RESUMEN
Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and ß-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.
Asunto(s)
Acetilesterasa/química , Glucuronatos/química , Oligosacáridos/química , Xilanos/química , Acetilación , Acetilesterasa/metabolismo , Aspergillus niger/enzimología , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Glicósido Hidrolasas/química , Hidrólisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Xilanos/metabolismoRESUMEN
Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper. Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture. In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles. Six lipase preparations were shown to be able to degrade steryl esters. Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities. The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit). Up to 80% of the steryl esters were degraded in aqueous dispersion. Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp. could be the most potential enzyme in industrial applications. The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6-10 and at temperatures up to 70 degrees C.
Asunto(s)
Esterasas/química , Lipasa/metabolismo , Candida/química , Ésteres del Colesterol/química , Ésteres del Colesterol/metabolismo , Chromobacterium/química , Estabilidad de Enzimas , Esterasas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Pseudomonas/química , Resinas de Plantas/metabolismo , Temperatura , MaderaRESUMEN
A well-known lipase, Lip 3 of Candida rugosa, was purified to homogeneity from a commercial lipase preparation, using hydrophobic interaction and anion exchange chromatography. Lip 3, which has been reported to act on cholesteryl esters, was also found to be active on plant-derived steryl esters. Lip 3 had optimal activity at pH 5-7 and below 55 degrees C. It was able to hydrolyse steryl esters totally in a clear micellar aqueous solution. However, the action on a dispersed colloidal steryl ester solution was limited and only about half of the steryl esters were degraded. The degree of hydrolysis was not improved by addition of fresh enzyme. The composition of released fatty acids and sterols was, however, almost identical to that obtained by alkaline hydrolysis, showing that all the different steryl esters were hydrolysed equally and that none of the individual components were responsible for incomplete hydrolysis. Thus, it appeared that the physical state of the colloidal steryl ester dispersion limited the action of Lip 3. Wood resins contain both triglycerides and steryl esters among the hydrophobic components, which create problems in papermaking. The simultaneous enzymatic hydrolysis of triglycerides and steryl ester is therefore of considerable interest and Lip 3 is the first enzyme reported to act on both triglycerides and steryl esters.
Asunto(s)
Candida/enzimología , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Fitosteroles/metabolismo , Candida/aislamiento & purificación , Candida/metabolismo , Catálisis , Ésteres del Colesterol , Ésteres/metabolismo , Hidrólisis , Lipasa/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.
Asunto(s)
Proteínas Fúngicas/genética , Amplificación de Genes , Eliminación de Gen , Isoformas de Proteínas/genética , Transformación Genética , Trichoderma/genética , Secuencia de Bases , Celulasa/biosíntesis , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Proteínas Fúngicas/aislamiento & purificación , Isoformas de Proteínas/aislamiento & purificación , Esporas Bacterianas , Trichoderma/enzimología , Trichoderma/crecimiento & desarrollo , Trichoderma/fisiologíaRESUMEN
There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.
Asunto(s)
Celulasa/metabolismo , Trichoderma/enzimología , Secuencia de Bases , Western Blotting , Celulasa/genética , Celulasa/aislamiento & purificación , Celulosa 1,4-beta-Celobiosidasa , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l(-1)) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Trichoderma/metabolismo , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Glucosa/metabolismoRESUMEN
The main alpha-glucuronidase (EC 3.2.1.131) of the fungus Schizophyllum commune was purified to homogeneity using standard chromatographic methods; anion exchange, hydrophobic interaction chromatography and gel filtration. The enzyme had a molecular mass of 125 kDa as determined by SDS-polyacrylamide gel electrophoresis and a pI value of 3.6 according to isoelectric focusing. The N-terminal amino acid sequence of the S. commune alpha-glucuronidase did not show any homology with other alpha-glucuronidases. It exhibited maximal activity at pH values from 4.5 to 5.5 and was stable for 24 h between pH 6 and 8 at 40 degrees C. The highest temperature at which the enzyme retained its full activity for 24 h at pH 5.8 was 40 degrees C. The alpha-glucuronidase of S. commune was able to remove almost all 4-O-methylglucuronic acid groups from water-soluble polymeric softwood arabinoglucuronoxylans. The action of the enzyme on birchwood acetyl-glucuronoxylan was limited due to the high amount of acetyl substituents. The degree of hydrolysis of partially soluble deacetylated glucuronoxylan did not exceed 50% of the theoretical maximum. However, together with a xylanase hydrolysing the xylan backbone the action of the alpha-glucuronidase of S. commune on glucuronoxylan was clearly enhanced. It was apparent that the enzyme was able to remove the 4-O-methylglucuronic groups mainly from soluble substrates.
Asunto(s)
Glicósido Hidrolasas/aislamiento & purificación , Polímeros/metabolismo , Schizophyllum/enzimología , Xilanos/metabolismo , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Especificidad por SustratoRESUMEN
Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion-exchange, hydrophobic-interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI-7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI-9.0 form of XYL II to pI-8.1 and pI-7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.
Asunto(s)
Trichoderma/enzimología , Xilosidasas/química , Xilosidasas/metabolismo , Secuencia de Aminoácidos , Endo-1,4-beta Xilanasas , Hidrólisis , Punto Isoeléctrico , Isoenzimas , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Xilosidasas/aislamiento & purificaciónRESUMEN
Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.
Asunto(s)
Acetilesterasa/química , Trichoderma/enzimología , Acetilación , Acetilesterasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fusarium/enzimología , Datos de Secuencia Molecular , Estructura Molecular , Penicillium/enzimología , Especificidad por SustratoRESUMEN
A beta-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg-1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4 5.0 and at 70 degrees C. The beta-mannosidase hydrolyzed beta-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2-6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-beta-D-mannopyranoside were 0.30 mM and 500 nkat mg-1, respectively. Hydrolysis of D-galactose substituted manno-oligosaccharides showed that the beta-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus beta-mannosidase belonging to family 2 of glycosyl hydrolases.
Asunto(s)
Aspergillus niger/enzimología , Manosidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Hidrólisis , Cinética , Manosidasas/aislamiento & purificación , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , beta-ManosidasaRESUMEN
The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBH I. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.
Asunto(s)
Celulasa/metabolismo , Proteínas Fúngicas/metabolismo , Trichoderma/enzimología , Adsorción , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa , Cromatografía Líquida de Alta Presión , Hidrólisis , Trichoderma/metabolismoRESUMEN
The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Trichoderma/enzimología , Celulasa/química , Celulosa/química , Celulosa 1,4-beta-Celobiosidasa , Hidrólisis , Isoenzimas/metabolismo , Cinética , Unión ProteicaRESUMEN
The effect of phenolic substitutions on the activity of an alpha-arabinofuranosidase from Aspergillus terreus was investigated using feruloylated oligosaccharides isolated from plant cell walls, equivalent oligosaccharides obtained through treatment with specific ferulic acid esterases, and a synthetic lignin-carbohydrate complex (LCC). Feruloyl substituents limited the hydrolysis of arabinoxylan and arabinan oligosaccharides but only if the feruloyl group was esterified to the terminal non-reducing arabinose. Somewhat surprisingly, the LCC-model compound, in which the arabinose residue is substituted with a bulky dilignol group, was degraded by the enzyme. This indicated that the enzyme is able to approach this linkage from the xylose side.
Asunto(s)
Aspergillus/enzimología , Glicósido Hidrolasas/metabolismo , Oligosacáridos/metabolismo , Secuencia de Carbohidratos , Pared Celular/química , Disacáridos/aislamiento & purificación , Disacáridos/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Plantas/química , Trisacáridos/aislamiento & purificación , Trisacáridos/metabolismoRESUMEN
Acetyl xylan esterase is involved in the biodegradation of hemicellulose. It cleaves O-acetyl groups from xylan, which is the most abundant hemicellulose in nature. The catalytic core of acetyl xylan esterase from T. reesei has been crystallized and X-ray diffraction data at 2.3 A collected. The crystal belongs to the triclinic space group P1 with unit-cell parameters a = 50.3, b = 62. 1, c = 40.0 A, alpha = 110.1, beta = 113.6 and gamma = 97.9 degrees. The asymmetric unit contains two molecules.
Asunto(s)
Acetilesterasa/química , Trichoderma/enzimología , Dominio Catalítico , Cristalización , Estructura Molecular , Peso Molecular , Difracción de Rayos XRESUMEN
Production of extracellular a-galactosidases by the filamentous fungus Penicillium simplicissimum (previously P. janthinellum) VTT-D-78090 was studied on different carbon sources. Steam-exploded oat husks were chosen as the best carbon source for enzyme production. Three a-galactosidases (AGL) were purified from the culture filtrate using ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The isoelectric points of AGLI, AGLII and AGLIII were 5.2, 4.4 and 7.0, and the molecular masses as determined by SDS/PAGE were 61, 84 and 61 kDa, respectively. All enzymes were glycosylated. The optimum pH for the activity of AGLI and AGLIII was between 3.0 and 4.5 and that of AGLII was between 4.0 and 5.0. AGLII was more stable and more resistant to product inhibition by galactose than the other two enzymes. AGLI and AGLIII were also inhibited by p-nitrophenol-a-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agl1, encodes 435 amino acids including the signal sequence. It showed similarity with the other a-galactosidases belonging to the glycosyl hydrolase family 27. The N-terminal amino acid sequence of AGLIII was also similar to the sequences of other members of family 27, whereas the N-terminus of AGLII was completely different from the sequences of other reported hydrolases.
Asunto(s)
Proteínas Bacterianas , Penicillium/enzimología , alfa-Galactosidasa/química , Secuencia de Aminoácidos , Clonación Molecular , Estabilidad de Enzimas , Proteínas Fúngicas/química , Galactosa/farmacología , Glicósido Hidrolasas/química , Isoenzimas/química , Cinética , Manosidasas/química , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad por Sustrato , beta-ManosidasaRESUMEN
Xylans and mannans contain different esterified substituents such as acetyl, feruloyl and p-coumaroyl side groups. The functions of hemicellulose-deacetylating esterases of Trichoderma reesei and Aspergillus oryzae are discussed in this paper. Both fungi produce multiple esterases and two different esterases were isolated from both T. reesei and A. oryzae. The enzymes differed significantly in their substrate specificities. Acetyl xylan esterase of T. reesei was highly active on polymeric xylan but was unable to remove acetyl substituents from glucomannan or phenolic substituents from wheat straw arabinoxylan. Another esterase, acetyl esterase from T. reesei, had activity only towards short oligomeric and monomeric acetates derived both from xylan and glucomannan. The acetyl glucomannan esterase of A. oryzae was most active towards polymeric glucomannan, but was also able to remove acetyl groups from xylan. The only esterase studied which was active against phenolic substituents in arabinoxylans was the feruloyl esterase from A. oryzae. Feruloyl esterase had the widest substrate specificity of the esterases studied. It was also able to act on acetyl groups both in xylan and in glucomannan. The simultaneous enzymic liberation of acetyl groups from xylan and glucomannan clearly enhanced the action of xylan- and mannan-degrading enzymes, thus increasing the hydrolysis yield significantly. However, none of the esterases was able to remove all acetyl substituents when acting alone and simultaneous action of two esterases was needed for complete deacetylation.
Asunto(s)
Acetilesterasa/metabolismo , Aspergillus oryzae/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Esterasas/metabolismo , Polisacáridos/metabolismo , Trichoderma/enzimología , Acetatos/metabolismo , Cromatografía Líquida de Alta Presión , Esterasas/aislamiento & purificación , Mananos/metabolismo , Especificidad por Sustrato , Xilanos/metabolismoRESUMEN
The substrate specificities of three Penicillium simplicissimum alpha-galactosidases, AGLI, AGLII, and AGLIII, were determined by using various isolated galactose-containing oligosaccharides and polymeric galacto(gluco)mannans. AGLI released galactose from melibiose and raffinose-family oligosaccharides but the amount of galactose released was decreased from 96% to 35% by the increasing chain length of the substrate from raffinose to verbascose. It was able to release galactose linked to the nonreducing end and less efficiently to the internal residues of the galactomanno-oligomers. AGLI was able to hydrolyze 60-92% of galactose from polymeric galacto(gluco)mannans alone but its action was facilitated by mannanase and beta-mannosidase. In addition, it was able to release about 10% of the galactose from softwood kraft pulp alone and about 22% in combination with mannanase. AGLII was highly specific toward small galactose-containing oligosaccharides in which the galactose is linked to the nonreducing end of the substrate. It released 90-100% of galactose present in melibiose, raffinose, stachyose, and verbascose; however, it was able to degrade polymeric substrates only in combination with mannanase and beta-mannosidase. AGLIII had only low activity toward the oligomeric substrates tested. It was able to release some galactose from the polymeric galacto(gluco)mannans alone, but its action was clearly enhanced by the backbone degrading enzymes.
Asunto(s)
Penicillium/enzimología , alfa-Galactosidasa/metabolismo , Aspergillus niger/enzimología , Galactosa/metabolismo , Isoenzimas/metabolismo , Mananos/metabolismo , Manosidasas/metabolismo , Melibiosa/análisis , Melibiosa/metabolismo , Oligosacáridos/metabolismo , Rafinosa/análisis , Rafinosa/metabolismo , Especificidad por Sustrato , Madera , beta-ManosidasaRESUMEN
A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulose-binding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporus cellulose-induced protein CEL1 whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene egl4. Polyclonal antibodies were prepared against EGIV produced in Escherichia coli and detected a 56-kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus.
Asunto(s)
Celulasa/metabolismo , Proteínas Fúngicas , Trichoderma/enzimología , Agaricus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Celulasa/química , Celulasa/genética , Celulosa 1,4-beta-Celobiosidasa , Clonación Molecular , ADN Complementario , Biblioteca de Genes , Genes Fúngicos , Modelos Estructurales , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trichoderma/genéticaRESUMEN
Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 (formerly F) and 11 (formerly G) differ in their action on 4-O-methyl-D-glucurono-D-xylan and rhodymenan, a beta-1,3-beta-1,4-xylan. Two high molecular mass EXs (family 10), the Cryptococcus albidus EX and XlnA of Streptomyces lividans, liberate from glucuronoxylan aldotetrauronic acid as the shortest acidic fragment, and from rhodymenan an isomeric xylotriose of the structure Xyl beta 1-3Xyl beta 1-4Xyl as the shortest fragment containing a beta-1,3-linkage. Low molecular mass EXs (family 11), such as the Trichoderma reesei enzymes and XlnB and XlnC of S. lividans, liberate from glucuronoxylan an aldopentauronic acid as the shortest fragment, and from rhodymenan an isomeric xylotetraose as the shortest fragment containing a beta-1,3-linkage. The structure of the oligosaccharides was established by: NMR spectroscopy, mass spectrometry of per-O-methylated compounds and enzymic hydrolysis by beta-xylosidase and EX, followed by analysis of products by chromatography. The structures of the fragments define in the polysaccharides the linkages attacked and non-attacked by the enzymes. EXs of family 10 require a lower number of unsubstituted consecutive beta-1,4-xylopyranosyl units in the main chain and a lower number of consecutive beta-1,4-xylopyranosyl linkages in rhodymenan than EXs of family 11. These results, together with a greater catalytic versatility of EXs of family 10, suggest that EXs of family 10 have substrate binding sites smaller than those of EXs of family 11. This suggestion is in agreement with the finding that EXs of family 10 show higher affinity for shorter linear beta-1,4-xylooligosaccharides than EXs of family 11. The results are discussed with relevant literature data to understand better the structure-function relationship in this group of glycanases.
Asunto(s)
Xilosidasas/metabolismo , Endo-1,4-beta Xilanasas , Hidrólisis , Oligosacáridos/metabolismo , Estructura Terciaria de Proteína , Xilanos/metabolismo , Xilosidasas/químicaRESUMEN
The di-, tri- and tetrasaccharides formed during Trichoderma reesei endo-beta-D-mannanase treatment of pine kraft pulp were studied. The oligosaccharides in the hydrolysate were fractionated using size-exclusion, anion exchange and activated carbon chromatography. The primary sequence of the purified oligomers was determined by two-dimensional NMR techniques. The T. reesei mannanase cleaves the beta-1,4-glycosidic linkage of D-mannosyl residues attached either to D-mannose or D-glucose. The D-mannosyl residue may also be substituted by a D-galactosyl group. The main disaccharide produced was mannobiose, but a significant amount of 4-O-beta-D-glucopyranosyl-D-mannopyranose (GlcMan) was also produced. After extensive hydrolysis the main trisaccharides produced were 4-O-beta-D-mannopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1Man2) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-D-mannopyranose (Glc2Man). Some mannotriose 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyra-nosyl-D-manno pyranose (GlcMan2) and 4-O-beta-D-glucopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1GlcMan) were also detected in the hydrolysate. The structures of two tetrasaccharides were studied. They appeared to be 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-4-O-beta-D- glucopyranosyl-D-mannopyranose (Glc3Man) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyranosyl-4-O-beta-D -glucopyranosyl-D-mannopyranose (GlcManGlcMan). According to the results obtained, the galactoglucomannan in pine contains regions in which two or three glucose units are linked together, which further means that it may contain regions with several successive mannose residues. The galactose side groups were found to be attached only to mannose.