A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus

Abstract

Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

“…AF013989), and Rhodothermus marinus ManA (31.1% identity and 48.9% similarity among 90 amino acids; accession no. X90947) (16). In particular, the putative catalytic domain of TxyA from strain XY-234 showed significant homology with ␤-1,3-xylanases such as Vibrio sp.…”

An endo-β-1,4-mannanase, AkMan, from the common sea hare Aplysia kurodai

“…-1,4-Mannanase has been isolated from bacteria (Araki, 1983 ;Akino et al, 1988;Talbot et al, 1990;Braithwaite et al, 1995;Nakajima and Matsuura, 1997;Li et al, 2000;Politz et al, 2000;Kansoh and Nagieb, 2004;Li et al, 2006), fungi (Johnson, 1990;Stalbrand et al, 1993;Kurakake et al, 2001;Puchart et al, 2004;Naganagouda et al, 2009), higher plants (Shimahara et al, 1975;Marraccini et al, 2001), and mollusks (Yamaura and Matsumoto, 1993;Yamaura et al, 1996;Xu et al, 2002a andOotsuka et al, 2006). Compared with bacterial and fungal enzymes, molluscan enzymes have not been well investigated, i.e., only four -1,4-mannanases have been investigated in Pomacea insularus (Yamaura and Matsumoto, 1993), Littorina brevicula (Yamaura et al, 1996), Mytilus edulis (Xu et al, 2002a and b), and Haliotis discus hannai (Ootsuka et.…”

Vitellibacter vladivostokensis gen. nov., sp. nov., a new member of the phylum Cytophaga–Flavobacterium–Bacteroides

“…These bacteria are heterotrophic, usually pigmented, motile by gliding or non-motile microorganisms, possess highly active enzymes and occupy different ecological niches (Reichenbach, 1992;Barbeyron et al, 2000;Humphry et al, 2001;Politz et al, 2000).…”