Isolation of Amylolytic Fungi and Characterisation of Amylase in Aspergillus niger KBA2 and Penicillium sp. BB3 from Starchy and Agro-Industrial Waste Locally Collected in Kuching and Samarahan, Sarawak, Malaysia

KAREN, HON JING YI (2025) Isolation of Amylolytic Fungi and Characterisation of Amylase in Aspergillus niger KBA2 and Penicillium sp. BB3 from Starchy and Agro-Industrial Waste Locally Collected in Kuching and Samarahan, Sarawak, Malaysia. Masters thesis, UNIVERSITI MALAYSIA SARAWAK.

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Abstract

Amylases are enzymes that catalyse hydrolysis of starch into simple sugars and the production of these enzymes largely depends on microbial sources, making it important to continuously screen for potential producers of amylases. Previous studies have reported on isolating amylolytic fungi from sources such as soil samples, farmlands and different types of waste. Agro-industrial and food wastes are major contributors to the generation of large amount of organic waste. Food is also largely wasted due to spoilage, causing spoiled food waste to be a significant issue worldwide. Despite the potential of these wastes as sources of amylolytic fungi, limited studies have explored their diversity and enzymatic properties under local context. The study therefore aims to isolate amylolytic fungi from various starchy waste, optimise amylase production in potent isolates and characterising the partial purified enzyme. Six types of wastes were collected for this study, which were sago pith waste (agro-industrial waste), washed rice water (food waste), and various spoiled foods such as potato, bread, banana cake and glutinous rice cake. Fungal isolation was conducted using serial dilution and direct plating on potato dextrose agar, followed by identification through morphological and molecular methods. Screening on starch agar was performed to identify amylolytic fungi and followed by the study of amylase activity through determining the enzymatic activity index (EAI). Potent isolates were then selected for study of initial medium pH and incubation period optimisation for amylase production. Amylase was then partial purified from two further chosen species through ammonium sulphate precipitation and molecular weight of the enzyme was determined through SDS-PAGE, while native-PAGE was done for investigating the hydrolytic activity. The partially purified amylase was also characterised to determine its optimal activity at different temperatures and pH values. A total of 41 isolates were obtained from the wastes, classified into 12 genera and 23 species.The Aspergillus genus, with 15 isolates from 9 different species, was the most prevalent and diverse genus, followed by Penicillium genus with 8 isolates of 3 species. Out of 41 isolates, 34 of them were extracellular amylase producers and subsequent study revealed that most members of Penicillium as well as several Aspergillus species had higher activity compared to other isolates. Therefore, further studies were focused on these two genera. Two isolates with the highest EAI were selected from each genus, namely Penicillium sp. BB3 (1.45±0.10), P. citrinum KBA4 (1.36±0.02), A. europaeus BNN1 (1.19±0.04) and A. niger KBA2 (1.16±0.02). Penicillium sp. BB3 and A. niger KBA2 stood out as the isolates which produced the highest value of amylase under optimised condition of pH 7 under a week of incubation and pH 5 under 5 days of incubation respectively. Amylase from strain BB3 was purified in 0.51-fold with a recovery of 26.47% while the enzyme from strain KBA2 was purified in 2.08-fold with a recovery of 28.85%. SDS-PAGE and native-PAGE revealed strain BB3 might be producing several isoforms or types of amylases, with estimated molecular weights of 80 kDa, 65 kDa, 42 kDa, 40 kDa and 35 kDa respectively. On the other hand, amylase of strain KBA2 formed a single band on both SDS-PAGE and native-PAGE with molecular weight of approximately 130 kDa. The enzyme of strain BB3 showed optimal activity under pH 7 and at a temperature of 40 °C, while amylase of strain KBA2 worked best at pH 5 and 50 °C. The results indicate that strain BB3 and KBA2 are promising amylase producers, with potential applications such as in food processing, biofuel production and waste management. Further studies should focus on aspects such as scaling up and genetic modification to enhance activity and stability of the enzyme for practical applications.

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