Characterization of an Endo-Processive-Type Xyloglucanase Having a β-1,4-Glucan-Binding Module and an Endo-Type Xyloglucanase from Streptomyces avermitilis

Abstract

e We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to ␤-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

“…Incubation of CjGH74 with XyG in this buffer at a range of temperatures over 10 min revealed that the recombinant enzyme had the highest activity at 65°C (Fig. 4B), which compares favourably with other characterized GH74 members [38][39][40][41][42][43][44][45].…”

“…charides with b-1,4-glucan backbone [42]. These findings suggest that bioinformatic analysis is not always sufficient to unambiguously predict CBM function, which emphasizes the importance of the careful biochemical and biophysical characterization of these modules.…”

“…1) to generate the oligosaccharides XXXG, XLXG, XXLG and XLLG, which differ in their degree of side-chain galactosylation (Figs 6 and 7). This is the most common cleavage pattern observed for GH74 endo-xyloglucanases [38,41,[48][49][50], although certain members are known to cleave between branched backbone glucosyl units [42,43,51,52].…”

Key amino acid residues for the endo‐processive activity of GH74 xyloglucanase

“…5B). Streptmyces GH74B [13], an endo‐dissociative‐type xyloglucanase, has three tryptophan residues analogous to W61, W64 and W318, but lacks a tryptophan residue corresponding to W319. The endo‐processive‐type enzymes Phanerochaete Xgh74B [15], Streptmyces GH74A [13], and Thermobifida Xeg74 [14, and our personal data (not shown)] have two tryptophan residues corresponding to W318 and W319, suggesting that both W318 and W319 are essential for the endo‐processive activity of GH74 xyloglucanases.…”

“…Several enzymes are involved in xyloglucan degradation, including endo‐β‐1,4‐glucanases ([8,11,13,15,19,28], reviewed in [10]). Some cellulases have been reported to hydrolyse not only cellulose but also xyloglucan as a substrate analogue [19].…”