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

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.

Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action on cellulose degradation

“…Microbial diversity in this habitat, as shown by clonal library analysis (24) and tagged 16S rRNA pyrosequencing (15) revealed novel and complex microbial communities with high metabolic potential on lignocellulose degradation. Activity screening based on target enzyme function has been demonstrated to be useful for identifying genes and gene clusters encoding various glycosyl hydrolases and auxiliary enzymes involved in biomass degradation from various environments, e.g., soil (25), rumen (26), and termite gut (10). On the one hand, activity-based screening is limited by differences among species in codon usage, transcription and translation signals, protein folding elements, and post-translational modification, which affect heterologous expression in E. coli host.…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…Recombinant T inact values 10-158C > T opt . Details can be found in [63,64,67,68]. Abbreviations: T inact , temperature at which enzyme are 50% inactive; T opt , optimum temperature for activity.…”

“…A large number of hyperthermophilic enzymes capable of hydrolyzing lignocellulosic biomass have been cloned, characterized, and expressed in E. coli and other hosts [60,[62][63][64], but only a few have been successfully expressed in various host plants (Table S1 in the supplementary material online). Comparative lignocellulosic-digestion studies with mesophilic and hyperthermophilic cellulases and hemicellulases have demonstrated that the functionally homologous hyperthermophilic enzymes can increase saccharification yields and shorten incubation times, and may eliminate the risk of downstream contamination by mesophilic microorganisms competing for fermentable sugars [65,66].…”

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

“…To date, microbial xylanases have been classified into nine glycoside hydrolase (GH) families: 5,8,10,11,16,26,30,43 and 62 based on the similarity of the catalytic domain sequences (www.CAZy.org). The true β-1,4-acting xylanases with a unique catalytic domain are restricted to GH families 5, 8, 10, 11 and 30.…”