Abstract
BackgroundThe alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes.Methodology/Principal FindingsMetagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C.Conclusion/SignificanceThis is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues.
Highlights
Hemicellulose represents the second most abundant renewable polymer of plant cell walls after cellulose, and xylan is the main constituent in lignocellulosic agro-residues
When 5.0 mg of 20–30 kb of high molecular weight metagenomic DNA was digested with Sau3AI and the fragments were ligated into p18GFP vector with an efficiency of 3.66104 clones per mg of DNA in constructing the library, the insert sizes were in the range of 3.0–8.0 kb with an average size of 5.5 kb
The sequence showed putative sequences of -35 (CACGCCA), -10 (TAAAAA) and ribosomal binding sites (AGGGG) at the upstream of xylanase gene followed by complete open reading frame (ORF) having ATG and TAA as start and stop codons, respectively (Figure S1)
Summary
Hemicellulose represents the second most abundant renewable polymer of plant cell walls after cellulose, and xylan is the main constituent in lignocellulosic agro-residues. The backbone of xylan is mainly composed of b-1,4-linked xylosyl residues along with various groups (arabinosyl, acetyl and glucuronosyl) in their side chains. These heterogeneous polysaccharides play a critical role in maintaining the cell wall integrity by making covalent and noncovalent bonds with cellulosic fibres and lignins [1,2]. The xylanases that withstand extreme conditions prevailing in the industrial processes are in high demand in paper pulp processing and feed industries. The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes
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