Molecular study on recombinant cold-adapted, detergent- and alkali stable esterase (EstRag) from Lysinibacillus sp.: a member of family VI

Cold-adapted esterases have potential industrial applications. To fulfil the global continuous demand for these enzymes, a cold-adapted esterase member of family VI from Lysinibacillus sp. YS11 was cloned on pET-28b (+) vector and expressed in E. coli BL21(DE3) Rosetta cells for the first time. The open reading frame (654 bp: GenBank MT120818.1) encodes a polypeptide (designated EstRag: 217 amino acid residues). EstRag amino acid sequence has conserved esterase signature motifs: pentapeptide (GFSQG) and catalytic triad Ser(110)-Asp(163)-His(194). EstRag 3D predicted model, built with LOMETS3 program, showed closest structural similarity to PDB 1AUO_A (esterase: Pseudomonas fluorescens); TM-align score program inferences. Purified EstRag to 9.28-fold, using Ni(2+)affinity agarose matrix, showed a single protein band (25 kDa) on SDS-PAGE, Km (0.031 mM) and Kcat/Km (657.7 s(−1) mM(−1)) on p-NP-C2. Temperature and pH optima of EstRag were 35 °C and 8.0, respectively. EstRag was fully stable at 5–30 °C for 120 min and at pH(s) 8.0–10.0 after 24 h. EstRag activity (391.46 ± 0.009%) was impressively enhanced after 30 min preincubation with 5 mM Cu(2+). EstRag retained full stability after 30 min pre-incubation with 0.1%(v/v) SDS, Triton X-100, and Tween-80. EstRag promising characteristics motivate performing guided evolution and industrial applications prospective studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11274-022-03402-5.