Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation

Abstract

The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.

FIG. 1.

Restriction enzyme map of the HindIII-to-EcoRI fragment encoding xynA (A) and modular structure of XynA and its derivatives (B). The white arrows indicate the coding sequences for XynA and XylA polypeptides. a.a., amino acids.

FIG. 2.

Thin-layer chromatography of hydrolysis products from xylooligosaccharides. Each xylooligosaccharide (xylobiose to xylopentaose, 3 mg of each; Megazyme) was incubated with purified enzyme (1 U) for 16 h, and the hydrolysates were analyzed by thin-layer chromatography (7). S, authentic oligosaccharides; X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose.

FIG. 3.

Identification of XynA in C. cellulovorans and expression of rXynA in E. coli. Gels were stained with Coomassie brilliant blue (A) or stained for xylanase activity (B). XynA proteins in immunoblot analyses were detected with a polyclonal rabbit antiserum raised against purified rXynA (C). Lanes: 1, cellulosomal fraction of C. cellulovorans; 2, noncellulosomal fraction of C. cellulovorans; 3, purified rXynA; 4, whole-cell proteins of E. coli XL1-Blue (Stratagene) harboring pX13; 5, whole-cell proteins of E. coli XL1-Blue; M, protein molecular mass standards. The proteins corresponding to the major components specified in the C. cellulovorans cellulosome (7) are indicated on the left of panel A.