Purification, Characterization, and Molecular Cloning of a Novel Keratan Sulfate Hydrolase, Endo-β-N-acetylglucosaminidase, from Bacillus circulans

Abstract

Keratan sulfate (KS) is degraded by various enzymes including endo-␤-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-␤-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with ϳ63-and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAc␤1-3Gal1 structure (endo-␤-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55°C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of ϳ200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function.

Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases

“…More generally, we now have six families of GHs encompassing enzymes degrading sulfated polysaccharides: families GH16 (κ-carrageenases, keratanases), GH79 (heparanases), GH82 (ι-carrageenases), a new GH family specific for the degradation of sulfated fucans [46] and two unclassified enzymes, Bacillus circulans keratanase II [47] and P. carrageenovora λ-carrageenase (the present study). With the exception of the GH16 family, all these families encompass enzymes that interact exclusively with anionic polysaccharides.…”

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

“…The CBM16 polypeptides constitute a large family of 59 members that are linked to diverse catalytic domains (17)(18)(19)(20)(21), suggesting that these CBMs possess distinct binding specificities. Until recently, the residues that are key to ligand binding in this family were unknown.…”

Keratan Sulfate Restricts Neural Plasticity after Spinal Cord Injury

“…This tube was very soft and thin so that we could minimize damage to the spinal cord. The osmotic mini-pumps (200 l of solution, 0.5 l/h, 14 d delivery; Alzet pump model 2002 [Durect]) were filled with K-II (0.05 U/200 l; purified from Bacillus circulans) (Yamagishi et al, 2003), C-ABC (0.05 U/200 l) (Seikagaku), or saline (as a vehicle control). To determine an appropriate dose of C-ABC, we examined several doses and found that at the doses of 0.05, 0.1, and 1.0 U/200 l, C-ABC showed a comparable effect on motor function recovery, while the dose of 0.025 U/200 l showed a less potent effect (supplemental Fig.…”

“…We purified K-II from B. circulans as previously described (Yamagishi et al, 2003). Although the extracts of B. circulans showed both keratanase and chondroitinase activities, our stepwise protocol completely abolished the chondroitinase activity and purified K-II (Table 2).…”

“…Although the extracts of B. circulans showed both keratanase and chondroitinase activities, our stepwise protocol completely abolished the chondroitinase activity and purified K-II (Table 2). Furthermore, it is known that the purified enzyme does not digest hyaluronic acid, heparan sulfate, or heparin (Yamagishi et al, 2003). This enzyme is thermo-stable; indeed, it retained its enzymatic activity for at least 120 h at 37°C (100 Ϯ 1.606% at 0 h vs 91.9 Ϯ 4.014% at 120 h).…”