title: Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.
abstract: Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.
authors.affiliation: Department of Applied and Environmental Microbiology, National Agricultural Research and Innovation Centre, Budapest, Hungary.
authors.forename: Ákos
authors.initials: Á
authors.lastname: Tóth
authors.affiliation: Department of Genetics and Applied Microbiology, University of Debrecen, Hungary.
authors.forename: Terézia
authors.initials: T
authors.lastname: Barna
authors.affiliation: Department of Genetics and Applied Microbiology, University of Debrecen, Hungary.
authors.forename: Erna
authors.initials: E
authors.lastname: Szabó
authors.affiliation: Department of Genetics and Applied Microbiology, University of Debrecen, Hungary.
authors.forename: Rita
authors.initials: R
authors.lastname: Elek
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute for Biochemistry, Martinsried, Germany.
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Hubert
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute for Biochemistry, Martinsried, Germany.
authors.forename: István
authors.initials: I
authors.lastname: Nagy
authors.affiliation: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Nagy
authors.affiliation: Department of Environmental Protection and Environmental Safety, Szent István University, Gödöllő, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Kriszt
authors.affiliation: Regional University Center of Excellence in Environmental Industry, Szent István University, Gödöllő, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Táncsics
authors.affiliation: Department of Applied and Environmental Microbiology, National Agricultural Research and Innovation Centre, Budapest, Hungary.
authors.forename: József
authors.initials: J
authors.lastname: Kukolya
mesh_terms: D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D003001:Cloning, Molecular; D015870:Gene Expression; D008361:Mannosidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; C007916:hemicellulose; D008361:Mannosidases