Phi29 DNA polymerase (DNAP) derives from bacteriophage Phi29 and replicates DNA under isothermal conditions by rolling circle amplification. It is a particularly interesting enzyme due to its outstanding processivity and low error rates in the range of one base-pair mismatch for every 10^(-5) to 10^(-6) nucleotides incorporated. Phi29 DNAP achieves...
Phi29 DNA polymerase (DNAP) derives from bacteriophage Phi29 and replicates DNA under isothermal conditions by rolling circle amplification. It is a particularly interesting enzyme due to its outstanding processivity and low error rates in the range of one base-pair mismatch for every 10^(-5) to 10^(-6) nucleotides incorporated. Phi29 DNAP achieves...
Published in Angewandte Chemie (International ed. in English)
In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs...
Natural evolution occurs when mutations to DNA confer an advantage to an organism facing a new or changing environment. However, this process is slow, occurring over millions of years. There is a great need in the chemicals and pharmaceutical industries among others, to be able to engineer proteins to catalyze specific reactions, bind new substrate...
The development of catalysts for faster, more selective, and more sustainable chemical reactions remains an outstanding goal in chemistry. While advancements in statistical models have greatly improved our predictive ability, physical models remain our most valuable tools for mechanistic understanding. Density functional theory (DFT) calculations p...
Published in Angewandte Chemie (International ed. in English)
Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non-complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolu...
Detailed biochemical characterization of natural and mutant enzymes provides essential clues to understand their mechanisms. There are, however, limits to the throughput of such approaches and they are not without errors. DNA polymerases have benefited from over 50 years of detailed study and remain not fully understood. As such, methods that allow...
Published in Bio-protocol
Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for ...
Published in Bioscience Reports
In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of unique functions. With the advent of genetic code extension technology, non-natural amino acids (nnAAs) are able to be incorporated into proteins in a site-specific or residue-specific...
Published in Journal of biomedical informatics
Accurately establishing the connection between a protein sequence and its function remains a focal point within the field of protein engineering, especially in the context of predicting the effects of mutations. From this, there has been a continued drive to build accurate and reliable predictive models via machine learning that allow for the virtu...
