An alkaline protease bmp mutant with high thermostability and its coding gene
A thermal stability and protease technology, applied in the field of genetic engineering, can solve the problems of poor thermal stability of BmP, easy to decompose and inactivate, and limit industrial application, and achieve the effect of improving stability and good thermal stability
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Embodiment 1
[0035] Embodiment 1, the cloning of bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP gene
[0036] The target gene was directly synthesized according to the reported alkaline protease gene sequence of Bacillus mohaiwei (Genebank: AY665611.1). Two primers (R: 5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F: 5'-TCTAGCGGCCGCTTATTGAGCG GCAGCTTCGAC-3') were designed according to the synthesized target gene to amplify the BmP gene of Bacillus mohaiwei alkaline protease. The amplified PCR product was purified and recovered, and connected to the expression vector phyP 43 L, get the expression vector phyP 43 L-BmP.
Embodiment 2
[0037] Embodiment 2, Rational Design Fixed-point Saturation Mutation
[0038] Alkaline protease BmP is modeled by homology modeling software to obtain the three-dimensional conformation map of BmP (such as figure 1 shown). Using bioinformatics software to perform minimum protein quantization calculations on the constructed BmP model, the 175th, 210th and 270th key amino acid positions were found. The effect of position 175, position 210 and position 270 on the thermostability of alkaline protease BmP was studied by saturation mutagenesis.
[0039] The process of site-specific saturation mutation is as follows: To construct a good phyP 43 L-BmP was used as a template, and the corresponding mutant primers were used for PCR amplification; the amplified PCR product was subjected to agarose electrophoresis, and the PCR product was purified and recovered. The original plasmid was decomposed with the restriction endonuclease DpnI, and the decomposed product was transformed into E....
Embodiment 3
[0043] Embodiment 3, combinatorial mutation and thermal stability analysis of mutant
[0044] A mutant named BmP-mut was finally obtained by combining single point mutations, which contained mutation sites T175I, G210Q and G270S. In order to accurately compare the thermal stability of the original alkaline protease BmP and the mutant BmP-mut, the corresponding proteases were firstly purified by nickel column purification. The thermal stability of the purified alkaline protease BmP and the mutant BmP-mut was measured at 75° C. in a water bath for 5 minutes. Finally, the retention rate of the mutant BmP-mut under this condition was determined to be 70%.
[0045] Table 2 Original alkaline protease BmP and combined mutant thermostability
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