A kind of esterase est-24 and its encoding gene and use
An est-24, esterase technology, applied in genetic engineering, plant genetic improvement, application and other directions, can solve problems such as soil and water pollution, environmental pollution, human health threats, and achieve good tolerance and good degradation effect. Effect
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Embodiment 1
[0038] Example 1: Construction of metagenomic library and screening of esterases
[0039] Extract the genome of the acid mine wastewater sample, after verifying that the metagenomic DNA meets the requirements through agarose gel electrophoresis, select a suitable endonuclease to digest the large fragments of metagenomic DNA fragments into small fragments of different lengths. After electrophoresis purification, T4 ligase was used to connect with PUC18 plasmid and transformed into Escherichia coli DH5α. Positive (white spot) clones were picked by blue-white spot screening and transferred to LB plate containing tributyrin for several days. The existence of hydrolysis circle was observed, and the subclones that could hydrolyze tributyrin to form a hydrolysis circle were picked, expanded, cultured, preserved, and sampled for sequencing.
Embodiment 2
[0040] Example 2: Determination and codon optimization of the open reading frame of the esterase Est-24 gene
[0041] Annotate the sequencing results in Example 1 with bioinformatics methods, analyze the boundaries of its open reading frames, the full-length nucleotide sequence is 1230bp (from the start codon to the stop codon), and the encoded amino acid sequence As shown in SEQID NO.2, there are a total of 409 amino acids. The analysis of rare codons contains a large number of rare codons of Escherichia coli, which may cause low expression or no expression during heterologous expression. , so it was codon-optimized for E. coli. The optimized nucleotide sequence is shown in SEQ ID NO.1. After codon optimization, the upstream restriction site Nde I and the downstream restriction site Hind III were added, and the full-length gene was synthesized by ligating the cloning vector pUC57 and transformed into E. coli Top10 strain.
Embodiment 3
[0042] Example 3: Construction of Esterase Est-24 Expression Vector
[0043] 3.1 Plasmid extraction and double digestion
[0044] The Top10 strain containing the pUC57 vector linked with the Est-24 sequence and the DH5α strain containing the pET30a empty vector were inoculated into LB liquid medium, and the plasmids were extracted after shaking overnight at 37°C. The pET30a empty vector and the pUC57 recombinant vector plasmid connected with the Est-24 sequence were double-enzyme digested with Nde I and Hind III restriction enzymes respectively. , buffer 3 μL, supplemented to 30 μL with double distilled water. After digestion, the target band of Est-24 and pET30a vector were purified and recovered by agarose gel electrophoresis.
[0045] The restriction endonuclease used for the above-mentioned double-enzyme digestion is the fast endonuclease produced by Thermo Fisher Company, the gel recovery kit from Omega Company is used for the purification and recovery after the restric...
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