Journal
JOURNAL OF POLYMERS AND THE ENVIRONMENT
Volume 18, Issue 2, Pages 141-147Publisher
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1007/s10924-010-0176-1
Keywords
Poly(3-hydroxybutyrate); PHB depolymerase; Aspergillus fumigatus 202; Thermotolerant fungus; Purification
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An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase produced by a thermotolerant fungal soil isolate, Aspergillus fumigatus 202, was purified and characterized. Maximum PHB depolymerase production was obtained at the end of 48 h with initial medium pH 7.0 and 45 A degrees C in Bushnell Haas Minerals medium containing PHB as sole source of carbon. The PHB depolymerase was purified using size exclusion chromatography to a fold purification of 20.62 and 61.62% yield. SDS-PAGE and isoelectric focusing revealed the molecular weight and pI of the purified enzyme as 63,744 Da and 4.2, respectively. N-terminal amino acid sequence of purified enzyme was HAXDAYLVK. This non-glycosylated enzyme was most active at pH 9.0 and 45 A degrees C. Purified enzyme was inactivated by N-bromosuccinimide and dithiothreitol suggesting the involvement of tryptophan residues and disulfide bonds at its active site. Nonionic detergents like Tween 20, Tween 80 and Triton X-100 inhibited the enzyme activity. Ions like Ca(+2) and Mg(+2) (5 mM) increased the enzyme activity 1.5 times. Fe(+2) effectively inhibited the enzyme activity to 88% whereas Hg(+2) completely inhibited the enzyme.
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