Rhizopus oryzae Lipase Mutants for High-Temperature Interesterification

The patent applies parameter changes by systematically mutating specific amino acid residues in the ROL enzyme structure. Multiple point mutations were introduced at key positions (such as Phe173, Gln197, Val209, Ser267) to alter the enzyme's thermal stability parameters, enabling it to maintain activity at temperatures up to 70°C while preserving catalytic function for structured TAG production

The patent creates a composite enzyme structure by combining multiple beneficial mutations into a single engineered ROL variant. The cumulative effect of several amino acid substitutions works synergistically to enhance thermostability, resulting in an enzyme that exhibits both high temperature resistance and maintained catalytic activity, effectively combining properties that were previously separate

The patent applies local quality by targeting specific local regions (amino acid positions) of the enzyme structure for mutation. Rather than random mutagenesis, specific residues known to influence thermal stability were selectively modified. This localized approach allowed precise improvement of thermostability at key structural positions while preserving the overall catalytic mechanism and active site functionality

The patent employs feedback through iterative screening and selection processes. Engineered ROL variants were subjected to high-temperature stability assays, and those showing both improved thermostability and retained activity were selected for further optimization. This feedback loop between stability testing and activity measurement guided the development of variants that simultaneously achieve both thermostability and productivity