Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 (LB130)

Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 Soumyadeep Chakraborty, Arun Goyal, Indian Institute of Technology, Guwahati, Assam, India A protein sequence with GeneBank accession number ABN53381.1 from Clostridium thermocellum ATCC 27405 belonging to family 1 polysacchaide lyase of CAZy database was scavenged out for this study. This sequence consisted of 352 aa N-terminal catalytic domain (PL1B) and a 123 aa family 35 carbohydrate binding domain designated as (CBM35) at the C-terminal. The catalytic domain PL1B was cloned in pET28a expression vector and expressed in E.coli as a soluble protein of molecular size 40 kDa. PL1B was active at alkaline pH on pectic substrates. Optimization of enzymatic reaction conditions revealed that PL1B shows 100% (3005 U/mg) activity at 50oC in 50 mM Tris-HCl buffer, pH 9.8. Highest activity was achieved on reaction with non-methylated polygalacturonic acid (PGA) and almost 75% of the tota...