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Title: Improvement of Aspergillus oryzae CMC1 Strain through y-ray Treatment for the Enhanced Production and Quality of a-Amylase
Authors: Azmat, Bushra
Keywords: Engineering & Technology
Issue Date: 2021
Publisher: Pakistan Institute of Engineering & Applied Sciences, Islamabad.
Abstract: Alpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto oligosaccharides and are important enzymes generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought-after properties for industrial use. Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and is considered safe for use in food industry. Hence, Koji strain development for the screening of potent mutants that are hyper-producers of thermostable α-amylases are preferable. A five-step process has been developed to improve super Koji (A. oryzae cmc1) strains through γ-ray treatment. This resulted in finding the best mutant (e.g. M100-6), which had a half-life of 52 min at 55 °C. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia showed that exposure to γ rays increased rigidity of the mycelium. Koji mutant M100-6 was grown on soluble starch in a 10 L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1. Growth kinetic parameters were: µ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1. The effect of temperature on growth kinetic parameters of Koji M100-6 determined that growth was strongly affected by varying the temperature. The amylase production was maximized at 30 °C (3658 U dl-1). Varying inoculum levels suggested that enzyme (amylase) production was maximized (3526 U dl-1) at 0.4 % (w/v) of the pack cell inoculum’s density, and the product yield Yp/x (189.83 U g-1) and specific product rate formation were highest at 0.3 % (w/v) inoculum density (9.87 U g-1 h-1). Molecular analysis of the amylase genes was also carried out. Mutations were not detected in Amy I and Amy III; however, four mutations were detected in Amy II. The apparent subunit molecular mass of the purified α-amylase product of Aspergillus oryzae mutant M100- 6 was 38 kDa. The optimum temperature and pH of the amylase was 55 °C and 5.50, respectively. Glutamic acid and histidine were lining the active site of α-amylase of A. oryzae M100-6. The values of Vmax, and Km determined by non-linear and linear xviii regression were found to be 871 U mg-1 min-1 and 2.5 mg soluble starch ml-1, and 365 U mg-1 min-1and 0.949 mg soluble starch ml-1, respectively. The enzyme was than entrapped in different beads. The 5 % alginate beads retained more than 90 % of activity in the first three cycles with an entrapment efficiency of 81.01 % and the temperature optimum was 55 °C. The entrapped enzyme exhibited stability up to a temperature of 62 °C. The beads were capable of hydrolyzing starch over a wide range of pH (e.g. 4.5-7.2). Optimal temperature of apo-amylase from Aspergillus oryzae mutant M100-6, for starch hydrolysis, was 55 °C, and it remained the same for Cobalt-bound α-amylase. In the case of calcium bound amylase, the optimal temperature was 60 °C. Optimum pH of apo-amylase from Aspergillus oryzae for starch hydrolysis fell within a range of 5.0-6.5, with maximum activity at pH 5.50. A similar trend was exhibited by cobalt-bound amylase. However, with the addition of CaCl2, the pH range become broader (e.g. 5.5 – 7.5) and the maximum enzyme activity was at pH 6.5. Both metals (e.g. cobalt and calcium) activated the amylase. Our five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ- ray-mediated mutagenesis. The development of thermostable α-amylases in Koji mutant M-100 6 has immense potential for application in the scarification process utilized in for maltose syrup production. Moreover, our five-step development process may be suitable for the improvement of productivity and thermal stability in other microbial enzymes.
Gov't Doc #: 22856
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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