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.
Highlights
The hemicellulose fraction of plant cell walls is mainly composed of xylan and mannan, and in case of leguminous plants high mannan content has been found in seeds
The primer design was based on the complete genome sequence of T. fusca TM51. 1362 bp DNA fragments were synthesized when T. fusca and T. alba genomic DNA served as template, but in case of T. halotolerans and T. cellulosilytica no PCR products were obtained in spite of extensive PCR optimization experiments
To capture endomannanase genes of T. halotolerans YIM90462 and T. cellulosilytica TB100, expression libraries were generated in Streptomyces lividans TK24 strain
Summary
The hemicellulose fraction of plant cell walls is mainly composed of xylan and mannan, and in case of leguminous plants high mannan content has been found in seeds. Biopolymers like cellulose or mannan are very promising raw materials for many industrial applications. Several studies have indicated that the most promising use of these biopolymers and their derivatives will be the health and food industry [2,3], since mannan degradation can produce a huge variety of biologically active oligosaccharides, which can be used as prebiotics. Galactomannan is composed of a homogenous backbone of β-1,4-linked mannose residues, that are branched with galactosyl residues, whereas galactoglucomannan has a heterogeneous backbone of β1,4-linked glucose and mannose residues; in some cases (mainly in softwoods) this backbone is acetylated. Endomannanases catalyze the random hydrolysis of the β -1,4-mannosidic backbone of the main mannan chain, α–galactosidases cleave the terminal α-1,6-linked D-galactosyl residues, and βmannosidases hydrolyze β-1,4-linked mannose residues from the non-reducing ends of various oligosaccharides [4,5]
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