Saccharification of cellulosic substrates byAspergillus niger cellulase

Chemical pre-treatment enhanced saccharification of agricultural lignocellulosic residues usingAspergillus niger cellulase by between 16 and 38%. Maximum saccharification (76%) of alkali-treated bagasse was with 0.5% substrate over 48 h using 0.3 U enzyme/ml.

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Résumé Le pré-traitement chimique augmente de 16 à 38% la saccharification de résidus agricoles ligno-cellulosiques lorsque l'on utilise la cellulase d'Aspergillus niger. On a obtenu la saccharification maximum (76%) de la bagasse traitée à l'alcali avec 0.5% de substrat en 48 h de traitement avec 0.3 unités d'enzyme par ml.

     

Isolation of mutants of Talaromyces emersonii CBS 814.70 with enhanced cellulase activity

By a combination of genetic mutation and modification of growth medium the cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4 etc.] activity of culture filtrates of Talaromyces emersonii CBS 814.70 has been increased four-fold to approximately 2 U ml^?1 and a productivity of 20–25 Ul^?1h^?1. At 50°C this system was completely stable for at least 24 h. At 60°C in static reaction mixtures 19% of the original activity was lost compared with 21% when mixtures were shaken. At 70°C the loss of activity after 4 h was 64% without shaking and 70% when shaken. The cellulase system from Trichoderma reesei was decidedly less stable than that of Talaromyces emersonii under each of the above conditions. The ability of each enzyme system, separately and together, to digest beet pulp was investigated.     

Mechanism of cellulase reaction on pure cellulosic substrates

Cellulase reaction mechanism was investigated with the use of following pure cellulosic substrates: Microcrystalline cellulose (Avicel), α‐cellulose (Sigma), filter paper, cotton, and non‐crystalline cellulose (NCC). NCC is amorphous cellulose prepared in our laboratory by treatment with concentrated sulfuric acid. When hydrolyzed with cellulase, NCC produces significant amount of cello‐oligosaccharides (COS) as reaction intermediates along with glucose and cellobiose. The COS produced by cellulase were categorized into two different moieties based upon their degree of polymerization (DP): low DP (less than 7) COS (LD‐COS) and high DP COS (HD‐COS). Endo‐glucanase (Endo‐G) reacts rapidly on the NCC reducing its DP to 30–60, after which the Endo‐G reaction with NCC ceases. HD‐COS is produced from NCC by the action of Endo‐G, whereas LD‐COS is produced by exo‐glucanase (Exo‐G). β‐Glucosidase (β‐G) hydrolyzes LD‐COS to produce cellobiose, but it does not hydrolyze HD‐COS. DP of NCC affects the action of Exo‐G in such a way that the overall yield is high for high DP NCC. This is in line with previous findings that substrate‐recognition by Exo‐G requires binding on β‐glucan chain with DP of 10 for the hydrolysis to take place. The individual cellulose chain residues within solid having DP less than 10 therefore remain unreacted. The percentage of the unreacted portion would be lower for high DP NCC, which results high overall conversion. The surface area and the number of reactive sites on the substrate facilitate adsorption of enzyme therefore the initial rate of the hydrolysis. The overall extent of conversion of cellulose, however, is controlled primarily by its inherent characteristics such as DP and crystallinity. Biotechnol. Bioeng. 2009;102: 1570–1581. © 2008 Wiley Periodicals, Inc.     

Anaerobic solid state fermentation of cellulosic substrates with possible application to cellulase production

Summary A solid state fermentation process was developed for the conversion of straw and cellulose under anaerobic conditions by a mixed culture of cellulolytic and methanogenic organisms. The bioconversion rate and efficiency were compared under mesophilic (35° C) and thermophilic (55° C) conditions. Cellulolytic activity was assayed in terms of sugar and overall soluble organic matter (chemical oxygen demand, COD) production. Maximum conversion rates were obtained under thermophilic conditions, i.e. 8.4 g and 14.2 g COD/kg·d, respectively, when wheat straw and cellulose were used as substrates. The cellulolytic activity of the reactor contents (23% dry matter), measured under substrate excess conditions, amounted to 50 g COD/kg·d. As a comparison, the activity of rumen contents (15% dry matter) measured by the same assay amounted to 150 g COD/kg·d. The anaerobic cellulases appeared to be substrate bound. This and the relative low activity levels attained, limit the perspectives of producing cellulase enzymes by this type of process.     

Studies on the biosorption of uranium by Talaromyces emersonii CBS 814.70 biomass

Residual biomass, produced by the thermophilic fungus, Talaromyces emersonii CBS 814.70, following growth on glucose-containing media, was examined for its ability to take up uranium from aqueous solution. It was found that the biomass had a relatively high observed biosorption capacity for the uranium (280 mg/g dry weight biomass). The calculated maximum biosorption capacity obtained by fitting the data to a Langmuir model was calculated to be 323 mg uranium/g dry weight biomass. Pretreatment of the biomass with either dilute HCl or NaOH brought about a significant decrease in biosorptive capacity for uranium. Studies on the effects of variation in temperature on the biosorptive capacity demonstrated no significant change in binding between 20°C and 60°C. However, a significant decrease in biosorptive capacity was observed at 5°C. Binding of uranium to the biomass at all temperatures reached equilibrium within 2 min. While the routine binding assays were performed at pH 5.0, adjustment of the pH to 3.0 gave rise to a significant decrease in biosorption capacity by the biomass. The biosorptive capacity of the biomass for uranium was increased when extraction from solution in sea-water was examined.     

Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.     

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.     

Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii

The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1' such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k(cat)/K(m)=2 x 10(5) m(-1) s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K(i)=1.5 nM), as well as a portion of the propeptide sequence, PT1 (K(i)=32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.     

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Modeling cellulase kinetics on lignocellulosic substrates

The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass to yield biofuel. This hydrolysis by cellulases, a heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion. To render the process economically viable, increases in hydrolysis rates and yields are necessary and require improvement both in enzymes (via protein engineering) and processing, i.e. optimization of reaction conditions, reactor design, enzyme and substrate cocktail compositions, enzyme recycling and recovery strategies. Advances in both areas in turn strongly depend on the progress in the accurate quantification of substrate–enzyme interactions and causes for the rate slowdown. The past five years have seen a significant increase in the number of studies on the kinetics of the enzymatic hydrolysis of cellulose. This review provides an overview of the models published thus far, classifies and tabulates these models, and presents an analysis of their basic assumptions. While the exact mechanism of cellulases on lignocellulosic biomass is not completely understood yet, models in the literature have elucidated various factors affecting the enzymatic rates and activities. Different assumptions regarding rate-limiting factors and basic substrate–enzyme interactions were employed to develop and validate these models. However, the models need to be further tested against additional experimental data to validate or disprove any underlying hypothesis. It should also provide better insight on additional parameters required in the case that more substrate and enzyme properties are to be included in a model.     

Characterisation of a Talaromyces emersonii thermostable enzyme cocktail with applications in wheat dough rheology

In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process.Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; β-glucosidase, xylan-1,4-β-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-β-xylanase, endo-arabinase, endo-glucanase, exo-β-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors’ knowledge.     

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K _m 1.66 mg mL⁻¹; V _max 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K _i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.     

Mode of action and properties of the beta-xylosidases from Talaromyces emersonii and Trichoderma reesei

Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant beta-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from Talaromyces emersonii (betaXTE) and Trichoderma reesei (betaXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both beta-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be beta-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of betaXTE and betaXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by betaXTE and betaXTR was 12.1% and 7.7%, respectively. Using the beta-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting beta-xylosidases and the endo-1,4-beta-xylanases and alpha-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two beta-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties.     

Cellulolytic activities ofChaetomium globosum on different cellulosic substrates

The effect of cellulosic substrates on the production of extra-cellular cellulases and their cellulolytic activity inChaetomium globosum has been studied in shake flask cultures. Production of endoglucanase, exoglucanase and filter paper cellulase was highest with pure cellulose whereas -glucosidase was maximally induced by wheat straw. A suitable pretreatment for wheat straw was peracetic acid followed by NaOH and that of bagasse with NaOH for saccharification.

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Résumé On a étudié l'effet de substrats cellulosiques sur la production de cellulases extracellulaires et leur activité cellulolytique chezChaetomium globosum en culture en flacons agités. La production d'endoglucanase, d'exoglucanase et de cellulase type papier-filtre est maximum avec la cellulose pure tandis que la -glucosidase est induite de manière maximum par la paille de froment. La paille de froment est traitée de manière adéquate pour la saccharification par l'acide peracétique suivi de NaOH, la bagasse, par NaOH.

     

Cloning,characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Production and characterization of monoclonal antibodies to the cellulases produced by Talaromyces emersonii CBS 814.70

Talaromyces emersonii CBS 814.70 produces a cellulase complex during growth on cellulose and lactose. The cellulase system consists of endoglucanase, exoglucanase and ß-glucosidase, and each type of enzyme exists as isoforms. In order to probe this complex system with respect to studying structure-function relationships of the components and also to pursue our studies on the genetic control of expression of the various components in the T. emersonii system, we decided to produce highly specific monoclonal antibodies to those cellulase components. Here we report on the production of the first three monoclonal antibodies to be made to the T. emersonii cellulase system. One of those antibodies is specific for endoglucanase and has been useful in demonstrating differences in the isoforms of endoglucanase produced during growth of the organism on lactose- and cellulose-containing media. The antibody is highly sensitive, being capable of detective less than 50 ng of endoglucanase in an ELISA system. The antibody also cross reacts with one major endoglucanase produced by Trichoderma reesei.