ATA TTT GGT TTT-3'

Using the genomic DNA of Bacillus lysosii as a template, the above specific primers are primers, and the UH enzyme gene is amplified.

PCR amplification was performed using the Takara kit, and 34 cycles were amplified using the instructions. The amplification conditions are as follows:

step	temperature	time
1	95 ° C	5 min
2	95 ° C	30 sec
3	55 ° C	30 sec
4	72°C	100 sec
5	72°C	10 min

The PCR product was separated on a 0.8% agarose gel and the DNA fragment was recovered to a size of 1.4 kb (see Figure 1). Show), by ligase with pMD19-T The vector was ligated, and the cloning vector pMD19-T-UH was constructed. The ligated product was transformed into Escherichia coli JM109, and the plasmid was extracted by shake flask culture. The enzyme was digested and sent to Shanghai Biotech Co., Ltd. to determine the nucleotide sequence, and the nucleotide sequence table was obtained. As shown in ID NO. 1, it is obtained by triplet codon translation as shown in SEQ ID NO. 2 of the amino acid sequence listing.

Example 2: Construction of expression vector and expression system

The cloning vectors pMD19-T-UH and pET20b(+) plasmids were Nde I and BamH I, respectively. After double digestion, the digested products were separated by 0.8% agarose gel electrophoresis, and DNA fragments of 1.4 kb and 3.7 kb were separately recovered, and ligase was ligated to construct pET20b(+)-UH. The expression vector was ligated into E. coli BL21 (DE3) competent cells, and the positive clones were screened by ampicillin plate. The recombinant plasmid was cultured and extracted by Nde I and BamH I. Double enzyme digestion was verified and sent to Shanghai Biotech Co., Ltd. for sequencing, which proved to contain the correct insert (as shown in Figure 2), thus obtaining the exogenous expression system of recombinant urethane hydrolase E. coli BL21(DE3)/pET20b(+)-UH.

Example 3: Recombinant expression of urethane hydrolase

Pick a genetically engineered strain E. coli BL21(DE3)/pET20b(+)-UH single colony, inoculated at 25 In mL, LB liquid medium containing 50 μg/mL ampicillin was incubated overnight at 37 °C with shaking. On the next day, transfer to the above liquid medium according to the 2% inoculum, and culture until the concentration of OD600=0.6 When IPTG was added to a final concentration of 0.1 mmol/L, the cells were cultured for 15 h, and the cells were collected by centrifugation, and the supernatant was removed by ultrasonication and centrifugation to determine the enzyme activity (such as the literature Zhao C, Kobashi). K (1994) Purification and characterization of iron-containing urethanase from Bacillus licheniformis. Biol Pharm Bull 17(6): Method for determining enzyme activity in 773-778) and by SDS-PAGE The amount of protein expression was measured. As can be seen from Figure 3, the lanes after induction were 50 kDa compared to the empty vector lanes to which no target bands were ligated. There is a target band consistent with the predicted size, which proves that the recombinant protein is expressed in this expression system, the protein expression amount is about 20% of the total protein amount, and the enzyme activity is 83.5 U/g wet cells.

Claims

A urethane hydrolase gene characterized by:

1) having the nucleotide sequence shown by SEQ ID NO: 1;

2) Encoding the protein of SEQ ID NO: 2.

2. As claimed in claim 1 The urethane hydrolase gene, characterized in that the urethane hydrolase gene is derived from Bacillus lysine (Lysinibacillus) Fusiformis).

3. A protein encoded by the urethane hydrolase gene of claim 1, the amino acid sequence of which is set forth in SEQ ID NO: 2 or in SEQ ID NO: A protein in which one or several amino acid residues are substituted, deleted or added and has the same enzymatic activity on the basis of the sequence shown.

4. A recombinant vector comprising the urethane hydrolase gene of claim 1.

5. A transgenic cell line comprising the urethane hydrolase gene of claim 1.

6. The use of the urethane hydrolase gene of claim 1 for the preparation of a recombinant urethane hydrolase.

7. Use of claim 1 The method for constructing a genetically engineered bacteria by the urethane hydrolase gene comprises the steps of: constructing a recombinant vector containing the urethane hydrolase gene, transforming the recombinant vector into Escherichia coli, and obtaining a recombinant gene The engineered bacteria are subjected to induction culture, and the culture liquid is separated to obtain a bacterial cell containing the recombinant urethane hydrolase.