Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
Glucoamylase (GA) is an exohydrolases, which release β-glucose units from the non-reducing ends of starch have industrial importance. The purposes of the study were to optimize glucoamylase production by Aspergillus flavus NSH9, to isolate and characterize amylase (glucoamylase and -amylase) gene a...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2016
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| Subjects: | |
| Online Access: | http://ir.unimas.my/id/eprint/30690/1/Kazi%20ft.pdf |
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| Summary: | Glucoamylase (GA) is an exohydrolases, which release β-glucose units from the non-reducing ends of starch have industrial importance. The purposes of the study were to optimize glucoamylase production by Aspergillus flavus NSH9, to isolate and characterize amylase (glucoamylase and -amylase) gene and to clone as well as to characterize the recombinant glucoamylase (rGA) heterologously expressed in Pichia pastoris. The highest activity of crude glucoamylase was observed in the fermentation media after 5 days of incubation at 25°C and at pH 5.0. Crude GA exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The molecular weight of the purified -amylase was estimated to be 54 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 50°C. Three complete open reading frames of amylase gene from A. flavus were identified; those encoded 493, 612 and 499 amino acids for GA1, GA2 and -amylase; respectively. The first 17, 19 and 21 amino acids were presumed to be the signal peptides for GA1, GA2, and -amylase; respectively. Both of the glucoamylases were assigned to glycosyl hydrolase family 15, and -amylase to glycosyl hydrolase family 13. All three cDNA were cloned into Pichia pastoris. The highest expression of recombinant glucoamylase (rGA1) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA1 was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C, and also thermostable. Low concentration of metal had a positive effect on rGA1 activity. GA2 was also successfully expressed and had significant raw starch degrading capacity. This two rGA might be used in the saccharification steps in the starch processing industry, due to its thermostability and raw starch degrading properties |
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