Recovery of Lignin peroxidase from Amauroderma rugosum using aqueous two-phase system / Zeinabsadat Masoudian

Masoudian, Zeinabsadat (2014) Recovery of Lignin peroxidase from Amauroderma rugosum using aqueous two-phase system / Zeinabsadat Masoudian. Masters thesis, University of Malaya.

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Abstract

A very important challenge in industrial biotechnology is development of an efficient downstream method for enzyme purification and separation due to the growing market of enzyme in biotechnology. Within the scope of this study, aqueous two-phase system based on imidazolium ionic liquid as an efficient downstream processing method was evaluated for primary purification of lignin peroxidase. The white-rot fungi Amauroderma rugosum is capable to produce lignin peroxidase as one of the ligninolytic enzymes. Lignin peroxidase (LiP) is an extracellular hem protein enzyme which is able to degrade lignin or lignin related aromatic compounds. The fungus cultivation in liquid media comprised of potato dextrose broth, 0.5% yeast and 1% sawdust gave the highest lignin peroxidase activity of 39.03±3.33 U/ml. The lignin peroxidase was then extracted by partitioning in aqueous two-phase system based on ionic liquid (IL-based ATPS) composed of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and di-potassium phosphate salt (K2HPO4). The effect of tie-line length (TLL), volume ratio (VR), pH and addition of more K2HPO4 on partitioning of LiP were studied. Experimental results showed that LiP was best partitioned in the ionic liquid rich top-phase with activity of 25.58±0.63U/ml, purification factor of 10.76±1.61 and 91.80%±0.30 of yield in the system with tie-line length (TLL) of 87.57 % (w/w), volume ratio (VR) at 1.17, pH 8 and with addition of more K2HPO4 up to 5.85g. It was indicated that hydrophobic interaction, electrostatic interaction and salting out effect were the driving force for partitioning of LiP into top phase. Importantly the enzyme could retain its activity in presence of ionic liquid. Thus the IL-based ATPS is expected to render a suitable method for bio-product separation.

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