Metal-dependent thermal stability of recombinant endo-mannanase (ManB-1601) belonging to family GH 26 from Bacillus sp. CFR1601
Affiliation:
1. Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570020, India;2. Department of Studies in Biochemistry, University of Mysore, Mysuru, India;3. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan;4. Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan;5. Biomass Engineering Program Cooperation Division, RIKEN, Yokohama, Kanagawa 230-0045, Japan;6. Laboratory of Environmental Molecular Biology, Graduate School of Yokohama City University, Yokohama, Kanagawa 230-0045, Japan;1. Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand;2. Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand (CASAF, NRU-KU, Thailand);3. Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria;1. Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570 020, India;2. Academy of Scientific and Innovative Research (AcSIR), CSIR-CFTRI Campus, Mysuru 570 020, India;1. Department of Microbiology, Federal University of Technology, PMB 704, Akure, Nigeria;2. Department of Biochemistry, Federal University of Technology, PMB 704, Akure, Nigeria;1. State Key Laboratory of Biobased Material and Green Papermaking, College of Bioengineering, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250100, PR China;2. College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China
Abstract:
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 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 87.4 kcal/mol, ΔH 86.7 kcal/mol, ΔS 186.6 cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47 kcal/mol, ΔH 45.7 kcal/mol, ΔS 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.
