pet-degrading enzyme mutants and their applications
A technology for degrading enzymes and mutants, applied in the field of enzyme engineering, can solve the problems that cannot be used to recycle colored or opaque plastics, are difficult to degrade, and have low degradation efficiency, and achieve good industrial prospects and improved degradation rates.
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Embodiment 1
[0033] Example 1 Source of PET degrading enzyme 7029PETase and construction of heterologous expression vector in E.coli BL21(DE3)
[0034] By analyzing the genome data in DSM7029 (ATCC 53080), a silently expressed gene was found in its genome, and its amino acid sequence and nucleic acid sequence were compared with the sequence of the reported PET degrading enzyme IsPETase, and it was found that they had certain homology The amino acid sequence similarity is 64.3%, and the nucleic acid sequence similarity is 72.6%. Therefore, it is predicted that the enzyme encoded by the gene is a PET degrading enzyme, and the gene is named 7029PETase, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence is shown in SEQ ID NO.9. After heterologous expression and in vitro activity detection, the PET degrading enzyme 7029PETase has the ability to degrade commercial PET at room temperature. The degradation process is as follows: figure 1 shown.
[0035] Use the genomic ...
Embodiment 2
[0048] Embodiment 2: Construction of PET degrading enzyme 7029PETase mutant
[0049] The wild-type 7029PETase was subjected to site-directed mutation using the overlap PCR method, and the recombinant vector pET22b-7029PETase of the wild-type 7029PETase was used as a template, and the front-end coding sequence of 7029PETase was amplified using primers 7029-L61T-1 and 7029-22b-1, and primer 7029 -L61T-2 and 7029-22b-2 amplify the back-end coding sequence and terminator sequence of 7029PETase, and introduce a nucleic acid sequence that mutates leucine at position 61 to threonine; finally use the product of overlap extension PCR as a template , using two primers 7029-22b-1 and 7029-22b-2 to amplify the coding sequence and terminator sequence of the mutant. The PCR program used in fragment amplification is the same as in Example 1, and the resulting mutant is named 7029PETase L61T , which is formed by mutating the 61st amino acid of 7029PETase from leucine to threonine, its amino ...
Embodiment 3
[0060] Example 3: Expression and purification of wild-type 7029PETase and its mutant proteins
[0061] The expression and purification process of wild-type 7029PETase is as follows:
[0062] 1) Streak the recombinant strain BL21-PETase on LB solid medium (containing ampicillin antibiotic 100 μg / ml) and culture overnight at 37°C;
[0063] 2) Pick the monoclonal cells in LB liquid medium (containing ampicillin 50 μg / ml) and culture overnight at 37°C and 200 rpm;
[0064] 3) Transfer the bacterial liquid obtained from overnight culture to 2L LB liquid medium (containing ampicillin antibiotic 50μg / ml) with an inoculum of 1%, and cultivate it at 37°C and 200 rpm until the OD600 is about 0.8, then add 0.1 mM IPTG Induction, then continue to culture at 18°C, 200 rpm for 16-18 h;
[0065] 4) Centrifuge the bacteria solution (4500 rpm, 15 min, 4°C), discard the supernatant, and resuspend the bacteria in 80 ml Buffer A (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.6);
[0066] ...
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