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Food Microbiology (FM)  |  Bioactive compounds and Physiological properties

Microbiol. Biotechnol. Lett. 2021; 49(1): 45-56

https://doi.org/10.48022/mbl.2003.03014

Received: April 1, 2020; Accepted: June 22, 2020

Studies on Improved Amylases Developed by Protoplast Fusion of Aspergillus species

Tolulope Modupe Adeleye1*, Sharafadeen Olateju Kareem1, Bankole Mobolaji Olufunmilayo1, Olusegun Atanda3, Michael Bamitale Osho3, and Olawale Dairo2

1Department of Microbiology, College of Biosciences, Federal University of Agriculture Abeokuta, P.M.B. 2240, Abeokuta, Nigeria 2Department of Agricultural Engineering, College of Engineering, Federal University of Agriculture Abeokuta, P.M.B. 2240, Abeokuta, Nigeria 3Department of Biological Sciences, College of Natural and Applied Sciences, McPherson University, Km 96, Lagos-Ibadan Expressway, Seriki Sotayo, P.M.B. 2094, Abeokuta, Nigeria

Correspondence to :
Tolulope Modupe Adeleye,
tolulopemodupeade@gmail.com

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: Protoplast fusion, Aspergillus species, amylase, solid state fermentation, optimization, response surface methodology

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