Metagenomik yöntem kullanılarak selüloz parçalayan yeni enzimlerin keşfedilmesi

Fossil fuel is a finite energy source and use of fossil fuels releases harmful chemicals to environment. To fulfill energy requirement, alternative energy sources that can replace fossil fuel must be developed. A high potential environmental friendly alternative is bio-fuel produced from cellulosic biomass. Despite of high number of biofuel researches, production of sustainable and economical biofuel remains a challenge due to recalcitrance of cellulosic materials. The major obstacle for industrial-scale production of biofuel is the inefficient degradation of plant material due to absence of enzymes that catalyze efficient hydrolysis at optimum conditions. Fully degradation of cellulose requires synergetic action of a complex of enzymes for optimized saccharification. Cellulases, which catalyze cellulolysis, are classified into three groups according to their structure and mechanism of action: endoglucanases, exoglucanases, and β-glucosidases. Unfortunately, commercial biocatalysis is insufficient due to the lack of enzymes with optimal performance for specific applications. A powerful approach, which draws attention lately, for discovery of new enzymes with optimum performance is screening of natural diversity. Microorganisms, which adapted to a wide range of environmental conditions, serve as a good source. Metagenomic approach, which comprises the direct extraction of genomic DNA from environmental samples, can prevent loss of diversity resulting from cultivation, and allows recovery of genomes of all microorganisms present in a given niche. In this study, the aim was identification of novel cellulolytic genes using metagenomic approach. Environmental samples used in the study comprised water samples from Dalaman Spring, Dalaman Lake, Emet Hot Spring, and Kuzuluk Hot Springs, straw samples that were incubated in water for enrichment of cellulolytic microorganisms, and grasshoppers obtained from TUBITAK MAM campus and commercial farm. The metagenomic DNA extraction and DNA clean-up (if required) were performed using commercial kits. The isolated DNAs were used for construction of metagenomic library with next-generation sequencing technology. Bioinformatic tools were used for assembly of reads, phylogenetic studies, and gene annotations. Total of 63 candidate cellulase genes were selected from annotated for further analysis. Candidate genes, which were amplified from metagenomic DNA, were cloned into E. coli BL21 (DE3) for production of recombinant protein. 22 of 63 candidate proteins was expressed successfully and screened for biochemical activity against cellulose. Two different substrate, CMC and pNPG, were used for screening of endoglucanases, exoglucanases, and β-glucosidases. Activity assay revealed one active enzyme against CMC, while no active protein that hydrolyzes pNPG was found.