To investigate the effect of alpha3 and alpha5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (deltabtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the deltaalpha3alpha5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the deltaalpha3alpha5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and deltaalpha3alpha5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and deltaalpha3alpha5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of deltaalpha3alpha5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and deltaalpha3alpha5 lipases by 2.1- and 2.5-fold, respectively.
Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Bacillus thermocatenulatus Lipase 2 (BTL2) is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide ((112)Ala-His-Ser-Gln-Gly(116)) was replaced with similar sequences ((207)Gly-Glu-Ser-Ala-Gly(211)) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in Escherichia coli, and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.
Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.
Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS-PAGE. The values of Km and Vmax, calculated by the Michaelis-Menten equation, were 1077muM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70 degrees C and pH 8.5. Activities at 50, 55 and 60 degrees C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5-10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% alpha-helix, 12.8% beta-sheet, 22.7% beta-turn and 27% random coil.
Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha5 helix is a region that causes disorder in BTL2 protein. So, the alpha5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Deltaalpha5-BTL2) has been expressed in Pichia pastoris yeast. The alpha5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.
Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide ((1)(1)(2)Ala-His-Ser-Gln-Gly(1)(1)(6)) was replaced with similar sequences ((2)(0)(7)Gly-Glu-Ser-Ala-Gly(2)(1)(1)) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 degrees C. The highest percentage of both lipases activity was observed at 60 degrees C and pH of 8.0. The ion Ca(2)(+) slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase ((1)(1)(2)Gly-Glu-Ser-Ala-Gly(1)(1)(6)) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.
