Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: Identification of the nucleophilic serine

Topakas, E; Moukouli, M; Dimarogona, M; Vafiadi, C; Christakopoulos, P

dc.contributor.author	Topakas, E	en
dc.contributor.author	Moukouli, M	en
dc.contributor.author	Dimarogona, M	en
dc.contributor.author	Vafiadi, C	en
dc.contributor.author	Christakopoulos, P	en
dc.date.accessioned	2014-03-01T01:33:30Z
dc.date.available	2014-03-01T01:33:30Z
dc.date.issued	2010	en
dc.identifier.issn	0175-7598	en
dc.identifier.uri	https://dspace.lib.ntua.gr/xmlui/handle/123456789/20457
dc.subject	Active site	en
dc.subject	Glucuronoyl esterase	en
dc.subject	Lignin-carbohydrate complex	en
dc.subject	Nucleophilic serine	en
dc.subject	Pichia pastoris	en
dc.subject	Sporotrichum thermophile	en
dc.subject.classification	Biotechnology & Applied Microbiology	en
dc.subject.other	Active site	en
dc.subject.other	Glucuronoyl	en
dc.subject.other	Lignin-carbohydrate complex	en
dc.subject.other	Pichia Pastoris	en
dc.subject.other	Sporotrichum thermophile	en
dc.subject.other	Carbohydrates	en
dc.subject.other	Cloning	en
dc.subject.other	Enzyme activity	en
dc.subject.other	Enzymes	en
dc.subject.other	Esters	en
dc.subject.other	Genes	en
dc.subject.other	Glucose	en
dc.subject.other	Lignin	en
dc.subject.other	Phosphatases	en
dc.subject.other	Yeast	en
dc.subject.other	Amino acids	en
dc.subject.other	esterase	en
dc.subject.other	fungal enzyme	en
dc.subject.other	glucuronoylesterase	en
dc.subject.other	unclassified drug	en
dc.subject.other	carbohydrate	en
dc.subject.other	catalysis	en
dc.subject.other	enzyme activity	en
dc.subject.other	fungus	en
dc.subject.other	gene expression	en
dc.subject.other	genetic analysis	en
dc.subject.other	genetic engineering	en
dc.subject.other	homology	en
dc.subject.other	lignin	en
dc.subject.other	mutation	en
dc.subject.other	pH	en
dc.subject.other	article	en
dc.subject.other	enzyme activity	en
dc.subject.other	gene identification	en
dc.subject.other	molecular cloning	en
dc.subject.other	nonhuman	en
dc.subject.other	nucleophilicity	en
dc.subject.other	nucleotide sequence	en
dc.subject.other	pH measurement	en
dc.subject.other	protein expression	en
dc.subject.other	Sporothrix	en
dc.subject.other	sporotrichum thermophile	en
dc.subject.other	temperature sensitivity	en
dc.subject.other	Amino Acid Sequence	en
dc.subject.other	Catalytic Domain	en
dc.subject.other	Cloning, Molecular	en
dc.subject.other	Consensus Sequence	en
dc.subject.other	Enzyme Stability	en
dc.subject.other	Esterases	en
dc.subject.other	Fungal Proteins	en
dc.subject.other	Glucuronates	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Mutagenesis, Site-Directed	en
dc.subject.other	Mutant Proteins	en
dc.subject.other	Pichia	en
dc.subject.other	Sequence Alignment	en
dc.subject.other	Sequence Homology, Amino Acid	en
dc.subject.other	Serine	en
dc.subject.other	Sporothrix	en
dc.subject.other	Temperature	en
dc.subject.other	Corynascus heterothallicus	en
dc.subject.other	Fungi	en
dc.subject.other	Pichia pastoris	en
dc.subject.other	Saccharomyces cerevisiae	en
dc.title	Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: Identification of the nucleophilic serine	en
heal.type	journalArticle	en
heal.identifier.primary	10.1007/s00253-010-2655-7	en
heal.identifier.secondary	http://dx.doi.org/10.1007/s00253-010-2655-7	en
heal.language	English	en
heal.publicationDate	2010	en
heal.abstract	A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4-10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction. © 2010 Springer-Verlag.	en
heal.publisher	SPRINGER	en
heal.journalName	Applied Microbiology and Biotechnology	en
dc.identifier.doi	10.1007/s00253-010-2655-7	en
dc.identifier.isi	ISI:000280036300018	en
dc.identifier.volume	87	en
dc.identifier.issue	5	en
dc.identifier.spage	1765	en
dc.identifier.epage	1772	en