PRODUCTION AND CHARACTERIZATION OF A RECOMBINANT BETA-1,4-ENDOGLUCANASE (GLYCOHYDROLASE FAMILY 9) FROM THE TERMITE Reticulitermes flavipes

摘要

Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) andprokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily btained in solubilized form and more active than the E. coli–expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993%w/v and 1.056 mmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5–7.5 and 50–60 deg C, is inhibited by EDTA, and displaysenhanced activity up to 70 deg C in the presence of CaCl_2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others.