Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

Abstract

Two xyloglucan-specific endo-␤-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 ␤-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 XXXG XXXG 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.

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

“…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].…”

Identification and Characterization of a Xyloglucan-Specific Family 74 Glycosyl Hydrolase from Streptomyces coelicolor A3(2)

“…Some cellulases (endo-1,4-␤-glucanases) have been reported to have hydrolytic activity against xyloglucan as well as major activities toward carboxymethyl cellulose (CMC) and barley ␤-glucan (2,22). A few specific xyloglucanases that either completely lack activity or display low levels of activity toward CMC and ␤-glucan have been discovered in recent years (7,9,10,15,19,23,25). These xyloglucanases represent a relatively new class of enzymes (6), belonging to the GH families 5, 12, and 74 (http://www.cazy.org/).…”

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

“…Some GH74 enzymes from fungi contain a CBM1 (15,16,22). In contrast, several bacterial GH74 enzymes contain a CBM2 and/or CBM3 (21,25,35). CBM1 and CBM3 as well as CBM2a are type A CBMs (3).…”

“…Endo-processive enzymes are found in Chrysosporium lucknowense, T. reesei (Hypocrea jecorina), Paenibacillus sp. KM21, and Phanerochaete chrysosporium (15,22,35). The Streptomyces coelicolor A(3) xyloglucanase Sco6545, belonging to the GH74 family, was recently cloned and characterized (9).…”

“…Celish microfibers were purchased from Daicel Chemical Industries, Ltd. (Tokyo, Japan), and chitin was purchased from Yaizu-Suisan (Yaizu, Japan). Various xyloglucan-derived oligosaccharides, such as XXX, XXXG, XLXG, XXLG, XLLG, GXXXG, XXXGXXXG, and XXXGXXXGXXXG, were prepared from TXG as previously described (33)(34)(35). G, X, and L refer to unbranched ␤-Glcp, ␣-Xylp-␤-1,6-Glcp, and ␤-Galp-␣-1,2-Xylp-␤-1,6-Glcp residues, respectively.…”