Research Article | Volume: 6, Issue: 1, January, 2016

Extraction, Purification and Characterization of Endo-Acting Pullulanase Type I from White Edible Mushrooms

Abeer N Shehata Doaa A Darwish Hassan MM Masoud   

Open Access   

Published:  Jan 26, 2016

DOI: 10.7324/JAPS.2016.600123
Abstract

Pullulanase (EC 3.2.1.41) has been isolated and purified from white edible mushrooms by ammonium sulphate precipitation (20-70%) followed by ion exchange chromatography (DEAE-cellulose) and gel filtration (Sephadex G 75-120), with final yield (20%) and purification fold (17.8). The molecular mass of pullulanase enzyme was 112 kDa as estimated by SDS-PAGE and the pI value was 6.2. The apparent Km and Vmax values for purified pullulanse on pulluan were 0.27 mM and 0.74 μM min-1 respectively. The activity was optimum at 40â—‹C and pH 6. Pullulanase showed moderate thermo-stability. A relative substrate specificity for hydrolysis of soluble starch, amylopectin and glycogen was 80, 60 and 30% respectively. Enzyme activity was highly activated by Fe+2, Mn+2 and Ca+2 ions, while the activity was inhibited by Hg+2 and Ag+ ions. Ethylenediaminetetraacetic acid (EDTA) and Dithiothreitol (DTT) were activated the enzyme activity. On contarary iodoacetate and sodium fluoride were inhibited the activity. HPTLC (High Performance Thin Layer Chromatography) plate showed that the purified pullulanase caused the complete hydrolysis of pullulan to maltotriose.


Keyword:     Pullulanase White edible mushrooms Purification Characterization HPTLC plate.


Citation:

Shehata AN, Darwish DA, Masoud HMM. Extraction, Purification and Characterization of Endo-Acting Pullulanase Type I from White Edible Mushrooms. J App Pharm Sci, 2016; 6 (01): 147-152.

Copyright:The Author(s). This is an open access article distributed under the Creative Commons Attribution Non-Commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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