Lipase is one of the most essential biocatalysts. It performs reactions in the aqueous and non-aqueous medium. It stimulates the aqueous degradation of triglycerides to glycerol and fatty acids. As a result of lipase’s chemical and mechanical properties, efforts were focused on it in the field of scientific and industrial research. Lipase enzymes are found in most organisms, such as animals, plants, yeasts, fungi, and bacteria. Microbial lipase enzymes have gained special industrial attention due to their ability to maintain their activity under extreme temperatures, pH and organic solvents, and chemical conditions. The fungal lipase emerges as a key enzymatic source due to its catalytic activity, low cost of production, and relative ease in genetic manipulation35.
The two most productive isolates for lipase in Aspergillus genus are A. niger MH078571.1, and A. niger MH079049.1. Both isolates were chosen to study their enzyme physiological and biochemical properties and their applicability in various industrial applications (Fig. 2).
A summary of the main steps conducted to validate the industrial application of using lipase enzymes produced by A. niger MH078571.1, and A. niger MH079049.1 to (A) degrade animal fat and (B) remove oil wastes36.
The enzyme tolerance to high temperatures and the stability of its activity is clearly beneficial in industrial processes. It contributes to raising the reaction rate and process yield by increasing the solubility of the reaction materials and products. It also displaces the balance in endothermic reactions and reduce bacterial contamination37.
The results showed that the enzyme activity’s temperature is the highest and more stable after 24 h of incubation is 45 °C for the enzyme produced by A. niger MH078571.1 and at 55 °C for the enzyme produced by A. niger MH079049.1. This would allow the enzyme in various commercial industries to be carried out at high temperatures (< 70 °C), such as lipid analysis, esterification, and Biodiesel production. Whereas the two enzymes lost most of their activity at 70 °C due to the denaturation of enzymatic proteins at high temperatures38.
These results were consistent with Falony et al.39, where they stated that lipase produced by A. niger had the optimum activity at 55 °C. Namboodiri et al.40 reported that the maximum activity of the producing lipase by Humicola lanuginose was at a temperature of 45 °C. They also reported that the thermal stability of the lipase enzyme was found at the temperature is 50 °C30.
Whereas most lipase activity produced by Aspergilli are at around 40 °C, for example, A. niger NCIM1207 lost 52% of the initial activity after an hour incubation at 50 °C41. Sundar and Kumaresapillai42 reported that the maximum enzymatic activity of lipase produced by A. niger is 40 °C; as 50 °C caused the enzyme to be denatured. Rashma and Shanmugam43 stated that the optimum temperature was 27 °C for the enzyme produced by A. Brasiliensis.
The optimum pH of the lipase activity produced by both strains under pH 8 was considered, where the enzyme is more active and stable over 24 h, and where the alkali and endothermic high temperature is very attractive to produce biodiesel and biopolymers in addition to their potential use in the production of chemicals Agricultural, cosmetic, detergent, flavoring and pharmaceutical preparations, and thus the lipase enzyme extracted from the two strains could be A. niger MH078571.1, and A. niger MH079049.1 the ideal candidate for various industrial and biotechnology applications44.
The results of the current study on enzyme activity at pH 8 were consistent with Malekabadia et al.29, while Cruege and Crueger45 stated that the optimum pH for a lipase enzyme was at 6. Falony et al.39 and Rashma and Shanmugam43 stated that the optimal activity of the enzyme is at pH 7, while41 reported that the activity of the enzyme lipase and its stability in the acidic environment at pH 3.
The lipase enzyme activity in various industrial processes depends significantly on the extent of the enzyme’s tolerance, and its activity in the presence of different organic solvents. The activation of the lipase can be clarified by the interaction of these organic solvents with the amino acid residues present in the cap that protects the catalytic site in the enzyme protein. Thus, maintaining the lipase is in its apparent state retains its elasticity, which facilitates its transportation to the active site and the response of the active site movement to the treatment of the reaction46.
The results of the study confirmed the lipase enzyme ability, produced by the two strains A. niger MH078571.1 and A. niger MH079049.1, to withstand high concentrations (50%) of different organic solvents; whereby the enzyme maintained 96.5% and 93.1% of their activity in the presence of 50% of acetone. These results were consistent with the results of14,30 at the same focus, while47,48 that the lipase lost most of its activity when incubated with various organic solvents.
Organic solvents can strongly affect living cells’ integrity and stability by binding to the cell membrane. Binding to cell membrane might disrupt its permeability leading to cellular metabolism damages, growth inhibition, and cell death49. In our experiment, we noticed that Lipase lost a large part (about 65%) of its efficacy in a 100% concentration of ethanol and acetone. This happens because the presence of organic solvents may cause drying by removing the water molecules from the enzyme circumference, which negatively affects its efficacy. Moreover, all organic solvents may cause denaturation of the amino acid residues present in an enzyme31. However, despite all these worse effects of organic solvents in living cells, there are organic solvent-tolerant bacteria capable of thriving in the presence of these toxic solvents50. The hydrolase-catalyzed perhydrolysis proceeds better in the present of organic solvent because of lower nucleophilic competition (H2O/H2O2) in these media.
Substances that affect surfactants are influences on enzyme activity in the industry, especially detergents. During washing, lipase enzymes must withstand useful cleaning materials in the presence of various surfactants along with their temperature and pH stability51.
In the presence of tween 80 at a concentration of 0.1%, it had a catalytic enzyme activity effect. The activity of the two enzymes increased by 103.4% and 112.4% for both enzyme strains A. niger MH078571.1 and A. niger MH079049.1, respectively. It is suggested that these abhorrent factors Water binds to the structure of the enzyme and changes occur in the formation of the enzyme, which increases the effective access to the substrate and increases the enzymatic activity gives promising advantages in the field of detergents52.
Bacha, et al.30, Das et al.31 and Malekabadia et al.29 all stated that tween 80 surfactant stimulated the enzymatic activity which is consistent with the obtained result. Zheng et al.1 and Sharma and Kanwar49 all supported the obtained results of SDS and tween 20 surfactants to have an inhibitory effect on enzyme activity. The inhibitory effect of these surfactants may be caused by disrupting the surfactants’ main structures, which corrupt enzyme activity53.
Dandavate et al.54 found that SDS and tween 20 both have a stimulating effect on enzyme activity, while the enzyme maintained 100% activity in the presence of surfactants, i.e. they did not affect the enzyme activity55.
Enzymes require metallic ions as common agents in various metabolic pathways. The results showed a slight increase on enzyme activity in the presence of both Zn+ and Mg+ at a concentration of 0.1% of 102% and in the presence of sodium 0.1% Na+ of 104.9% for the two enzymes produced by strains A. niger MH078571.1 and A niger MH079049.1, respectively Sahoo et al.56 also obtained the catalytic result of the enzyme in the presence of the same ions, while Yang et al.57 stated that magnesium has an inhibitory effect on enzymatic activity.
Concentration gave 1% an inhibitory effect of the enzyme in all the tested elements, while in concentration 0.1%, the effect of K+ and EDTA was the most inhibiting on the enzyme produced by A. niger MH078571.1, while Ca+ and EDTA was the most inhibiting of the enzyme produced by A. niger MH079049.1. The reason may be that these mineral ions bind to lipase in inactive sites instead of active sites, which reduces enzyme activity and efficacy31, while1 for EDTA has a catalytic role on the lipase enzyme.
After studying the physiological and biochemical properties of the enzyme and knowing the optimal conditions in which the enzyme is more active and stable, the optimum temperature was checked to maintain and stabilize the enzymatic activity as long as possible. For use in industrial applications later, the optimum degree for maintaining the raw enzyme was − 80%. The enzyme produced by the A. niger strain MH078571.1 sustained 75.31% of its activity, while the enzyme produced by A. niger MH079049.1 retained 73.04% of its activity, and Souza et al.28 all obtained similar results, as the enzyme maintained 90% and 80% of its activity, respectively.
The possibility of producing lipase enzyme from the two strains from oily waste was studied for the oils used to reduce the production cost in addition to reducing and disposing of waste in a way that does not harm the environment. From the results, we find that it was possible to produce the enzyme in all types of tested oily waste, where the optimum production was in the presence of Potato oil followed by motor oil. Vegetable frying oil, chicken and fish frying oil58 produced the lipase enzyme by A. niger using the oily waste of the palm oil.
The optimal time for production was also studied. It was found that it is also 3 days, where an increase in the period caused a significant decrease in the enzymatic activity as a result of the critical diminishing nutrients present in the environment by increasing the period and not being compensated, in addition to the secondary metabolism considered to be inhibiting enzyme production. Therefore, Industrial waste is a promising candidate for use in the industrial applications involved in enzyme production and biotechnological transformations3.
The optimum conditions for enzyme activity and stability were determined. Then the possibility of its application in various applications was tested. The effectiveness of the enzyme and its activity in the presence of powder and liquid commercial detergents was studied at a concentration of 0.1% and 1%, as the lipase enzymes used in detergents need to be active and stable in Alkaline environments (pH 8–11) you encounter in severe washing conditions. Among the results obtained, it was found that in the presence of powder detergents, the concentration of 0.1% of the Omo powder for the enzyme produced by A. niger MH078571.1, and 1% of Ariel A. A. niger MH079049.1, while the liquid detergent 0.1% Ferry was the most appropriate. To maintain the enzymatic activity, while Bacha et al.30 found that the activity of the enzyme ALA1 was 100% in the presence of Ariel for powder detergents, while Dac was the optimal liquid detergent for the activity of the enzyme and its activity was the best when compared with the commercial enzyme Lipolase in the presence of Various detergents, as lipase KM12 maintained 95% of its activity in the presence of many commercial detergents29.
These results also agreed with Das et al.31 where it tested the enzyme’s ability to clean the peanut oil stain used for deep frying. Das et al.31 emphasized that the enzyme was able to enhance the ability of detergents to remove stains; however, they stated that the effectiveness of removing oil stains in the presence of detergent and enzyme was the same in cold and hot water.
A previous study concluded that a lipase produced by Fusarium oxysporum increased the cleaning efficacy with various commercial detergents59. Hemachander and Puvanakrishnan60 also confirmed that the presence of detergents with a lipase produced by Ralstonia pickettii increased the effectiveness of removing stains by 24–27% compared to its treatment with only detergents.
Fat biodegradation is a critical characteristic of lipase enzymes. It was found that analytic enzymes such as lipase enzymes can solve environmental issues of fat pollutants in a safer and cheaper way61. In this study, the lipolysis property was studied by the lipase Grease both chicken and sheep to know the ability of the enzyme to Lipolysis fats in each of them and found that the enzyme managed to Lipolysis the fat mass within 6 days of incubation at optimal temperatures for each enzyme, while the enzyme produced by L. plantarum managed to lipolysis fats within 3 days33, from the results obtained, this enzyme can be applied to removing fats in the medical field as well as Lipolysis fats in water Sanitation and water pollution prevention33.
In this study, Biochemical characterizations of lipase enzyme activity and stability for the two highest lipase producer strains were examined A. niger MH078571.1 and A. niger MH079049.1. Lipase production of two isolates was studied on medium contains waste oil. The optimal conditions for lipase activity were as follows: 50% of acetone as organic solvents, 0.1% of tween 80 as surfactants. Furthermore, mg2+ and zn2+ ions enhanced lipase activity of A. niger MH078571.1 while Na2+ and cu2+ enhanced enzyme activity of A. niger MH079049.1. Lipase activity was tested for industrial applications such as integrating the enzyme with different detergents. Moreover, animal natural animal fat degradation with crude enzyme was tested using chicken and sheep fats.
As a result of these findings, the crude fungal lipase produced by both A. niger MH078571.1 and A. niger MH079049.1 strains has been purified. The purified lipases were tested to identify their molecular sizes and properties. This project, which is currently in progress, aims to determine purified lipases’ sequences and related genes through BLAST.
