This work focused on two enzymes involved in rapid nucleic acid extractions.

The first step was defining the cleavage profile of the protease EA1 using both an Escherichia coli-derived peptide library to determine cleavage following an extended digestion and synthetic peptides for kinetic assays.

The second was engineering an enzyme of interest that could be added to the extraction chemistry to further improve the nucleic acid extraction workflow.

The extraction of nucleic acids from various sample types is a critical first step for many downstream applications.

Extraction typically starts with a cell lysis step and is followed by isolation and clean-up of the desired nucleic acids. This process can be time-consuming, particularly when dealing with samples that are difficult to lyse and have laborious clean-up steps. Cell lysis can be achieved through physical and chemical methods, such as by sonication or by the addition of a lysis buffer. Enzymatic lysis and clean-up of samples is also common and is usually facilitated by addition of the proteinase K enzyme. An alternative to proteinase K is the thermophilic metalloprotease, EA1. The EA1 protease has an optimal temperature of 75°C and has been used for the rapid extraction of nucleic acids from various samples. Previous characterisation of EA1 determined the thermal stability, metal ion coordination, and activity of the protease. However, investigation into which amino acid sequences EA1 prefers to cleave had not been conducted. Instead, cleavage preference has been assumed to be similar to thermolysin, a closely related and well-characterised metalloprotease. The broad characterisation of EA1 cleavage was performed through the digest of a proteome-derived peptide library. Quantitative mass spectrometry with iTRAQ labelling was used to investigate the cleavage preference of both EA1 and thermolysin. These results showed strong similarities of cleavage preference, with both proteases having strong preference for cleaving at the N-terminal side of leucine. To further compare the proteases, kinetic assays were performed with a FRET-reporter peptide substrate. These peptides compared rates of cleavage preceding a leucine, valine, phenylalanine, isoleucine, methionine, or alanine residue. This experiment showed greater differences between the proteases than did the limit digested analysed by mass spectrometry, particularly with thermolysin having a much greater catalytic efficiency for leucine residues than EA1. Addition of a second enzyme into the extraction chemistry is desirable but can be ineffective due to the protease activity of EA1. For some applications, EA1 must be removed or inactivated prior to proceeding with enzyme-based downstream applications. The goal of this work is to engineer the enzyme of interest to prevent cleavage by EA1, thus avoiding the necessity of EA1 removal or inactivation. Positions of proteolysis by EA1 were identified in the enzyme of interest and site-saturation mutagenesis was used to alter the exposed loop containing the initial EA1 cleavage site. A library containing multiple copies of 3 x 10⁵ different variant genes was subject to selection for protease-resistant activity of the enzyme of interest.