Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain KM21

Abstract

Two xyloglucan-specific endo-beta-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley beta-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

FIG. 1.

Purification schemes for XEG5 and XEG74 from Paenibacillus sp. strain KM21.

FIG. 2.

SDS-PAGE of purified XEG5 and XEG74. Lane 1, purified XEG5; lane 2, purified XEG74. MW, molecular mass marker.

FIG. 3.

Schematic illustrating the modular architecture of XEG5 and XEG74. XEG5 contains an N-terminal signal sequence (striped) followed by a GH5 catalytic domain. XEG74 contains an N-terminal signal sequence (striped) followed by a GH74 catalytic domain, an X2 module, and a CBM3 region.

FIG. 4.

Analysis of the final products resulting from the complete digestion of tamarind seed xyloglucan by XEG74. The reaction products were analyzed by HPLC (A) and MALDI-TOF MS (B).

FIG. 5.

HPLC and MALDI-TOF MS analysis of the digestion products of the xyloglucan oligosaccharide XXXGXXXG. (A to C) HPLC profiles of XXXGXXXG (A) and the digestion products by XEG5 (B) and XEG74 (C). (D to F) MALDI-TOF MS analysis of the digestion products by XEG74. (D) Product A in panel C; (E) product B in panel C; (F) all of the products of XXXGXXXG digestion by XEG74.

FIG. 6.

Substrate specificity of XEG74 toward xyloglucan oligosaccharides. Xyloglucan oligosaccharides were incubated with recombinant XEG74. The resulting digestion products were quantified and analyzed by HPLC and MALDI-TOF MS. The cleavage sites and ratios are indicated.

FIG. 7.

Viscosimetric analysis. Tamarind seed xyloglucan was incubated with XEG5 or XEG74. After various incubation times, the specific viscosity was calculated and the hydrolysis ratio was determined by measuring the reducing power. The reducing power obtained following complete digestion with excess enzyme and incubation time was normalized to 100%. Open circles, XEG5; closed circles, XEG74.

FIG. 8.

Analysis of xyloglucan hydrolysis products by gel filtration chromatography. Tamarind seed xyloglucan was incubated with XEG5 (A) or XEG74 (B) for various incubation times, and the reaction products were applied to a gel filtration column.