Fermentation of pectin by a newly isolated Clostridium thermosaccharolyticum strain

Abstract By enrichment on pectin a thermophilic anaerobic bacterium was isolated. This strain, identified as Clostridium thermosaccharolyticum , was capable of fast growth on pectin (μ_max 0.58 h⁻¹) forming acetate, butyrate, hydrogen, carbon dioxide, methanol and traces of ethanol. The optimum temperature for growth was 58°C and the optimal pH was 6. The initial breakdown of pectin was catalysed by methylesterase and polygalacturonate hydrolase activity; no polygalacturonate lyase activity was found.     

Properties of polygalacturonate and cell cohesion in apple fruit cortical tissue

Examination of the hydrodynamic properties of polygalacturonate fractions from unripe and ripe apple tissue suggested that the wall bound fraction was degraded during ripening but that the soluble fraction was not. Esterification of cell wall preparations with CH₂N₂ caused solubilisation of polygalacturonate. Acid MeOH caused more extensive solubilization, but this reagent hydrolysed arabinofuranosyl linkages. Both reagents reduced the cohesion of EtOH extracted apple tissue. This effect could also be achieved by treatment with sodium polyphosphate at pH 4 but not by EDTA or chaotropic agents. Free carboxyl groups on polygalacturonate probably maintain cell cohesion through co-operative binding of Ca²⁺ ions. The integrity of primary wall structure is thought to depend upon non-covalent bonding between cellulose, protein and polygalacturonate.     

Catabolism of sulphoquinovosyl diacylglycerol by an enzyme preparation from Phaseolus multiflorus

An enzyme which will deacylate sulphoquinovosyl diacylglycerol (SQDG) has been partially purified from the leaves of runner bean (Phaseolus multiflorus). No monoacyl intermediate was observed and the acyl hydrolase was more active towards unsaturated molecular species of SQDG than towards saturated species. The major peak of activity of SQDG acyl hydrolase, separated on both DEAE-cellulose and Sephadex columns, also contained galactolipid acyl hydrolase activity. The distribution of these activities together with substrate competition and inhibitor experiments indicated that at least part of the SQDG acyl hydrolase activity was due to an enzyme that also hydrolysed galactolipids.     

添加保护剂促进碱性果胶酶的工业保藏稳定性

保藏稳定性对于商品酶具有重要的实际意义。通过对碱性果胶酶的保护剂进行研究,最终筛选得到最优复合保护剂配方为：Tween201mL／L,六偏磷酸钠1．5g／L,明胶5g／L,甘油5％（口～）,CaCl22g／L;热稳定性提高18倍。此外在常温保藏时,在酶液中添加0．3‰山梨酸钾和0．3‰苯甲酸钠,能有效提高保藏效果,减少酶液染茵、混浊和发臭,最终常温保藏90d后酶活保留率为84．5％,达到工业化保藏的要求。     

Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group

Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes. The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis. The presence of the hydrolase was also confirmed by enzyme assay. In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P. diminuta and P. vesicularis. An absence of cytosine deaminase activity was found when assaying extracts of the two type strains. Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P. vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P. diminuta growth on dihydrothymine as a nitrogen source.     

Effect of 2,4-dichlorophenoxyacetic acid treatment on the soluble and cell wall bound protein in Cichorium intybus root tissue

Measurements have been made of protein and activities of enzymes bound to the cell wall of chicory root tissue. It has been shown that promotion of cell enlargement brought about by treatment with the highly active growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) does not arise from bound enzyme released from the wall. Experiments with inhibitors of protein synthesis suggest that the enhanced invertase and hydrolase activity found in soluble protein extracts after 2,4-D treatment arises from the synthesis of small amounts of protein.     

Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (DeltaGalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in DeltaGalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of DeltaGalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of DeltaGalA.     

Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor alpha into the bronchoalveolar lavage (BAL) fluid, which was not accompanied by epithelial cell injury. The treatment, however, did not change significantly the levels of anandamide and the related compound palmitoylethanolamide in the cell-free fraction of the BAL fluid. The activities of the anandamide synthetic enzymes N-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity.     

Involvement of an Intracellular Oligogalacturonate Hydrolase in Metabolism of Pectin by Clostridium thermosaccharolyticum

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initial steps of pectin degradation by Clostridium thermosaccharolyticum, were shown to be induced on the polymeric substrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose. The digalacturonate and trigalacturonate produced by the extracellular pectin methylesterase-polygalacturonate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducible oligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from the polygalacturonate hydrolase and characterized. Its temperature optimum was 65°C, and its pH optimum was 6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65°C. The maximum reaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was released by hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modified Entner-Doudoroff route.     

Metabolism of polymethylgalacturonate in apple fruit cortical tissue during ripening

Changes in the composition and metabolism of polymethylgalacturonate were followed in ripening apples. After the onset of ethylene production and fruit softening total polygalacturonate decreased and the water soluble fraction increased. No change was detected in the overall degree of esterification but the esterification of the water soluble fraction increased. Incorporation of radioactivity from methionine-[¹⁴C] into Me groups on polygalacturonate continued during ripening but incorporation from inositol-[³H] decreased sharply. Cell separation probably depends upon the removal of low ester polygalacturonate from the middle lamella by exopolygalacturonase; the continued incorporation from methionine-[¹⁴C] is probably due to synthesis of new polymethylgalacturonate.     

Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (～ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.     

High-performance liquid chromatographic determination of lipoamidase (lipoyl-X hydrolase) activity with a novel substrate, lipoyl-6-aminoquinoline

An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been developed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) through lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time-course, the dependence on enzyme and substrate concentrations were performed by using LAQ and a model enzyme (milk lipoamidase). Moreover, this method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate, we demonstrated that LAQ hydrolase was present in some specific tissues: LAQ hydrolase was solely present in the grey matter and not in the white matter in the human cerebrum. Furthermore, LAQ hydrolase activity was shown to increase in human liver cancer. Thus, this enzyme assay method is expected to be applicable to the tissue distribution study and also to the basic research on human diseases such as cancer.     

A new structural class of S-adenosylhomocysteine hydrolase inhibitors

Effective inhibitors of S-adenosylhomocysteine hydrolase hold promise towards becoming useful therapeutic agents. Since most efforts have focused on the development of nucleoside analog inhibitors, issues regarding bioavailability and selectivity have been major challenges. Considering the marine sponge metabolite ilimaquinone was found to be a competitive inhibitor of S-adenosylhomocysteine hydrolase, new opportunities for developing selective new inhibitors of this enzyme have become available. Based on the activities of various hybrid analogs, SAR studies, pharmacophore modeling, and computer docking studies have lead to a predictive understanding of ilimaquinone’s S-adenosylhomocysteine hydrolase inhibitory activities. These studies have allowed for the design and preparation of simplified structural variants possessing new furanoside bioisosteres with 100-fold greater inhibitory activities than that of the natural product.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

Structural and functional insights into peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.     

Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus

Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000.     

Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.     

Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture

Summary The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, andα-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.     

Subcellular Distribution and Developmental Expression of Cholesterol Ester Hydrolases in Fetal Rat Brain Cultures

Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19- to 21-day-old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two- to three-fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.     

Retinyl ester hydrolases in retinal pigment epithelium

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.