Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus niger and other black aspergilli

Wild type Aspergillus niger isolates from different biotopes from all over the world were compared to each other and to the type strains of other black Aspergillus species with respect to growth and extracellular enzyme profiles. The origin of the A. niger isolate did not result in differences in growth profile with respect to monomeric or polymeric carbon sources. Differences were observed in the growth rate of the A. niger isolates, but these were observed on all carbon sources and not specific for a particular carbon source. In contrast, carbon source specific differences were observed between the different species. Aspergillus brasiliensis is the only species able to grow on D-galactose, and A. aculeatus had significantly better growth on Locus Bean gum than the other species. Only small differences were found in the extracellular enzyme profile of the A. niger isolates during growth on wheat bran, while large differences were observed in the profiles of the different black aspergilli. In addition, differences were observed in temperature profiles between the black Aspergillus species, but not between the A. niger isolates, demonstrating no isolate-specific adaptations to the environment

A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed

Expression of Trichoderma reesei β-Mannanase in Tobacco Chloroplasts and Its Utilization in Lignocellulosic Woody Biomass Hydrolysis

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40°C to 70°C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6–7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families

Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review

Lignocelluloses are often a major or sometimes the sole components of different waste streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these materials is the first step for either digestion to biogas (methane) or fermentation to ethanol. However, enzymatic hydrolysis of lignocelluloses with no pretreatment is usually not so effective because of high stability of the materials to enzymatic or bacterial attacks. The present work is dedicated to reviewing the methods that have been studied for pretreatment of lignocellulosic wastes for conversion to ethanol or biogas. Effective parameters in pretreatment of lignocelluloses, such as crystallinity, accessible surface area, and protection by lignin and hemicellulose are described first. Then, several pretreatment methods are discussed and their effects on improvement in ethanol and/or biogas production are described. They include milling, irradiation, microwave, steam explosion, ammonia fiber explosion (AFEX), supercritical CO2 and its explosion, alkaline hydrolysis, liquid hot-water pretreatment, organosolv processes, wet oxidation, ozonolysis, dilute-and concentrated-acid hydrolyses, and biological pretreatments

Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level

<p>Abstract</p> <p>Background</p> <p><it>Rhizopus oryzae </it>is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses.</p> <p>Results</p> <p>Carbohydrate Active enzyme (CAZy) annotation of the <it>R. oryzae </it>identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs) and a high number of glycosyl transferases (GTs) and carbohydrate esterases (CEs). A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars), chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of <it>R. oryzae </it>to its environment.</p> <p>Conclusions</p> <p>CAZy analyses of the genome of the zygomycete fungus <it>R. oryzae </it>and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.</p

Galactoglucomannan-degrading enzymes from Aspergillus niger

Galactoglucomannan and galactomannan are among the most abundant plant polysaccharides in nature. The central enzymes involved in their degradation, i.e., endo-1,4-beta-mannanase (beta-mannanase, EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), and alpha-galactosidase (EC 3.2.1.22) were purified from the filamentous fungus Aspergillus niger and characterized with respect to physical properties and substrate specificity. The beta-mannanase degraded polymeric ivory nut mannan to mainly mannobiose and mannotriose, and NMR analysis of the hydrolysis of mannopentaose showed that it acts by the retaining mechanism. Unlike some other beta-mannanases it probably lacks a cellulose-binding domain, since it was unable to adsorb on cellulose. The preferred substrates for the beta-mannosidase (MndA) were linear manno-oligosaccharides, but it also hydrolyzed terminal non-reducing mannose residues from polymeric mannan and galactomannan. It was able to cleave up to, but not beyond, a galactose side group. Two alpha-galactosidases (AglB and AglC) with different substrate specificities were purified. AglC had a strong preference for terminal non-reducing galactose residues, whereas AglB hydrolyzed galactose residues linked to terminal as well as internal residues of the substrate main chain. The cooperation of individual enzymes during degradation of polymeric mannan, galactomannan, and galactomanno-oligosaccharides were investigated. The genes encoding AglC and MndA were cloned, characterized, and overexpressed in A. niger. On the basis of sequence comparisons, AglC could be assigned to family 36 of the glycosyl hydrolases and MndA could be assigned to family 2. The expression patterns of three alpha-galactosidase genes (aglA, aglB, and aglC) and a beta-mannosidase gene (mndA) during growth of A. niger on different carbon sources were studied by Northern analysis. The aglB and mndA genes were expressed early and in high levels on galactomannan, indicating that the corresponding enzymes play a key role in the degradation of this polymer

Multiple alpha-galactosidases from Aspergillus niger: purification, characterization, and substrate specificities

Enzymes with α-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major α-galactosidase forms (α-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. α-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. α-Gal I is a completely different enzyme that belongs to family 36. α-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. α-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. α-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2–5, whereas α-gal I was most stable at pH 5–6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. α-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. α-Gal I released galactose from locust bean gum only in combination with A. niger β-mannosidase. Kinetic experiments showed that α-gal I hydrolyzed p-nitrophenyl-α-Image-galactopyranoside and melibiose more efficiently than α-gal II-IV. The distinct hydrolytic and biochemical properties of α-gal I and α-gal II-IV further signifies the difference between α-galactosidases of family 27 and 36