Héctor Francisco Terenzi

Xylanases from fungi: properties and industrial applications

Xylan is the principal type of hemicellulose. It is a linear polymer of β-D-xylopyranosyl units linked by (1–4) glycosidic bonds. In nature, the polysaccharide backbone may be added to 4-O-methyl-α-D-glucuronopyranosyl units, acetyl groups, α-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-β-xylanase and β-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.

Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa

A cAMP‐activatable Ca2+‐dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild‐type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post‐translational regulatory mechanisms, in particular cAMP‐dependent protein kinase‐mediated phosphorylation.

Effect of Carbon Source and pH on the Production and Secretion of Acid Phosphatase (EC 3.1.3.2) and Alkaline Phosphatase (EC 3.1.3.1) in Neurospora crassa

Summary: Growth conditions (carbon source and pH of the medium) affected the level and distribution of derepressed acid and alkaline phosphatases in Neurospora crassa. Regardless of the pH, the production of both acid and alkaline phosphatases was stimulated by sucrose. Irrespective of the carbon source used, at pH values higher than 7.4 the secretion of alkaline phosphatase was stimulated, while the production and secretion of acid phosphatase was restricted. The converse was true at pH values lower than 5.7. The secretion of one of these enzymes did not exclude the simultaneous secretion of the other at pH values of around 7.0. These results suggest that the pH of the medium may play an important role in the survival of the organism in phosphorus-limited environments.

A Neurospora crassa morphological mutant showing reduced adenylate cyclase activity.

Summary A Neurospora crassa morphological mutation at the crisp locus is related to a low specific activity of adenylate cyclase. This observation was made in two different strains: FGSC No. 329 and FGSC No. 326, both carrying the crisp mutation (allele N. B 123). Crosses of these strains to others having the wild type allele yield isolates with the crisp phenotype all of them having low adenylate cyclase specific activity in crude extracts. The other phenotypes obtained showed wild type levels of specific activity. Addition of 3′, 5′ dibutyryl cyclic AMP to the FGSC No. 329 cultures increased four-fold mycelial growth rate and stimulated the formation of aerial hyphae. Simultaneously, uniform conidiation on the surface of the slant was inhibited by the cyclic AMP derivative.

Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].

Effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in Neurospora crassa.

Neurospora crassa conidiospore germlings exposed to a heat shock (30–45°C) rapidly accumulated trehalose and degraded glycogen, even in the presence of cycloheximide. This phenomenon was also rapidly reversible upon return of the cells at 30°C. Trehalose accumulation at 45°C demanded an exogenous source of carbon and either glucose or glycerol fulfilled such requirement. Experiments with the cyclic AMP‐deficient cr‐1 mutant suggested that the effects of temperature shifts on trehalose level were independent of cAMP metabolism. Cells exposed at 45°C under conditions permissive for trehalose accumulation (i.e. in the presence of an assimilable carbon source) also acquired thermotolerance.

Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings

To understand the molecular mechanisms induced by stress that contribute to the development of tolerance in eukaryotic cells, the filamentous fungus Aspergillus nidulans has been chosen as a model system. Here, the response of A. nidulans germlings to heat shock is reported. The heat treatment dramatically increased the concentration of trehalose and induced the accumulation of mannitol and mRNA from the catalase gene catA. Both mannitol and catalase function to protect cells from different reactive oxygen species. Treatment with hydrogen peroxide increased A. nidulans germling viability after heat shock whilst mutants deficient in catalase were more sensitive to a 50 degrees C heat exposure. It is concluded that the defence against the lethal effects of heat exposure can be correlated with the activity of the defence system against oxidative stress.

β-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Production of thermostable invertases by Aspergillus caespitosus under submerged or solid state fermentation using agroindustrial residues as carbon source

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.