Characterization of a novel subfamily 1.4 lipase from Bacillus licheniformis IBRL-CHS2: Cloning and expression optimization

Abstract

This study focuses on a novel lipase from Bacillus licheniformis IBRL-CHS2. The lipase gene was cloned into the pGEM-T Easy vector, and its sequences were registered in GenBank (KU984433 and AOT80658). It was identified as a member of the bacterial lipase subfamily 1.4. The pCold I vector and E. coli BL21 (DE3) host were utilized for expression, with the best results obtained by removing the enzyme's signal peptide. Optimal conditions were found to be 15°C for 24 h, using 0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The His-tagged lipase was purified 13-fold with a 68% recovery and a specific activity of 331.3 U/mg using affinity purification. The lipase demonstrated optimal activity at 35°C and pH 7. It remained stable after 24 h in 25% (v/v) organic solvents such as isooctane, n-hexane, dimethyl sulfoxide (DMSO), and methanol, which enhanced its activity. Chloroform and diethyl ether inhibited the lipase. The enzyme exhibited the highest affinity for p-nitrophenol laurate (C12:0) with a Km of 0.36 mM and a Vmax of 357 μmol min-1 mg-1. Among natural oils, it performed best with coconut oil and worst with olive oil. The lipase was stable in the presence of 1 mM and 5 mM Ca2⁺, K⁺, Na⁺, Mg2⁺, and Ba2⁺, but its activity decreased with Zn2⁺ and Al3⁺. Non-ionic surfactants like Triton X-100, Nonidet P40, Tween 20, and Tween 40 boosted activity, while Sodium Dodecyl Sulfate (SDS) inhibited it. This lipase's unique properties, particularly its stability in organic solvents, make it suitable for applications in organic synthesis and various industries.

Fig 1. Molecular characterization and phylogenetic analysis of LipA_{B.licheniformis}.

(A) shows the nucleotide and amino acid sequences of LipA_{B.licheniformis}. The signal peptide is identified in green, and the start and stop codons are indicated in blue. The conserved pentapeptide sequence of LipA_{B.licheniformis} is enclosed in a box, and the inferred catalytic residues Ser, Asp, and His are highlighted in yellow and labeled in red. (B) displays the amino acid sequence alignment of LipA_{B.licheniformis} (NCBI accession number: AOT80658) with lipases from other Bacillus sp. belonging to subfamily 1.4. These include Bacillus licheniformis (CAB95850), Bacillus licheniformis RSP-09 (ACB38749), B. subtilis Lip A (AAA22574), B. pumilus (AAR84668), and B. amyloliquefaciens (AGO17775). The conserved pentapeptide is highlighted in grey and shown in bold. The putative catalytic triad residues, Ser, Asp, and His, are denoted in red and underlined. Similar amino acids in the sequences are denoted with asterisks (*). The signal peptide for LipA_{B.licheniformis} is indicated within a box.

Fig 2. Displayed all the result of SDS-PAGE analysis with Lane M as BenchMark^TM Protein Ladder and Lane NC is induced E. coli BL21 (DE3) harbouring empty pCold.

(A,B) MLipA_{B.licheniformis} expression in E. coli BL21 (DE3) harbouring pCold-MLipA_{B.licheniformis} (A) and solubility of the expressed lipase (B); Lane UI is uninduced E. coli BL21 (DE3) harbouring pCold- MLipA_{B.licheniformis}; Lane 1 is crude cell extract; Lane 2 is soluble proteins; Lane 3 is inclusion bodies. (C) Expression of MLipA_{B.licheniformis} in E. coli BL21 (DE3) harbouring pCold-MLipA_{B.licheniformis} after induction with different IPTG concentrations at 15°C. Lane1 is induction with 0 mM IPTG; Lane 2 is induction with 0.2 mM IPTG; Lane 3 is induction with 0.4 mM IPTG; Lane 4 is induction with 0.6 mM IPTG; Lane 5 is induction with 0.8 mM IPTG; Lane 6 is induction with 1.0 mM IPTG. (D) Time course analysis of the expression of MLipA_{B.licheniformis} in E. coli BL21 (DE3) harbouring pCold- MLipA_{B.licheniformis}. Lane 1 is expression at 0 hour; Lane 2 is expression after 4 hours; Lane 3 is expression after 8 hours; Lane 4 is expression after 12 hours; Lane 5 is expression after 16 hours; Lane 6 is expression after 20 hours; Lane 7 is expression after 24 hours; Lane 8 is expression after 28 hours; Lane 9 is expression after 32 hours. (E) Purification of recombinant MLipA_{B.licheniformis} by IMAC technique. Lane 1 is the diluted soluble crude lysate; Lane 2 is unbound proteins; Lanes 3–10 are wash fractions; Lanes 11–15 are eluate fractions containing purified lipase MLipA_{B.licheniformis}.

Fig 3. Displays a tributyrin substrate plate indicating zones of hydrolysis caused by purified MLipA_{B.licheniformis}, as indicated by arrows in the image.

No clearance zone was observed around the negative control disc.

Fig 4. Temperature profile and stability of MLipA_{B.licheniformis}.

(A) Temperature profile of MLipA_{B.licheniformis} (♦) and temperature stability study (▲) of the lipase. To study the stability of lipase, the purified lipase was incubated in various temperatures for 1 hour prior to lipase assay. Residual activity was assayed at the optimum temperature, 35°C. Specific lipase activity at 35°C was set as 100% (361.6 U/mg) for determination of optimum temperature while for stability studies, the specific activity of lipase without pre-incubation (325.7 U/mg) was set as 100%. (B) Stability study of the lipase at 30°C, 35°C and 40°C. To study the stability of lipase, the purified lipase was incubated in the mentioned temperatures for 24 hours and aliquots were taken at specified intervals. Residual activity was assayed at the optimum temperature, 35°C. Specific lipase activity without pre-incubation (325.7 U/mg) was set at 100%. Symbols used are: (♦) for 30°C, (■) for 35°C and (▲) for 40°C.

Fig 5. pH profile and stability of MLipA_{B.licheniformis}.

(A) pH profile of MLipA_{B.licheniformis} (♦) and pH stability study of the lipase (▲). For stability study, the purified lipase was incubated for 1 hour in 35°C in suitable buffers. Residual activity was assayed at the optimum temperature, 35°C. Specific lipase activity at pH 7 was set as 100% (321.2 U/mg) for optimum pH while for pH stability, the activity of lipase without pre-incubation (325.7 U/mg) was set at 100%. (B) Stability study of MLipA_{B.licheniformis} at pH 6, pH 7 and pH 8. For stability study, the purified lipase was incubated for 24 hours in 35°C in 0.1M phosphate buffer (pH 6 and pH 7) and 0.1M Tris-Cl buffer (pH 8). Aliquots were taken at specified intervals. Residual activity was assayed at the optimum temperature, 35°C. The specific activity of lipase without pre-incubation (325.7 U/mg) was set at 100%. Symbols used are: (♦) for pH 6, (■) for pH 7 and (▲) for pH 8.

Fig 6. Substrate specificity and Relative activity of MLipA_{B.licheniformis}.

(A) illustrates the MLipA_{B.licheniformis} activity using various substrates. The experiments were carried out following standard assay protocols, with the substrate in the reaction replaced by different p-NP esters. The activity relative to lipase activity using p-NP laurate (C12) as the substrate was determined, with a value of 100% (318.5 U/mg). (B) displays the relative activity of MLipA_{B.licheniformis} on different natural oils. The specific lipase activity of 36.6 U/mg when using olive oil as the substrate (control) was established as 100%.

Fig 7. The impact of metal ions on MLipA_{B.licheniformis} activity.

The purified lipase was incubated with various additives for 1 hour at 35°C and 100 rpm. The activity was assayed after the incubation and relative activity was calculated against the control reaction (without any additives) set as 100% (324.4 U/mg).

Fig 8. The effect of effector molecules on MLipA_{B.licheniformis} activity.

The purified lipase was incubated in the presence of the various additives for 1 hour at 35°C and 100 rpm. The activity was assayed after the incubation and relative activity was calculated against the control reaction (without any additives) set as 100% (328.3 U/mg).

Fig 9. The effect of surfactants on MLipA_{B.licheniformis} activity.

The purified lipase was incubated in the presence of the various additives for 1 hour at 35°C and 100 rpm. The activity was assayed after the incubation and relative activity was calculated against the control reaction (without any additives) set as 100% (322.1).