title: An acidothermophilic functionally active novel GH12 family endoglucanase from Aspergillus niger HO: purification, characterization and molecular interaction studies.
abstract: Endoglucanase (EG) from Aspergillus niger HO was sequentially purified through ultrafiltration, ion exchange and size exclusion chromatography to homogeneity, with an overall recovery of 18 %. The purified EG was a monomeric protein with a molecular weight of about 55 kDa. The enzyme was optimally active at pH 3.5 and 70 °C with a half life (t1/2) of 3 h and Km value of 2.5 mg/ml. Metal ions, such as Ca(2+) and Co(2+) helped in enzyme induction, while Hg(2+) and Cu(2+) strongly inhibited the enzyme activity. Peptide mass fingerprinting results revealed that the purified EG is a novel enzyme that belongs to family 12 of glycoside hydrolase (GH12). Molecular docking studies indicated the presence of Glu116 and Glu204 as important determinant residues for the functional interaction with carboxymethylcellulose and showed hydrogen bonding with Asp99, Glu116, Glu204 and hydrophobic interactions with Trp22, Val58, Tyr61, Phe101, Met118, Trp120, Pro129, Ile130, Thr160 and Phe206. Hydrolysis of 2 % CMC with purified acidothermophilic EG at its optimum temperature and pH resulted in complete hydrolysis within 2 h yielding 18 % cellotriose, 72 % cellobiose and 10 % glucose as evident from HPLC analysis. In comparison to most of the EGs reported in literature, EG from A. niger HO exhibited higher thermostability. The acidothermophilic nature of this enzyme makes it potentially useful for industrial applications.
authors.affiliation: Central Institute of Post Harvest Engineering and Technology, P.O. PAU, Ludhiana, 141 004, Punjab, India.
authors.forename: Rekha
authors.initials: R
authors.lastname: Rawat
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.forename: Bhupinder Singh
authors.initials: BS
authors.lastname: Chadha
authors.forename: Dinesh
authors.initials: D
authors.lastname: Kumar
authors.forename: Harinder Singh
authors.initials: HS
authors.lastname: Oberoi
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D062105:Molecular Docking Simulation; D008970:Molecular Weight; D013696:Temperature; D014462:Ultrafiltration
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium