Two extremely thermostable xylanases of the hyperthermophilic bacterium Thermotoga maritima MSB8 (original) (raw)
Related papers
2006
The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynADC; XynADA1C and XynADNC) with increased maximum activities (V max ) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynADNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear b-1,4-glucans and mixed-linkage b-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynADC and the lowest affinity with XynADNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylanbinding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.
Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima
Journal of Bacteriology, 2008
The cellular localization and processing of the endo-xylanases (1,4--D-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane ("toga") of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cellbound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.
Molecular and biotechnological aspects of xylanases
Fems Microbiology Reviews, 1999
Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. Recently cellulase-free xylanases have received great attention in the development of environmentally friendly technologies in the paper and pulp industry. In microorganisms that produce xylanases low molecular mass fragments of xylan and their positional isomers play a key role in regulating its biosynthesis. Xylanase and cellulase production appear to be regulated separately, although the pleiotropy of mutations, which causes the elimination of both genes, suggests some linkage in the synthesis of the two enzymes. Xylanases are found in a cornucopia of organisms and the genes encoding them have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Sequence analyses of xylanases have revealed distinct catalytic and cellulose binding domains, with a separate non-catalytic domain that has been reported to confer enhanced thermostability in some xylanases. Analyses of three-dimensional structures and the properties of mutants have revealed the involvement of specific tyrosine and tryptophan residues in the substrate binding site and of glutamate and aspartate residues in the catalytic mechanism. Many lines of evidence suggest that xylanases operate via a double displacement mechanism in which the anomeric configuration is retained, although some of the enzymes catalyze single displacement reactions with inversion of configuration. Based on a dendrogram obtained from amino acid sequence similarities the evolutionary relationship between xylanases is assessed. In addition the properties of xylanases from extremophilic organisms have been evaluated in terms of biotechnological applications.
Xylanase production by Thermomonospora curvata
Journal of Applied Microbiology, 1992
F.J. STUTZENBERGER AND A.B. BODINE. 1992. The thermophilic actinomycete, Thermomonospora curvata, produced <1 xylanase U/mg dry cell weight during growth in minimal medium with soluble sugars, 3–7 U/mg on purified xylan or cellulose and 28 U/mg on cotton fibres. The optimal growth temperature for xylanase production was 55°C. Cell-bound xylanase decreased from about 30% of total activity in early culture to about 2% in stationary phase. Fractionation of extracellular proteins by isoelectric focusing and size exclusion chromatography yielded three endoxylanases (XI, X2 and X3) with pI and mol. wts of pH 4.2, 7.1 and 8.4 and 36, 19 and 15 kDa respectively. X1, X2 and X3 had similar pH optima (7.8, 7.2 and 6.8) and Km for xylan (2.5, 1.4 and 2.0 mg/ml) respectively, but differed in their thermostability; half-lives at 75°C were 21 h for X1, 151 h for X2 and 302 h for X3.
Molecular Microbiology, 1995
A segment of Ttiermotoga maritima strain I\/ISB8 chromosomal DNA was isolated which encodes an endo-1,4-(J-o-xy!anase, and the nucleolide sequence of the xylanase gene, designated xynA, was determined. With a half-life of about 40 min at 9O'C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (--120 kDa) modular enzyme composed of an W-termina! signal peptide and five domains, in the order A1-A2-B-C1-C2. By comparison witb other xylanases of family 10 of glycosyl hydrolases, the central -340-amino-acid part (domain B) of XynA represents the catalytic domain. Tbe Nterminal -150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal -.170amino-acid repeated domains (C1-C2). Cellulosebinding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulosebinding domain (CBD) and thus are CBDS of a novel type. Structurally related protein segments whicb are probably also CBDs were found in other multidomain xylanolytic enzymes. Deletion of the Wterminal repeated domains or of all the non-catalytic domains resulted In substantially reduced tbermostability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme. It is argued that tbe multidomain organization of some enzymes may be one Received
Applied and Environmental Microbiology, 1995
An unusual cell-associated -1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70؇C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80؇C. The K m and k cat values when oat spelt xylan was used were 3 mg/ml and 26,680 U/mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cellassociated enzyme activity appeared in the S-layer fraction.
Microbiology, 1999
The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120567 Da. A comparison of the deduced amino acid sequence of xylanase C with known /I-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology t o family 10 xylanases. A domain homologous t o family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology t o thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar t o that of enzymes from mesophilic microorganisms. Maximum activity was found at 45OC, and the enzyme was only stable at 55 O C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous t o thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 O C instead of 4 5 OC, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 OC.
The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120567 Da. A comparison of the deduced amino acid sequence of xylanase C with known /I-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology t o family 10 xylanases. A domain homologous t o family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology t o thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar t o that of enzymes from mesophilic microorganisms. Maximum activity was found at 45OC, and the enzyme was only stable at 55 O C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous t o thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 O C instead of 4 5 OC, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 OC.