Characterization and sequence of a Thermomonospora fusca xylanase

Abstract

TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. The enzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed by plasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after 18 h at 75°C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The Vmax values for TfxA and the fragment are 600 and 540 ,umol/min/mg, respectively, while the Kms are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequences of 12 other xylanases from seven different organisms and belongs to family G. Two glutamic acid residues, previously identified as essential for catalytic activity in BaciUlus pumilus XynA, are conserved in all 13 proteins. TfxA is the only thermophilic xylanase in family G as well as one of only two family-G xylanases to contain a binding domain. Xylanases cleave the 3-1,4 linkage of xylan, a polymer with a linear backbone of P-1,4-D-xylopyranoside residues, which are commonly substituted with acetyl, arabinosyl, and glucuronosyl residues. Xylan is a major component of hemicellulose from monocots and is usually associated with the cellulose and lignin components of plant cell walls. Numerous bacteria and fungi grow on xylan as a carbon source by using a variety of enzymes, including exoxylanases, endoxylanases, ,B-xylosidases, c-glucuronidases, a-arabinofuranosidases, and esterases (3, 5, 30, 33). Zymogram analysis of isoelectric focusing gels has revealed six protein bands with xylanase activity from Thermomonospora fusca BD21 (1) and four from T. fusca YX (9). The gene that codes for one of these, xynA, has been cloned and

Production of cutinase byThermomonospora fuscaATCC 27730

“…Most Thermomonospora species are moderate thermophiles exhibiting good growth at 50°C with some strains capable of growth up to 60°C. The thermophiles T. fusca and T. curvata produce cellulose-and xylose-degrading enzymes (Wilson 1988;Bachmann and McCarthy 1991;Irwin et al 1994;Stutzenberger 1994;Lin and Stutzenberger 1995) as might be expected, based on their natural habitats. In this study, the ability of Thermomonospora species to produce cutin-degrading enzymes or cutinases was determined.…”

The methylotrophic yeast as a host for the expression and production of thermostable xylanase from the bacterium

“…pastoris is capable of adding N-and O-linked carbohydrate moieties to secreted proteins [20]. N-glycosylation occurs at the amide nitrogen of Asn-residues, when found in the consensus Asn-Xaa-Ser/Thr, and the N-linked oligosaccharides formed in P. pastoris are generally in the range (Man [8][9][10][11][12][13][14] GlcNAc 2 ) which is shorter than those of S. cerevisiae [47]. Despite this shorter length, it is still possible to determine N-glycosylation by catalysing hydrolysis of linkages in the N-linked oligosaccharide (by Endo H or PNGase F), followed by separation on SDS-PAGE in order to demonstrate shifts in mobility.…”

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli

“…Xylanase from Neocallimastix frontalis does not require glycosylation for enzyme activity and has been produced in its active form by E. coli (Durand et al 1996). Xylanase (E2) from T. fusca is lightly glycosylated and the cloned protein in S. lividans lacks the natural glycosylation (Irwin et al 1994).…”

“…The supernatant¯uid after centrifugation, referred to as intracellular extract, was assayed for xylanase activity. Xylanase activity was determined as described earlier (Irwin et al 1994). Protein was determined by the method of Bradford (1976).…”

“…The N-terminal domain (Domain A) of xynA showed signi®cant homology to other xylanases (59% sequence identity with xynA of Bacillus circulans (Yang et al 1988) and Bacillus subtilis (Lapidus et al 1997), 57% identity with the xylanase I precursor of Aeromonas caviae (Kubata et al 1992), 52% identity with xynD of Cellulomonas ®mi (Clarke et al 1996), 52% identity with Thermomonospora fusca (Irwin et al 1994) and Bacillus stearothermophilus (Cho and Choi 1997), and 50% identity with xynC of Streptomyces lividans (Georis 1996)). The most striking homology was the 59% identity between the catalytic domain of P. stipitis xylanase and a xylanase from B. circulans.…”