Recombinant expression vector and recombinant bacterium of ferulic acid esterase bpfae gene, and method for recombinant expression
A technology of ferulic acid esterase and expression vector, which is applied in the field of genetic engineering and can solve problems such as efficiency improvement
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Embodiment 1
[0050] Embodiment 1: BpFae bioinformatics analysis
[0051] Predict the theoretical molecular weight and isoelectric point of ferulic acid esterase BpFae online through (http: / / www.expasy.ch); predict whether there is a signal peptide in protein BpFae through Signal P analysis; through MEGA X and ESPript 3( http: / / espript.ibcp.fr ) for multiple sequence alignment; the phylogenetic tree was also constructed by MEGA X using the Neighbor-Joining method; the three-dimensional structure of the protein BpFae was constructed based on the homology modeling method using Discovery Studio software. Use PyMOL (http: / / pymol.org) to render and display the 3D model of BpFae.
[0052] Through analysis, it was found that there was an open reading frame (ORF) of 1719bp, encoding a hypothetical 573 amino acid ferulic acid esterase, named BpFae. Its molecular weight was predicted online to be 59.04kDa, and its pI was 5.09. SignalP 4.1 analysis showed that BpFae had a signal peptide of 19 am...
Embodiment 2
[0053] Embodiment two: BpFae clone expression
[0054] 1. Cloning of BpFae gene
[0055] In the early stage, the whole genome sequencing and functional gene annotation of the strain B.pyrrocinia B1213 were carried out. The total DNA of the strain was extracted using a bacterial genome extraction kit. Primer F / R (Table 1) was designed according to the gene sequence encoding BpFae protein for cloning the target gene. Genomic DNA was used as a template to amplify the target gene BpFae. PCR reaction system (50μL) contains 5μL LA-Taq buffer, 5μL dNTP (2.5mM), 1μL genomic DNA, 1μL F primer (10mM), 1μL R primer (10mM), 0.5μL LA-Taq polymerase (2.5UμL –1 ), add water to 50 μL. Amplification conditions: pre-denaturation at 95°C for 5 min, 30 cycles: denaturation at 94°C for 30 s, annealing at 65°C for 1.5 min, extension at 72°C for 1 min, and extension at 72°C for 10 min. The PCR amplification product was detected by agarose gel electrophoresis with a mass fraction of 1%, and then...
Embodiment 3
[0073]Example 3: Optimization of Induced Expression Conditions
[0074] 1. Optimization of IPTG-induced enzyme production conditions
[0075] Using IPTG as an inducer, the fermentation conditions for the production of BpFae by the recombinant strain pGEX-4T-1-BpFae were optimized in order to increase the expression level of BpFae. Several factors were mainly optimized such as medium type, initial pH value, IPTG concentration, inoculum size, induction timing, induction temperature, shaker speed, and induction time (Table 1).
[0076] Table 1 Factors and levels of optimization of IPTG-induced enzyme production conditions
[0077] factor Level Medium type LB,TB,LBBM,LBBNM,LBBMG,LBBSMG,MX,TB-GN and SOB IPTG concentration (mM) 0.025, 0.05, 0.1, 0.25, 0.5 and 1 Inoculation amount (%, v / v) 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 initial pH 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 Induction temperature (℃) 18, 20, 22, 24, 26, 28 and 30 Shaker speed(...
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