Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

Kourtoglou, E; Anasontzis, GE; Mamma, D; Topakas, E; Hatzinikolaou, DG; Christakopoulos, P

dc.contributor.author	Kourtoglou, E	en
dc.contributor.author	Anasontzis, GE	en
dc.contributor.author	Mamma, D	en
dc.contributor.author	Topakas, E	en
dc.contributor.author	Hatzinikolaou, DG	en
dc.contributor.author	Christakopoulos, P	en
dc.date.accessioned	2014-03-01T01:35:27Z
dc.date.available	2014-03-01T01:35:27Z
dc.date.issued	2011	en
dc.identifier.issn	0141-0229	en
dc.identifier.uri	https://dspace.lib.ntua.gr/xmlui/handle/123456789/21057
dc.subject	Biochemical characterization	en
dc.subject	Constitutive expression	en
dc.subject	Fusarium oxysporum	en
dc.subject	Phosphoglucomutase	en
dc.subject	Purification	en
dc.subject.classification	Biotechnology & Applied Microbiology	en
dc.subject.other	Acid buffers	en
dc.subject.other	Anion exchange	en
dc.subject.other	Apparent k	en
dc.subject.other	Aspergillus nidulans	en
dc.subject.other	Biochemical characterization	en
dc.subject.other	Constitutive expression	en
dc.subject.other	Constitutive promoters	en
dc.subject.other	Divalent cation	en
dc.subject.other	Divalent metal ion	en
dc.subject.other	Elevated level	en
dc.subject.other	Fusarium oxysporum	en
dc.subject.other	Fusarium oxysporums	en
dc.subject.other	Gel-filtration chromatography	en
dc.subject.other	Iso-electric points	en
dc.subject.other	Kinetic constant	en
dc.subject.other	Monomeric structures	en
dc.subject.other	Native enzymes	en
dc.subject.other	Optimal temperature	en
dc.subject.other	pH range	en
dc.subject.other	Phosphate buffers	en
dc.subject.other	Phosphoglucomutase	en
dc.subject.other	Ping pong	en
dc.subject.other	Purification procedures	en
dc.subject.other	Wild types	en
dc.subject.other	Gel permeation chromatography	en
dc.subject.other	Manganese	en
dc.subject.other	Metal ions	en
dc.subject.other	Purification	en
dc.subject.other	Enzymes	en
dc.subject.other	3 n morpholino propanesulfonic acid	en
dc.subject.other	calcium ion	en
dc.subject.other	magnesium ion	en
dc.subject.other	manganese	en
dc.subject.other	nickel	en
dc.subject.other	phosphoglucomutase	en
dc.subject.other	sulfonic acid derivative	en
dc.subject.other	unclassified drug	en
dc.subject.other	article	en
dc.subject.other	enzyme activity	en
dc.subject.other	enzyme analysis	en
dc.subject.other	enzyme purification	en
dc.subject.other	enzyme structure	en
dc.subject.other	Fusarium oxysporum	en
dc.subject.other	gel filtration chromatography	en
dc.subject.other	isomerization	en
dc.subject.other	kinetics	en
dc.subject.other	nonhuman	en
dc.subject.other	protein expression	en
dc.subject.other	temperature sensitivity	en
dc.subject.other	wild type	en
dc.subject.other	Biotechnology	en
dc.subject.other	Catalysis	en
dc.subject.other	Enzyme Stability	en
dc.subject.other	Fusarium	en
dc.subject.other	Gene Expression Regulation, Fungal	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Kinetics	en
dc.subject.other	Phosphoglucomutase	en
dc.subject.other	Plasmids	en
dc.subject.other	Temperature	en
dc.subject.other	Emericella nidulans	en
dc.subject.other	Fusarium oxysporum	en
dc.title	Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.enzmictec.2010.10.007	en
heal.identifier.secondary	http://dx.doi.org/10.1016/j.enzmictec.2010.10.007	en
heal.language	English	en
heal.publicationDate	2011	en
heal.abstract	The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45 degrees C but the enzyme became unstable at temperatures above 40 degrees C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co2+. The apparent K-m for Co2+ was found to be 10 mu M. The enzyme was also active with other divalent metal ions such as Mn2+, Mg2+, Ni2+ and Ca2+ but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 mu mol mg(protein)(-1) min(-1); k(cat), 44.2 min(-1); K-m(G1P), 0.10 mM: K-m(G1,6diP), 1.03 mu M; k(cat)/K-m(G1P), 443 mM(-1) min(-1) and k(cat)/K-m(G1,6diP), 42,860 mM(-1) min(-1). The enzyme was considered to follow a Ping Pang substituted enzyme or enzyme isomerization mechanism. (c) 2010 Elsevier Inc. All rights reserved.	en
heal.publisher	ELSEVIER SCIENCE INC	en
heal.journalName	Enzyme and Microbial Technology	en
dc.identifier.doi	10.1016/j.enzmictec.2010.10.007	en
dc.identifier.isi	ISI:000288310400003	en
dc.identifier.volume	48	en
dc.identifier.issue	3	en
dc.identifier.spage	217	en
dc.identifier.epage	224	en