International Journal of Biological Macromolecules 107 (2018) 2131–2140 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Molecular characterization and bioinformatics studies of a lipase from Bacillus thermoamylovorans BHK67 Abhishek Sharma, Khem Raj Meena, Shamsher S. Kanwar ∗ Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171 005, India a r t i c l e i n f o Article history: Received 8 April 2017 Received in revised form 29 September 2017 Accepted 14 October 2017 Available online 16 October 2017 Keywords: Purification Multimeric nature MALDI-TOF-MS Molecular modeling a b s t r a c t A bacterium isolated from a hot-water spring identified as Bacillus thermoamylovorans BHK67 success- fully produced a thermotolerant extracellular alkaliphilic lipase. The lipase was purified to homogeneity by anion exchange chromatography with 15-fold purification and 12.1% yield. The lipase appeared to be a hexameric protein as it possessed a single band of Mr 25 kDa in SDS PAGE and 150 kDa in Native PAGE. DLS analysis of purified Bacillus thermoamylovorans BHK67 lipase (BTL) also showed the molec- ular integrity, homogeneity and stability of the enzyme. The purified lipase showed maximum activity at pH 7.5 with a half-life of 10.5 h at 55 ◦ C. Kinetic study of purified lipase by Lineweaver-Burk plot provided K m (7.7 mM), V max (90.9 U/mL/min), K cat (227.3 s -1 ) and K spec (29.4 mM s -1 ) for substrate p- nitrophenylpalmitate.The purified lipase also showed astonishing stability following exposure to ethanol, n-propanol, iso-propanol, n-butanol and DMSO. Amino acid characterization of BTL by MALDI-TOF-MS showed considerable resemblance with lysophospholipase L1 related esterase of Lactobacillus ozensis DSM 23829. Experimental coupled molecular modeling postulated a structure-activity correlation of BTL as a probable contender in degradation of xenobiotic compounds, biocatalysis, biotransformation of compounds, synthesis of optically active compounds, foodstuff industry, anticancer therapeutics etc. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Lipases (Glycerol ester hydrolase; EC 3.1.1.3) are most incredible biocatalysts exploited in industrial biotechnology [1–4]. Ther- mophilic lipases are multitalented biocatalysts, as they are often stable over a broad range of pH, elevated temperature and in the presence of organic solvents [1]. Thermophilic lipases have higher impedance to chemical denaturation and thus they act as suitable tools for use at industrial scale where relatively high reaction tem- peratures and/or organic solvents are needed to achievebiocatalysis [5]. Lipases have tremendous applications in food additives, chem- icals, detergents, wastewater treatment, cosmetics, paper & pulp conditioning, pharmaceuticals and leather industries [3,6–9]. Purification of a protein is very crucial when working with enzymes because it contributes the biological material obligatory for structural, functional and kinetic studies. The extra-cellular microbial enzyme can be purified from the cell-free fermentation broth by successive fractionation, concentration and chromatogra- phy techniques [10–12]. The thermotolerant enzymes are always in ample demand to perform catalysis in reactions that need higher ∗ Corresponding author. E-mail address: kanwarss2000@yahoo.com (S.S. Kanwar). temperature to keep reactants in liquefied state. An orthologus approach of structure prediction using SWISS MODEL server has been used to generate 3D protein models. Homology modeling is at present the most truthful computational method to generate trustworthy structural models and is consistently used in many biological applications. Lipases contain a catalytic triad of a ser- ine, histidine and aspartate at their active site [11]. The serine is involved in covalent catalysis and the histidine activates the ser- ine to undergo the nucleophilic attack [13]. In the present study B. thermoamylovorans lipase active site residues most likely sustain the spatial geometry for optimal activity. The lipase production by Bacillus thermoamylovorans has been previously reported following optimization of a few physico-chemical conditions that included temperature, time and pH [14]. However, no attempt was made to purify and/or characterize the lipasefrom Bacillus thermoamylovo- rans. In the current study the thermophilic B. thermoamylovorans BHK67 strain was used topurify an extracellular alkaline thermo- tolerant lipase that was found to be a homo-multimeric protein. https://doi.org/10.1016/j.ijbiomac.2017.10.092 0141-8130/© 2017 Elsevier B.V. All rights reserved.