Maria de Lourdes Teixeira de Moraes Polizeli

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.

Purification and properties of a thermostable extracellular β-D-xylosidase produced by a thermotolerant Aspergillus phoenicis

A β-D-xylosidase was purified from cultures of a thermotolerant strain of Aspergillus phoenicis grown on xylan at 45°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 pI of 3.7. Optima of temperature and pH were 75°C and 4.0–4.5, respectively. The activity was stable at 60°C and had a Km of 2.36 mM for p-nitrophenyl-β-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. Journal of Industrial Microbiology & Biotechnology (2001) 26, 156–160.

Screening of filamentous fungi for production of enzymes of biotechnological interest

Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the Sao Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C-8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of Sao Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.

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].

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.

Engineering Bifunctional Laccase-Xylanase Chimeras for Improved Catalytic Performance

Background: Rational design methods can be used to create chimeric enzymes with novel catalytic combinations. Results: Bifunctional enzymes combining xylanase and laccase activities showed enhanced catalytic activity and stability. Conclusion: Formation of an inter-domain interface alters enzyme conformation that enhances catalytic performance of the chimera. Significance: Deeper understanding of structural principles of protein fusion can improve the design of novel catalysts. Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built replacing the wild-type xynA gene by a thermostable variant (xynAG3) previously obtained by in vitro molecular evolution. Kinetic measurements demonstrated that the pH and temperature optima of the catalytic domains in the chimeras were altered by less than 0.5 pH units and 5 °C, respectively, when compared with the parental enzymes. In contrast, the catalytic efficiency (kcat/Km) of the laccase activity in both chimeras was 2-fold higher than for the parental laccase. Molecular dynamics simulations of the CotA-XynA chimera indicated that the two domains are in close contact, which was confirmed by the low resolution structure obtained by small angle x-ray scattering. The simulation also indicates that the formation of the inter-domain interface causes the dislocation of the loop comprising residues Leu-558 to Lys-573 in the laccase domain, resulting in a more accessible active site and exposing the type I Cu2+ ion to the solvent. These structural changes are consistent with the results from UV-visible electronic and EPR spectroscopy experiments of the type I copper between the native and chimeric enzymes and are likely to contribute to the observed increase in catalytic turnover number.

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology

BackgroundThe search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.ResultsXylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.ConclusionsMpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg2+ (27%), Zn2+ (21%), Ba2+ (8%) and Mn2+ (5%).Km and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus.

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.