Purification and characterization of detergent stable alkaline lipase from Bacillus safensis TKW3 isolated from Tso Kar brackish water lake

Abstract

Extensive and escalating research has been directed towards halozymes derived from halophiles thriving in extreme hypersaline environments, owing to their myriad industrial applications. These extremophiles have evolved various physiological and metabolic adaptations to endure such extremes, enhancing their industrial potential. Being a potential source of lipases, halophiles of extreme niches have emerged as a emerging research area. This interest has been fueled by the recognition that extreme environments serve as rich reservoirs of diverse cold-active alkaliphilic enzymes.

Methods: Bacillus safensis TKW3, isolated from brackish Lake Tso Kar of the Ladakh region, India, produced cold-adapted haloalkaliphilic lipase halozyme. The current study focused on the purification and biochemical characterisation of lipase derived from halophilic bacteria.

Results: The lipase enzyme, purified to homogeneity, exhibited a molecular mass of 28 kDa as confirmed by SDS-PAGE analysis. The purification process yielded a purification fold of 12.01 and a final recovery rate of 29.9%. It demonstrated optimal activity at 30 °C and pH 9. The enzyme was evaluated and demonstrated to exhibit stability over a broad temperature range spanning from 5 °C to 55 °C, as well as a wide pH range of 7.0 to 9.0. Due to its stability across a diverse spectrum of pH values, surfactants, metal ions, and inhibitors, the enzyme appeared to hold significant promise for application within the leather and detergent sectors. Upon undergoing detergent compatibility tests spanning diverse temperature ranges, the lipase showcased compatibility with various commercial detergents, thereby presenting itself as an attractive candidate for inclusion in detergent formulations within the industry.

Conclusions: The lipase from B. safensis TKW3 exhibits promising attributes, including alkali stability, halophilicity, and a wide spectrum of substrate specificity, rendering it an attractive option for incorporation into detergent formulations within the detergent industry. As far as we are aware, this is the first report on the purification and characterization of lipase enzyme from bacterial halophiles in a Tso Kar brackish lake.

Keywords: Alkaline; B. safensis; Cold-adapted; Detergent compatible; Halophilic; Lipase.

Figure 1. SDS PAGE profile for determination of molecular weight and native page for determination of lipase activity.

(A) 12% SDS PAGE showing different fractions of purified protein. Lane 1: Dialyzed, Lane 2: 75% ammonium sulphate precipitate, Lane 3: crude, Lane 4: Empty, Lane 5: Molecular weight Marker, Lane 6: Purified fraction. (B) The zymogram analysis of the purified enzyme exhibited distinct red bands on the gel, indicating enzyme activity.

Figure 2. Effect of temperature on the lipase activity of B. safensis TKW3.

All experiments were performed in triplicates and standard deviation was calculated The optimal temperature of the purified lipase enzyme from B. safensis TKW3 was determined to be 30 °C. The enzyme exhibited remarkable stability over a wide temperature range, from 5 °C to 55 °C. However, a significant drop in stability of the purified lipase was observed beyond 55 °C.

Figure 3. Effect of pH on the lipase activity of B. safensis TKW3.

All experiments were performed in triplicates and standard deviation was calculated. The purified lipase exhibited maximum activity at pH 9.0, with activity decreasing to approximately 32% at pH 11.0 and 15% at pH 12.0. pH stability analysis revealed that the purified lipase from B. safensis TKW3 retained about 90% activity within the pH range of 7.0 to 9.0. However, activity decreased to 52% at pH 10.0.

Figure 4. Effect of NaCl on the lipase activity of B. safensis TKW3.

All experiments were performed in triplicates and standard deviation was calculated. The purified lipase enzyme from B. safensis TKW3 demonstrated increased activity with rising NaCl concentrations, peaking at 3 M. However, further increases in salt concentration led to a decline in activity. Optimal activity of the purified enzyme was observed at 3 M NaCl concentration. Additionally, the enzyme exhibited stability over a wide range of salt concentrations, ranging from 0.5 to 6.0 M.

Figure 5. Effect of surfactants on the enzyme activity of B. safensis TKW3.

All experiments were performed in triplicates and standard deviation was calculated The addition of surfactants significantly increased lipase activity. SDS was the most effective surfactant, enhancing enzyme activity the most, followed by Tween-80. The activity increased by 47% and 129% with Triton X-100 and Tween-80, respectively. However, a decrease in activity by approximately 26.5% was observed with Tween-20. Strong anionic detergents like SDS and SLS doubled the enzyme activity compared to the control.

Figure 6. Effect of inhibitors and metal ions on the enzyme activity of B. safensis TKW3.

(A) Effect of inhibitors; (B) effect of metal ions. All experiments were performed in triplicates and standard deviation was calculated Effect of inhibitors on lipase activity. Lipase activity was assayed after incubation with various inhibitors at concentrations of 2 and 5 mM. ß-mercaptoethanol drastically reduced activity, while DTT showed a significant decrease. EDTA exhibited a notable increase in activity at both concentrations. Panel (B) shows the impact of metal ions on lipase activity. Various monovalent and divalent cations were tested for their effect on lipase activity. Cu²⁺ showed a substantial reduction in activity compared to other metal ions, resulting in a 74% decrease in enzyme activity.

Figure 7. Substrate specificity of B. safensis TKW3.

All experiments were performed in triplicates and standard deviation was calculated Various p-NPEs with acyl chains of different lengths (C2 to C14) were tested to assess substrate preference. Short-chain fatty acids exhibited minimal activity, resulting in 18% relative initial activity. However, the initial hydrolysis of C4 to C6 fatty acid esters reached approximately 87%.

Figure 8. Detergent compatibility of B. safensis TKW3.

(A) All experiments were performed in triplicates and standard deviation was calculated. The compatibility of B. safensis TKW3 lipase with various commercial laundry detergents at different temperatures was checked. Lipase activity without detergent was set as 100%. Surf Excel exhibited the highest activity across a wide temperature range (5 °C to 45 °C), with peak activity at 30 °C. Tide maintained over 90% activity at all temperatures except at 30 °C where it showed an 11% enhancement. Ariel exhibited compatibility at lower temperatures but showed decreased activity at higher temperatures. (B) Washing performance of B. safensis TKW3 lipase on oil-stained cloth pieces: (a) cloth washed with distilled water (control), (b) cloth stained with vegetable oil washed with distilled water and detergent, (c) cloth stained with vegetable oil washed with distilled water, purified lipase & detergent.