Ethyl carbamate hydrolase gene, protein coded thereby and application
A urethane and gene-encoded technology, applied in the fields of hydrolase, application, genetic engineering, etc., can solve the problems of cumbersome protein purification steps and small enzyme production by wild bacteria
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0025] Example 1: Cloning of the UH gene from the genome of Lysinibacillus fusiformis
[0026] Inoculate a single colony of Lysinibacillus fusiformis activated by streaking on the plate in LB liquid medium, and collect the bacterium when it is in the logarithmic growth phase after culturing for about 15 hours. Refer to the steps of the Genome Extraction Kit to extract its genomic DNA. Design specific primers according to the protein sequence alignment results, including forward primer 1 and reverse primer 2, forward primer 1 plus Nde I, 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 UH enzyme gene is amplified by using the genomic DNA of Lysinobacillus fusiformis as a template and the above-mentioned specific primer as a primer. For PCR amplification, a kit from Takara Company was used, referring to its instruc...
Embodiment 2
[0032] Embodiment 2: Construction of expression vector and expression system
[0033]The cloning vectors 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 the DNA fragments of 1.4kb and 3.7kb were recovered respectively , through ligase ligation reaction, construct pET20b(+)-UH expression vector, the ligation product was transformed into Escherichia coli BL21(DE3) competent cells, and the positive clones were obtained by ampicillin plate screening, and the recombinant plasmid was cultured and extracted, and passed through Nde I and BamH I double-enzyme digestion verification and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to prove that it contains the correct insert (such as figure 2 shown), so as to obtain the exogenous expression system E.coli BL21(DE3) / pET20b(+)-UH of the recombinant ethyl carbamate hydrolase.
Embodiment 3
[0034] Embodiment 3: Recombinant expression of ethyl carbamate hydrolase
[0035] Pick a single colony of genetically engineered bacteria E.coli BL21(DE3) / pET20b(+)-UH, inoculate it in 25 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture it overnight at 37°C with shaking. The next day, transfer to the above liquid medium according to 2% inoculation amount, and cultivate to the bacterial concentration OD 600 =0.6, add IPTG to a final concentration of 0.1mmol / L induction, cultivate for 15h, collect the thalli by centrifugation, and get the supernatant by ultrasonic breaking and centrifugation, and measure the enzyme activity (such as the literature Zhao C, Kobashi K (1994) Purification and Characterization of iron-containing urethanase from Bacillus licheniformis. Biol Pharm Bull17 (6): 773-778 method for measuring enzyme activity) and detect protein expression by SDS-PAGE. From image 3 It can be seen from the figure that compared with the empty vector lane n...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More