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Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.
Microbiol Mol Biol Rev 2000 Sep
PMID:Thermophilic fungi: their physiology and enzymes. 1097 22

The type XIII xylan-binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was found to be structurally similar to the ricin B chain which recognizes the non-reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575-585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose-binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose-based polysaccharides. Moreover, it was found that the sugar-binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar-binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar-binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.
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PMID:Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86. 1102 66

Two Euphausia superba Dana endo-1.4-beta-xylanases (A, and B), hydrolysing xylan in the same manner as the enzyme classified as EC 3.2.1.8, were isolated and purified. (2) The enzymes were distinguished by their molecular mass and charge, affinities towards the oat xylan (Km of 4.1 and 7.7 mg ml(-1), respectively), values of activation energy in oat xylan hydrolysis (35.5 and 42.5 kJ mol(-1), respectively), as well as the way in which they split the substrate. (3) In vitro they showed the same optimal temperature (37-40 degrees C), optimal pH (5.7-6.0), very low thermostability, and were stabilized and activated by Ca2+ and Mg2+ ions, as well as by some unidentified substances with molecular mass less than 17 kDa, present in crude extracts of krill.
Comp Biochem Physiol B Biochem Mol Biol 2000 Nov
PMID:Purification and characterization of two endo-1,4-beta-xylanases from Antarctic krill, Euphausia superba Dana. 1112 63

The objectives of this study were to evaluate the effect of diet on the colonisation by Campylobacter jejuni of the chick caeca, and to determine whether the viscosity of the intestinal contents and mucin carbohydrates were altered by the diet. The diets investigated were maize based, wheat-based or wheat-based supplemented with xylanase. The xylanase-supplemented diet reduced the viscosity and lowered the numbers of Camp. jejuni. Feeding the enzyme-supplemented diet increased the amount of neutral and sulphated mucins in the goblet cells of the small and large intestines and caecum. An abundance of sulphated and carboxylated mucins was seen in the surface goblet cells of the large intestine with the maize- and wheat-based diets. Both the diet supplemented with xylanase and the maize diets increased crypt-surface glycosylation of the sialic acid residues. The analysed data from the combined sites showed significant differences in the amount of neutral and acidic mucins when comparing the wheat and the wheat plus xylanase diets. However, no changes were shown in the staining intensity of sulphated mucins between the three diets. Significant differences in the glycosylation of sialic acid and in the N-acetylglucosamine residues were shown between dietary groups. These results provide evidence that the wheat diet supplemented with xylanase leads to greater changes in the mucin composition and carbohydrate expression of goblet cell glycoconjugates, which are associated with a reduction in intestinal viscosity and decreased numbers of Camp. jejuni.
Cell Mol Life Sci 2000 Nov
PMID:Diet influences the colonisation of Campylobacter jejuni and distribution of mucin carbohydrates in the chick intestinal tract. 1113 Jan 83

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.
J Mol Biol 2002 Feb 08
PMID:Crystal structures of the sugar complexes of Streptomyces olivaceoviridis E-86 xylanase: sugar binding structure of the family 13 carbohydrate binding module. 1182 3

Two alkaline xylanases designated as "A" and "C", respectively, were isolated from the culture filtrates of the alkalophilic Bacillus grown on a wheat bran-yeast extract medium. The two xylanases occurred in the culture filtrate in a ratio of 10:90. These xylanases were purified to homogeneity on a CM-Sephadex matrix followed by further separation of Xylanase "A" on a phenyl sepharose column and preparative electrophoresis. The two xylanases differed considerably in their physico-chemical properties, kinetics and in their mode of action. Xylanase "C" had a molecular weight of 25,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was a cationic protein with a pI of 8.9. In contrast xylanase "A" had a molecular weight of 45,000 with a pI of 5.3. The two xylanases showed distinct differences in their hydrolysis pattern. Xylanase "A" produced comparatively larger amounts of small molecular weight oligosaccharides and xylose namely xylotriose (X(3)), xylobiose (X(2)) and xylose even in the initial stages of hydrolysis (2 and 5 h) while xylanase "C" produced negligible amounts of X(2) and no xylose for the same period of incubation. At 24 h only traces of xylose was produced by xylanase "C" while substantial amounts of the monomer was produced by xylanase A in 24 h. Xylanase "A" had a broad pH optimum ranging from pH 6.0-10.0 at 40-60 degrees C while xylanase "C" had an optimum pH of 8.0 at 40-60 degrees C. Xylanases "A" and "C" differed in their K(m) and V(max) values. Xylanase "A" had a K(m) of 1.67 mg/ml and a V(max) of 3.85 x 10(2) micromol/ml/min, whereas xylanase "C" had a K(m) of 10 mg/ml and a V(max) of 1.43 x 10(4) micromol/ml/min.
J Biochem Mol Biol Biophys 2002 Oct
PMID:Characterization of alkaline thermoactive cellulase-free xylanases from alkalophilic Bacillus (NCL 87-6-10). 1238 68

The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.
J Biochem Mol Biol Biophys 2002 Oct
PMID:Cloning of high activity xylanase gene from Bacillus pumilus PJ19. 1238 74

Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs.
J Mol Biol 2003 Mar 28
PMID:Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains. 1263 60

Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man(1) to Man(4). MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.
J Mol Microbiol Biotechnol 2003
PMID:O-glycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris. 1267 59

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation.
J Mol Biol 2003 Apr 25
PMID:Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. 1269 50


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