Pandya, Jyoti (2002) Purification and characterisation of amylolytic enzymes from Lipomyces starkeyi. PhD thesis, University of Greenwich.

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Abstract

A purification scheme has been developed for the extracellular carbohydrases; a­glucosidase and a-amylase, secreted by the ascosporogenous soil yeast Lipomyces starkeyi NCYC 1436.

Growth and optimum enzyme yield conditions were determined with cultures of L. starkeyi grown at 30°C on a medium containing 2% soluble starch, 1 % yeast extract and 1 % Bactopeptone, appropriate enzyme assay procedures having been devised. Both enzymes were initially precipitated from the cell free supernatant by an 85%(w/v) ammonium sulphate precipitation. Many different chromatographic media were then assessed, but the choice of a hydrophobic (Phenyl Sepharose CL-4B) column had the advantage of utilizing the ammonium sulphate precipitate with minimum sample preparation. The pellet was adjusted to lM salt and adsorbed to the hydrophobic column. Elution of the two activities was carried out by a series of decreasing salt washes. Although fractionation of the two enzymes was not complete, the column was effective in eliminating a substantial quantity of inactive material, whilst maintaining good recoveries of approximately 59% for a-glucosidase and 72% for a-amylase. Both enzymes were substantially purified using ion exchange chromatography ( Q­Sepharose fast flow medium), with a-glucosidase being apparently close to electrophoretic homogeneity; elution of the two enzyme activities was carried out at 4°C, using a linear gradient with sodium acetate buffer, pH 5.0.

Individual HIC fractions purified by ion exchange showed between a 0.3 to 24.6-fold increase in purity for a-glucosidase, and between a 2.8 to 7.6 increase in purity for a­amylase. However, by selecting the most active a-glucosidase fraction isolated after running the 0.01M HIC fraction on ion exchange, the purification factor rose to 83.4. Several properties of the purified a-:glucosidase enzyme were investigated. The molecular weight of the a-glucosidase was determined by electrophoresis under denaturing and non-denaturing conditions, after ion exchange chromatography. A single major band was detected by SDS-PAGE with an estimated molecular weight of 93,000 + 5,000 Daltons, and under native conditions, the molecular weight was estimated at 162,000 Daltons. Specific enzyme activity staining of the native gel confirmed that the single band was an a-glucosidase. However, isoelectric focusing of the purified Lipomyces starkeyi a-glucosidase, detected three bands - within the pl range of 4.6-5.0. Further characterisation studies revealed the pH and temperature optimum of the purified L. starkeyi a-glucosidase at 4.5 and 55°C respectively. Km and Vmax measurements using a range of substrates suggested that the a­glucosidase had a broad substrate specificity but showed a marked preference for substrates with a-1,4 linkages such as soluble starch, PNPG, and a-1,3 linkages, such as n1gerose.

SDS and native gel electrophoresis ( combined with specific activity staining) revealed 3 distinct a-amylase activities, and the molecular weights were determined as 82,000, 68,000 and 48,500 Daltons.

Item Type:	Thesis (PhD)
Additional Information:	uk.bl.ethos.616522
Uncontrolled Keywords:	enzymes; soil yeast; glucosidases
Subjects:	Q Science > QP Physiology
Pre-2014 Departments:	School of Science
Last Modified:	26 Sep 2018 15:42
URI:	http://gala.gre.ac.uk/id/eprint/11899

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