A Broad Temperature Active Lipase Purified From a Psychrotrophic Bacterium of Sikkim Himalaya With Potential Application in Detergent Formulation

Abstract

Bacterial lipases with activity spanning over a broad temperature and substrate range have several industrial applications. An efficient enzyme-producing bacterium Chryseobacterium polytrichastri ERMR1:04, previously reported from Sikkim Himalaya, was explored for purification and characterization of cold-adapted lipase. Optimum lipase production was observed in 1% (v/v) rice bran oil, pH 7 at 20°C. Size exclusion and hydrophobic interaction chromatography purified the enzyme up to 21.3-fold predicting it to be a hexameric protein of 250 kDa, with 39.8 kDa monomeric unit. MALDI-TOF-MS analysis of the purified lipase showed maximum similarity with alpha/beta hydrolase (lipase superfamily). Biochemical characterization of the purified enzyme revealed optimum pH (8.0), temperature (37°C) and activity over a temperature range of 5-65°C. The tested metals (except Cu²⁺ and Fe²⁺) enhanced the enzyme activity and it was tolerant to 5% (v/v) methanol and isopropanol. The Km and Vmax values were determined as 0.104 mM and 3.58 U/mg, respectively for p-nitrophenyl palmitate. Bioinformatics analysis also supported in vitro findings by predicting enzyme's broad temperature and substrate specificity. The compatibility of the purified lipase with regular commercial detergents, coupled with its versatile temperature and substrate range, renders the given enzyme a promising biocatalyst for potential detergent formulations.

Keywords: Chryseobacterium polytrichastri ERMR1:04; bioinformatics analysis; broad temperature activity; detergent formulation; lipase; purification.

Figure 1

Optimization of culture conditions for the production of ERMR1:04 lipase. The bacterial isolate was grown in mineral-based production media for 60 h at 150 rpm. The optimized condition for lipase production was determined by checking the relative activity of the lipase produced at different experimental parameters, and the maximum was set to 100%. Lipase activity was determined using 50 mM tris buffer pH 8 at 37°C. Effect of (A) different substrate types (olive oil, sunflower oil, sapium oil, and rice bran oil) on the production of lipase. The bacterial isolate was grown at 20°C pH 7 having 1.5% (v/v) oil. Maximum lipase production was observed in rice bran oil; (B) different temperature (10, 15, 20, and 28°C) on lipase production in 1.5% (v/v) rice bran oil pH 7. Maximum lipase production was observed at 20°C; (C) different pH (6–9) on lipase production. The bacterial isolate was grown in 1.5% (v/v) rice bran oil at 20°C. Maximum lipase production was observed at pH 7; (D) different substrate percentage (0.5–2.5% v/v) on lipase production. The bacterial isolate was grown at 20°C pH 7 having rice bran oil as a substrate. Maximum lipase production was observed in 1% (v/v) rice bran oil.

Figure 2

Electrophoretic pattern of lipase, (A) SDS-PAGE analysis of ERMR1:04 lipase at each purification step. The proteins were separated on 12% SDS-PAGE gel and then stained with Coomassie Blue R-250. M represents molecular weight marker (Pierce™ Unstained Protein MW Marker, Thermo Scientific); L1: crude cell-free extract of ERMR1:04; L2: size exclusion chromatography (Sephadex G-100) purified protein fraction; L3: hydrophobic interaction chromatography (Octyl-Sepharose fast flow) purified lipase; (B) zymographic analysis of purified lipase in the presence of substrate MUF-butyrate in 12% SDS-PAGE; (C) standard curve between log [MW] vs. Rf of proteins moved on SDS-PAGE (12%). The linear relationship between protein MW markers and migration distance showed reliability in predicting MW of lipase; (D) native-PAGE (10%) analysis of purified lipase. M represents the molecular weight marker; L represents hydrophobic interaction chromatography (Octyl-Sepharose fast flow) purified lipase fraction.

Figure 3

Peptide mass spectra of purified lipase obtained from MALDI-TOF-MS. The x-axis denotes m/z ratio and the y-axis denotes intensity (a.u) of peptides. The amino acid sequence represents the alpha/beta hydrolase of Solibacillus sp. to which the query sequence matched. The residues in bold are the ones identical with the query sequence.

Figure 4

Effect of (A) different temperature (5–65°C) on lipase activity. The enzyme assay was performed at a different temperature in 50 mM Tris buffer pH 8. Maximum enzyme activity was observed at 37°C and the enzyme was active in the temperature range of 5–65°C; (B) different pH (7–9) on lipase activity. The enzyme assay was performed at different pH in 50 mM tris buffer. Maximum activity was observed at pH 8; (C) metal ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Fe²⁺) at 5 and 10 mM concentration on lipase activity. The enzyme assay was performed after pre-incubation with metal ions for 30 min at 37°C in 50 mM tris buffer. All the metal ions except (Cu²⁺ and Fe²⁺) increased lipase activity.

Figure 5

Effect of (A) organic solvents (5% (v/v) Butanol, Isopropanol, Acetonitrile, and Methanol) on lipase activity. The enzyme assay was performed after pre-incubation with tested solvents for 30 min at 37°C in 50 mM tris buffer. Lipase retained 80% of activity in isopropanol and methanol while activity was reduced considerably in the presence of butanol and acetonitrile; (B) inhibitors (1, 5, 10 mM EDTA, DTT, PMSF) on lipase activity. The enzyme assay was performed after pre-incubation with tested solvents for 30 min at 37°C in 50 mM tris buffer. All the tested inhibitors decreased lipase activity. (C) Kinetic study of purified lipase was studied using the Lineweaver–Burk plot. The kinetic parameters (K_m and V_max) were studied by varying pNPP concentration from 0.025 to 0.3 mM. The observed K_m and V_max values were 0.104 mM and 3.58 U/mg, respectively.

Figure 6

Detergent compatibility test of purified lipase checked on commercially available detergents (1% (v/v) Tide, Surf, Aerial, and Wheel). Lipase assay was performed after incubating the enzyme in the presence of different detergents at a temperature ranging from 5 to 60°C for 30 min in tris buffer. All the detergents showed good compatibility with lipase as the activity increased in the presence of tested detergents.

Figure 7

(A) 3D model of GDSL lipase built by modeler v9.20; (B) model assessment by PROCHECK tool. It describes the quality of the model and shows that 97% region of Ramachandran plot falls in allowed quadrants, thereby specifying a good model quality.

Figure 8

Molecular docking simulation study of lipase with different fatty acids. It is used to predict the binding affinity of the enzyme toward different fatty acids which acts as inducers in lipase production. (A) Docking study in the presence of linoleic acid. The binding energy is −7.4 kcal/mol; (B) docking study in presence of palmitic acid, the binding energy is −6.5 kcal/mol; (C) docking study in presence of oleic acid, the binding energy is −6.1 kcal/mol. It is observed that maximum binding energy is observed in the presence of linoleic acid so it acts as a better inducer for ERMR1:04 lipase production.

Figure 9

Molecular docking simulation study of lipase with substrate p-nitrophenyl palmitate. It is used to predict the interaction pattern between the active site of protein and ligand. The binding affinity for p-nitrophenyl palmitate is (−7.6 kcal/mol), thereby specifying a good interaction with the lipase.

Figure 10

(A) Backbone RMSD comparison of lipase protein at various temperature (−5, 5, 20, 37, 40, 60°C). It shows that protein remains stable throughout 5 ns with insignificant fluctuation in protein backbone at 60°C. The protein did not break down at any interval across the time; (B) RMSF comparison of lipase protein at various temperature; (C) radius of gyration comparison of lipase protein at various temperature. It shows the minor fluctuation in lipase structure at 60°C over 2 ns but overall protein structure remains stable.