fulltext.study @t Gmail

Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus

Paper ID Volume ID Publish Year Pages File Format Full-Text
18675 42732 2006 9 PDF Available
Title
Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus
Abstract

In this report the ORF PF2001 from the hyperthermophilic archaeon Pyrococcus furiosus was identified and its protein sequence was characterized in silico, revealing characteristics of the conserved domains of the dipeptidyl aminopeptidases, hydrolases of the α/β superfamily, esterases and lipases. In order to understand its function, the ORF PF2001 without the 60 bp of the 5′-terminus, responsible for encoding a signal peptide (PF2001Δ60), was cloned and expressed in Escherichia coli BL21(DE3) pLysS, and the recombinant enzyme was characterized for esterase, lipase and protease activities. The total protein extract from E. coli harboring the plasmid containing the ORF PF2001Δ60 exhibited its highest activity towards the substrate 4-methylumbelliferyl-heptanoate (C7) and lower activities towards 4-methylumbelliferyl-acetate (C2) and 4-methylumbelliferyl-palmitate (C16). The enzyme was thermostable for 120 min at 75 °C and was completely inhibited by 1 mM PMSF. A theoretical structural model was constructed by comparative modeling using as template a prolyl oligopeptidase from Sus scrofa. Although no protease activity was detected a putative catalytic triad (Ser149, Asp233 and His264) with high similarity to the template was identified. The structural characteristics that confer enzymatic specificity to the P. furiosus enzyme are discussed. Taken together the data strongly suggest that ORF PF2001 from P. furiosus is responsible for encoding a novel esterase.

Keywords
Esterases; Lipases; Proteases; Comparative modeling; Homology modeling; Extremozymes; Thermophilic enzymes; Archaea
First Page Preview
Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 39, Issue 5, 4 September 2006, Pages 1128–1136
Authors
, , , , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering