Covalent immobilization of bacterial alkaline protease and its application in poultry feed
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Date
2023-06
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CCSHAU, Hisar
Abstract
Proteases are enzymes that hydrolyse peptide bonds in proteins. Proteases represent 60% of the enzyme market and around 66% of the proteases are derived from the microbial sources (Hadedy et al., 2023). Microorganisms act as an important source of enzymes because of their vast distribution, high reproduction, and potential to be genetically altered for higher productivity. Both bacteria (Bacillus subtilis, Bacillus licheniformis etc.) and fungi (Aspergillus oryzae, Aspergillus niger etc.) are capable of producing large amounts of protease enzymes. In industries, enzymes lack reusability during hydrolytic process, which influences overall economy of the process. This problem can be addressed by immobilization of enzyme on inert material (Wu et al., 2020). Immobilization convert enzyme from soluble form to insoluble form thus enhances stability and reusability. The immobilized enzyme has many advantages over the use of free enzymes such as: increased functional efficiency, reusability, increased product stability etc. The hydrolysis of poultry feed with protease aids in the digestion of dietary proteins in the gastrointestinal system by breaking down ingested proteins into their constituent amino acids for absorption.In the present study, protease enzyme was produced from bacterial isolate KTP9 under submerged fermentation having activity 17.43 IU/mL unit and 3.626 mg/ml of protein content under submerged fermentation. Ammonium sulphate precipitation increased the specific activity from 4.80 to 22.45 IU/ ml with 4.67 purification fold. Bacterial KTP9 free protease have optimum pH 8.0, temperature 35°C and found stable upto 28 days on storage at 4°C. The covalent immobilization of protease over aluminium oxide beads was performed. The immobilization yield and immobilization efficiency were found as 40.54% and 89.72%, respectively. Optimum conditions of immobilization were 1% Glutaraldehyde as linker with 1h and 1.5h cross-linking and coupling time, respectively. Upon immobilization, shift in temperature (35°C to 45°C) and pH optima (8.0 to 10.0) was observed as compared to free protease. The immobilized protease was found to retain 50% residual activity upto 35 days of storage at 4°C. Enzyme after immobilization can be reused upto 6 cycles with 50% residual activity. The immobilized protease showed 76% protein hydrolysis in 120 minutes as compare to bacterial KTP9 free protease.