Isolation, Screening and Molecular Characterization of Keratinolytic Protease Producing Bacteria and Potential Application of the Enzyme as Detergent Additive and for Leather Dehairing Process

Abstract

Improving various industries such as leather and detergent processing from an environmental sustainability point of view would be beneficial as these sectors offer income generation and employment and can boost the economy of the country and the region. However, the conventional leather dehairing process results in the disposal of large amounts of waste and chemicals that pollute water, soil, and air, causing serious health problems of the community. It is thus urgently needed to use alternative non-polluting processes. In recent years, the use of biological catalysts in industrial processes has received a great deal of attention, mainly for environmental sustainability reasons. Keratinolytic proteases are currently of interest in a wide variety of industries, as they are environmentally friendly hydrolytic enzymes, cultivated using cheap and available agricultural wastes, and used in different industrial applications including in leather dehairing and as detergent additives. Microorganisms capable of producing keratinolytic proteases from Ethiopian traditional leather tanning environments are of great potential; however, they have not been well studied. Therefore, the main objective of this study was to isolate, screen, and molecularly identify keratinolytic protease producing bacteria, produce and characterize the enzyme, and evaluate its potential application as detergent additive and for leather dehairing processes. Isolation and screening of potential isolates were performed using skim milk agar (SMA) and feather minimal medium (FMA), and identification of the best isolate was performed using 16S rRNA sequencing. Optimization of physicochemical parameters for enzyme production was studied using one variable at a time (OVAT) and statistical approaches such as Plackett‒Burman Design (PBD) and Response Surface Methodology (RSM) to maximize the production of keratinolytic protease from the potent bacterial isolate. Finally, the extracted crude keratinolytic protease produced by SmF was used as a detergent additive and for skin dehairing. The environmental compatibility assessment of the effluents from enzyme and lime-sulfide based depilation were evaluated. A total of 224 bacterial isolates were isolated and screened. Among all these isolates, ES5 was the most potent one producing keratinolytic protease. Molecular characterization of the isolate ES5 revealed it to be B. subtilis, and it was finally named B. subtilis ES5. The sequence of this isolate has been deposited in GenBank under accession number OQ034477. The gene encoding keratinase was validated with a fragment length of approximately 1100 bp. Fourier transform infrared spectroscopy (FTIR) revealed that various functional groups were released during feather biodegradation. Of the seven factors evaluated, incubation time, temperature and feather concentration significantly influenced keratinase production using statistical optimization using PBD. RSM-based optimization revealed a P value of <0.0001 for the model, suggesting the importance and possible practical applicability of the model in keratinase production. Overall, statistical optimization revealed a keratinase yield of 317.60±1.21 U/mL, demonstrating a 1.50fold

production enhancement. Crude keratinase showed optimal activity at pH 8 and temperature of 45

℃. Among the metal ions tested, Ca xii 2+ and Mg 2+ improved keratinase activity by 132.56 and 111.78%, respectively, at 5 mM. β-Mercaptoethanol enhanced keratinolytic

protease activity, whereas ethylenediaminetetraacetic acid (EDTA) significantly inhibited the activity, suggesting that the enzyme belongs to a member of the metalloprotease family. The activation energy (

, Gibbs free energy (

), entropy ( ), enthalpy (

), free energy of substrate binding (

), free energy of transition state formation (

, and temperature coefficient (

for casein hydrolysis were 17.59 KJ/mol, 66.11 KJ/mol, 160.88 J/mol/k, 14.95 KJ/mol, 2.61 KJ/mol, 9.21 KJ/mol, and 1.62, respectively. The thermal denaturation energy of the enzyme was 110.83 KJ/mol. The cleaning performance of the enzyme was comparable to that of commercial detergents, and their combination showed superior performance. Biochemical oxygen demand (BOD 5 ), chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), and overall pollution load were reduced by 42.61, 70.63, 93.59, 94.90, and 75.43%, respectively using keratinolytic protease-based dehairing compared with conventional lime-sulfide based dehairing. The production of the enzyme using cheap substrate in a short period of time demonstrates the pivotal advantages of this enzyme in terms of eco-friendly processing and sustainable waste disposal. Thermodynamic parameters indicate that the enzyme is relatively thermostable. Crude keratinase from B. subtilis ES5 showed relative stability for alkaline pH, metal ions and compatibility with surfactants and detergents, revealing that it could be considered a promising detergent additive. Furthermore, the improved properties of enzymatically dehaired hides suggest that crude keratinase can be used in environmentally friendly holistic leather manufacturing to avoid contamination problems associated with the use of chemicals with improved leather quality.