Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Production,purification, and properties of an α-l-arabinofuranosidase from Cihomitus squalens

A white-rot fungus Dichomitus squalens, when grown on 1% wheat-straw glucuronoarabinoxylan under aerated submerged conditions, secreted an α-l-arabinofuranosidase (4.3 nkat/mL). The enzyme was purified 70-fold by ammonium sulfate precipitation, chromatofocusing on PBE 94, gel filtration on Ultrogel AcA 54, rechromatofocusing on PBE 94, and lectin affinity chromatography on Concanavalin A-Ultrogel. The enzyme is a glycoprotein having a molecular weight of 60,000 and a pI of 5.1. The enzyme exhibited maximal activity at pH 3.5 and at 60°, and was fully inactivated within 30 min at 70°. The K_m value for p-nitrophenyl α-l-arabinofuranoside was 1.64mm. The α-l-arabinofuranosidase liberated arabinose from sugar-beet arabinan, wheat-straw and oat-spelt arabinoxylans, and wheat-bran heteroxylan, and was inactive towards gum arabic.     

Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae

A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced β-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a β-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30°C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K_m (30°C, pH 5.5) for soluble starch was 9 g liter⁻¹.     

Biochemical and Proteomic Characterization of a Novel Extracellular β-Glucosidase from Trichoderma citrinoviride

β-Glucosidases are of pivotal importance in bioconversion of carbonic biomass into fermentable and other useful metabolites, food industry, biotransformation, glyco-trimming of metabolome, etc. Trichoderma citrinoviride when grown on delignified Lantana camara produced a β-glucosidase and secreted it out in the medium. The extracellularly secreted β-glucosidase of T. citrinoviride was homogeneity purified and then characterized for its kinetic properties and proteomic characteristics. The 90 kDa enzyme was monomeric in nature, optimally active at pH 5.5 and the catalytic reaction rate was highest at 55°C. Uniquely, the enzyme was insensitive to inhibition by glucose (up to 5 mM). It also possessed catalytic ability of transglycosylation, as it could catalyze conversion of geraniol into its glucoside. MALDI-TOF assisted proteomic analysis revealed its high degree of sequence similarity with family 3 glycoside hydrolases.     

Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1

Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme ??45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70°C, respectively. The metal ions Na⁺, K⁺, Ca²⁺, and Co²⁺ showed partial inhibition of the activity, while Mn²⁺ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55?C70°C. It presented the highest stability after incubation at 55°C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60°C for 60 min as well as at 65 and 70°C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli.     

Investigation on the immobilization of Pseudomonas isoamylase onto polysaccharide matrices

This study examined Pseudomonas isoamylase immobilized onto polysaccharide matrices, among which included agarose, cellulose, and raw corn starch. For chemical binding of polysaccharides activated with tosyl chloride, a high specific activity of 23144?U/g-starch was obtained as compared with matrices of cellulose and agarose with 3229?U/g-cellulose and 84?U/g-agarose, respectively. For raw corn starch, isoamylase desorption occurred when the immobilized enzyme by physical adsorption was subjected to 0.05?M acetate buffer with pH?5.2 at 40?°C; this is despite the considerable affinity between the enzyme and the matrix. In contrast, no detectable activity leached from the matrix for chemical binding, regardless of whether maltose, i.e. an affinity species to isoamylase, was added. For immobilized starch-isoamylase, its optimal activity performance was obtained in broader pH?ranges of 3.5–5.5 and 5?°C higher than those of the free enzymes. More specifically, the free enzyme's activity markedly decreased within five hours while the immobilized starch-isoamylase exhibited a fairly stable behavior over a three day incubation period at 40?°C. After 175 days of storage at 4?°C, the residues of relative activity of 75% and 45% were obtained with respect to immobilized and free isoamylases, respectively.     

Conversion of cellulosic materials to ethanol

The plant cell wall can be regarded as a giant bag-like macromolecule in which crystalline bundles of cellulose are embedded in a covalently linked matrix of hemicellulose and lignin. This heterologous polymer represents the dominant form of biomass on earth and a formidable challenge for solubilization and bioconversion. Bioconversion of lignocellulose requires the saccharification of both the hemicellulose and cellulose. Hemicellulose is composed of a mixture of sugars and can be readily hydrolysed by dilute acid at 140°C to produce a syrup containing pentoses and hexoses. However, no organisms in nature rapidly and efficiently convert both pentoses and hexoses into a single product of value. Our laboratory has developed such an organism by genetic engineering. Recombinant strains of Gram-negative bacteria (Escherichia coli or Klebsiella oxytoca or Erwinia sp.) have been constructed in which genes encoding the ethanol pathway from Zymomonas mobilis (pdc and adh) were inserted into the chromosome. These strains now efficiently convert all of the component sugars of hemicellulose and (cellulose) into ethanol. The saccharification of cellulose is more difficult and more complex. An enzymatic approach is preferred but at least three classes of enzymes are needed: endoglucanase, exoglucanase, and β-glucosidase. Klebsiella oxytoca and Erwinia sp. possess the native ability to transport and metabolize cellobiose (also cellotriose, xylobiose, and xylotriose), minimizing the need for added β-glucosidase. K. oxytoca strain P2, an ethanol-producing recombinant, has been evaluated in simultaneous saccharification and fermentation experiments to determine optimal conditions and limits of performance. Temperature was varied between 32 and 40°C over a pH range of 5.0–5.8 with 100 g 1⁻¹ of crystalline cellulose (Sigmacell 50, Sigma Chemical Company, St. Louis, MO) as the substrate and commercial cellulase (Spezyme CE; Genencor, South San Francisco, CA). A broad optimum for fermentation was observed which allowed the production of over 44 g ethanol 1⁻¹ (82–87% of the maximum theoretical yield). Two optimal saccharification and fermentation conditions were identified for fermentation yield, pH 5.2 at 35°C and pH 5.5 at 32°C, which produced 47 g ethanol 1⁻¹ in 144 h (0.48 g ethanol (g cellulose) ⁻¹). Although yields were reduced at the lowest cellulase levels tested (2–5 filter paper units (g cellulose)⁻¹), ethanol production per unit enzyme was much higher.     

Immobilization of trypsin on polysaccharide film from Anacardium occidentale L. and its application as cutaneous dressing

Polysaccharide obtained from Anacardium occidentale L. gum was used for trypsin entrapment using cellulose (gaze) as a support and this preparation was applied as cutaneous wound healing. Trypsin release in vitro and the influence of pH and temperature on activity, stability and storage time of entrapped enzyme were evaluated. The preparation showed that it was still capable to release enzyme even after 48 h. Entrapped enzyme presented an optimal pH and temperature of 8.6 and 55 °C, respectively. Also, it was stable at high temperature (45 °C for 60 min) and wide range of pH, retaining 80% of its initial activity when stored for 28 days at 25 °C. Histopathological analysis of mice skin wound healing under the entrapped trypsin preparation treatment showed an acceleration of fibroblast proliferation, neovascularization of granulation tissue and stimulating effect on the epithelium formation compared to the skin wound under the treatment using preparations without trypsin. These results demonstrate that the trypsin–polysaccharide–cellulose preparation could be used in cutaneous dressing applications for wound healing.     

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri,Turkey

A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in A?ri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28^th hour of incubation and the highest α-amylase activity was obtained at the 40^th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn²⁺ (123%), whereas it was strongly inhibited 1.5 mM by Hg²⁺. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Inorganic carbon and pH effect on growth and lipid productivity of Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag photobioreactors

There has been considerable interest in cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. However, almost all mass cultures of algae are carbon-limited. Therefore, to reach a high biomass and oil productivities, the ideal selected microalgae will most likely need a source of inorganic carbon. Here, growth and lipid productivities of Tetraselmis suecica CS-187 and Chlorella sp were tested under various ranges of pH and different sources of inorganic carbon (untreated flue gas from coal-fired power plant, pure industrial CO₂, pH-adjusted using HCl and sodium bicarbonate). Biomass and lipid productivities were highest at pH 7.5 (320?±?29.9 mg biomass L^?1 day^?1and 92?±?13.1 mg lipid L^?1 day^?1) and pH 7 (407?±?5.5 mg biomass L^?1 day^?1 and 99?±?17.2 mg lipid L^?1 day^?1) for T. suecica CS-187 and Chlorella sp, respectively. In general, biomass and lipid productivities were pH 7.5?>?pH 7?>?pH 8?>?pH 6.5 and pH 7?>?pH 7.5?=?pH 8?>?pH 6.5?>?pH 6?>?pH 5.5 for T. suecica CS-187 and Chlorella sp, respectively. The effect of various inorganic carbon on growth and productivities of T. suecica (regulated at pH?=?7.5) and Chlorella sp (regulated at pH?=?7) grown in bag photobioreactors was also examined outdoor at the International Power Hazelwood, Gippsland, Victoria, Australia. The highest biomass and lipid productivities of T. suecica (51.45?±?2.67 mg biomass L^?1 day^?1 and 14.8?±?2.46 mg lipid L^?1 day^?1) and Chlorella sp (60.00?±?2.4 mg biomass L^?1 day^?1 and 13.70?±?1.35 mg lipid L^?1 day^?1) were achieved when grown using CO₂ as inorganic carbon source. No significant differences were found between CO₂ and flue gas biomass and lipid productivities. While grown using CO₂ and flue gas, biomass productivities were 10, 13 and 18 %, and 7, 14 and 19 % higher than NaHCO₃, HCl and unregulated pH for T. suecica and Chlorella sp, respectively. Addition of inorganic carbon increased specific growth rate and lipid content but reduced biomass yield and cell weight of T. suecica. Addition of inorganic carbon increased yield but did not change specific growth rate, cell weight or content of the cell weight of Chlorella sp. Both strains showed significantly higher maximum quantum yield (F_v/F_m) when grown under optimum pH.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

Secretion of alpha-amylase by Rumex virus tumors in vitro; properties and assay

The existence of an extracellular α-amylase, which is secreted by virus tumors from the roots of Rumex acetosa grown in vitro, has been demonstrated and the properties of the enzyme have been studied. As far as the authors are aware, this is the first enzyme demonstrated to be secreted from intact cells of higher plants.An assay for the quantitative determination of this enzyme is presented. This assay is based on the spectrophotometric determination of the decrease in color of the starch-iodine complex due to the activity of the amylase on the starch substrate.Among the factors discussed which affect the density of the starch-iodine complex are iodine concentration, temperature, and ionic strength.The optimum pH for activity of this enzyme is 4.6, both in acetate and citrate-phosphate buffers.At pH 4.6 in 0.02 M acetate buffer, the Q₁₀ for the hydrolysis of starch by the enzyme is 1.6 in the range of 20–40 °C. The activation energy in this range calculated by the Arrhenius equation is 8000 cal./ mole.This α-amylase is protected by calcium and is sensitive to low pH values as are the cereal α-amylases.The energy of activation for the heat denaturation of the enzyme is 43,000 cal./mole, as calculated by the Arrhenius equation.     

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.     

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome

From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited?≤?55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3–1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (>?1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg²⁺ may adopt the function of Ca²⁺. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Isolation of a Cellodextrinase from Bacteroides succinogenes

An enzyme which released the cellobiose group from p-nitrophenyl cellobioside was isolated from the periplasmic space of Bacteroides succinogenes grown on Avicel crystalline cellulose in a continuous cultivation system and separated from endoglucanases by column chromatography. The molecular weight of the enzyme was approximately 40,000, as estimated by gel filtration. The enzyme has an isoelectric point of 4.9. The enzyme exhibited low hydrolytic activity on acid-swollen cellulose and practically no activity on carboxymethyl cellulose, Avicel cellulose, and cellobiose, but it hydrolyzed p-nitrophenyl lactoside and released cellobiose from cellotriose and from higher cello-oligosaccharides. These data demonstrate that the enzyme is a cellodextrinase with an exotype of function.     

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

Background

Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.

Results

A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14?days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2?days and then maintained a steady state of 4% of the total biomass for the next 12?days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14?days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.

Conclusions

Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1?day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.