title: Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli.
abstract: The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6-8.5) and temperature (25-45 °C) ranges. Protein activity was stimulated by the metal ions Mg(+2), K(+), and Ca(+2) and strongly inhibited by Mn(+2). HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20% of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)3, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.
authors.affiliation: Faculty of Biology, Department of Functional Biology and Health Sciences, University of Vigo, 36310, Vigo, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: García-Fraga
authors.forename: Abigaíl F
authors.initials: AF
authors.lastname: da Silva
authors.forename: Jacobo
authors.initials: J
authors.lastname: López-Seijas
authors.forename: Carmen
authors.initials: C
authors.lastname: Sieiro
mesh_terms: D059001:Aquatic Organisms / Q000201:enzymology* / Q000235:genetics; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019617:Halobacterium salinarum / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D012965:Sodium Chloride; D002688:Chitinases