Thermostable cholesterol esterase mutant OPE S109P/S271P as well as preparation method thereof
An OPE-S109P, cholesterol esterase technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of huge screening workload, time-consuming and laborious, low positive mutation rate, etc., to improve operational stability, reduce production costs, and extend shelf life. effect of cycle
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Embodiment 1
[0027] Embodiment 1, cholesterol esterase gene ope Synthesis and construction of related vectors and expression systems
[0028] According to the NCBI database Ophiostoma piceae Cholesterol esterase gene sequence (Genbank: AY899847), 3D structure data (4BE4 and 4BE9) in the PDB database identified Ophiostoma piceae Cholesterol esterase mature peptide chain and its amino acid sequence. Based on codon usage database (http: / / www.kazusa.or.jp / codon / ) Pichia pastoris Codon usage frequency distribution table to analyze the codon usage of cholesterol esterase. The codon-optimized cholesterol esterase gene sequence is shown in SEQ ID NO.4, and the encoded mature peptide sequence is shown in SEQ ID NO.3 (the encoded mature cholesterol esterase contains 537 amino acid residues). Cholesterol esterase gene ( ope gene) to entrust a commercial company to synthesize the whole gene. The pPIC9K plasmid is used as the expression vector in the gene synthesis service, and the restrict...
Embodiment 2
[0029] Example 2, Screening and Determination of Mutation Sites
[0030] Use YASARA software to optimize the 3D structure of cholesterol esterase (PDB files: 4BE4 and 4BE9); use FoldX, Rosetta and PoPMuSIC software for the optimized 3D structure files, and combine the free energy change value of thermostable mutants (ΔΔG<0) , Construction of an electronic library of thermostable cholesterol esterase mutants. Use YASARA software to analyze the 3D structural characteristics of each cholesterol esterase one by one, and screen out cholesterol esterase mutants with unreasonable 3D structures; from the mutants with reasonable structures, screen out mutants whose mutation sites are located at the β-turn angle, and further Carry out the following analysis: ①Calculate the average value of z_B-factor of the four amino acid residues constituting the β-turn angle at 300K. Mutants whose average value of z_B-factor of the β-turn at the mutation site is greater than zero are selected as candi...
Embodiment 3
[0033] Embodiment 3, utilizing genetic engineering technique primer mutation site
[0034] With pPIC9K- ope The plasmid was used as a template for site-directed PCR amplification to construct the above-mentioned mutants. The PCR amplification system is 50 μL, including: 0.5 μL PrimeSTAR HS DNA polymerase, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 1.5 μL plasmid template (100 ng / μL), high temperature sterilized ultra Make up to a total volume of 50 μL with pure water. The primer pairs used in PCR amplification are listed in the table below.
[0035] The PCR amplification program was as follows: after denaturation at 95°C for 5 min, the amplification cycle was entered, that is, denaturation at 95°C for 30 s, annealing for 30 s, extension at 72°C for 10 min, a total of 25 cycles, and finally extension at 72°C for 7 min. The PCR product was detected by electrophoresis, and its band was single and clear. The annealing temperature used for PCR amplification ...
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