Studies on the Chitinolytic Enzymes of Black-koji Mold

The mode of degradation of glycol chitin and chitin by two enzyme fractions separated from Aspergillus niger was investigated. One of the enzyme rapidly cleaved the endo-β-glucosaminidic bonds in the polysaccharide chain, forming chitodextrin and oligosaccharides, while the other produced monosaccharide as a main product in the degradation. The successive action of the two enzymes was also examined. Intermediate products in the enzymatic degradation were surveyed using paper and column chromatography. Also, the over-all pattern of degradation of glycol chitin and chitin by the chitinase system of Aspergillus niger was discussed.     

Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4

Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves alpha-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acids in the backbone of rhamnogalacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from Aspergillus aculeatus has been determined to 1.5 A resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non-catalytic domains from other carbohydrate active enzymes.     

Mapping the polysaccharide degradation potential of Aspergillus niger

ABSTRACT: BACKGROUND: The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. RESULTS: Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. CONCLUSIONS: The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.     

Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus

A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, 1H and 13C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.     

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily‐accessible hosts, such as Pichia pastoris, can help in revealing these effects.     

The utilisation of galactose by an Aspergillus nidulans mutant lacking galactose phosphate-UDP glucose transferase and its relation to cell wall synthesis

Summary A mutant of Aspergillus nidulans lacking galactose phosphate-UDP glucose transferase could not grow on galactose but incorporated this sugar into cell constituents when supplied with another carbon source. 75% of the radioactivity taken up was found in the galactose and glucose monomers of the hyphal wall. Most of the remaining label was in a cytoplasmic polysaccharide and in free galactose and galactose phosphate. The composition of the cytoplasmic polysaccharide resembled that of the wall polymers. These findings are taken to indicate that enzymes not connected with the Leloir pathway can activate and epimerise galactose and that polymeric wall precursors may be present in the cytoplasm. The specific labelling obtained with galactose was combined with radioautography to show that glucose and galactose containing polymers are incorporated into the hyphal wall at the growing tip.     

Allescheria boydii and Aspergillus fumigatus Skin Test Antigens

May, Lewis K. (Woman's Medical College of Pennsylvania, Philadelphia), Ralph A. Knight, and H. William Harris. Allescheria boydii and Aspergillus fumigatus skin test antigens. J. Bacteriol. 91:2155-2157. 1966.-Protein and polysaccharide fractions were extracted from culture filtrates of Allescheria boydii and Aspergillus fumigatus by the methods of Seibert and of Heidelberger, and injected intradermally into guinea pigs previously infected with these fungi. The diameter of erythema and induration was determined at 8, 24, and 48 hr. The protein and polysaccharide antigens yielded specific skin reactions in homologously infected guinea pigs. Erythema appeared at 8 hr with both the protein and polysaccharide antigens. At this time, the polysaccharide skin tests showed erythema and a central blanched wheal. A similar wheal was not observed with the protein. The erythema of the polysaccharide reaction began fading at 24 hr, whereas the protein reaction remained unchanged through 48 hr with both antigens. In guinea pigs, the area of erythema was more constant and thus easier to measure than was induration.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Characterization of an Aspergillus nidulans L-arabitol dehydrogenase mutant

Abstract The degradation pathway for L-arabinose, which consists of a sequence of alternating reduction and oxidation reactions prior to ultimate phosphorylation, was studied in Aspergillus nidulans wild-type as well as in an L-arabinose non-utilizing mutant. The inability of the mutant to use L-arabinose was caused by the absence of L-arabitol dehydrogenase activity. The effect of the mutation on polyol accumulation patterns was studied upon growth on various carbon sources. The presence of L-arabinose resulted in intracellular accumulation of arabitol in this mutant. Moreover, the mutant secreted arabitol under these conditions and, in contrast to the wild-type, featured enhanced expression of enzymes involved in L-arabinose catabolism as well as of extracellular glycosyl hydrolases involved in degradation of the plant cell wall polysaccharide L-arabinan.     

Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi

Aspergillus niger and Aspergillus oryzae were co-cultivated with each other and with Magnaporthe grisea or Phanerochaete chrysosporium, respectively. Enzyme assays for plant polysaccharide and lignin-degrading enzymes showed that co-cultivation can improve extracellular enzyme production. Highest ??-glucosidase, ??-cellobiohydrolase, ??-galactosidase, and laccase activities were found for A. oryzae in combination with other fungi, in particular with P. chrysosporium. Highest ??-xylosidase activity was obtained when A. niger was co-cultivated with P. chrysosporium. SDS-PAGE protein profiles demonstrated that A. niger and A. oryzae contributed most to the overall enzyme activities found in the culture medium of the mixed cultivations. These data demonstrate that co-cultivation of two major industrial fungi, A. niger and A. oryzae, results in improved production of biotechnologically relevant enzymes.     

The stability of extracellular β-glucosidase fromAspergillus niger is significantly enhanced by non-covalently attached polysaccharides

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes ofAspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of β-glucosidase. The polysaccharide-β-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free β-glucosidase. The β-glucosidase-polysaccharide complex was 18-, 36-, 40-, and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free β-glucosidase. The activation energy of polysaccharide-complexed β-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/K _m) for 4-nitrophenyl β-d-glucopyranoside between polysaccharide-free and polysaccharide-complexed β-glucosidase. We suggest that the function of these polysaccharides secreted by fungi includingA. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.     

GH10 XynF1 and Xyn11A: the predominant xylanase identified in the profiling of extracellular proteome of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> LC1

Advanced techniques of enzyme production and purification have become prerequisite due to their diverse industrial applications. There is an utmost requirement for screening of new strains capable of synthesising industrially useful enzymes. The present study reports the production and profiling of extracellular proteins expressed by the newly isolated strain of a filamentous fungus, Aspergillus oryzae LC1. The extracellular enzyme production was done by submerged fermentation using Mendel’s and Sternberg’s medium (MSM), and its optimisation was done using one factor at a time (OFAT). The presence of xylanase was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. In addition, the profiling of extracellular proteome of Aspergillus oryzae LC1 was carried out by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). In this study, media optimisation showed 5.7-fold increase in xylanase activity. The multiple bands observed in zymography revealed the presence of various forms of xylanase. A total of 73 proteins were identified in LC-MS/MS analysis. Functional classification showed that the hydrolytic enzymes consisted of 48% glycoside hydrolase, 11% proteases, 1% polysaccharide lyase and esterase’s, 9% oxidoreductases and 30% other proteins. A total of 26 families of glycosidic hydrolase were detected with other protein families such as serine peptidase, S, LysM, G-D-S-L, M35, carboxyl esterase (CE1), pectate lyase (PL) and oxidoreductases. Among the huge diversity of synergistically acting biomass cleaving enzymes, endo-1, 4-β xylanase with isoforms: xyn F1, xyn B, β xylanase and xyn 11A belonging to GH10 family covered the major portion of the total percentage of identified proteins. As per our knowledge, this is the first report of extracellular proteome analysis of Aspergillus oryzae LC1 suggesting its capability for recombinant expression and evaluation in hemicellulose deconstruction applications.     

Isolation and chemical characterization of compounds isolated fromAspergillus flavus conidia

Three types of heterogenous preparations and four types of preparations of polysaccharide nature were obtained in studies aimed at the isolation of active compounds fromAspergillus flavus conidia bearing their biological stimulatory activity. Extraction with trichloroacetic acid at 0 °C yielded a preparation in which the protein component predominated over the polysaccharide moiety at a ratio of 3: 1. In the preparation isolated from the phenolic phase of the phenol—water mixture at 68 °C the protein polysaccharide ratio was 1 : 1. In the material extracted in the aquoues phase and in that obtained by extraction with acetic acid at 100 °C the polysaccharide portion highly predominated (8 : 1 and 7 : 1 respectively).     

N+注入选育黑曲霉益生菌及其突变菌株产酶条件的研究

以益生菌株黑曲霉AN01为材料,经N 多次诱变得突变益生菌株AN03。结果表明,出发益生菌株AN01酸性蛋白酶、纤维素酶和果胶酶的酶活分别由原来的71.6Ug、141.7Ug和264.8Ug相继提高到996.5Ug、940.4Ug和906.5Ug。突变益生菌株AN03经传5代培养,产酶特性稳定。试验还研究了变突变益生菌株AN03最佳产酶条件,培养基为每升含麸皮105g,玉米芯105g,豆粕105g,氯化铵16g,pH5.0。30℃培养4d。     

Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences

We cloned two genes for alkaline pectate lyase, pel-4A and pel-4B, from alkaline pectinase-producing alkaliphilic Bacillus sp. strain P-4-N. The pel-4B gene product Pel-4B was purified to homogeneity and characterized. The purified enzyme had an isoelectric point of pH 9.6 and a molecular mass of 35 kDa, values close to those of the pel-4A gene product Pel-4A. The pH and temperature optima for activity were as high as 11.5 and 70 degrees C, respectively, which are the highest among the pectate lyases reported to date. The mature Pel-4B (304 amino acids; 33,868 Da) was structurally related to the enzymes in the polysaccharide lyase family 1 and showed 35.6% identity with Pel-4A on the amino acid level. It showed significant homology to other pectate lyases in the same family, such as the enzymes from alkaliphilic Bacillus sp. strains KSM-P7 and KSM-P103 and the fungi Aspergillus nidulans and Colletotrichum gloeosporioides f. sp. malvae.     

Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.