Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme

Glycogen debranching enzyme (GDE) in mammals and yeast exhibits α-1,4-transferase and α-1,6-glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; α-1,4-glycosyltransferase activity with the N-terminal domain and α-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-β-cyclodextrin (Gn-β-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1–644) showed α-1,4-transferase activity on maltotetraose (G4) and G4-β-CD, yielding various lengths of (G)_n. Surprisingly, the N-terminal domain also exhibited α-1,6-glucosidase activity against G1-β-CD and G4-β-CD, producing G1 and β-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1–644) alone has both α-1,4-transferase and the α-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes.     

Cellulase formation by Aspergillus nidulans

The optimum pH, temperature and concentration of the substrate, carboxymethyl-cellulose (CMC), for the production of cellulases by Aspergillus nidulans were found to be 3.05, 37°C and 1%, respectively. When grown on CMC under optimum conditions, it produced the three components of the cellulase complex, exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase, both in cell free as well as cell-associated states. The enzyme yields in shake cultures were lower than those obtained during stationary cultivation. Among the defined substrates, lactose emerged as the best inducer for exo-glucanase and endo-glucanase, while β-glucosidase was best induced by pectin. Endo-glucanase production increased significantly when A. nidulans was grown on insoluble delignified lognocellulosic substrates, with the maximum being on paddy straw.It appears that the synthesis of individual components of the cellulase system of A. nidulans may not be regulated in a strictly coordinated manner.     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

The effect of seaweed composite flour on the textural properties of dough and bread

Seaweeds as food and seaweed-derived food flavors, colors, and nutrients are attracting considerable commercial attention. In the baking industries, hydrocolloids are of increasing importance as bread making improvers, where their use aims to improve dough handling properties, increase the quality of fresh bread, and extend the shelf life of stored bread. Seaweeds contain a significant amount of soluble polysaccharides and have the potential function as a source of dietary fiber. In this study, red seaweed (Kappaphycus alvarezii) powder was incorporated (2–8 %) with wheat flour and used to produce bread. The effect of seaweed composite flour on dough rheological properties and the quality of bread was investigated using various techniques. Farinograph tests were applied to determine the effect of seaweed powder on the rheological properties of wheat flour dough, while texture profile analysis (TPA) was used to measure the textural properties of dough as well as the final product. The results showed that the additions of seaweed powder (2–8 %) increased the water absorption of the dough. TPA results showed that the addition of seaweed powder decreased stickiness properties. Bread produced with seaweed composite flour showed higher values of firmness.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.     

Characterization of hemicellulases from thermophilic fungi

The thermophilic fungi Thermomyces lanuginosus, Malbranchea cinnamomea, Myceliophthora fergusii and the thermotolerant Aspergillus terreus were cultivated on various carbon sources, and hemicellulolytic and cellulolytic enzyme profiles were evaluated. All fungi could grow on locust bean galactomannan (LBG), Solka floc, wheat bran and pectin, except T. lanuginosus, which failed to utilize LBG for growth. Different levels of cellulase and hemicellulase activities were produced by these fungal strains. Depending on the carbon source, variable ratios of thermostable hydrolytic enzymes were obtained, which may be useful in various applications. All strains were found to secrete xylanolytic and mannanolytic enzymes. Generally, LBG was the most efficient carbon source to induce mannanase activities, although T. lanuginosus was able to produce mannanase only on wheat bran as a carbon source. Xylanolytic activities were usually highest on wheat bran medium, but in contrast to other investigated fungi, xylanase production by M. fergusii was enhanced on pectin medium. Preliminary thermostability screening indicated that among the investigated species, thermotolerant glycosidases can be found. Some of the accessory activities, including the α-arabinosidase activity, were surprisingly high. The capability of the produced enzymes to improve the hydrolysis of lignocellulosic pretreated substrate was evaluated and revealed potential for these enzymes.     

Carbohydrate-hydrolyzing enzyme ratios during fungal degradation of woody and non-woody lignocellulose substrates

Understanding the order that enzymes are secreted during lignocellulosic degradation is relevant both to better understanding basic fungal degradation mechanisms and to industrial attempts to control reactions for biofuels production and other bioprocessing technology. Much is known about the enzymes that are produced and their effect on individual substrates, but little is known about temporal variation and relative enzyme activity on different lignocellulosics substrates. Wood decay fungi Trametes versicolor and Postia placenta were grown in liquid culture with different substrates (aspen, pine, corn stover, prairie grass and alfalfa) over a 16-week period. Samples of liquid media were taken every 2 weeks for endoglucanase, β-glucosidase and xylanase activity measurement. Endoglucanase:β-glucosidase:xylanase ratios varied for both fungi over the sampling period. T. versicolor showed large differences in cellulase enzyme (total cellulase:endoglucanase:β-glucosidase) composition when grown on woody substrates compared with non-woody substrates; there were also difference between the two wood types. This research presents evidence that the ratio of carbohydrate-hydrolyzing enzymes secreted by fungi is not influenced solely by lignin:carbohydrate content of the substrate and other factors including cell anatomy and constituent composition have some control on enzyme production. This provides a useful and broad survey of natural adaptations to various plant tissues relevant to bioenergy and general bioprospecting.     

Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum

The enzymes produced by two thermophilic fungi claimed to produce heat-stable cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been compared to those of two mesophilic fungi on the basis of the following criteria: polysaccharolytic spectrum, heat and pH effects on stability and on activity of the different enzymes, and the ability to hydrolyse raw natural substrates. The cellulases produced by one of the thermophiles, Sporotrichum cellulophilum, appeared to be as heat-labile as those from the mesophile Trichoderma reesei; moreover, the former enzyme preparation is the least efficient of the four tested. Thielavia terrestris enzymes are the most thermostable; on the basis of the other properties tested, T. terrestris enzymes are comparable to, or in some cases better than, those from mesophilic strains. However, the differences are not so great as to compensate for the much lower productivity of T. terrestris compared to the improved T. reesei and Penicillium sp. strains.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

A recruited protease is involved in catabolism of pyrimidines

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-β-alanine to β-alanine, is catalyzed by two β-alanine synthase (βASase, EC 3.5.1.6) subfamilies. We show that the “prototype” eukaryote βASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-βA compared with a representative of fungal βASases, the yeast Saccharomyces kluyveri βASase, which has a high K_m value (71 mM). S. kluyveri βASase is specifically inhibited by dipeptides and tripeptides, and the apparent K_i value of glycyl-glycine is in the same range as the substrate K_m. We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal βASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.     

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.     

Granular Formulations of Steinernema carpocapsae (strain All) (Nematoda: Rhabditida) with Improved Shelf Life

Shelf life (nematode survival) of Steinernema carpocapsae (strain All) nematodes at 21 C in "Pesta" granules, made by a pasta-like process, was increased from 8 to 26 weeks by incorporating low concentrations of formaldehyde. Pesta samples containing an average of 427,000 nematodes/g were prepared with wheat flour (semolina or bread flour), kaolin, bentonite, peat moss, nematode slurry, and formaldehyde (0-1.4% w/w) and were dried to a water content of 23.6-26.9%. Nematodes emerged from Pesta (S. carpocapsae) granules when placed in water or on moist filter paper. Incorporation of 0.2% w/w formaldehyde (nominal; 0.05% by analysis) was optimum for increasing nematode survival in semolina-based Pesta, and also inhibited fungal growth on the granules. Bread flour Pesta samples prepared by formaldehyde addition to the nematode slurry prior to dough preparation, rather than by addition to a mixture of dry ingredients, had longer shelf life. Nematodes recovered from granules made with 0.2% formaldehyde and stored 20 weeks at 21 C caused 100% mortality of wax moth (Galleria mellonella) larvae.     

Production of thermostable hydrolases (cellulases and xylanase) from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK: a potential fungus

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20 % inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90 %, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH^? scavenging, FRAP and in vivo antioxidant capacity against H₂O₂-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Some Bacillus thuringiensis strains secrete β-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of β-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of β-exotoxin production. With the aim of improving the fly β-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel β-exotoxin bioassay. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between β-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for β-exotoxins.     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.