Ancestral sequence reconstruction and spatial structure analysis guided alteration of longer-chain substrate catalysis for Thermomicrobium roseum lipase

Thermomicrobium roseum DSM 5159 lipase (TrLip) is an enzyme with marked thermostability and excellent solvent resistance. However, TrLip reveals relatively high catalytic efficiency on short-chain substrates but poor activity against mid-long or long-chain fatty acids, which would limit its industrial application. In this study, ancestral sequence reconstruction (ASR), a common engineering tool for the evolutionary history of protein families, was employed to identify the natural evolutionary trends within 5 Å around the catalytic center. Two mutation libraries were constructed, one for the catalytic center and the other for the pocket flexibility. A total of 69 mutants were expressed and purified in the Escherichia coli expression system to determine the kinetic parameters, and W219G could significantly enhance the catalytic efficiency against substrates with 12-, 16- and 18-carbon side chains. In addition, the double mutant W219G/F265M could further catalyze the breakdown of the above three substrates up to 6.34-, 4.21- and 4.86-folds compared to the wild-type TrLip, while the initial pH and thermostability were maintained. Through bioinformatics analysis, the significantly enhanced catalytic efficiency against longer-side chain substrates should be associated with the reduction of steric hindrance. With the outstanding stability and the promoted activity, TrLip should be of great potential in chemical and food industry.