Isolation, overexpression and characterization of an alkaline˗stable lipase KV1 from acinetobacter haemolyticus

Abstract

The study reports the purification and comprehensive biochemical characterization of a novel lipase KV1 (LipKV1) from Acinetobacter haemolyticus strain KV1. Strain KV1 was identified as Acinetobacter haemolyticus based on results of 16S rDNA sequencing, phylogenetic and BIOLOG. The intracellular wild-type LipKV1 was purified to offer specific activity of 32 U/mg with an estimated relative molecular mass of 37 kDa. The PCR product of LipKV1 revealed that the retrieved sequence contained the proposed complete lipase gene sequence at nucleic acid positions 1~954. The purified wild-type LipKV1 exhibited a maximum relative activity at 40°C and pH 8.0. The lipase was activated (112-128%) in Na+, Ca2+, K+ and Mg2+ and the enzyme hydrolyzed a wide range of oils with tributyrin (140%) being the preferred ones. Reducing (PMSF, DTT, β-mercaptoethanol) and chelating (EDTA) agents significantly inhibited the LipKV1 relative activity (p < 0.05). Surfactants Tween 20-80 (110-143%) significantly enhanced the relative activity (p < 0.05). Gene encoding intracellular lipase was cloned to produce a large quantity of the recombinant LipKV1. The lipase which contained His-tag was expressed in Esherichia coli BL21 (DE3) cells using pET-30a as expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG concentration, post-induction temperature and post-induction time) were performed. All parameters were significant (p < 0.05) in influencing the expression of LipKV1, rendering a 70% increase in enzyme production at optimum induction conditions. The expressed recombinant LipKV1 was purified using Ni-affinity chromatography, to a specific activity of 233.4 U/mg and an estimated relative molecular mass of 39 kDa. The recombinant LipKV1 exhibited a maximum activity at 40°C and pH 8.0. Homology modeling of the lipase structure was carried out based on the template structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus, which shares a 58% sequence identity to LipKV1. The LipKV1 model comprised a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three conserved active-site residues, namely Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the active site of LipKV1 modeled at pH 8.0 revealed an aromatic platform responsible for the substrate recognition and preference towards tributyrin. The binding modes from the docking simulation appear to correlate well with the experimentally determined hydrolysis pattern, for which pH 8.0 is optimum and tributyrin being the preferred substrate. A low Km value (0.6 mM) for tributyrin further verifies the high affinity of LipKV1 for the substrate. Biophysical characterization of recombinant LipKV1 protein using ultaviolet-visible (UV-Vis) spectroscopy, circular dichroism (CD), fluorescence spectroscopy, ANS fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) indicated that the lipase retains its secondary structure and good folding at alkaline pH conditions (pH 8.0 and pH 12.0) and at 40C. Alkaline-stable enzymes such as LipKV1 are therefore, useful in biotechnology-based industries in order to shorten production time, minimizing energy consumption and preventing undesired chemical transformations.