Selective Aqueous Biocatalysis of <scp>d</scp> -Panthenol via Engineered Pantothenate Synthetase

d-panthenol (provitamin B5) is widely used in personal-care products, pharmaceutical formulations, and feed applications, yet industrial production still relies mainly on chemical condensation. Here, we develop an aqueous biocatalytic route using a pantothenate synthetase (PS106) from <i>Rhodococcus</i> sp. N106. Recombinant PS106 catalyzed ATP-driven coupling of sodium d-pantoate with 3-amino-1-propanol in water at room temperature. Protein engineering increased the pantoate <i>k</i>cat/<i>K</i>_m from 4.87 M^-1·s^-1 in WT to 10.36 and 23.62 M^-1·s^-1 in V75G and A155 V, respectively, and lowered the apparent <i>K</i>_0.5 for 3-amino-1-propanol (WT, 156.47 mM). PS106 showed strong dependence on Mg²⁺ in the reaction system and followed Michaelis-Menten kinetics for ATP (<i>K</i>_m = 3.822 mM) and pantoate (<i>K</i>_m = 13.20 mM). Process optimization (Plackett-Burman → steepest-ascent → RSM) identified total substrate concentration, enzyme loading, and pH, producing 4.24 mM d-panthenol (2.67 μmol in 630 μL) in 1 h. These results identify A155 V as a promising variant for improved ATP and pantoate utilization, but downstream product recovery and PMI/E-factor optimization still require further work.