Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp. (18th April 2017)

• Record Type: Journal Article
• Authors: Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, Jaime
• Is Part Of: Carbohydrate research. Volume 443/444(2017)
• Page Start: 42
• Page End: 48
• Publication Date: 2017-04-18
• Subjects: Penicillium purpurogenum -- Esterases -- Pectin -- Heterologous expression -- Pichia pastoris; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2017.03.014
• Languages: English
• ISSNs: 0008-6215

Abstract

Abstract: The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum . Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris . The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). Graphical abstract: Highlights: Penicilium purpurogenum is grown on sugar beet pulp. Esterase activity is analyzed by zymograms in fractions of culture supernatant. Potential esterases are identified in active fractions by mass spectrometry. Genes of these potential esterases are identified in the genome of the fungus. Three esterases are expressed in Pichia pastoris, purified and characterized.