Amol Vaijanathappa Shivange

• The thesis focuses on directed evolution and structure function relationship of phytase from Yersinia mollaretii (Ymphytase). The “key beneficial” mutations identified in the directed evolution have been iteratively combined. Ymphytase variant with 54% improvement in thermostability (58°C for 20 min) and 200 U/mg improved activity was achieved. MD simulations results showed decreased overall Ymphytase flexibility in thermostable variant with slight increase in active site loop flexibility. The decreased flexibility might be due to improved intra-protein interactions like hydrogen bonds (G187S, K289E) and salt-bridge interaction (T77K). Three conceptually novel methods for protein engineering have been developed in part-III (Chapter 6 – 8). Combinatorial assembly of site saturation test in protein segment (ProCASTing), a sequence independent method was developed for parallel site saturation of more than one consecutive site (4 to 8) in any part of the protein. Multisite combinatorial assembly of site saturation test (OmniCASTing), practically simple method was developed for parallel site saturation of more than one site (five) regardless of positions in the gene. Using OmniCASTing, a variant with improved thermostability, pH stability and activity was obtained. Three properties improvement might be due to cooperative effects between the new combinations of mutations compare to parent combination. Protein consensus based surface engineering (ProCoS) method combining computational analysis and molecular biology tools was developed. The utility of ProCoS method has been demonstrated by surface engineering of Ymphytase that yielded a variant with 34 amino acid substitution (20% negative polar amino acids) and 3.8 fold improvement in pH stability (pH 2.8). Two hypotheses have been proposed in part-IV (chapter 8 – 9) and validated by experimental evidences.