Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601

A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (Mw ∼39 kDa, specific
activity 10,461.5 ± 100 IU/mg). Endo-mannanase gene (manb-1601, 1083 bp, accession No. KM404299)
was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of ␣/␤ 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 quintessen-
tial for stabilization of ManB-1601 structure as holoenzyme (Ea 87.4 kcal/mol, �H 86.7 kcal/mol, �S
186.6 cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted
ManB-1601 (Ea 47 kcal/mol, �H 45.7 kcal/mol, �S 64.7 cal/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.