Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: A hybrid enzyme being suitable for xylooligosaccharides production

Abstract

The hybrid xylanase gene Xyn2-A2 was previously constructed and expressed in Pichia pastoris. In this study, the hybrid recombinant enzyme (PTXC2) was prepared from a 2-L bioreactor and subsequently characterized. The produced xylanase was estimated by SDS-PAGE to be 26 kDa and practically free of cellulolytic activity. The optimal temperature and pH of PTXC2 was 65 °C and pH 6.0, respectively. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30 min incubation at this temperature. The specificity of PTXC2 towards different natural substrates was evaluated. PTXC2 was highly specific towards xylans tested but exhibited low activities towards cellulosic substrates, such as gellan gum (9.7%), Avicel (1.5%) and carboxymethylcellulose (CMC, 1.2%). The apparent K m values of oat-spelt xylan and birchwood xylan was 1.6 and 1.8 mg/mL, respectively. Analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as an endo-xylanase with xylotriose as the main hydrolysis products. These attributes should make the enzyme an attractive applicant for various applications, such as large-scale production of xylooligosaccharides.