A xylan-decomposing Gram-positive bacterium, Cellulosimicrobium cellulans DY-8, was isolated from the gut of a wood-feeding longicorn beetle, Moechotypa diphysis. To amplify a partial fragment of the GH 10 β-1,4- xylanase (XylC) gene of strain DY-8, two degenerated oligonucleotide primers were designed based on strictly conserved regions (WDVVNE and ITELLDV) in the GH family 10 xylanolytic enzymes. The full gene (1488-bp) of XylC, which was predicted to encode a protein consisting of 495 amino acids with a molecular mass of 52.0 kDa and a calculated pI of 6.49, was cloned by repeated DNA walking and nested PCR protocols. The results of a protein blast survey showed that XylC was a β -1,4-xylanase comprised of an N-terminal catalytic GH10 domain (from Ser48 to Leu338) and a C-terminal RICIN domain (from Tyr359 to Leu492). This overall structure of XylC was 57% identical to that of Actinoplanes sp. SE50/110 β -1,4-xylanase (Accession number: YP_006265966), which has not yet been biochemically characterized.

A xylanolytic microorganism, strain DY-7, was isolated from the gut of the mole cricket, Gryllotalpa orientalis. The result of phylogenetic analysis based on its 16S rDNA sequence revealed that the isolate was a Gram-positive bacterium belonging to the genus Streptomyces. The cloned gene (1350-bp) encoding a GH family 10 β -1,4-xylanase (XylA) from Streptomyces sp. strain DY-7 was overexpressed in Escherichia coli BL21 and its gene products were characterized. The hydrolysis activities of rXylA and rXylAΔCBD II against xylosidic materials were maximum at pH 5.5 and 65oC. However, deletion of CBD II in the C-terminus region of XylA significantly increased the thermal stability of the enzyme at high temperatures above 50oC. The xylanolytic activity of rXylA was slightly enhanced in the presence of 1 mM Mn2+ and 5 mM sodium azide but it was completely inactivated by 1 mM Hg2+ and 5 mM N-bromosuccinimide. rXylA was capable of efficiently decomposing various xylosidic compounds, PNP-cellobioside, and PNP-xylopyranoside, whereas other hexose-based compounds were insensitive to the enzyme. The specific activities of rXylA toward oat spelts xylan and PNP-cellobioside were 649.8 U/mg and 328.1 U/mg, respectively. Enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) resulted in the production of xylobiose (>75%) as the main hydrolysis product together with a small amount (4%<) of xylose as the final hydrolysis product.

The extracellular GH11 β-1,4-xylanase (XylY) gene (633-bp) of Paenibacillus sp. strain KYJ-16 was molecularly cloned by repeated DNA walking and nested PCR method. The xylY gene was predicted to encode an extracellular protein consisting of 611 amino acids with a nesuced molecular mass of 23 kDa and a calculated pI of 9.55. Protein blast search revealed that the enzyme consisted of a putative catalytic domain, which is homologous to a catalytic GH11 domain. The highest sequence identity (92%) was obtained as the catalytic GH11 domain of XylY was compared to that of Paenibacillus sp. strain HGF5 (GenBank accession number: EGG35584) that has not yet been characterized. Enzymatic properties of the recombinant His-tagged enzyme (rXylY) overexpressed in E. coli BL21 harboring pET-28a(+)/xylY will be also presented.

The gene (2,304-bp) encoding a novel xylanolytic enzyme (XylD) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 β-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylDΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg-1) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylDΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-β-1,4-xylanase activity of XylDΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50 mM xylobiose.