title: Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.
abstract: A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India.
authors.forename: Sharmili
authors.initials: S
authors.lastname: Jagtap
authors.forename: Mala
authors.initials: M
authors.lastname: Rao
mesh_terms: D000192:Actinomycetales / Q000378:metabolism*; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002482:Cellulose / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D044643:Glucan 1,4-beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014990:Xylans / Q000737:chemistry*
chemical_list: D014990:Xylans; D002482:Cellulose; D044643:Glucan 1,4-beta-Glucosidase