A new member of family 8 polysaccharide lyase chondroitin AC lyase (PsPL8A) from Pedobacter saltans displays endo- and exo-lytic catalysis

Abstract

Chondroitin lyases are therapeutically important enzymes. The functional aspects of a chondroitin AC lyase (PsPL8A) from Pedobacter saltans DSM12145 were investigated. PsPL8A was cloned in to pET28a(+) vector, expressed in E. coli BL-21(DE3) cells exhibited a molecular size of approximately, 77kDa. PsPL8A displayed maximum activity with chondroitin 4-sulphate, C4S (489Umg−1) followed by chondroitin 6-sulphate, C6S (214Umg−1) and hyaluronic acid (43.2Umg−1). PsPL8A was maximally active at 39°C and pH 7.2. 100mM Na+ and 20mM Ca2+ ions enhanced the activity of PsPL8A by 2-fold. The time dependent TLC analysis of PsPL8A degraded products of C4S revealed the presence of higher degree of polymerization (DP) chondroitin sulphate (CS) oligosaccharides at initial stage, but after 1h, only ΔC4S disaccharide was produced as the major product. This result displayed that PsPL8A follows initially a concomitant endo- and exo-lytic mode which finally shifted to exolytic mode of catalysis. The oligosaccharides released were identified as di-, hexa-, octa- and dodeca-saccharide by ESI–MS analysis. The ΔC4S disaccharide showed a peak at m/z 458 (ESI–MS) while in MS/MS mode it gave the peak at m/z 300. ESI–MS/MS, 1H- and 13C- NMR analyses confirmed the structure of ΔC4S disaccharide product obtained after 24h reaction of C4S with PsPL8A. The enzyme reported in present study can be used for cancer mitigation, spinal cord injury treatment and CS oligosaccharides production which act as anti-inflammatory agents. This is the first study reporting the cloning and expression of chondroitin AC lyase from Pedobacter saltans DSM 12145.