Optimization and immobilization of amylase produced by Aspergillus terreus using pomegranate peel waste

Amylases are amongst the most important hydrolytic enzymes that are used in numerous industrial uses reaching for food to pharmaceuticals. Immobilization of enzymes can proposal several assistances as reusability and retrieval from their products improve strength under both operating and storing environments. Marine fungal isolate was recovered from red sea water at Sharm El-Sheikh Province and was tested for amylase activity using different agricultural wastes as substrate. It was found that pomegranate peel was the best substrate for amylase production (339 U/ml). Thus, it was subjected for identifying by 18S rDNA gene. The phylogenetic analysis results indicated that this fungal isolate belonged to Aspergillus species with similarity of 99% and named as Aspergillus terrus SS_RS-NE. Its nucleotide sequences were deposited in NCBI GenBank under accession no. of MN901491. Some parameters affecting amylase activity using pomegranate peel as substrate were studied. The results denoted that, the highest amylase activity of 340.69 U/ml using 1.5% pomegranate peel at 30 °C, pH 6.0 on 5 days incubation time by Aspergillus terreus. The produced crude enzyme was partially purified with 80% ammonium sulfate followed by dialysis. The enzyme activity was 1246 U/ml and 2411 U/ml employing ammonium sulfate precipitation and dialysis respectively. The partially purified amylase was immobilized with 2% sodium alginate and the results showed the highest immobilized enzyme yield was 92.8%. The characterizations of immobilized amylase were studied and the results indicated that, the maximal immobilized amylase activity was 2522.5 U/ml with 2% starch as a substrate at optimum pH value of 6.5, temperature at 60 °C and 10 min reaction time in comparison to maximal free amylase enzyme at pH 5, 50 °C after 40 min. The results also indicated the immobilized amylase was stable at 60 °C for 20 min. Aspergillus terrus SS_RS-NE (MN901491) was isolated and genetically identified. It has the ability to produce amylase enzyme using pomegranate peel waste with a yield of 339 U/ml. The crude enzyme was partially purified by ammonium sulfate followed by dialysis. The maximal immobilized amylase activity of 2522.5 U/ml was obtained under optimized some culture conditions and medium nutrient parameters.