title: Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase from Geobacillus sp. WBI.
abstract: An endo-β-1,4-xylanase gene xynA of a thermophilic Geobacillus sp. WBI from "hot" compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni-NTA chromatography. The purified enzyme (47 kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 °C. The enzyme retained 100% of its activity when incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The K(m) and V(max) of the enzyme were 0.9 mg ml(-1) and 0.8 µmol ml(-1) min(-1), respectively. In molecular dynamics simulation at 338 K (65 °C), the enzyme was found to be stable. At an elevated temperature (450 K) specific α-helix and β-turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T-6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali-thermostability.
authors.affiliation: Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India.
authors.forename: Suranjita
authors.initials: S
authors.lastname: Mitra
authors.forename: Bidhan Chandra
authors.initials: BC
authors.lastname: Mukhopadhyay
authors.forename: Anisur Rahaman
authors.initials: AR
authors.lastname: Mandal
authors.forename: Ananta Prasad
authors.initials: AP
authors.lastname: Arukha
authors.forename: Kuheli
authors.initials: K
authors.lastname: Chakrabarty
authors.forename: Gourab Kanti
authors.initials: GK
authors.lastname: Das
authors.forename: Pran Krishna
authors.initials: PK
authors.lastname: Chakrabartty
authors.forename: Swadesh Ranjan
authors.initials: SR
authors.lastname: Biswas
mesh_terms: D000468:Alkalies; D003001:Cloning, Molecular; D003198:Computer Simulation; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D012988:Soil Microbiology; D014990:Xylans / Q000378:metabolism*
chemical_list: D000468:Alkalies; D014990:Xylans; D043364:Endo-1,4-beta Xylanases
keywords: Alkali-thermostable; Geobacillus; Molecular dynamics; Overexpression; Xylanase