Search

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and also electronic considerations at the copper active site., Copper-dependent lytic polysaccharide monooxygenases (LPMOs) oxidatively cleave polysaccharides. Here the authors present a structure-function characterization of fungal LPMOs, showing that a particular LPMO cleaves xylan using a mechanism that involves an alternative copper coordination geometry.

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [ 3 H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [ 3 H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA · liter −1 · h −1 and from 0.76 to 0.23 nmol of 3 H-FFA · liter −1 · h −1 , respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26μ g of C · liter −1 · h −1 ). However, the ratio of MUF-oleate activities to [ 3 H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.

The present paper attempts to explain why enzyme production in solid-state fermentation (SSF) is higher than in submerged fermentation (SmF). Recent work done in our laboratory [Biotechnol. Lett. 22 (2000) 1255; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 271; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 296] related to the production of invertase, pectinases and tannases, by Aspergillus niger grown by SSF and SmF is reviewed. To do such a comparative study, logistic and Luedeking–Piret equations are used in order to estimate the values of the following coefficients: maximal specific growth rate (μM), maximal biomass level (XM), enzyme/biomass yield (YP/X) and secondary rate of production, or breakdown (k). It is shown that enzyme productivity is proportional to group, μMYP/XXM, corrected by a function of ν=k/YP/XμM. In all three cases of enzyme production studied, productivity using a SSF system was higher than in SmF. Studies with invertase resulted in higher values of μMXM. Studies with pectinases resulted in higher values of YP/XXM. Studies with tannases resulted in higher YP/X and less negative values of k. Finally, a reaction–diffusion model is presented to try to explain such differences based on micrographic measurements of mycelial aggregates for each kind of fermentation system.

Summary Cassava Manihot esculenta Crantz (Euphorbiaceae) is distinguished by the presence of Cyanid compounds in leaves, stems and tubers. In plant's tissues,—CN group is linked with D—glucose to form cyanogenic glucosides: mainly linamarin. In Congo, the cassava mealybug Phenacoccus manihoti Matile—Ferrero (Homoptera: Pseudococcidae) is the major pest of cassava. This insect is living mainly on genus Manihot. This oligophagy of P. manihoti towards cassava suggests that the mealybug is adapted to secondary compounds of its host plant, and more particularly to linamarin. This specialization supposes the presence of an enzymatical fittings adjusted. In fact, our results shows that this insect has a linamarase (participating in hydolysis of linamarin), an endogenous and an inductive enzyme in cassava mealybug. As other insects, P. manihoti has an enzymatical fittings hydolysing linamarin. In this insect, the use of hydolysis products is discussed.

The fate of a racemic lactic acid polymer (PLA50) was investigated by allowing parallelepiped plates of PLA50 to age in liquid medium containing a mixed culture of Fusarium moniliforme and Pseudomonas putida at 30°C. Microbial activity was monitored by measuring pH, lactic acid formation by high-performance liquid chromatography, and esterase activity in supernatant. Degradation of the plates was monitored by weighing, size exclusion chromatography, and visual examination. Under the selected conditions, pH increased up to 8, whereas it remained constant in the microorganism-free control. No significant lactic acid formation or esterase activity was detected in the supernatant during the experimental period (32 weeks). For the first 17 weeks, the water absorption rate of the plates was the same in both the microbial and the control media. Subsequently, plates absorbed more water in microbial than in control media. Typical surface/center differential degradation of plates was observed until 17 weeks, but it reduced progressively. At 32 weeks, specimens appeared completely disintegrated in the microbial medium. PLA50 plates were also buried in the soil for 8 weeks. After recovery, plates were allowed to degrade 8 weeks under controlled conditions. Scanning electron microscopy of these plates showed the development of some filamentous fungi at the surface and into the bulk of plates. Five fungal strains were isolated which appeared to be able to assimilate PLA50 oligomers in mixed cultures. It was assumed that PLA50 plates had been first degraded by chemical hydrolysis, followed by the bioassimilation of degradation by-products in both experiments. © 1996 John Wiley & Sons. Inc.

Las xilanasas son endoglucanasas capaces de degradar xilano. Estas enzimas se pueden aplicar en varios procesos industriales como en la degradación de residuos agrícolas para obtener azúcares, los cuales se fermentan para producir etanol. La aplicación industrial, sin en embargo, requiere estabilidad principalmente a elevadas temperaturas, por ello existe gran interés en el estudio de microorganismos termófilos. En la presente investigación, se estudió la producción de xilanasas en un sistema de inmovilización con alginato de calcio de la bacteria FT3 anaeróbica termófila, aislada de la Región altiplánica de Bolivia. Los resultados muestran que en un cultivo en batch suplementado con cloruro de amonio como fuente de nitrógeno y xilano como fuente de carbono, la actividad xilanolítica fue de 0,2 UI/mL, y un rendimiento de producción de 0,004 mUI de xilanasa/g de xilano. Esta actividad fue incrementada a 1,2 UI/mL (0,009 mUI/g), en cultivo en batch con células encapsuladas en alginato cálcico de alta viscosidad al 2%. En la optimización de fuentes de carbono y nitrógeno, la actividad incrementó únicamente en el sistema de células encapsuladas, mientras que en el de células libres esto parece no influir. Es así, que las condiciones óptimas de cultivo en base a las fuentes carbono y nitrógeno fueron residuos de soya y extracto de levadura, a una concentración de 5 g/L y 0,2 g/L respectivamente, obteniéndose una actividad de 1,8 UI/mL (0,1 mUI/g). El sistema inmovilizado fue ensayado en cultivo continuo que permitió la producción de la enzima lbre de células. Se logró incrementar la actividad hasta 1,3 UI/mL (0,13 mUI/g). El cambio de NH4Cl por extracto de levadura como fuente de nitrógeno en cultivo continuo incrementó la actividad enzimática en el bioreactor hasta 3,3 UI/mL (0,15 mUI/g). Así mismo, el cambio de la fuente de carbono de xilano por hidrolizado de residuos de soya no incrementó la actividad enzimática, sin embargo ésta se mantuvo estable. (résumé d'auteur)

Forty-four fungal strains capable of growing at temperatures above 50 C were isolated from different samples of soil and coconut residues collected in Mexican tropical and subtropical regions. These thermophilic and thermotolerant fungal strains were identified by microscopical analysis using standard procedures. Three species were identified: Rhizomucor pusillus (Lindt)Schipper (19 strains), Rhizopus microsporus van Tieghem (6 strains), and Aspergillus fumigatus Fresenius (19 strains). Four strains identified as Rhizopus microsporus were ascribed to the variety rhizopodiformis; however, the other two strains showed new characteristics which require further analysis, such as a homothallic sexual reproduction. Aspergillus fumigatus was found in coconut residues as a common contaminant during the isolation of other thermophilic species. Strains were isolated from samples containing a high content of lipids (mainly from coconut coprah), and accordingly extracellular lipase biosynthesis was directly confirmed in Petri dishes for every strain.

Alpha-amylases from the intestinal cavity of two tilapia species, #Oreochromis niloticus$ (ONI-AMY) and #Sarotherodon melanotheron$ (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly : 56 600 (ONI-AMY) vs. 55 500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct pI : 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by alpha, Beta, and gamma-cyclodextrin (alpha-, Beta- and gamma-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. Beta- and gamma-CD are stronger inhibitors than alpha-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the alpha-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food. (Résumé d'auteur)

The glycolytic enzymatic activities in the gut of the endogeic earthworm #Hormogaster elisae$ from El Molar (Madrid, Spain) were studied in order to determine its digestive capacity and to assess its alimentary regime. Most endogeic earthworms have weak enzymatic complement and they usually establish mutualistic relationships with soil microflora to digest some organic compounds. Therefore, the intestinal wall tissues were cultured in vitro to assess the origin of the glycolytic enzymes found in the gut and enzymatic activities were measured in both cultured tissues and culture media. #H. elisae$ had a wide but not very strong enzyme complement, since all substrates were degraded but most of them at a low rate. This species cannot produce cellulase and mannamase, so for the digestion of these substrates it probably uses the digestive enzymatic capabilities of the ingested microflora. (Résumé d'auteur)

This study compares the origin and activities of glycolytic enzymes present in the gut contents of some geophagous adult earthworms and demonstrates that all the species did not develop mutualistic relationships with the ingested microorganisms to digest soil organic matter. Although the glycolytic activities were rather weak in the earthworms studied, they possess quite a complete enzymatic system that allows to degrade root and fungal substrates available in soils. #Millsonia anomala$ Omo. from Lamto (Côte d'Ivoire) and #Hormogaster elisae$ Alvarez from El Molar (Madrid) developed a strong mutualistic earthworm-microflora digestion system for hydrolysing cellulose : cellulase produced by inested microflora is able to degrade cellulose to cellobiose, cellobiase released by the worm itself breaks down cellobiose into D-glucose. Both enzymes found in intestinal contents of #Pontoscolex corethrurus$ Müll. from Palma Sola, Veracruz (Mexico) were produced by ingested soil microflora since they were not found in gut tissue culture. On the contrary, #Polypheretima elongata$ Perr. from Sainte Anne (Martinique), #Hyperiodrilus africanus$ Bedd. and #Dichogaster terrae nigrae$ Omo. et Vld. from Lamto (Cîte d'Ivoire) possess a complete enzymatic system for hydrolyzing cellulose. They could synthesize cellulolytic enzymes, i.e. cellulase and cellobiase by themselves. In the course of their digestion, geophagous earthworms seem to display variable adaptive characters which are undoubtedly linked to the different ecological categories and niches. (Résumé d'auteur)

Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org ) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.

Ferulic, caffeic, p-coumaric and chlorogenic acids are classified as hydroxycinnamic acids, presenting anticarcinogenic, anti-inflammatory and antioxidant properties. In this work, enzymatic extraction has been studied in order to extract high value-added products like hydroxycinnamic acids from coffee pulp. A commercial pectinase and enzyme extract produced by Rhizomucor pusillus strain 23aIV in solid-state fermentation using olive oil or coffee pulp (CP) as an inducer of the feruloyl esterase activity were evaluated separately and mixed. The total content (covalently linked and free) of ferulic, caffeic, p-coumaric and chlorogenic acids was 5276 mg per kg of coffee pulp. Distribution was as follows (in %): chlorogenic acid 58.7, caffeic acid 37.6, ferulic acid 2.1 and p-coumaric acid 1.5. Most of the hydroxycinnamic acids were covalently bound to the cell wall (in %): p-coumaric acid 97.2, caffeic acid 94.4, chlorogenic acid 76.9 and ferulic acid 73.4. The content of covalently linked hydroxycinnamic acid was used to calculate the enzyme extraction yield. The maximum carbon dioxide rate for the solid-state fermentation using olive oil as an inducer was higher and it was reached in a short cultivation time. Nevertheless, the feruloyl esterase (FAE) activity (units per mg of protein) obtained in the fermentation using CP as an inducer was 31.8 % higher in comparison with that obtained in the fermentation using olive oil as the inducer. To our knowledge, this is the first report indicating the composition of both esterified and free ferulic, caffeic, p-coumaric and chlorogenic acids in coffee pulp. The highest yield of extraction of hydroxycinnamic acids was obtained by mixing the produced enzyme extract using coffee pulp as an inducer and a commercial pectinase. Extraction yields were as follows (in %): chlorogenic acid 54.4, ferulic acid 19.8, p-coumaric acid 7.2 and caffeic acid 2.3. An important increase in the added value of coffee pulp was mainly due to the extraction of chlorogenic acid.