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KMID : 0545120110210040412
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Journal of Microbiology and Biotechnology
2011 Volume.21 No. 4 p.412 ~ p.420
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Evidence of an Alternative Route of Cellobiase Secretion in the Presence of Brefeldin A in the Filamentous Fungus Termitomyces clypeatus
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Samudra Prosad Banik
Swagata Pal
Sudeshna Chowdhury
Shakuntala Ghorai
Suman Khowala
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Abstract
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Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 μg/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50oC, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.
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KEYWORD
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Termitomyces clypeatus, Spitzenkorper, Brefeldin A, Cellobiase, Filamentous fungi, Fungal secretory pathway
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