RESUMEN
Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC(50)=19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC(50)=60-80 mM), and weakly inhibited by glucose (IC(50)=350-380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).
Asunto(s)
Celobiosa/farmacología , Celulasa/antagonistas & inhibidores , Celulosa/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Glucosa/farmacología , Hypocrea/enzimología , Trichoderma/enzimología , Calorimetría , Celulasa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/antagonistas & inhibidores , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólisis , Microbiología Industrial , Concentración 50 InhibidoraRESUMEN
ß-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10-15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.
Asunto(s)
Celobiosa/metabolismo , Celulasas/aislamiento & purificación , Celulasas/metabolismo , Hongos/enzimología , Glucosa/metabolismo , Glicosilación , Hidrólisis , CinéticaRESUMEN
An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the ß-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.
Asunto(s)
Técnicas Biosensibles , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Celulosa/metabolismo , Proteínas Fúngicas/metabolismo , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/metabolismo , Celulosa/análisis , Cromatografía Líquida de Alta Presión , Electrodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Proteínas Fúngicas/química , Hidrólisis , Cinética , Límite de Detección , Phanerochaete/enzimología , Análisis de Regresión , Reproducibilidad de los Resultados , Trichoderma/enzimologíaRESUMEN
The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.
Asunto(s)
Celulasa/química , Celulosa/química , Proteínas Fúngicas/química , Trichoderma/enzimología , Hidrólisis , SolubilidadRESUMEN
BACKGROUND: The well-studied cellulase mixture secreted by Trichoderma reesei (anamorph to Hypocrea jecorina) contains two cellobiohydolases (CBHs), cellobiohydrolase I (TrCel7A) and cellobiohydrolase II (TrCeI6A), that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs) of variable lengths with respect to their binding and inhibition of both TrCel7A and an enzyme variant without the cellulose-binding domain (CBM). RESULTS: We studied the binding of XOSs to TrCel7A by isothermal titration calorimetry. We found that XOSs bind to TrCel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of TrCel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number. CONCLUSIONS: On the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of TrCel7A with phosphoric swollen cellulose as a substrate.
RESUMEN
The glycoside hydrolase family 5 (GH5) endo-ß-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards ß-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer ß-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-ß-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.
Asunto(s)
Aspergillus nidulans/enzimología , Glicosilación , Manosidasas/genética , Manosidasas/aislamiento & purificación , Aspergillus nidulans/química , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Secuencia de Carbohidratos , Clonación Molecular , Hidrólisis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.
Asunto(s)
Glucanos/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Almidón/metabolismo , alfa-Amilasas/química , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/citología , Arabidopsis/metabolismo , Calorimetría , Glucanos/genética , Glucanos/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Solanum tuberosum/química , Solanum tuberosum/citología , Solanum tuberosum/metabolismo , Resonancia por Plasmón de Superficie , Nicotiana/química , Nicotiana/citología , Nicotiana/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Two ß-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl ß-D-xylopyranoside (pNPX) and ß-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K(m) decreased with increasing ß-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different ß-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved ß-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, ß-D-Xylp-(1â4)-D-Lyxp, ß-D-Xylp-(1â6)-D-Galp, ß-D-Xylp-(1â4)-α-D-Glcp-(1â3)-ß-D-Fruf, ß-D-Xylp-(1â4)-α-D-Glcp-(1â2)-ß-D-Fruf, and ß-D-Xylp-(1â6)-ß-D-Fruf-(2â1)-α-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 ß-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.
Asunto(s)
Aspergillus nidulans , Oligosacáridos/síntesis química , Proteínas Recombinantes/química , Xilosa/análogos & derivados , Xilosa/síntesis química , Xilosidasas/química , Catálisis , Disacáridos/síntesis química , Conformación Molecular , Estructura Molecular , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Estereoisomerismo , Trisacáridos/síntesis química , Xilosidasas/biosíntesis , Xilosidasas/aislamiento & purificaciónRESUMEN
In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.
Asunto(s)
Calorimetría/métodos , Endo-1,4-beta Xilanasas/metabolismo , Pruebas de Enzimas/métodos , Aspergillus/enzimología , Benzotiazoles/química , Benzotiazoles/metabolismo , Ácidos Cumáricos/metabolismo , Endo-1,4-beta Xilanasas/química , Hidrólisis , Hidroxibenzoatos/metabolismo , Cinética , Oxidación-Reducción , Quinolinas/metabolismo , Ácido Salicílico/química , Ácido Salicílico/metabolismo , Termodinámica , Xilanos/metabolismoRESUMEN
Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming ß-glucosyl disaccharides with ß-(1â4)- regioselectivity from five monosaccharides as well as branched ß-glucosyl trisaccharides with ß-(1â4)-regioselectivity from three (1â6)-linked disaccharides. CtCDP showed strict ß-(1â4)-regioselectivity and catalysed linear chain extension of the three ß-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two ß-glucosyl oligosaccharide product series to represent novel compounds, i.e. ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â2)-D-glucopyranose, and ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of ß-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridium thermocellum/enzimología , Glucosiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Biocatálisis , Celobiosa/biosíntesis , Celobiosa/química , Celulosa/análogos & derivados , Celulosa/biosíntesis , Celulosa/química , Cromatografía Líquida de Alta Presión , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Dextrinas/biosíntesis , Dextrinas/química , Estabilidad de Enzimas , Glucosiltransferasas/química , Glucosiltransferasas/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Oligosacáridos/química , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Estereoisomerismo , TemperaturaRESUMEN
The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1-->6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-D-Galp-(1-->6)-D-Galp, alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->beta2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-D-galactopyranoside (40 mm), alpha-(1-->6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, L-arabinose, L-fucose and L-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and L-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-D-Galp-(1-->6)-D-Manp; alpha-D-Galp-(1-->6)-beta-D-Glcp-(1-->4)-D-Glcp; alpha-D-Galp-(1-->6)-beta-D-Galp-(1-->4)-D-Fruf; alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->alpha1)-D-Glcp; and alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Fruf.
Asunto(s)
Aspergillus nidulans/enzimología , Biocatálisis , Oligosacáridos/biosíntesis , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Conformación de Carbohidratos , Clonación Molecular , Escherichia coli/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Oligosacáridos/química , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Temperatura , alfa-Galactosidasa/biosíntesis , alfa-Galactosidasa/aislamiento & purificaciónRESUMEN
The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and fluorogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of -2.5 kJ mol(-1), an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.
Asunto(s)
Calorimetría/métodos , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Catalasa/metabolismo , Celulosa/metabolismo , Pruebas de Enzimas/métodos , Glucosa Oxidasa/metabolismo , Hidrólisis , Cinética , Especificidad por Sustrato , TermodinámicaRESUMEN
A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmbeta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1-->4)-Glcp (maltose), alpha-Glcp-(1-->4)-GlcNp (maltosamine), alpha-Glcp-(1-->4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1-->4)-Manp, alpha-Glcp-(1-->4)-Xylp and alpha-Glcp-(1-->4)- L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.
Asunto(s)
Glucosiltransferasas/metabolismo , Lactobacillus acidophilus/metabolismo , Polisacáridos/metabolismo , Transporte Biológico , Glucósidos/metabolismo , Lactobacillus acidophilus/enzimología , Conformación Molecular , Especificidad por SustratoRESUMEN
There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.
Asunto(s)
Pruebas de Enzimas/métodos , Colorantes Fluorescentes/metabolismo , Fluorometría/métodos , Glicósido Hidrolasas/metabolismo , Glicosiltransferasas/metabolismo , Nasturtium/enzimología , Arabidopsis/enzimología , Biocatálisis , Celulasa , Glicósidos/química , Glicósidos/metabolismo , Hidrólisis , Cinética , Especificidad de Órganos , Oxazinas/química , Oxazinas/metabolismo , Espectrometría de Fluorescencia , Especificidad por Sustrato , Factores de Tiempo , Trichoderma/enzimologíaRESUMEN
Reorganization and degradation of the wall crosslinking and seed storage polysaccharide xyloglucan by glycoside hydrolase family 16 (GH16) endo-transglycosylases and hydrolases are crucial to the growth of the majority of land plants, affecting processes as diverse as germination, morphogenesis, and fruit ripening. A high-resolution, three-dimensional structure of a nasturtium (Tropaeolum majus) endo-xyloglucanase loop mutant, TmNXG1-DeltaYNIIG, with an oligosaccharide product bound in the negative active-site subsites, has been solved by X-ray crystallography. Comparison of this novel complex to that of the strict xyloglucan endo-transglycosylase PttXET16-34 from hybrid aspen (Populus tremula x tremuloides), previously solved with a xylogluco-oligosaccharide bound in the positive subsites, highlighted key protein structures that affect the disparate catalytic activities displayed by these closely related enzymes. Combination of these "partial" active-site complexes through molecular dynamics simulations in water allowed modeling of wild-type TmNXG1, TmNXG1-DeltaYNIIG, and wild-type PttXET16-34 in complex with a xyloglucan octadecasaccharide spanning the entire catalytic cleft. A comprehensive analysis of these full-length complexes underscored the importance of various loops lining the active site. Subtle differences leading to a tighter hydrogen bonding pattern on the negative (glycosyl donor) binding subsites, together with loop flexibility on the positive (glycosyl acceptor) binding subsites appear to favor hydrolysis over transglycosylation in GH16 xyloglucan-active enzymes.
Asunto(s)
Glucanos/metabolismo , Glicósido Hidrolasas/química , Glicosiltransferasas/química , Nasturtium/enzimología , Proteínas de Plantas/química , Xilanos/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Simulación por Computador , Cristalografía por Rayos X , Glucanos/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nasturtium/química , Nasturtium/genética , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Triptófano/química , Xilanos/químicaRESUMEN
A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on Glc 4 backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per- O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon p K a values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with p K a values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon p K a values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Glucanos/química , Glicósido Hidrolasas/química , Glicósidos/síntesis química , Glicósidos/química , Cinética , Estructura Molecular , Oligosacáridos/química , Xilanos/químicaRESUMEN
Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16-34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal=Galbeta(1-->4), G=Glcbeta(1-->4), and X=Xylalpha(1-->6)Glcbeta(1-->4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (DeltaG(0)) for the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the beta(1-->4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.
Asunto(s)
Glicosiltransferasas/metabolismo , Populus/enzimología , Termodinámica , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/química , Hidrólisis , Espectrometría de Masas , Oligosacáridos/química , Oligosacáridos/metabolismo , Populus/química , Coloración y EtiquetadoRESUMEN
High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula x Populus tremuloides). Production of the loop deletion variant Tm-NXG1-DeltaYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.
Asunto(s)
Pared Celular/metabolismo , Evolución Molecular , Glicósido Hidrolasas/química , Proteínas de Plantas/química , Tropaeolum/enzimología , Catálisis , Cromatografía en Gel , Clonación Molecular , Cristalografía por Rayos X , ADN Complementario/metabolismo , Eliminación de Gen , Glucanos , Cinética , Datos de Secuencia Molecular , Mutagénesis , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligosacáridos/metabolismo , Filogenia , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Electricidad Estática , Relación Estructura-Actividad , Especificidad por Sustrato , XilanosRESUMEN
The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.
Asunto(s)
Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Catálisis , Dominio Catalítico , Clostridium thermocellum/enzimología , Glucanos/química , Glucanos/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Xilanos/química , Xilanos/metabolismoRESUMEN
Cellulose constitutes an important raw material for many industries. However, the superb load-bearing properties of cellulose are accompanied by poor chemical reactivity. The hydroxyl groups on cellulose surfaces can be reacted but usually not without loss of fiber integrity and strength. Here, we describe a novel chemoenzymatic approach for the efficient incorporation of chemical functionality onto cellulose surfaces. The modification is brought about by using a transglycosylating enzyme, xyloglucan endotranglycosylase, to join chemically modified xyloglucan oligosaccharides to xyloglucan, which has a naturally high affinity to cellulose. Binding of the chemically modified hemicellulose molecules can thus be used to attach a wide variety of chemical moieties without disruption of the individual fiber or fiber matrix.