Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

Abstract

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.

Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes

“…A similar trend was seen previously for the recombinant RfCell-1 endoglucanase of R. flavipes, which possesses a well defined calcium binding motif and is inhibited by the calcium chelator EDTA (Zhou et al, 2010). Additionally, another recombinant enzyme corresponding to the R. flavipes salivary laccase RfLacA binds calcium and also is inhibited by EDTA (Coy et al, in review).…”

“…6). Calcium chloride was previously found to enhance temperature stability for the recombinant RfCell-1 endoglucanase of R. flavipes (Zhou et al, 2010). Recombinant RfBGluc-1 activity was relatively stable at 40 C and below, and showed only w10% reductions in activity after 60 min at 40 C (Fig.…”

Saccharification of Agricultural Lignocellulose Feedstocks and Protein-Level Responses by a Termite Gut-Microbe Bioreactor

“…The higher CBH activity measured in CS-fed guts might be due to qualitative differences in cellulose composition between CS and SR. Additionally, CBH (GHF7) enzymes in R. flavipes are primarily produced by gut symbionts (Tartar et al, 2009), and higher activity of CBH in CS-fed guts therefore suggests significant participation by symbionts in CS saccharification. In contrast, endoglucanase and β-glucosidase, which are primarily hostderived (Tartar et al, 2009;Scharf et al, 2010;Zhou et al, 2010), had similar activity across guts from termites fed on different diets. An alternative explanation for the observed differences in CBH activity between feedstocks is that, as noted above, protein degradation/inhibition was more substantial with SR feeding and/or SR impacts on symbiont mortality were more significant.…”

“…Tubes were vented by melting a pinhole in the assay tube lids. All assay conditions are based on extensive preliminary work showing circum-neutral gut pH in R. flavipes, digestive enzyme pH optima around 7, and enzyme stability at 37°C (Zhou et al, 2007(Zhou et al, , 2010Tartar et al, 2009;Coy et al, 2010;Scharf et al, 2010Scharf et al, , 2011aSethi et al, 2013). A range of feedstock amounts was compared (0.2, 0.4, 0.8, 1, and 2% w/v).…”

“…There is ample evidence of termites having endogenous cellulases that aid in cellulose digestion independent of symbiont action (Watanabe et al, 1998;Zhou et al, 2007), but clearly a synergistic collaboration exists between the termite and its symbiota that achieves efficient lignocellulose digestion (Scharf et al, 2011a,b). Several GH families have been identified in termites with activity toward various cellulosic substrates (Tokuda et al, 1997;Zhou et al, 2007Zhou et al, , 2010Scharf et al, 2010). In contrast, little is known about lignin breakdown mechanisms of wood-feeding insects (Geib et al, 2008), which is a limiting step in biofuel production (Yang and Wyman, 2008).…”