Xylan is the principal type of hemicellulose. It is a linear polymer of beta-D-xylopyranosyl units linked by (1-4) glycosidic bonds. In nature, the polysaccharide backbone may be added to 4-O-methyl-alpha-D-glucuronopyranosyl units, acetyl groups, alpha-L-arabinofuranosyl, etc., in variable proportions. An enzymatic complex is responsible for the hydrolysis of xylan, but the main enzymes involved are endo-1,4-beta-xylanase and beta-xylosidase. These enzymes are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect, seeds, etc., but the principal commercial source is filamentous fungi. Recently, there has been much industrial interest in xylan and its hydrolytic enzymatic complex, as a supplement in animal feed, for the manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp, ethanol and xylitol production. This review describes some properties of xylan and its metabolism, as well as the biochemical properties of xylanases and their commercial applications.

A beta-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45 degrees C. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass 132 kDa by gel filtration and SDS-PAGE. Treatment with endoglycosidase H resulted in a protein with a molecular mass of 104 kDa. The enzyme was a glycoprotein with 43.5% carbohydrate content and exhibited a pl of 3.7. Optima of temperature and pH were 75 degrees C and 4.0-4.5, respectively. The activity was stable at 60 degrees C and had a Km of 2.36 mM for p-nitrophenyl-beta-D-xylopiranoside. The enzyme did not exhibit xylanase, cellulase, galactosidase or arabinosidase activities. The purified enzyme was active against natural substrates, such as xylobiose and xylotriose.

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

No abstract

No abstract

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic beta- D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of beta- D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial beta-xylosidase induced by xylan was purified using a procedure that included heating at 50 degrees C, ammonium sulfate fractioning (30-75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified beta- D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60 degrees C and 5.0, respectively. beta- D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60 degrees C and has half-lives of 11 and 30 min at 65 degrees C in the absence or presence of calcium, respectively. The purified beta- D-xylosidase hydrolyzed p-nitrophenol-beta- D-xylopyranoside and p-nitrophenol-beta- D-glucopyranoside, exhibiting apparent K(m) and V(max) values of 1.3 mM, 88 micromol min(-1) protein(-1) and 0.5 mM, 20 micromol min(-1) protein(-1), respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true beta- D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most beta-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum beta-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.

The filamentous fungus Aspergillus caespitosus was a good producer of intracellular and extracellular invertases under submerged (SbmF) or solid-state fermentation (SSF), using agroindustrial residues, such as wheat bran, as carbon source. The production of extracellular enzyme under SSF at 30°C, for 72h, was enhanced using SR salt solution (1:1, w/v) to humidify the substrate. The extracellular activity under SSF using wheat bran was around 5.5-fold higher than that obtained in SbmF (Khanna medium) with the same carbon source. However, the production of enzyme with wheat bran plus oat meal was 2.2-fold higher than wheat bran isolated. The enzymatic production was affected by supplementation with nitrogen and phosphate sources. The addition of glucose in SbmF and SSF promoted the decreasing of extracellular activity, but the intracellular form obtained in SbmF was enhanced 3-5-fold. The invertase produced in SSF exhibited optimum temperature at 50°C while the extra-and intracellular enzymes produced in SbmF exhibited maximal activities at 60°C. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50°C.

No abstract