Structural Effects on Enzymatic Activity of Bacteriophage T 4 Lysozyme upon Adsorption to Colloidal Silica

approved by : The enzymatic activities of bacteriophage T4 lysozyme mutants with different structural stabilities were measured in the presence and absence of colloidal silica nanoparticles using a spectrophotometric technique and an agar plate bioassay. Spectrophotometric technique is based on the continuous reading of the change in turbidity of a bacterial suspension, while agar plate bioassay is based on the comparison of the clear zone width developed by T4 lysozymes. Wild type (isoleucine at position 3) T4 lysozyme along with two structural stability mutants Ile 3oCys and Ile 3oTrp were produced from Escherichia coil cells bearing the desired lysozyme genes. The three lysozyme variants differed by a single amino acid residue at position three. Each substitution resulted an altered structural stability, quantified by a difference in free energy of unfolding relative to wild type (-2.8 Kcal/mol and 1.2 Kcal/mol for Ile 3-0Trp mutant and Ile 3 --oCys mutant, respectively). Two different substrates E. coil cell walls and Micrococcus lysodeikticus cell walls, were used to measure the activity of each free lysozyme and lysozyme-particle complexes. For a ratio of 1:1 protein to particle with a 90 minute adsorption time, all the T4 lysozyme Redacted for Privacy mutants lost part of their enzymatic activity. The extent of enzymatic activity reduction is related to the stabilities of the proteins. The mutants with lower stability had lower enzymatic activity and a greater tendency to lose their enzymatic activity upon adsorption. Structural Effects on Enzymatic Activity of Bacteriophage T4 Lysozyme upon Adsorption to Colloidal Silica by