Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB. (33/2543)

An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45 degrees C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The K(m) and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 microM as acetyl content in the polymer) and 36.0 microM, and 66.8 and 39.1 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.  (+info)

A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi. (34/2543)

Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 degrees C and pH 6.5. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in lambdaZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A") was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 degrees C and a pH optimum of 6.5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A" cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A") bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A" consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.  (+info)

Heparin binding peptides co-purify with glycosaminoglycans from human plasma. (35/2543)

Glycosaminoglycans (GAGs) are complexed with plasma proteins and proteolysis of plasma reduced the protein-GAG ratio about 140-fold. After dialysis, analysis by gradient PAGE revealed heparinase-1-sensitive GAGs, thus suggesting that heparin could be among the plasma GAGs. However, after dialysis most of the plasma GAGs were still not 'free'. PAGE of peptides resistant to proteolysis showed high molecular weight bands on the two sides of the dialysis membrane despite the 3.5 kDa molecular weight cut-off. Progressive dilution of the sample allowed passage of peptides appearing as high molecular weight bands in the diffusate. We interpret this phenomenon as the presence of low molecular weight peptides that aggregate when concentrated. Peptides on both sides of the membranes bound heparin.  (+info)

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A. (36/2543)

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.  (+info)

Cellulose microfibrils: visualization of biosynthetic and orienting complexes in association with the plasma membrane. (37/2543)

Cellulose microfibril biosynthesis, assembly, and orientation in the unicellular green alga, Oocystis, is visualized in association with a linear enzyme complex embedded in the B face of the plasma membrane. Granule bands of the A face and complementary ridges of the B face are postulated to assist in the orientation of recently synthesized microfibrils. A model for microfibril synthesis and orientation is proposed and correlated with current hypotheses regarding cellulose biosynthesis in higher plants.  (+info)

Preparation of oligomeric beta-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a trichoderma reesei cellulase. (38/2543)

Oligoglycosyl (allyl, 2,3-dihydroxypropyl, ethyl, 2-hydroxyethyl, and methyl) beta-glycosides were generated by endo -transglycosylation reactions catalyzed by commercially available Trichoderma reesei cellulase. A polymeric donor substrate (xyloglucan or cellulose) was incubated with the enzyme in an aqueous solution containing 20% of the acceptor alcohol (allyl alcohol, glycerol, ethanol, ethylene glycol, and methanol, respectively). The products of these reactions included oligomeric alkyl beta-glycosides and reducing oligosaccharides. The high yield of alkyl beta-glycosides may be explained by the resistance of the xyloglucan beta-glycosides to cellulase-mediated hydrolysis. The resistance of the oligoxyloglucan beta-glycosides to endo glucanase catalyzed hydrolysis supports the hypothesis that productive binding of the glycan substrate depends on its interaction with enzyme subsites on both sides of the cleavage point, leading to distortion of the ring geometry of the residue whose glycosidic bond is cleaved. Oligoxyloglucan beta-glycosides were purified by a combination of gel-permeation and reversed-phase HPLC and were structurally characterized by MS and NMR spectroscopy. These results demonstrate that novel oligosaccharide beta-glycosides can be efficiently produced by enzyme-catalyzed fragmentation/transglycosylation reactions starting with a polysaccharide donor substrate. This class of reactions may represent a convenient source of beta-glycosides to be used as synthons for the rapid synthesis of complex glycans.  (+info)

Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. (39/2543)

BACKGROUND: Mucins play an important protective role in the colonic mucosa. Luminal factors modulating colonic mucus release have been not fully identified. AIM: To determine the effect of some dietary compounds on mucus discharge in rat colon. METHODS: An isolated vascularly perfused rat colon model was used. Mucus secretion was induced by a variety of luminal factors administered as a bolus of 1 ml for 30 minutes in the colonic loop. Mucin release was evaluated using a sandwich enzyme linked immunosorbent assay supported by histological analysis. RESULTS: The three dietary fibres tested in this study (pectin, gum arabic, and cellulose) did not provoke mucus secretion. Luminal administration of sodium alginate (an algal polysaccharide used as a food additive) or ulvan (a sulphated algal polymer) induced a dose dependent increase in mucin discharge over the concentration range 1-25 mg/l (p<0.05 for 25 mg/l alginate and p<0.05 for 10 and 25 mg/l ulvan). Glucuronic acid and galacturonic acid, which are major constituents of a variety of fibres, produced significant mucin secretion (p<0.05). Hydrogen sulphide and mercaptoacetate, two sulphides produced in the colonic lumen by microbial fermentation of sulphated polysaccharides, did not modify mucin secretion. Among the short chain fatty acids, acetate (5-100 mM) induced a dose dependent release of mucus (p<0.05 for 100 mM acetate). Interestingly, butyrate at a concentration of 5 mM produced colonic mucin secretion (p<0.05), but increasing its concentration to 100 mM provoked a gradual decrease in mucus discharge. Propionate (5-100 mM) did not induce mucin release. Several dietary phenolic compounds (quercetin, epicatechin, resveratrol) did not provoke mucus discharge. CONCLUSIONS: Two algal polysaccharides (alginate and ulvan), two uronic acids (glucuronic acid and galacturonic acid), and the short chain fatty acids acetate and butyrate induce mucin secretion in rat colon. Taken together, these data suggest that some food constituents and their fermentation products may regulate the secretory function of colonic goblet cells.  (+info)

Comparison of cellulose diacetate and polysulfone membranes in the outcome of acute renal failure. A prospective randomized study. (40/2543)

BACKGROUND: Whether the nature of haemodialysis (HD) membranes can influence the outcome of acute renal failure (ARF) remains debatable. Recent studies have suggested that dialysis with bioincompatible unsubstituted cellulosic membranes is associated with a less favourable patient outcome than dialysis with biocompatible synthetic membranes. Since we generally use a modified cellulosic membrane with substantially lower complement- and leukocyte-activating potential than cuprophane, for dialysis of patients with ARF, and because there are no data in the literature regarding the influence of modified cellulosic membranes on the outcome of patients with ARF, we compared the outcome of ARF patients dialysed either with cellulose diacetate or with a synthetic polysulfone membrane. We also investigated the potential role of permeability by comparing membranes with high-flux versus low-flux characteristics. METHODS: This prospective, randomized, single centre study included 159 patients with ARF requiring HD. Patients were stratified according to age, gender, and APACHE II score and then randomized in chronological order to one of three dialysis membranes: low-flux polysulfone, high-flux polysulfone and meltspun cellulose diacetate. RESULTS: Aetiologies of ARF and the prevalence of oliguria were similarly distributed among the three groups. There was no significant difference between the three groups for survival (multivariate Cox's proportional hazards model, P=0.57), time necessary to recover renal function (P=0.82), and number of dialysis sessions required before recovery (P=0.86). Multivariate analysis showed that survival was significantly influenced only by the severity of the disease state (APACHE III score, P<0.0001), but not by the nature of the dialysis membrane (P=0.57) or the presence of oliguria (P=0.24). CONCLUSIONS: Among patients with ARF requiring HD survival and recovery time are not significantly influenced by the use of either meltspun cellulose diacetate or the more biocompatible high-flux or low-flux polysulfone. Dialysis using modified cellulose membranes is just as effective as dialysis using synthetic polysulfone membranes, but at a lower cost. In addition, the flux of the membrane did not influence patient outcome.  (+info)