Biochemical and biophysical description of a novel glycoside hydrolase family 6 from sugarcane soil metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. Considering that  there is an urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass and that cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoglucanase family GH6 (SCMGH6) identified from sugarcane soil metagenome. Sequence analysis revealed that SCMGH6 gene of 909 bp encodes a mature protein of 302 amino acids. The deduced amino acids of SCMGH6 showed 45% sequence identity with the 1,4-beta-glucanase of Mycobacterium neoaurum, displaying no significant sequence homology to already known cellulases.  The recombinant enzyme was mainly active on β-glucan and lichenan substrates with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. The optimum temperature and pH for this enzyme were respectively 40°C and 6.0, and enzyme activity were not affected by presence of CuSO₄ or FeCl₃, however it was significantly reduced by the presence of EDTA and CoCl_2. The K_m and V_max towards β-glucan was 2.15mg.mL^-1 and 214.8 U.mg^-1 respectively. Secondary structure and thermal stability of SCMGH6 were assessed by far-UV CD spectrum analysis that indicates an α-helix protein with Tm of 44.5°C. The properties of SCMGH6 make this enzyme an attractive candidate for large-scale expression and further trials seeking applications in biotechnological processes.