title: Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601.
abstract: A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (Mw ∼39kDa, specific activity 10,461.5±100IU/mg). Endo-mannanase gene (manb-1601, 1083bp, accession No. KM404299) was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of α/β proteins in the far-UV CD. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122-152 and 181-212) was observed in endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stabilization of ManB-1601 structure as holoenzyme (Ea 87.4kcal/mol, ΔH 86.7kcal/mol, ΔS 186.6cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47kcal/mol, ΔH 45.7kcal/mol, ΔS 64.7cal/k/mol). EDTA treatment of ManB-1601 not only lead to transitions in both secondary and tertiary structure but also promulgated the population of conformational state that unfolds at lower temperature. ManB-1601 followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity.
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570020, India.
authors.forename: Praveen Kumar
authors.initials: PK
authors.lastname: Srivastava
authors.affiliation: Department of Studies in Biochemistry, University of Mysore, Mysuru, India.
authors.forename: Appu Rao
authors.initials: AR
authors.lastname: Appu Rao G
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570020, India. Electronic address: mkapoor@cftri.res.in.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Kapoor
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013816:Thermodynamics; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Bacillus sp.; Endo-mannanase; GH family 26; Metal-binding site; Thermal stability