Metagenomic approach seeks novel enzymes beneficial to microbial biotechnology through the screening of metagenomes from complex environments, with crucial steps: cloning and the expression of selected genes in unpretentious bacterial or other types of strains. Glycoside hydrolases degrade complex plant polysaccharides and belong to a group of one of the most demanded industrially relevant enzymes. In this thesis we focused on isolation and characterization of putative GH10 identified in compost metagenome (xyl6). We successfully isolated xylanase with molecular mass approx. 40 kDa and activity against beechwood xylan and arabinoxylan. Recombinant xylanase revealed optimal xylanolytic activity against beechwood xylan at 50 °C and pH 8. Xylanolytic activity of Xyl6 increased in the presence of 5 mM and decreased in the presence of 10 mM Co2+, Fe3+, Mn2+ or Ca2+. The latter concentration of Cu2+ completely inhibited the enzyme activity. Enzyme activity also decreased when exposed to NaCl above 100 mmol/L. Exposure to 10 % (v/v) methanol, ethanol or acetone, as well as 20 mM EDTA or SDS was shown to decrease xylanolytic activity of Xyl6. Furthermore, enzyme was not stable at a temperature above 50 °C or in acidic conditions (below pH 6). Xylanolytic activity increased when exposed to reducing agents such as 2 mM DTT or 10 mM ß-mercaptoethanol. The recombinant xylanase exhibited Km and Vmax values of 1,8 ± 0,3 mg/mL and 787,2 ± 12,9 μmol/min/mg for beechwood xylan at optimal conditions, and turnover number (kcat) value of 526,2 ± 17,4 s-1. Enzyme also proved successful in the degradation of natural hemicellulose-rich substrates, such as wheat or barley bran, thus has great potential for future use in industrial applications.
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