Kcat Enzymatic advances in enzyme engineering algorithms hybridized with the Quantum Mechanics (QM) approach to derive functional hotspots and substitutions. The Site Saturated Mutagenesis (SSM) technology based on 7D Grid technology performs SSM to substitute targeted hotspots with any other naturally occurring amino acid to produce libraries of single-residue substitutions. The Probe-based Hotspots (PHP) technology based on 7D Grid technology uses probes on targeted hotspots that mimic the chemical properties of naturally occurring amino acids in the enzyme to produce libraries of the doublet to quadruplet substitutions. Both SSM and PHP variant libraries are screened against Kcat Enzymatic’s proprietary six exhaustive parameters to derive the focused libraries of enzyme variants. These two techniques are most efficient and relatively faster than any other conventional method for predicting and substituting the hotspots across the protein.