Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by Aspergillus fumigatus 202 |
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Authors: | Rachana Bhatt K. C. Patel Ujjval Trivedi |
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Affiliation: | (1) BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, 388 120, Gujarat, India; |
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Abstract: | 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 °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 °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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