## 4 Discussion  
Due to their biotechnological potential, lipolytic enzymes are attracting enormous attention [24; 25]. Most lipases used in industry are microbial enzymes that originate from fungal or bacterial sources. Bacterial lipolytic enzymes II family are widespread, display broad substrate specificities, and have a diverse range of hydrolytic functions, such as carboxylase, lipase, protease, thioesterase, lysophospholipase, and acyltransferases activities [22]. Numerous family II esterases have been characterized. The thermophilic bacterium _Bacillus_ sp. K91 encodes an esterase, Est19, which is highly stable in alkaline solutions, denaturants, and detergents; is stable at 60 \({}^{\circ}\)C; and has deacetylase activity in semisynthetic antibiotics [26]. Recombinant EstL5 from _G. thermodentirificans_ T2 was characterized. EstL5 displayed 24% of its highest activity at 0 \({}^{\circ}\)C and exhibited a unique cold-adaptation feature [27; 28]. A novel GDSL esterase 115 was activated by Tween-20, -60, and Triton X-100 and inhibited by 1 mM SDS [29]. To date, there have been few reports on the natural characteristics and structural features of GDSL family biases. In this study, LipGt6, a new thermophilic lipase, from _G. thermolevoorans_ H9, was cloned, purified, and characterized. The sequences were processed using DNAMAN Version 6.0 (Lynnon Biosoft, San Ramon, CA, USA). LipGt6 has 85.77% similarity with _G. thermocatemulatus_ Lip29 [14]. However, they have different substrate preferences. Lip29 exhibited the maximum activity towards long-chain fatty acids (C12-C16) and almost no activity towards short-chain fatty acids. The recombinant LipGt6 exhibited the highest activity towards medium- and long-chain fatty acids (C8-C14). LipGt6 has 12.6% sequence similarity with the lipase Lip486 [17]. Both Lip29 and Lip486 are reported lipases that belong to the GDSL family. Sequence comparison analysis of LipGt6, a GDSL family lipase, revealed a Gly-Asp-Ser-(Leu) [GDS(L)] motif that contained an active-site serine residue. Site-directed mutagenesis was used to substitute these three amino acid residues of the catalytic triad (Ser47, Asp221, and His224) with alanine. The activity of the D221A mutant was unaffected, whereas the activity of the S47A and H224A mutants was the same as that of the negative control, indicating that the S47A and H224A mutants were inactive. Typically, the  
Figure 6: Three-dimensional modelling and mutant activity of LipGt6. (**a**) The tertiary structure of LipGt6 was modelled based on that of an esterase from _G. thermodentirificans_ (PDB: 7E16). The catalytic triads are shown (Ser47, Asp221, His224). The \(\beta\)-sheet structures and \(\alpha\)-helices are colored yellow and red, respectively. (**b**) Retention enzyme activity of the S47A, D221A, and H224A mutants. Data points in Figure 6b are the average of triplicate experiments, and error bars represent the standard deviation. catalytic triad involves a histidine, an aspartic residue that lies three positions upstream of the active-site histidine, and an important serine [30], as observed in _G. thermocatenulatus_ Lip29 [14] and _Bacillus_ sp. Est19 [26]. [PERSON] and [PERSON] [31] proposed that the aspartic acid residue is Asp-116, which is located in block III, and the catalytic triad of the enzyme consists of Ser-16, Asp-116, and His-291. The specific lipolytic activity of LipGt6 may involve an unknown catalytic motif. More structural information is needed to establish whether LipGt6 proteins share a common architecture of their catalytic triad (or dyad) in addition to the similarity of their sequences [32]. In this study, the lipase LipGt6 had a molecular weight of 28 kDa, and its optimal temperature and pH were 50 \({}^{\circ}\)C and 9.0, respectively. The enzyme was also thermostable up to 70 \({}^{\circ}\)C. Optimum temperature and pH for _Bacillus stearothermophilus_ P1 lipase were 55 \({}^{\circ}\)C and pH 8.5 and it was highly stable in the temperature range of 30-65 \({}^{\circ}\)C [33]. [PERSON] et al.