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  Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches

Toth, A., Barna, T., Szabo, E., Elek, R., Hubert, A., Nagy, I., et al. (2016). Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches. PLoS One, 11(5): e0155769. doi:10.1371/journal.pone.0155769.

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Toth, Akos1, Author
Barna, Terezia1, Author
Szabo, Erna1, Author
Elek, Rita1, Author
Hubert, Agnes2, Author              
Nagy, Istvan2, Author              
Nagy, Istvan1, Author
Kriszt, Balazs1, Author
Tancsics, Andras1, Author
Kukolya, Jozsef1, Author
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1external, ou_persistent22              
2Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              

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Free keywords: THERMOSTABLE BETA-MANNANASE; PROTEIN FAMILIES DATABASE; HALOTOLERANS YIM 90462(T); STREPTOMYCES-LIVIDANS; PICHIA-PASTORIS; HETEROLOGOUS EXPRESSION; THERMOMONOSPORA-FUSCA; DEGRADING ENZYMES; GENE CLONING; SP NOV.
 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 TB100(T) and T. halotolerans YIM90462(T)). 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 degrees C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of K-M and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70 degrees 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.

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Language(s): eng - English
 Dates: 2016
 Publication Status: Published online
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
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Title: PLoS One
Source Genre: Journal
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Publ. Info: San Francisco, CA : Public Library of Science
Pages: - Volume / Issue: 11 (5) Sequence Number: e0155769 Start / End Page: - Identifier: ISSN: 1932-6203
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000277850