Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.     

Expression and characterization of Ca2+-independent lipase from Bacillus pumilus B26

A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M_r) of 19,225. Based on the M_r and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 °C and 8.5, respectively. The lipase B26 showed a ‘Ca²⁺-independent thermostability and catalytic activity’. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca²⁺-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C₁₄–C₁₈) and triolein (C_18:1) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.