Insights into controlling role of substitution mutation, E315G on thermostability of a lipase cloned from metagenome of hot spring soil

Rational mutagenesis was performed (at the vicinity of the active site residues D317 and H358 of a mature polypeptide) to investigate the role of amino acids in the thermostability/activity of a lipase enzyme. The single variant enzyme created with E315G (lip M2) mutation near one of the active site residue (D317) found to be an important residue in controlling the thermal stability, the variant with E315G mutation demonstrated biochemical properties similar to that of native lipase. However, we found that this mutation strongly affected the activity and stability of the lip M1 mutant, reported in our previous study (Sharma et al. in Gene 491:264–271, 2012b). The dual mutant with E315G/N355K mutation in the Wt showed small increase in the protein thermostability compared to the native lipase, however, the thermostability of the mutant lip M1 was reduced several fold. Presumably, E315G (lip M2) mutation reverted the thermostability evolved by N355K (lip M1). The native and variant enzymes also displayed large variation in enzyme kinetics and their preference for pNP-esters (substrates). We further generated 3D models and studied the loop modelling of the WT and variants. Interestingly, loop region Leu314-Asn321 showed structural flexibility on introducing E315G mutation in the native lipase. On the other hand, lysine in mutant N355K exhibited side chain conformational changes in the loop Thr353-His358 which resulted in its H-bonding with Glu284. In addition, replacing glutamic acid by glycine at 315 position in lip M3 distorted the electrostatic interactions between Glu315 and Lys355 in the flexible loop region Leu314-Asn321. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13205-013-0142-4) contains supplementary material, which is available to authorized users.