AbstractsBiology & Animal Science

Rational design of the cold active subtilisin-like serine protease VPR towards higher activity and thermostability

by Kristinn Ragnar Óskarsson 1991

Institution: University of Iceland
Year: 2015
Keywords: Lífefnafræði
Record ID: 1221959
Full text PDF: http://hdl.handle.net/1946/21648


This research project builds on research previously done on the subtilisin-like serine proteinase VPR, from a psychrotrophic Vibrio species and its structural homologue aqualysin I (AQUI) from the thermophile Thermus aquaticus. We set out to design a mutant of VPR using site directed mutagenesis that would be both more stable against heat denaturation and retain the high activity of the wild type enzyme. Starting with two different templates, one being a C-terminal truncated form of the enzyme (VPRΔC), containing two proline mutations, N3P/I5P, close to the N-terminus of the protein, which had shown increased stability but loss of catalytic activity. The ∆C truncated form was produced by introducing a mutation as a stop codon at C277 to imitate the structure of AQUI in more detail. The other template contained the ∆C mutation and additional six mutations on a loop that may act as a hinge for movements that are postulated to be important for catalysis. The A116T/Q117R/A119H/S120R/G121R/S123A (6x) mutant had shown an increase in activity without losing stability to any degree. On top of these templates two mutations were added; N15D and Q142K. The N15D mutation had been shown to introduce a salt bridge yielding higher stability but with no detrimental effects on activity. The Q142K exchange on the other hand increased significantly the catalytic activity of the enzyme. Thus, we attempted to improve stability of the two mutants by introducing the N15D mutation, while the Q142K mutations was added with the purpose of increasing catalytic activity. The VPR∆C/N3P/I5P/N15D/Q142K mutant was a success, giving an 8°C rise in the Tm, a 10°C rise to the T50% and the catalytic activity was slightly higher than that of the wild type enzyme. The VPR∆C/6x/N15D/Q142K led to a 3°C rise in both Tm and T50%. To examine the effects of the N3P/I5P mutation on the flexibility of the structure, fluorescence quenching with acrylamide was preformed comparing AQUI, VPR∆C and VPR∆C/N3P/I5P. The results indicated that the environment of Trp6 in VPR∆C/N3P/I5P is not as accessible as in VPR∆C probably due to tighter packing of the N-terminus. Rannsóknasjóður Háskóla Íslands