Ancestral Nitrilase\nMining and Semi-Rational Engineering\nfor Enhanced Thermal Stability in Rapeseed Meals-Derived Nitriles\nDegradation

Rapeseed meal (RSM), a protein-rich\nbyproduct, holds\npotential\nas a high-quality animal feed, but nitrile compounds derived from\nglucosinolates (GSLs) in RSM pose a toxicity risk. Nitrilases, enzymes\nthat hydrolyze toxic nitriles to carboxylic acids, offer a potential\nsolution for detoxification. However, the low thermal stability of\nnitrilases restricts their industrial applicability. We herein identified\neight ancestral nitrilases through sequence-based mining using 6803NIT\nas a probe enzyme. Among these, ancestral enzyme A1 exhibited the\nhighest specific activity (58.3 U/mg) and half-life (<i>t</i><sub>1/2</sub> = 3.5 h at 40 °C). To enhance thermal stability,\nwe engineered a quadruple mutant A1M_4C, which exhibited a 4.7-fold\nincrease in half-life (<i>t</i><sub>1/2</sub> = 16.3 h)\nand a 2-fold increase in specific activity (118.5 U/mg). Kinetic analysis\nrevealed a reduction in <i>K</i><sub>m</sub> from 14.9 to\n10.5 mM and an increase in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> from 1.9 to 4.37 s<sup>–1</sup>·mM<sup>–1</sup>. Mechanistic studies indicated that enhanced stability\nin A1M_4C was due to increased hydrogen bonding and stronger amino\nacid interactions. Simulated feed pelletization at 90 °C for\n2 min showed that A1M_4C acquired a 22.2-fold improvement toward nitriles\ndegradation over wild-type A1. These findings demonstrate the potential\nof ancestral enzyme mining to develop thermostable nitrilases for\nindustrial feed applications.