Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

Four types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designatedbglA13 ,bglA16 ,bglA51 , andbglM2 , were found in the cDNA library ofNeocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias toStreptococcus equinus . The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) ofbglA16 andbglM2 suggest that these two genes were duplicated recently, whereasbglA13 andbglA16 , which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes fromN. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes ofStreptococcus equinus ,Ruminococcus albus 7, andN. patriciarum J11 were cloned and expressed byEscherichia coli . The recombinant β-glucanases cloned fromS. equinus ,R. albus 7, andN. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higherk cat values than others. These results support the notion that duplicated β-glucanase genes, namely,bglA16 andbglM2 , increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms inN. patriciarum J11.