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1.
Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically 总被引:1,自引:0,他引:1
The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes beta-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these 'soluble'-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley beta-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound. 相似文献
2.
Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans. 相似文献
3.
Kurokawa J Hemjinda E Arai T Kimura T Sakka K Ohmiya K 《Applied microbiology and biotechnology》2002,59(4-5):455-461
The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548–554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature
form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks
a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9
catalytic domains. CelT devoid of the dockerin domain (CelTΔdoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTΔdoc showed strong activity toward carboxymethylcellulose (CMC) and barley
β-glucan, and low activity toward xylan. The V
max and K
m values were 137 μmol min–1 mg–1 and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain
or family 3c CBM.
Electronic Publication 相似文献
4.
Alina Iulia Chiriac Edith Marleny Cadena Teresa Vidal Antonio L. Torres Pilar Diaz F. I. Javier Pastor 《Applied microbiology and biotechnology》2010,86(4):1125-1134
Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9),
a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn31,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have
been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not
affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited
binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on
crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small
ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble
sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of
domains GH9–CBM3c, although the slightly reduced processivity of truncated form GH9–CBM3c suggests a minor contribution of
domains Fn31,2 or CBM3b, not contained in it, on processivity of endoglucanase Cel9B. 相似文献
5.
Kitago Y Karita S Watanabe N Kamiya M Aizawa T Sakka K Tanaka I 《The Journal of biological chemistry》2007,282(49):35703-35711
The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 A. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a beta-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu(186) and Glu(359), are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a (1)H NMR experiment using the reduced cellooligosaccharide as a substrate. 相似文献
6.
7.
CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose. 总被引:3,自引:0,他引:3
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L Gal C Gaudin A Belaich S Pages C Tardif J P Belaich 《Journal of bacteriology》1997,179(21):6595-6601
The gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum, was cloned and overexpressed in Escherichia coli. Four different forms of the protein were genetically engineered, purified, and studied: CelGL (the entire form of CelG), CelGcat1 (the catalytic domain of CelG alone), CelGcat2 (CelGcat1 plus 91 amino acids at the beginning of the cellulose binding domain [CBD]), and GST-CBD(CelG) (the CBD of CelG fused to glutathione S-transferase). The biochemical properties of CelG were compared with those of CelA, an endoglucanase from C. cellulolyticum which was previously studied. CelG, like CelA, was found to have an endo cutting mode of activity on carboxymethyl cellulose (CMC) but exhibited greater activity on crystalline substrates (bacterial microcrystalline cellulose and Avicel) than CelA. As observed with CelA, the presence of the nonhydrolytic miniscaffolding protein (miniCipC1) enhanced the activity of CelG on phosphoric acid swollen cellulose (PASC), but to a lesser extent. The absence of the CBD led to the complete inactivation of the enzyme. The abilities of CelG and GST-CBD(CelG) to bind various substrates were also studied. Although the entire enzyme is able to bind to crystalline cellulose at a limited number of sites, the chimeric protein GST-CBD(CelG) does not bind to either of the tested substrates (Avicel and PASC). The lack of independence between the two domains and the weak binding to cellulose suggest that this CBD-like domain may play a special role and be either directly or indirectly involved in the catalytic reaction. 相似文献
8.
Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein. 相似文献
9.
Westereng B Ishida T Vaaje-Kolstad G Wu M Eijsink VG Igarashi K Samejima M Ståhlberg J Horn SJ Sandgren M 《PloS one》2011,6(11):e27807
Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency. 相似文献
10.
A hyperthermophilic -1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region. 相似文献
11.
The catalytic domain of cellulase CelA, a family 8 glycohydrolase from C. thermocellum, has been crystallized in the orthorhombic space group P212121 with unit cell dimensions a = 50.12 Å, b = 63.52 Å, c = 104.97 Å. The diffraction pattern extends beyond 1.5 Å resolution. © 1996 Wiley-Liss, Inc. 相似文献
12.
Abstract The anaerobic degradation of microcrystalline cellulose by thermostable cellulolytic enzyme complexes from Clostridium thermocellum JW20 (ATCC 31449) was monitored. For quantitative investigations as enzyme-coupled spectrophotometric assay has been developed. The assay allows for the evaluation of the release of cellubiose-/glucose-units from native cellulose. Kinetic studies revealed that the anaerobic breakdown of crystalline cellulose (CC) at 60°C follows Michaelis-Menten kinetics K m CC values have been determined for different aggregation states of the cellulolytic complex. The presented assay seems well suited to screen for CC-degrading enzymes of various sources, and to further explore the mechanism of CC-breakdown. 相似文献
13.
Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii 总被引:1,自引:0,他引:1
Abstract Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Suprisingly, not all fungal cellulase components exhibit this property, and no such synergism has been observed so far between fungal and bacterial cellulases.
The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca2+ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellum cellulase to effect the breakdown of cellulose in the absence of Ca2+ and DTT. enhanced activity is observed if Ca2+ and DTT are present.
This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum . 相似文献
The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca
This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum . 相似文献
14.
Evernia prunastri (L.) Ach. produces a cellulase system capable of solubilizing crystalline cellulose. The enzyme is secreted into the incubation medium in response to the presence of 0.5% (w/v) cellobiose. Cellulase secretion is pH dependent, the maximum occurring at an initial pH of 9.0, which is rapidly decreased by the action of the lichen. The effect of cellobiose on the cellulase system is inhibited by cycloheximide and 8-azaguanine. The cellulasc activity increases with the metabolic reactivation that is initiated by thallus hydration. Several functions of cellulase in this lichen are discussed. 相似文献
15.
Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum 总被引:6,自引:0,他引:6
P Tomme S Chauvaux P Béguin J Millet J P Aubert M Claeyssens 《The Journal of biological chemistry》1991,266(16):10313-10318
Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme. 相似文献
16.
Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a C. thermocellum cipA mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The in vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases. 相似文献
17.
Abstract A protein that degrades pneumococcal walls containing choline, but not ethanolamine, in the teichoic acids has been isolated and purified from supernatants obtained from cultures of Clostridium acetobutylicum . The analyses of the degradation products of [3 H]choline-labeled cell walls treated with this enzyme indicated that the purified protein, showing an apparent M r of 115 000, is an N-acetylmuramyl- l -alanine amidase. Our results also suggest that C. acetobutylicum contains choline in its cell wall. 相似文献
18.
Mechthild Pohlschroeder Susan B. Leschine Ercole Canale-Parola 《Archives of microbiology》1994,161(1):17-24
Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H2, CO2, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded. 相似文献
19.
Sequence fingerprint and structural analysis of the SCOR enzyme A3DFK9 from Clostridium thermocellum
Robert Huether Zhi‐Jie Liu Hao Xu Bi‐Cheng Wang Vladimir Z. Pletnev Qilong Mao William L. Duax Timothy C. Umland 《Proteins》2010,78(3):603-613
We have identified a highly conserved fingerprint of 40 residues in the TGYK subfamily of the short‐chain oxidoreductase enzymes. The TGYK subfamily is defined by the presence of an N‐terminal TGxxxGxG motif and a catalytic YxxxK motif. This subfamily contains more than 12,000 members, with individual members displaying unique substrate specificities. The 40 fingerprint residues are critical to catalysis, cofactor binding, protein folding, and oligomerization but are substrate independent. Their conservation provides critical insight into evolution of the folding and function of TGYK enzymes. Substrate specificity is determined by distinct combinations of residues in three flexible loops that make up the substrate‐binding pocket. Here, we report the structure determinations of the TGYK enzyme A3DFK9 from Clostridium thermocellum in its apo form and with bound NAD+ cofactor. The function of this protein is unknown, but our analysis of the substrate‐binding loops putatively identifies A3DFK9 as a carbohydrate or polyalcohol metabolizing enzyme. C. thermocellum has potential commercial applications because of its ability to convert biomaterial into ethanol. A3DFK9 contains 31 of the 40 TGYK subfamily fingerprint residues. The most significant variations are the substitution of a cysteine (Cys84) for a highly conserved glycine within a characteristic VNNAG motif, and the substitution of a glycine (Gly106) for a highly conserved asparagine residue at a helical kink. Both of these variations occur at positions typically participating in the formation of a catalytically important proton transfer network. An alternate means of stabilizing this proton wire was observed in the A3DFK9 crystal structures. Proteins 2010. © 2009 Wiley‐Liss, Inc. 相似文献
20.
Xiao-Zhou Zhang Zuoming Zhang Zhiguang Zhu Noppadon Sathitsuksanoh Yunfeng Yang Y.-H. Percival Zhang 《Applied microbiology and biotechnology》2010,86(2):525-533
Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose
hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully
expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol−1 on Avicel and 35.9 U μmol−1 on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products.
By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not
significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48.
Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed
for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous
Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to
be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step. 相似文献