Expression and characterization of a lipase EstA from Bacillus subtilis KM-BS for application in bio-hydrolysis of waste cooking oil
- Ohapi
- 1 thg 4, 2024
- 3 phút đọc
Abstract
A lipase EstA from Bacillus subtilis KM-BS was expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme achieved high activity (49.67 U/mL) with protein concentration of 1.29 mg/mL under optimal conditions at the large-scale expression of 6 h and post-induction time at 30 °C using 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The optimal temperature and pH of the purified enzyme were at 45–55 °C and pH 8.0 − 9.0, respectively. Activity of the purified enzyme was stable in the presence of 1 mM Ca2+; stimulated by 1 mM Mg2+ and Mn2+, and inhibited by Fe3+. A significant amount of fatty acids was released during the hydrolysis of waste cooking oil under the catalysis of purified lipase, indicating that this recombinant lipase showed promise as a suitable candidate in industrial fields, particularly in biodiesel and detergent sector.
Introduction
The growing population, industrialization, and civilization are all contributing to an increase in the need for clean water. Large amounts of industrial wastewater now require effective treatment to safeguard the environment [1]. Lipids such as fats, oil, and grease (FOG) are present in large amounts in wastewater discharged by the food processing, restaurant, dairy, oil refining, meat processing, tannery, cosmetics, and pharmaceutical industries [2,3]. FOG content in home wastewater ranges from 50 to 100 mg/L while in oil refineries, food packaging facilities, and dairy effluent the range is 110–264 mg/L, 100–1000 mg/L, and 50–100 mg/L, respectively [4,5]. Discharge of treated or partially treated lipids from the aforementioned manufactures results in the formation of an oily layer on the water surface, which hinders the absorption of oxygen and sunlight and has detrimental impacts on aquatic ecosystems. Wastewater flow and treatment processes are hindered when the oil droplets combine with suspended particles in wastewater and obstruct sewer systems. Cooking oils are also subjected to oxidation and change in their physical characteristics when heated to high temperatures [6]. Waste cooking oil (WCO) contaminates land and water resources and its degradation has become a significant challenge. Most WCO is dumped into rivers, drainage systems, and landfills causing environmental damage.
Physical, mechanical, and chemical methods have all been used to treat oil contamination. Treating the oil at high temperatures (160 − 200 °C) for an extended period creates very offensive conditions and causes fatty acids to oxidize, producing an unpleasant flavor and odor [7]. A safe and efficient method for removing WCO or oil contamination from the environment is urgently required. The biological process of WCO degradation uses living things to degrade organic materials. Organic substances are transformed or degraded by the microbes or enzymes that they produce. Numerous aerobic and anaerobic bacteria have been used to treat WCO. Besides, enzymes that are environmental friendly biocatalysts are very specific and efficient. Enzymes act best in moderate conditions such as temperature, pH, and other factors [5,8].
Lipase produced by a variety of bacteria and fungi and is useful in bioremediation. Triacylglycerol acylhydrolases, which include lipases, are a class of ubiquitous enzymes [5]. Lipase (EC 3.1.1.3) can hydrolyze triacylglycerol into glycerol and fatty acids, thus it has numerous industrial applications. No extra cofactor is required for enzymatic reaction. As reported previously, some lipases were very effective, even in the presence of various chemicals such as acids, bases, solvents, detergents, and salts, as well as under different environmental conditions (temperature, pH) [9]. The negative impacts of fat and oil residues can be minimized by the use of hydrolytic microbial enzymes. In this study, the lipase from B. subtilis KM-BS was cloned and optimized for efficient heterologous protein expression in E. coli BL21 (DE3) cells. This purified recombinant lipase was biochemically characterized and used for the bio-hydrolysis of waste cooking oils.
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