Protein Improvement: Hermodynamic And Kinetic Stabilities

1048 words - 4 pages

hermodynamic and kinetic stabilities contribute to the thermostability of an enzyme. Thermal stability is a critical property for many proteins
biotechnological applications as it implies longer life-times and frequently higher tolerance to the presence of organic co-solvents, extreme pH
values and high salt concentration or pressures (Vania Brissos e.t al. 2014). Directed evolution through the error-prone PCRs is the procedure to
carry out iterative mutagenesis for the protein parent genes. Enzyme azoreductase can reduce azo dye whose compounds are linked to bladder cancer in
humans and to nuclear anomalies in intestinal epithelial cells in mice. Azoreductase require two cycles of NADPH-dependent reduction of FMN to FMNH
for reducing the azo substrate. In a study conducted by Vania Brissos et al (2014), kinetic stability (mutant 1B6) and thermal activity (mutant
of Azoreductase (encoded by ppAzoR gene) from Pseudomonas putida MET94 is improved through directed evolution. Mutant 1B6 is improved through
introduction of random mutations that disturbed hydrophobic patches and increased the surface net charge of the protein, while mutation in 2A
variant leads to the improved structure robustness occurring in residues that are mostly involved in strengthening the solvent-exposed loops or the
inter-dimer interactions of the folded state

Mutation in the ppAzoR gene was generated by using error prone PCR with varied MnCl2 concentration and primers with NdeI and BamHI restriction sites
First round of mutagenesis are performed and the PCR products are purified and treated with RE and cloned into pET21-a before overexpression in E
coli KRX cells and first generation library is constructed and screened. Plasmids harbouring the beneficial mutations from the first screening are
pooled and used as templates for the second round of PCR. Recombination via DNA shuffling was performed using established methods and selected ppAzoR
gene coding variant parent from the first generation library screening were amplified by PCR using primers pET21D and pET21R and digested with DNaseI
in the thermocyclers and digestion was stopped using EDTA and normal PCR steps are proceeded. The PCR assembly product is then amplified again using
primer based on ppAzoR gene and purified, cloned into pET21a and transformed in E. coli KRX cells for the second generation library construction then
screening. Site directed is also done using selected 2A1 and 1B6 variant(contained random mutation resulted from epPCR as template and primers with
A1 + Y179H mutation and 1B6+A46P mutation are used to generate mutants for third generation mutant library. These libraries were constructed to
attain 1–3 amino acid changes of crude mutants for kinetic stability and thermal activity exhibiting either the highest initial activity or
highest thermostability during the screening. Mutants were re-screened to get rid of false positives. The presence of the desired mutations in the
resulting plasmids is...

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