A kind of esterase est112-2 and its coding gene and application
An esterase and gene technology, applied to esterase EST112-2 and its encoding gene and application fields, can solve the problems of energy consumption, severe reaction process, large difference in properties and the like
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Embodiment 1
[0040] Example 1: Design of primers for esterase gene EST112-2 and determination of open reading frame boundaries
[0041] The genomic DNA of Psychrobacter sp. 112 was extracted, and after being verified by 16S rRNA, it was submitted to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. Using bioinformatics means to annotate the genome, analyze the esterase gene, and determine the open reading frame of the esterase gene EST112-2, which has a base of 1401bp, and its nucleotide sequence is as shown in SEQ ID NO.1 It shows that the esterase EST112-2 encoded by it has 466 amino acids, and the specific sequence is shown in SEQ ID NO.2. Through the signal analysis tool http: / / www.cbs.dtu.dk / services / SignalP / , it is found that There is no signal peptide, and the primer information is as follows: Forward primer: 5'-CAC GGATCCATGTCTCACTCAACCATATCGTCCAATACC-3', the underlined part is the restriction site of BamH I; reverse primer: 5'-CCG CTCGAG TTAGGCCGCTAACTGCCC-3', the underli...
Embodiment 2
[0042] Embodiment 2: Cloning and vector construction of esterase gene EST112-2
[0043] 2.1PCR amplification
[0044] The primer (forward primer: 5'-CAC) designed in embodiment 1 GGATCC ATGTCTCACTCAACCATATCGTCCAATACC-3'; reverse primer: 5'-CCG CTCGAG TTAGGCCGCTAACTGCCC-3') was sent to Shanghai Bioengineering Co., Ltd. to synthesize primers. The synthesized primers were diluted to 10 μM with TE, and the genomic DNA of Psychrobacter sp. 112 was extracted as a DNA template, and the PCR reaction shown in Table 1 was established. system:
[0045] Table 1 PCR reaction system
[0046]
[0047]
[0048] Use the following PCR amplification program to amplify the esterase gene EST112-2: a. Denaturation at 95°C for 5 min; b. Denaturation at 95°C for 1 min, annealing at 55-65°C for 0.5 min, extension at 72°C for 3 min 30 s, for 30 cycles; c. Extend at 72°C for 10min and cool to 10°C.
[0049] The PCR product was electrophoresed in 1% agarose gel for 20 min at 120V, and observ...
Embodiment 3
[0060] Embodiment 3: High expression of esterase EST112-2 in Escherichia coli BL21 (DE3)
[0061] 3.1 Preparation of Escherichia coli BL21(DE3) Competent Cells
[0062] a. Inoculate Escherichia coli BL21(DE3) strain into 5mL LB liquid medium, shake overnight at 37°C, 250rpm;
[0063] b. Inoculate the Escherichia coli BL21 (DE3) bacterium liquid in the liquid culture tube after overnight shaking culture in the LB shake flask according to the inoculation amount of 1% volume ratio, and shake it at 37° C. for 3 hours (≥ 300 rpm) to obtain the original culture;
[0064] c. Rapidly cool the cultured shake flask to 0°C in ice water, divide the original culture into ice-precooled centrifuge tubes (50 mL), and place on ice for several minutes;
[0065] d. Centrifuge at 4000rpm for 10 minutes at 4°C to recover the cells, and dry the residual liquid in air (rapidly);
[0066] e. 10mL 0.1M CaCl pre-cooled with ice 2 Resuspend the cells and centrifuge at 4000rpm for 10min at 4°C to reco...
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