Esterase mutant with improved thermal stability and application thereof
A thermostable and mutant technology, applied in the field of bioengineering, can solve the problems of insufficiency and poor thermostability of EstWY enzyme, and achieve the effect of high efficiency and simple purification
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Embodiment 1
[0039] The cloning of embodiment 1 wild EstWY enzyme ENND-TOP gene
[0040] The wild-type EstWY enzyme gene was codon-optimized in Escherichia coli as the host, and was synthesized by Suzhou Jinweizhi Co., Ltd., through the upstream primer 5'-ACTGCT CATATG ACCGATCCGACCTTTGATGCA-3' (underlined base is the recognition site of restriction endonuclease NdeI) and downstream primer 5'-TCAGCT CTCGAG TTAATGGCCCAGTGCTTTA-3' (the underlined base is the restriction endonuclease XhoI recognition site) to amplify the target gene. Toyobo’s KOD high-fidelity polymerase is used for PCR amplification. The amplification conditions are: 95°C for 2min, then 55°C 20sec, 100sec at 72°C, a total of 30 cycles, and finally 10min at 72°C.
[0041] After the reaction, the PCR product was detected by 1.5% agarose gel electrophoresis, and a 1.0 kb band was obtained, the length of which was in line with the expected result. According to the standard operation of the kit, recover and purify the target f...
Embodiment 2
[0042] Example 2 Expression, purification and activity determination of ENND-TOP
[0043] The engineered bacteria in the glycerol tube were inoculated into a 5mL LB medium test tube containing 100ug / mL Kan at a volume ratio of 1%, and cultured at 37°C and 220rpm for 12h. Transfer the 5mL bacterial liquid to a 1L LB medium shake flask containing 50ug / mL Kan, culture at 37°C 220rpm for about 2h, make the OD600 reach about 0.8, add 0.1mM IPTG inducer, and induce culture at 25°C 220rpm for 12-18h . The Escherichia coli cell suspension harvested after fermentation is ultrasonically disrupted, and after one-step Ni-NTA affinity chromatography treatment, the target protein with a purity of more than 95% can be obtained.
[0044] The EstWY enzyme assay method is as follows: 20 mM p-nitrophenylbutyrate is used as the reaction substrate, and the buffer system is 50 mM Tris-HCl buffer solution (pH9.0), and the enzyme activity is measured in real time. In the range of 30-60°C, measure t...
Embodiment 3
[0045] Example 3 Multiple Sequence Alignment and Consensus Analysis of ENND-TOP Homologous Proteins
[0046] The specific operation is as follows:
[0047] 1. Enter the homepage of the Pfam database (http: / / pfam.xfam.org / ), enter the amino acid sequence of FPOX-E in the SEQUENCE SEARCH tool to search. The server directly feeds back the amino acid sequence alignment results of the entire family of the protein, and displays the abundance of various amino acids at each site in the form of a histogram. The website can also automatically generate the consensus sequence of the protein family.
[0048] 2. Input the amino acid sequence of ENND-TOP in the NCBI protein database or Pfam database, and use the Blast tool to find all protein sequences with an identity greater than 40% with the target protein sequence. The repeated identical sequences were deleted, the remaining sequences were organized into fasta. format, and input into Clustalx1.83 software for multiple sequence alignmen...
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