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Protein engineering has been used to alter the stability of proteins for several decades withmuch success, one approach being to introduce two cysteine residues that together form adisulfide bridge. The disulfide bridge can increase the Gibbs free energy of the transitionstate, thus increasing energy difference between the folded state and the unfolding transitionstate, leading to increased kinetic stability of the protein. Subtilisin Carlsberg is a serineprotease that has widespread applications within the industry but has also been tried in biogasprocesses to increase the biomethane yield from proteinaceous substrates. Subtilisin’s activitylifetime was found to be short in the biogas process, which prompted the need to increase theenzyme’s kinetic stability, meaning that the introduction of a disulfide bridge could be asolution. The aim of this project was to increase the kinetic stability of Subtilisin Carlsbergwith the use of introduced disulfide bridges.The production of Subtilisin Carlsberg has traditionally been done using the source organismBacillus Licheniformis, but here a successful method for expressing Subtilisin, and fourdisulfide variants of it, as an inclusion-body protein is presented. Also, a method forpurifying and refolding the protein under denaturing conditions is presented with a significantprotein yield.Thermal stability analysis of the WT enzyme and its four variants (A24C/S86C,N122C/A227C, K12C/E270C, V26C/A231C) was performed using NanoDSF, and showedthat the thermal stability was practically unchanged for A24C/S86C at 67.9 ℃, decreased by5.6 ℃ for N122C/A227C, increased by 8.2 ℃ for K12C/E270C, and increased by 11.5 ℃ forV26C/A231C.The kinetic stability of Subtilisin and its variants was analysed using stopped-flowmeasurements of the proteins’ denaturation rate at various GuHCl concentrations. The resultsshowed that N122C/A227C and V26C/A231C were more kinetically stable than the WTenzyme, while A24C/S86C and K12C/E270C were less stable. N122C/A227C had anactivation energy for unfolding of 5.217 kJ/mol higher than WT Subtilisin. V26C/A231C hadan activation energy for unfolding of 1.220 kJ/mol higher than WT Subtilisin. The resultsthereby show that two disulfides bond mutations achieved the desired outcome of increasedkinetic stability. Thereby, the aim of the project was fulfilled.

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