Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

Abstract

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80؇C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa). xynA was preceded by a putative promoter at ؊35 (TTAAT) and ؊10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 38 to 96% similarity to sequences of family F ␤-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences (59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.

Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov., and emendation of the genus Thermoanaerobacterium.

“…Catalase activity was negative for both isolates, which is consistent with the genera Thermoanaerobacter (Cayol et al, 1995 ;Holt et al, 1994) and Thermoanaerobacterium (Holt et al, 1994 ;Lee et al, 1993b ;Liu et al, 1996a). Neither isolate reduced nitrate or produced indole.…”

“…These bacteria are likely to exhibit a surface-layer protein (see Fig. 2) as in related organisms (Liu et al, 1996a ;Brechtel & Bahl, 1999), which is supported by the presence of an S-layer-like protein region observed at the C-terminal region of a mannanase gene that has been cloned and sequenced from strain KMTHCJ T (Cann et al, 1999). Strain mel2 T grew readily on agar surfaces, whereas strain KMTHCJ T did not grow readily on nutrient agar plates, but could be maintained on agar slants.…”

“…Several thermostable enzymes have either been purified or cloned from the members of this group of organisms. For example, polysaccharide-hydrolysing enzymes from this group are thermostable endoxylanases (Lee et al, 1993a ;Liu et al, 1996a) which can be used in biomass conversion and the pulp and paper industry.…”

Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase.

“…The phenotypic characteristics of isolate FH1 were also consistent with those of the Thermoanaerobacterium species, though none of the latter has been reported to produce dextranase. Members of this genus, however, are known for their ability to produce exoenzymes such as amylases, xylanases, and cellulases, which are active at temperatures above 65°C (Ganghofer et al, 1998;Lee et al, 1993;Liu et al, 1996;Shao and Wiegel, 1995;Spreinat and Antranikian, 1992). The T. thermosaccharolyticum type strain DSM571 showed no growth with dextran as the sole carbon and energy source under the employed conditions (Table 1).…”

The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together

“…They are found not only in various S-layer proteins but also in many other surface-associated proteins, e.g. the cellulosomes (Bayer et al, 2004) or other surface-associated enzymes (Liu et al, 1996). In the case of Bacillus anthracis, the aetiological agent of anthrax, the molecular basis of the interaction between SLH domains and SCWPs was established by elucidating their binding properties (Mesnage et al, 2000).…”