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Ethyl carbamate hydrolase gene, protein coded thereby and application

A technology of urethane and hydrolase, which is applied in the fields of hydrolase, application, and genetic engineering, and can solve the problems of cumbersome protein purification steps and small enzyme production by wild bacteria

Active Publication Date: 2013-12-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, wild bacteria have not been widely used in the food industry due to the small amount of enzyme production, cumbersome protein purification steps, and some unsafe strains. system is important

Method used

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  • Ethyl carbamate hydrolase gene, protein coded thereby and application
  • Ethyl carbamate hydrolase gene, protein coded thereby and application
  • Ethyl carbamate hydrolase gene, protein coded thereby and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of UH gene from the genome of Bacillus fusiformis

[0026] A single colony of Bacillus fusiformis activated by streaking on the plate was inoculated into LB liquid medium and cultured for about 15 hours in the logarithmic growth phase, and the bacteria were collected. Refer to the steps of the genome extraction kit to extract the genomic DNA. Design specific primers based on the protein sequence alignment results, including forward primer 1 and reverse primer 2, forward primer 1 plus Nde I, and reverse primer 2 plus BamH I, as follows:

[0027] Forward primer 1: 5'-GGA ATT CCA TAT GAT GCG GAC ATT GCT GTA CGT-3'

[0028] Reverse primer 2: 5'-CGG GAT CCT TAG ATA TTA GCA AAA ATA TTT GGT TTT-3'

[0029] The genomic DNA of Bacillus fusiformis lysine was used as a template, and the above-mentioned specific primers were used as primers to amplify the UH enzyme gene. For PCR amplification, Takara kit was used, and 34 cycles were amplified by referring to its instru...

Embodiment 2

[0032] Example 2: Construction of expression vector and expression system

[0033] The cloning vector pMD19-T-UH and pET20b(+) plasmids were digested with Nde I and BamH I respectively, and the digested products were separated by 0.8% agarose gel electrophoresis, and DNA fragments of 1.4kb and 3.7kb were recovered respectively After the ligase ligation reaction, the pET20b(+)-UH expression vector was constructed, the ligation product was transformed into E. coli BL21(DE3) competent cells, the positive clone was obtained by the ampicillin plate selection, and the recombinant plasmid was cultured and extracted. After Nde I and BamH I double enzyme digestion verification and sent to Shanghai Shenggong Biological Company for sequencing to prove that it contains the correct insert (such as figure 2 As shown), the exogenous expression system of recombinant urethane hydrolase E.coli BL21(DE3) / pET20b(+)-UH was obtained.

Embodiment 3

[0034] Example 3: Recombinant expression of carbamate hydrolase

[0035] A single colony of genetically engineered bacteria E.coli BL21(DE3) / pET20b(+)-UH was picked and inoculated in 25mL LB liquid medium containing 50μg / mL ampicillin, and cultured overnight at 37°C with shaking. Transfer to the above liquid medium according to the 2% inoculum volume on the next day, and cultivate to the bacteria concentration OD 600 =0.6, add IPTG to a final concentration of 0.1mmol / L for induction, culture for 15h, collect the bacteria by centrifugation, break the wall by ultrasound, take the supernatant by centrifugation, and measure the enzyme activity (such as Zhao C, Kobashi K (1994) Purification and Characterization of iron-containing urethanase from Bacillus licheniformis. Biol Pharm Bull17(6): 773-778 in the method for determining enzyme activity) and detect protein expression by SDS-PAGE. From image 3 It can be seen that compared with the empty vector lane without the target band conn...

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Abstract

The invention discloses an ethyl carbamate hydrolase gene UH, a protein coded thereby and an application. The ethyl carbamate hydrolase gene UH has a nucleotide sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO.2. A gene engineering strain constructed by the UH gene provided by the invention has the enzyme activity of 83.5U / g of wet cell; and a wide application foundation is brought.

Description

Technical field [0001] The present invention discloses a carbamate hydrolase gene and its encoded protein, in particular a carbamate hydrolase gene derived from fusiform lysine bacillus and its encoded protein. Background technique [0002] Ethyl carbamate (English name: urethane or ethyl carbamate, abbreviated as EC), also known as urethane, urethane, is an intermediate of medicines, pesticides, and spices, or used in the production of sleeping pills, sedatives, injection cosolvents and printing and dyeing Industrial colorants can also be used in biochemical research. In addition, urethane itself can be used as a medicine, has anti-cancer properties, and is used to treat multiple myeloma and chronic leukemia. [0003] In the 1940s, Nettleship experiments proved that urethane has carcinogenic effects. Mainly can cause lung cancer, lymphoma, liver cancer, skin cancer and so on. As a by-product, urethane is widely present in fermented foods (such as bread, yogurt, cheese, soy sauc...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/18C12N15/63C12N5/10C12N1/21
CPCC12N9/18C12Y301/01001
Inventor 陈坚方芳李京京刘庆涛堵国成
Owner JIANGNAN UNIV
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