Specific xyloglucanases as a new class of polysaccharide-degrading enzymes

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.     

Newer uses of microbial enzymes in food processing

Microbial enzymes are widely used in food processing: many new enzymes and enzyme processes acting on nearly all types of organic food components — starch, sugars, proteins, fats, fibers, and flavour compounds — have come into the industry during the 1980s and their application has a major impact on enzyme technology in general. The particular roles of immobilized enzymes and genetic engineering in food enzymology are briefly discussed.     

The distribution of polysaccharide-degrading enzymes in the bovine rumen digesta ecosystem

The location and level of activity of the principal polysaccharidases and glycoside hydrolases involved in the degradation of plant structural and storage polysaccharides were monitored in microbial populations isolated from liquid and particulate phases of bovine rumen digesta. The three principal subpopulations, and their constituent subgroups studied, all contained polysaccharide depolymerizing enzymes; however, the specific activities of the enzymes that degraded the plant cell wall structural polymers were highest within the adherent particle-associated populations. Separate functional groups of organisms could be recongnized in the particle-associated population by their distinctive enzyme profiles.     

Application of enzymes for textile fibres processing

This review highlights the use of enzymes in the textile industry, covering both current commercial processes and research in this field. Amylases have been used for desizing since the middle of the last century. Enzymes used in detergent formulations have also been successfully used over the past 40 years. The application of cellulases for denim finishing and laccases for decolourization of textile effluents and textile bleaching are the most recent commercial advances. New developments rely on the modification of natural and synthetic fibres. Advances in enzymology, molecular biology and screening techniques provide possibilities for the development of new enzyme-based processes for a more environmentally friendly approach in the textile industry.     

The enzyme as drug: application of enzymes as pharmaceuticals

Enzymes as drugs have two important features that distinguish them from all other types of drugs. First, enzymes often bind and act on their targets with great affinity and specificity. Second, enzymes are catalytic and convert multiple target molecules to the desired products. These two features make enzymes specific and potent drugs that can accomplish therapeutic biochemistry in the body that small molecules cannot. These characteristics have resulted in the development of many enzyme drugs for a wide range of disorders.     

Phages as friends and enemies in food processing

1. Download : Download high-res image (93KB)
2. Download : Download full-size image

     

Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.     

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.     

Application of dried sewage sludge as phenol biosorbent

The aim of this work was to determine the potential application of dried sewage sludge as a biosorbent for removing phenol from aqueous solution. Results showed that biosorption capacity was strongly influenced by the pH of the aqueous solution with an observed maximum phenol removal at pH around 6-8. Biosorption capacity increased when initial phenol concentration was increased to 110 mg/L but beyond this concentration, biosorption capacity decreased suggesting an inhibitory effect of phenol on biomass activity. Biosorption capacity decreased from 94 to 5 mg/g when biosorbent concentration was increased from 0.5 to 10 g/L suggesting a possible competitive effect of leachable heavy metals from the sludge. The effect of Cu2+ on biosorption capacity was also observed and the results confirmed that the phenol biosorption capacity decreased when concentration of Cu2+ in the sorption medium was increased up to 15 mg/L. Desorption of phenol using distilled deionized water was less than 2% suggesting a strong biosorption by the biomass.     

Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.     

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

Effect of an algicidal product fromOscillatoria late-virens and of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) on growth, photosynthesis, and mouse toxicity inMicrocystis PCC 7820 was examined. Their lethal concentrations abolished photosystem (PS)-II reactions and eventually bleached and detoxified the cyanobacterium. Although loss of protein, chlorophyll, and toxicity were also induced by sublethal antibiotic doses, photosynthetic activities remained unchanged and developed antibiotic tolerance. These effects could be duplicated in natural conditions, implying utility of the natural algicide in control of toxic cyanobacteria.     

Production of polysaccharide-degrading enzymes by saprophytic fungi from glyphosate-treated flax and their involvement in retting

The fungi present on glyphosate-treated flax plants were isolated. Cladosporium herbarum, Epicoccum nigrum, Botrytis cinerea and yeasts occurred most frequently immediately after glyphosate treatment but as retting progressed the frequency of occurrence of Fusarium culmorum, Alternaria alternata and a Phoma sp. increased. Many of the fungi isolated from retting flax were also present as epiphytes on healthy flax stems. Glyphosate was shown to be fungitoxic in vitro but it had only a very slight effect on fungi colonising the flax. The application of sucrose and urea to flax 1 wk after glyphosate treatment resulted in more rapid fungal colonisation of the stems, but did not significantly enhance retting. When grown on sterilised flax stem sections, fungi known to be saprophytic on flax produced polysaccharide-degrading enzymes. All seven fungi tested produced polygalacturonase, pectin-lyase and xylanase. The greatest cellulase activity was present in stem tissues inoculated with F. culmorum and the Phoma sp. while no cellulase was detected in tissue inoculated with B. cinerea, a Mucor sp. or a Penicillium sp. Extracts from flax inoculated with the cellulolytic fungi caused the solubilisation of native cellulose. Pectinases, xylanase and cellulase were also detected in naturally-colonised senescing and dead flax stems. Stems which had been treated with a sucrose solution tended to contain the greatest enzyme activity.     

A simple preparation of an enzyme reactor producing nicotinamidemononucleotide

Summary An enzyme reactor which produces nicotinamidemononucleotide is easily prepared by adsorption of NAD pyrophosphatase to phosphocellulose. The separation and purification of the mononucleotide is achieved in a single chromatographic step. The spectrophotometric data of purified NMN and its cyanide adduct were redetermined.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Synthetic peptides as substrates for processing enzymes in the bovine pituitary

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additional cleavage, following glutamic acid residues, was also seen.     

Histochemical study of enzyme systems in frozen dried tissue

Summary Cytochrome oxidase, succinic dehydrogenase, phosphorylase and branching enzymes were consistently demonstrated in frozen dried paraffin embedded dog myocardium if the appropriate solvent for deparaffinization was used. No modification of the incubating medium was necessary for cytochrome oxidase. For succinic dehydrogenase a 0.8 M succinate solution was used instead of the usual 0.2 M solution and phenazine methosulfate had to be added to the incubating medium. No primer or activators were necessary for demonstration of phosphorylase and branching enzyme activities.This work was supported by grant number HE-07605-06 from the National Heart Institute, National Institutes of Health, Bethesda, Md.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Synthesis of malformin by an enzyme preparation from Aspergillus niger

Yukioka, M. (University of Hawaii, Honolulu), and T. Winnick. Synthesis of malformin by an enzyme preparation from Aspergillus niger. J. Bacteriol. 91:2237-2244. 1966.-An enzyme fraction derived from disrupted Aspergillus cells was able to utilize each of the component labeled amino acids of malformin for the synthesis of this cyclic pentapeptide. The process was stimulated by adenosine triphosphate, K(+), and Mg(++), and was optimal at approximately pH 8.5. It was not affected by inhibitors of protein synthesis (ribonuclease, chloramphenicol, puromycin). There is evidence that cysteine, rather than cystine, was incorporated into peptide linkage, so that the disulfide bridge of malformin was formed subsequently. Although only the d isomers of cysteine and leucine occur in the malformin molecule, the l, as well as the d form of these amino acids, was readily utilized by the enzyme preparation. As in the case of several other microbial peptide systems, it appears that the d enantiomorph can arise from the l isomer at an intermediate stage of polypeptide synthesis.