Cloning, expression and evaluation of a GH10 alkali xylanase from termite gut microbiomes

MON, MARIA L.; MARRERO DIAZ RUBEN; TALIA PAOLA

Bogotá

Congreso; II Congreso Internacional ISME Latinoamericano; 2021

International Society for Microbial Ecology

In recent years, interest has increased in the development of robust biocatalysts both for the hydrolysis of lignocellulosic biomass and for the synthesis of value-added bioproducts. Termites are among the most efficient lignocellulose decomposers on earth, with hydrolysis efficiencies of up to 90%. This capacity to degrade lignocellulose relies on both mechanical and enzymatic machinery of the termite host, together with the action of intestinal symbiont enzymes. This metabolic potential makes termites an ideal target to search for microbial lignocellulosic enzymes that might be used in the textile, food, animal feed, paper and biofuel industries.The aim of this work was the molecular cloning, heterologous expression and activity evaluation of a endo-1,4-beta-xylanase (Xyl10B) from termite gut microbiome. Xyl10B was identified in a previous study by our group. The BLAST analysis of the amino acid sequence of Xyl10B against Genbank showed 59% identity and 97% coverge with a GH10 from an uncultured bacterium (AGS53960). In addition, a comparative computational modeling was performed using ITASSER. That structural-based modeling analysis indicated that this xylanase possesses a typical eight-fold TIM-barrel characteristic of GH10. The complete Xyl10B coding sequence was cloned into the plasmid pET-28b(+). The recombinant protein expressed with a N-terminal 6xHis fusion tag and purified by immobilized metal affinity chromatography. By SDS-PAGE a protein of 46 KDa (Xyl10B), in agreement with the expected molecular weight, was obtained and identified. The recombinant purified enzyme showed xylanase activity by hydrolysis of beechwood xylan and generation of reducing sugars. According to enzymatic activity assays, Xyl10B had a specific endo-1,4-beta-xylanase of 255 IU/mg of enzyme, with an optimum activity at around 50 °C and pH 8. Enzymes adapted to very specific and regulated environments are very promising candidates for hemicellulose degradation within the constraints of a biotechnological process.