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Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 (LB130)
Author(s) -
Chakraborty Soumyadeep,
Goyal Arun
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb130
Subject(s) - clostridium thermocellum , pectate lyase , chemistry , cellulase , biochemistry , cleavage (geology) , enzyme , pectin , lyase , glycoside hydrolase , pectin lyase , pectinase , biology , paleontology , fracture (geology)
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 total activity was found with methylated citrus pectin. As this enzyme showed highest activity on non‐methylated PGA hence it was named as pectate lyase. The cleavage mechanism of PL1B towards pectic substrates was determined after analysis of the oligosaccharide produced. The reaction was setup with PGA and citrus pectin separately under optimized conditions of pH and temperature and samples was collected at different time interval at 0 min up to 60 min. Analysis of the reaction products by thin layer chromatography (TLC) revealed that unsaturated di and tri‐galacturonate was produced after enzymatic cleavage together with other oligosaccharides. This finding elucidated that PL1B cleaves within the polysaccharide chain and hence it is an endo cleaving enzyme; thus PL1B can be named as endo‐pectate lyase. Enzymatic degradation products were further analyzed by HPEAC and mass spectrometry.