Expression and analysis of Endo Beta-1,4-Mannanase of Aspergillus Fumigatus in heterologous hosts

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2007

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Duruksu, Gökhan

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Extracellular endo-1,4-b-mannanase (EC 3.2.1.78) gene of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was cloned and transformed into Aspergillus sojae (ATCC 11906) and Pichia pastoris GS115. High level of expression was achieved in both expression systems. Attempts to produce heterologous mannanase in Arabidopsis thaliana, suitable for large scale production, were not successful. Comparison of the expression levels of heterologous mannanase reveals that A. sojae is a better expression system than P. pastoris with respect to extracellular mannanase activity. The production of mannanase in A. sojae (AsT1) after 3 days of incubation reached 204 U/ml in YpSs containing 1 % glucose. In P. pastoris (PpT1), highest production was observed after 10 hrs of induction with methanol (61 U/ml). Expressed enzymes were purified and analyzed. Both enzymes have specific activity c. 349 U/mg protein with pH and temperature optimum of c. 4.5 and c. 60 °C for mannanases from AsT1 and c. 5.2-5.6 and c. 45 °C for mannanases from PpT1. A truncated form of mannanase (MAN-S) deleted at amino acids from P291 to P368, which still displayed hydrolytic activity was also isolated and characterized. MAN-S has pH and temperature optimum of c. 6.5-8.0 and c. 60 °C. During incubation of the mannanase on locust bean gum, transglycosylation reactions, in which longer or rare prebiotic oligosaccharides could be produced catalyzed by glycolysis, was detected. The products of hydrolytic activity of the enzyme on various carbohydrates were analyzed by PACE and MALDI-TOF. Accordingly, hexamannose and smaller oligosaccharides were characterized.

Subject Keywords

Genetics., Aspergillus fumigatus., Aspergillosis., Aspergillus fumigatus, Aspergillosis

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http://etd.lib.metu.edu.tr/upload/3/12609092/index.pdf
https://hdl.handle.net/11511/17435

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

G. Duruksu, “Expression and analysis of Endo Beta-1,4-Mannanase of Aspergillus Fumigatus in heterologous hosts,” Ph.D. - Doctoral Program, Middle East Technical University, 2007.