title: Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus.
abstract: Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for β-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, β-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 μM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.
authors: Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil.
authors: Luciana
authors: L
authors: Graciano
authors: Juliana Moço
authors: JM
authors: Corrêa
authors: Fabíola Giovanna Nesello
authors: FG
authors: Vieira
authors: Adilson
authors: A
authors: Bosetto
authors: Eduardo Alexandre
authors: EA
authors: Loth
authors: Marina Kimiko
authors: MK
authors: Kadowaki
authors: Rinaldo Ferreira
authors: RF
authors: Gandra
authors: Rita de Cássia Garcia
authors: Rde C
authors: Simão
mesh_terms: D000595:Amino Acid Sequence; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014990:Xylans / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014990:Xylans; D043364:Endo-1,4-beta Xylanases