Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.
Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.
Hong
Sung-Woon
SW
College of Pharmacy, Kyung Hee University, Seoul, Korea.
Kim
Byung-Taek
BT
Shin
Ho-Young
HY
Kim
Wan-Suk
WS
Lee
Keun-Sook
KS
Kim
Yeong-Shik
YS
Kim
Dong-Hyun
DH
D001421:Bacteria, Anaerobic / Q000201:enzymology; D001439:Bacteroides / Q000201:enzymology*; D019765:Chondroitin ABC Lyase / Q000302:isolation & purification / Q000378:metabolism*; D002808:Chondroitin Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000382:microbiology; D007700:Kinetics; D013379:Substrate Specificity
D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D019765:Chondroitin ABC Lyase