Kn. Gu et al., PURIFICATION, CHARACTERIZATION AND SPECIFICITY OF CHONDROITIN LYASES AND GLYCURONIDASE FROM FLAVOBACTERIUM-HEPARINUM, Biochemical journal, 312, 1995, pp. 569-577
The chondroitin lyases from Flavobacterium heparinum (Cytophaga hepari
nia) have been widely used in depolymerization of glycosaminoglycan an
d proteoglycan chondroitin sulphates. Oligosaccharide products derived
from chondroitin sulphate can be further degraded by glycuronidases a
nd sulphatases obtained from the same organism. There has been no repo
rted purification of these enzymes to homogeneity nor is there any inf
ormation on their physical and kinetic characteristics. The absence of
pure enzymes has resulted in a lack of understanding of the optimal c
onditions for their catalytic activity and their substrate specificity
. This has limited the use of these enzymes as reagents for preparatio
n of oligosaccharides for structure and activity studies. Reproducible
schemes to purify a chondroitin AC lyase, a glycuronidase and chondro
itin B lyase from Flavobacterium heparinum to apparent homogeneity are
described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glyc
uronidase [chondro-(1 --> 3)-glycuronidase, no EC number] and chondroi
tin B lyase (chondroitinase B, no EC number) have M(r) values (assesse
d by SDS/PAGE) of 74 000, 41 800 and 55 200 respectively, and isoelect
ric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05
respectively. Chondroitin lyase AC and B contain pyroglutamic acid at
their N-termini precluding their analysis by Edman degradation. Deblo
cking with pyroglutamate aminopeptidase facilitated the determination
of their N-terminal sequences. The kinetic properties of these enzymes
have been determined as well as the optimum conditions for their cata
lytic activity. The specificity of the glycouronidase, determined usin
g 17 different disaccharide substrates, shows that it only acts on uns
ulphated or 6-O-sulphated 1 --> 3 linkages. The chondroitin lyases are
both endolytic enzymes, and oligosaccharide mapping shows their expec
ted specificity towards the chondroitin and dermatan sulphate polymers
.