Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Studies on Improved Amylases Developed by Protoplast Fusion of Aspergillus species

  • Adeleye, Tolulope Modupe (Department of Microbiology, College of Biosciences, Federal University of Agriculture Abeokuta) ;
  • Kareem, Sharafadeen Olateju (Department of Microbiology, College of Biosciences, Federal University of Agriculture Abeokuta) ;
  • Olufunmilayo, Bankole Mobolaji (Department of Microbiology, College of Biosciences, Federal University of Agriculture Abeokuta) ;
  • Atanda, Olusegun (Department of Biological Sciences, College of Natural and Applied Sciences, McPherson University) ;
  • Osho, Michael Bamitale (Department of Biological Sciences, College of Natural and Applied Sciences, McPherson University) ;
  • Dairo, Olawale (Department of Agricultural Engineering, College of Engineering, Federal University of Agriculture Abeokuta)
  • Received : 2020.03.31
  • Accepted : 2020.06.29
  • Published : 2021.03.28
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Abstract

Improved amylases were developed from protoplast fusants of two amylase-producing Aspergillus species. Twenty regenerated fusants were screened for amylase production using Remazol Brilliant Blue agar. Crude enzyme extracts produced by solid state fermentation of rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized to increase the amylase activity of the parents and selected fusants using rice bran medium and solid state fermentation. Analysis of this optimization was completed using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activity assays conducted at room temperature and 80℃ demonstrated that Aspergillus designates, T5 (920.21 U/ml, 966.67 U/ml), T13 (430 U/ml, 1011.11 U/ml) and T14 (500.63 U/ml, 1012.00 U/ml) all exhibited improved function making them the preferred fusants. Amylases produced from these fusants were observed to be active over the entire pH range evaluated in this study. Fusants T5 and T14 demonstrated optimal activity under acidic and alkaline conditions, respectively. Fusants T13 and T14 produced the most amylase at 72 h while parents TA, TC and fusant T5 produced the most amylase after 96 h of incubation. Response surface methodology examinations revealed that the enzyme from fusant T5 was the optimal enzyme demonstrating the highest activity (1055.17 U/ml) at pH 4 and a temperature of 40℃. This enzyme lost activity with further increases in temperature. Starch hydrolysis using fusant T5 gave the highest yield of glucose (1.6158 g/100 ml). The significant activities of the selected fusants at 28 ± 2℃ and 80℃ and the higher sugar yields from cassava starch hydrolysis over their parental strains indicate that it is possible to improve amylase activity using the protoplast fusion technique.

Keywords

Acknowledgement

The authors acknowledge the staff of the Virology Unit of the Institute of Tropical Agriculture (IITA), Ibadan, Nigeria, where the molecular characterization of the isolates was carried out.

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