Purification, characterization, and molecular cloning of a novel keratan sulfate hydrolase, endo-beta-N-acetylglucosaminidase, from Bacillus circulans.
Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- 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 4GlcNAcbeta1-3Gal1 structure (endo-beta-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 degrees C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 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.
Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan.
Kiwamu
K
Yamagishi
Kiyoshi
K
Suzuki
Kyoko
K
Imai
Hideo
H
Mochizuki
Kiyoshi
K
Morikawa
Mamoru
M
Kyogashima
Koji
K
Kimata
Hideto
H
Watanabe
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001407:Bacillus / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D002356:Cartilage / Q000378:metabolism; D002417:Cattle; D002809:Chondroitin Sulfates / Q000737:chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate / Q000737:chemistry; D006820:Hyaluronic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007632:Keratan Sulfate / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012754:Sharks; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D002809:Chondroitin Sulfates; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D006820:Hyaluronic Acid; D007632:Keratan Sulfate; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009844:Oligosaccharides; D010455:Peptides; D001616:beta-Galactosidase; C026093:keratan-sulfate endo-1,4-beta-galactosidase