Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase

While thermophilic enzymes have thermostability desired for broad industrial applications, they can lose activity at ambient temperatures far from their optimal temperature. Engineering cold activity into thermophilic enzymes has the potential to broaden the range of temperatures resulting in significant activity (i.e., decreasing the temperature dependence of k_cat). Even though it has been widely suggested that cold activity, which results from active site flexibility, is at odds with thermostability, which results from enzyme rigidity, directed evolution experiments have shown that these properties are not mutually exclusive. In this study, rational protein engineering was used to introduce glycine as flexibility inducing mutations around the active site of Geobacillus thermocatenulatus lipase (GTL). Two mutants were found to have enhanced specific activity compared to wild-type at temperatures between 283 K and 363 K with p-nitrophenol butyrate but not with larger substrates. Kinetics assay revealed both mutations resulted in psychrophilic traits, such as lower activation enthalpy and more negative entropy values compared to wild type in all substrates. Furthermore, the mutants had significantly improved thermostability compared to wild type enzyme, which proves that it is feasible to improve the cold activity without trade-off. Our study provides insight into the enzyme cold adaptation mechanism and design principles for engineering cold activity into thermostable enzymes.