• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.284-292
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• 2011

An endocellulase-free multienzyme complex was produced by a thermophilic anaerobic bacterium, Thermoanaerobacterium thermosaccharolyticum strain NOI-1, when grown on xylan. The temperature and pH optima for growth were $60^{\circ}C$ and 6.0, respectively. The bacterial cells were found to adhere to insoluble xylan and Avicel. A scanning electron microscopy analysis showed the adhesion of xylan to the cells. An endocellulase-free multienzyme complex was isolated from the crude enzyme of strain NOI-1 by affinity purification on cellulose and Sephacryl S-300 gel filtration. The molecular mass of the multienzyme complex was estimated to be about 1,200 kDa. The multienzyme complex showed one protein on native PAGE, one xylanase on a native zymogram, 21 proteins on SDS-PAGE, and 5 xylanases on a SDS zymogram. The multienzyme complex consisted of xylanase, ${\beta}$-xylosidase, ${\alpha}$-L-arabinofuranosidase, ${\beta}$-glucosidase, and cellobiohydrolase. The multienzyme complex was effective in hydrolyzing xylan and corn hulls. This is the first report of an endocellulase-free multienzyme complex produced by a thermophilic anaerobic bacterium, T. thermosaccharolyticum strain NOI-1.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1077-1086
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• 2016

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn²⁺ enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp¹⁷¹, His²⁵⁴, and His²⁷¹) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60℃ and pH 8.0 and towards DHA at 60℃ and pH 6.0. DHA reduction was the dominant reaction, with a lower K_m(DHA) of 1.08 ± 0.13 mM and V_max of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a K_m(glycerol) of 30.29 ± 3.42 mM and V_max of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60℃. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg⁴³, both related to dinucleotide binding.

• KSBB Journal
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• v.20 no.6
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• pp.431-437
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• 2005

Molecular methods were employed to investigate microorganisms in a thermophilic continuous stirred tank reactor(CSTR) used for continuous $H_2$ production. The reactor was inoculated with heat-treated anaerobic sludge and fed with a glucose-based medium. Denaturing gradient gel electrophoresis showed dynamic changes of bacterial populations in the reactor during 43 days of operation. Gas composition was constant from approximately 14 days but population shift still occurred. Populations affiliated with Fervidobactrium gondwanens and Thermoanaerobacterium thermosaccharolyticum were dominant on 21 and 41 days, respectively. Keeping pH of the medium at 5.0 could suppress methanogenic activity that was detected during initial operation period. $CH_4$ and mcrA detected in the samples obtained from the reactor or inoculum suggested the heat treatment condition employed in this study is not enough to remove methanogens in the inoculum. PCR using primer sets specific to 4 main orders of methanogens suggested that major $H_2$-consuming methanogens in the CSTR belong to the order Methanobacteriales.

• KSBB Journal
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• v.20 no.6
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• pp.425-430
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• 2005

Thermophilic $H_2$ was produced for 1 year using a bench-scale continuous stirred tank reactor(CSTR). The CSTR was inoculated with anaerobically digested sludge after heat treatment and fed with a glucose-based medium. The reactor showed relatively short start-up period(30 days) and high maximal $H_2$ yield(2.4 mol $H_2/mol$ glucose). Keeping pH 5.0 or less suppressed methanogenic activity. Bacteria affiliated with Thermoanaerobacterium thermosaccharolyticum kept being dominant from approximately 40 days as determined by DGGE. Environmental perturbation(pH or temperature) caused the decrease of biomass concentration in the reactor and the instability of reactor performance, $H_2$ production rate and $H_2$ yield. The unstable performance was accompanied with high concentration of lactate in the effluent. Taken together, the poor recovery of CSTR after perturbations could be partly explained by low biomass concentration and/or metabolic shift of the major population in the CSTR.