A Xylanase Gene Directly Cloned from the Genomic DNA of Alkaline Wastewater Sludge Showing Application Potential in the Paper Industry

Abstract

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55°C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

“…[48] and Plectosphaerella cucumerina [41]. The inhibition of xylanase activity by Cu 2+ is similar to the majority of the xylanases [13], [39], [45], [49]. The stimulatory effect of EDTA could be due to a different catalytic mechanism as reported in xylanases of Aspergillus niger and Fusarium proliferatum [42], [49].…”

“…Detergents exerted a slight stimulatory effect on the recombinant xylanase which is a common feature of the other xylanases. However, rMxyl was inhibited by SDS as in other xylanases [39], [46], [49].…”

“…Cations (Mg 2+ , Sn 2+ and Fe 2+ ) stimulated the rMxyl activity like those of B. subtilis AMX-4 and an uncultured microbe [39], [40]. Even at 1 mM, Hg 2+ and Mn 2+ significantly inhibited the activity.…”

“…The activity and stability of rMxyl at higher pH are the crucial properties of xylanases for their applicability in paper processing industry. Xylanases of G. thermoleovorans , Bacillus firmus , B. stereothermophilus T-6 exhibited optimum activity at pH at 8.0 or above [30], [37], [39]. The shelf life of rMxyl is more than 3 months at 4°C, which retains greater than 90% activity.…”

“…Even at 1 mM, Hg 2+ and Mn 2+ significantly inhibited the activity. The inhibition of xylanase by Hg 2+ suggests the presence of tryptophan residues that oxidize indole ring, thereby inhibiting the xylanase activity [39], [41], [42], [43]. Xylanases from Streptomyces olivaceoviridis A1, Streptomyces sp.…”