Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum

In medium supplemented with chondroitin sulfate, Flavobacterium heparinum s ynthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC numb er), into its periplasmic space. Chondroitinase AC preferentially depolymer izes chondroitin sulfates A and C, whereas chondroitinase B degrades only d ermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B). They were found to be separated by 5.5 kb on the chromosome off: heparinum, transcribed in the same orientation, but not lin ked to any of the heparinase genes. In addition, the synthesis of both enzy mes appeared to be coregulated. The cslA and cslB DNA sequences revealed op en reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The matu re forms of chondroitinases AC and B are comprised of 678 and 481 amino aci d residues and have calculated molecular masses of 77,169 and 53,563 Da, re spectively. Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coil. Partially pur ified recombinant chondroitinases AC and B exhibit specific activities simi lar to those of chondroitinases AC and B from F. heparinum.