Production and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes.
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) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli-expressed enzyme. K(m) and V(max) values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 micromol/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 degrees C, is inhibited by EDTA, and displays enhanced activity up to 70 degrees 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.
Zhou
Xuguo
X
Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32611-0620, USA.
Kovaleva
Elena S
ES
Wu-Scharf
Dancia
D
Campbell
James H
JH
Buchman
George W
GW
Boucias
Drion G
DG
Scharf
Michael E
ME
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002480:Cellulase / Q000096:biosynthesis* / Q000378:metabolism; D003124:Colorimetry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D020049:Isoptera / Q000378:metabolism*; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D002480:Cellulase