Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

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.

     

Cellulase production from the corn stover fraction based on the organ and tissue

In this study, the biodegradation of fractionated corn stover in solid-state fermentation by Trichoderma reesei YG3 was investigated. Fractions of miscellaneous cells (MC) and fasciculi (FC) tissue from leaf, shell or core were separated using the combined pretreatment method of carding classification after steam explosion. The highest enzyme activities including the filter paper activity, endoglucanase, exoglucanase and β-glucosidase activities, weight loss rate of dry material and biodegradation rate were all observed in the MC tissue fraction from the leaf, which was more nutritious, while lowest activity was observed in the FC tissue fraction from the shell. The maximum filter paper activity and weight loss rate of the dry material were 4.56 and 1.89 times the minimum and the cellulose and hemicellulose biodegradation rates were 51.22 and 39.38% versus 23.85 and 26.51%, respectively. These variances maybe attributed to the heterogeneity of the component in the fractions. A higher weight loss rate corresponded to higher enzymatic activities, whereas cellulose biodegradation was not proportional to cellulase activities. Hemicellulose biodegradation was much slower than cellulose degradation. Here, we demonstrated the importance of fractionation in component biodegradation and utilization of straw.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

Pretreatment of cellulosic wastes to increase enzyme reactivity

The enzymatic hydrolysis of cellulose to glucose is generally a slow reaction. Different pretreatments, such as ball milling to a ?200 mesh or swelling in 1–2% NaOH are reported to increase the reactivity considerably. In this work a fiber fraction from cattle manure was treated in an autoclave for 5–30 min at temperatures ranging from 130–200°C. The reactivity of the cellulose, measured by incubating samples with a commercial cellulase preparation for one hour at 50°C and pH 4.8, was increased by a factor of 4–6 compared to NaOH treatment and 10–12 compared to untreated fiber. The increased reaction rate is probably mostly due to an increase in cellulose availability to enzymatic attack, as structural hemicellulose is hydrolyzed and removed during the treatment. Sugars, produced by hemicellulosis, hydrolysis, will react further to give caramelization products. These side reactions were shown to be suppressed by short treatment times. The treated fiber could support growth of a mixed culture of Trichoderma viride and Candida utilis only after washing, indicating the formation of water soluble inhibitory products during treatment. The treatment with high-temperature steam can probably be used also with other cellulosic materials to increase reactivity. This may be an attractive way to prepare low-valued wastes such as manure fibers, straw, stalks, or corn cobs for fermentation processes to increase the protein content or for use directly as ruminant animal feed.     

Evaluation of chemical pre-treatment for biodegradation of agricultural lignocellulosic wastes byAspergillus niger

Summary Chemical pre-treatment with NaOH, NaClO₂ or peracetic acid of wheat straw, bagasse, corn cobs and groundnut shells increased cellulose utilization, cellulase activity and protein content ofAspergillus niger AS-101 used as a test biodegradative microorganism.

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Resumen El pre-tratamiento químico con NaOH, NaClO2 o àcido peracético de paja de trigo, bagazo, mazorcas de maíz y cáscaras de cacahuete, incrementó la utilización de celulosa, la actividad celulásica y el contenido en proteínas deAspergillus niger AS-101.

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Résumé Le prétraitement chimique par NaOH, NaClO₂ ou par l'acide peracétique de la paille de froment, de la bagasse, des épis de maïs ou des coquilles d'arachide, augmente l'utilisation de la cellulose, l'activité de la cellulase et le contenu en protéines d'Aspergillus niger AS-101, utilisé comme souche test dans la biodégradation par les microorganismes.

     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

Co-fermentation of cellulose/xylan using engineered industrial yeast strain OC-2 displaying both β-glucosidase and β-xylosidase

We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the β-glucosidase (BGL) and β-xylosidase (XYL) genes, and a gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked β-glucosidase and β-xylosidase activities. Notably, the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes.     

Use of mutation strategies applied toAspergillus terreus ATCC 52430 to obtain mutants with improved cellulase productivity

Summary Mutation studies were carried out onAspergillus terreus ATCC 52430 to increase cellulase production. Optimal treatment conditions for increasing cellulase activity by UV mutagenesis were observed by exposure of spores to a UV germicidal lamp, wavelength 250 nm at a distance of 30 cm for 1 min. Nitrosoguanidine mutagenesis was optimal with respect to development of high cellulase mutants when spores were exposed to 500 ug/ml nitrosoguanidine for 1 h. Mutants were isolated on agar containing Walseth cellulose and the catabolite repressor, glycerol. Successive UV and nitrosoguanidine treatments resulted in isolation of strain UNG1-40 having 3.5, 4.6 and 3.3-fold increases in filter paper, β-glucosidase and carboxymethyl cellulase activity, respectively, compared toA. terreus ATCC 52430.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol

Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10⁶ spores ml⁻¹, average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds⁻¹) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l⁻¹). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.     

Yersinia kristensenii: A new species of enterobacteriaceae composed of sucrose-negative strains (formerly called atypicalYersinia enterocolitica orYersinia enterocolitica-Like)

Induction of cellulase was observed inFusarium sp. with reduction in lag period by lactose-pregrown cells as compared with glucose-pregrown cells. Insoluble cellulose (Sigmacell) induced maximum cellulase production in the induction medium. Supplementation of the culture growing on cellulose by cellobiose or glucose resulted in increased cellular growth and decreased cellulase production. Stepfeeding of cellobiose to the culture growting on carboxymethyl cellulose resulted in decreased cellulase production. Significant cellulase activity was detected in the culture filtrate of cells growing on Sigmacell supplemented with glucose, only when the glucose disappeared from the medium. This suggests that cellulase production may in part be regulated by catabolite repression.     

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H₃PO₄-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all. Received: April 28, 1997 / Accepted: May 24, 1997     

Production of protein and cellulase byAspergillus niger in solid state culture

Summary Single-cell protein (SCP) and cellulase production byAspergillus niger AS-101, grown on alkalitreated corn cobs, was studied under various cultural conditions. The maximum yields of SCP and cellulase, under solid-state fermentation were obtained when the culture was incubated at pH 5.5 for 18 d at 30°C with 12% substrate.

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Effect des conditions de culture sur la production de protéines et de cellulases par Aspergillus niger en milieu solide

Résumé La production de protéine d'origine unicellulaire (POU) et de cellulase parAspergillus niger AS-101, cultivé sur des épis de maïs traités à l'alcali, est étudiée sous diverses conditions de culture. Les rendements maximum en POU et en cellulase par fermentation en milieu solide sont obtenus lorsque la culture est incubée à pH 5.5 pendant 18 jours à 30°C et avec une concentration en substrat de 12%.

     

Enzymatic degradation of steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) by cellulosic-substrate induced cellulases

The cellulase production by Trichoderma viride, cultivated on different substrates, namely steam-pretreated Lespedeza, filter paper, microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC), was studied. Different cellulase systems were secreted when cultivated on different substrates. The cellulolytic enzyme from steam-pretreated Lespedeza medium performed the highest filter paper activity, exoglucanase and endoglucanase activities, while the highest β-glucosidase activity was obtained from the enzyme produced on filter paper medium. The hydrolytic potential of the enzymes produced from different media was evaluated on steam-pretreated Lespedeza. The cellulase from steam-pretreated Lespedeza was found to have the most efficient hydrolysis capability to this specific substrate. The molecular weights of the cellulases produced on steam-pretreated Lespedeza, filter paper and MCC media were 33, 37 and 40 kDa, respectively, and the cellulase from CMC medium had molecular weights of 20 and 43 kDa. The degree of polymerization, crystallinity index and micro structure scanned by the scanning electron microscopy of degraded steam-pretreated Lespedeza residues were also studied.     

Chemical composition,digestibility and feeding value of maize cobs

The digestibility of treated maize cobs was studied using Romney Marsh wether sheep in a 2 × 3 factorial design; maize cobs were ground through a 10- or 6-mm screen and three chemical treatments were applied to each grinding: distilled water (control); sodium hydroxide (4.5 g per 100 g cob dry matter); and Magadi soda (9.0 g per 100 g cob dry matter). Cobs were treated for 24 h using one litre of solution per kg of maize cobs.The digestion coefficient of crude protein was lowered (P< 0.01) by finer grinding of the maize cobs. Dry matter digestibility coefficients were 44.7, 54.2 and 61.6% for maize cobs treated with water, NaOH and Magadi soda, respectively. Digestibility of energy, cell walls and cellulose was increased more by Magadi soda than by NaOH. Digestibility of acid detergent fibre (ADF) was depressed (P< 0.05) by NaOH but improved (P< 0.05) by Magadi soda. Chemical treatment lowered (P< 0.01) the digestibility of crude protein from 28.0 to 17.0% (NaOH) or to 18.7% (Magadi soda). Grinding and chemical treatment of maize cobs interacted (P< 0.01) in their effects on digestibility of crude protein.Friesian cattle, grazed on a predominantly Nandi Setaria (Setaria sphacelata) and Silver Leaf Desmodium (Desmodium uncinatum) pasture, and supplemented with water-, NaOH- and Magadi soda-treated maize cobs, gained 0.41, 0.55 and 0.51 kg per day, respectively. Daily maize cob intake was higher on NaOH-treated than on water- and Magadi soda-treated cobs. Cattle on NaOH- and Magadi soda-treated cobs required more than 1 month to adapt to the cobs and show superior gain.     

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Background  

The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.     

Strain improvement of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C-30 for increased cellulase production

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml⁻¹, a CMCase activity of 54.2 U ml⁻¹, a β-glucosidase activity of 0.39 U ml⁻¹, and a fungal biomass of 12.6 mg ml⁻¹. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml⁻¹). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.