Identification of 'Haemophilus somnus' by rapid tests for pre-formed enzymes

In rapid tests for pre-formed enzymes, all strains of ' Haemophilus somnus ' examined gave positive reactions for alkaline phosphatase, cytochrome oxidase, β-glucosidase, β-glucuronidase and hippurate hydrolase but were negative for α-mannosidase, β-galactosidase and β-xylosidase, tributyrin esterase, cystine aminopeptidase and γ-glutamyl aminopeptidase. The pattern of results differed from those given by Actinobacillus, Haemophilus, 'Histophilus', Pasteurella and Taylorella species.     

Enzyme Activity Profiles in Mouse Teratocarcinomas

Abstract. Nine tumour lines with different developmental capacities were derived from spontaneous as well as from one induced teratocarcinoma: three teratocarcinoma-derived rhabdomyosarcomas TDR 602, TDR 694, and TDR 114; two teratocarcinoma-derived neuroblastomas TDN 2151 and TDN 2283; two teratocarcinoma-derived endodermal tumours TDE 274 and TDE 113; one multipotential teratocarcinoma OTT 2289, and one undifferentiated teratocarcinoma OTT 2158. Quantitative analyses of ten catabolic enzymes, i.e. alkaline and acid phosphatase, α- and β-galactosidase, α- and β-glucosidase, α-mannosidase, α-fucosidase, β-glucuronidase, and hexosaminidase were carried out at the 20-cell level, and specific enzyme activity profiles were established for each of the tumour lines studied. These profiles may be used for the biochemical identification of a tumour type at the single cell level in addition to morphological and biological criteria.     

Changes in Particulate Neuraminidase Activity During Normal and Staggerer Mutant Mouse Development

Abstract: The activity of particulate neuraminidase (sialidase, EC 3.2.1.18) in wild-type mice and the neurological mutant Staggerer was studied during development. Peak activity of this enzyme was observed at postnatal day 3 (P3) in three tissues of normal mice: cerebellum, cerebrum, and liver. In Staggerer, however, neuraminidase peak activity was observed at P27 in the cerebellum, whereas the activity was close to normal in Staggerer cerebrum and liver. Activities of other glycosidases in Staggerer (α-glucosidase (pH 3.7), α- glucosidase (pH 6.0), N -acetyl-β-hexosaminidase, β-glucosidase, and β-galactosidase) did not show significant variation compared with wild-type at P27 in any of the three tissues. This indicates that the late activity peak of particulate neuraminidase activity in the Staggerer cerebellum is neuraminidase-specific and not due to a general increase of lysosomal enzymes.     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (β-glucuronidase, β-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6–8. All three enzymes were influenced by pH, as exemplified by β-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6–6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with β-glucuronidase, which showed maximal activity at pH 6–0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8–0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

Ref cell hydrolases: Glycosidase activities in human erythrocyte plasma membranes

Summary Human erythrocyte plasma membranes were found to contain the following glycosidases: α- and β-glucosidase, α- and β-galactosidase, α- and β-fucosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylosidase and α-mannosidase. All the enzymes except β-fucosidase had activity interpreted to be on the external surface of the plasma membrane. The enzymes had optimum pH values of 5.2 to 5.0 and temperatures of 37 to 40°C. The enzymes were not greatly activated by divalent cations but Hg⁺⁺ and Pb⁺⁺ were inhibitory. The enzyme extract of the human erythrocyte plasma membranes liberated carbohydrate from intact red cells, which lead to the speculation that the glycosidases might function to modify the erythrocyte plasma membrane. The author is a Research Career Development Awardee of the National Institute of General Medical Sciences.     

Lysosomal Hydrolases in Acanthamoeba sp.

SYNOPSIS. Homogenates of axenic Acanthamoeba sp. had ribonuclease, phosphatase, proteinase, α-glucosidase, β- N -acetylglucosaminidase, and β-glucuronidase activities; all had acid pH optima. After isopycnic density-gradient centrifugation all showed the same relatively broad and unimodal distribution pattern, with a peak at a density of 1.17. This differs from the distribution of mitochondrial malate dehydrogenase and of peroxisomal urate oxidase and catalase. The findings are believed to reflect the occurrence of lysosomes in Acanthamoeba.     

Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.     

SIALOGLYCOPROTEINS AND SEVERAL GLYCOSIDASES IN DEVELOPING RAT BRAIN

Abstract— The amount of sialoglycoproteins expressed as μmol of sialic acid per g of lipid-free residue remained fairly constant in developing rat brain. However, the activity of various enzymes which may be involved in glycoprotein metabolism varied in an inconstant fashion during the period of development. The specific activity of a neuraminidase increased, N -acetyl-β-glucosaminidase remained relatively constant, while the specific activities of α-mannosidase and α-fucosidase decreased.     

A study of the enzymatic profile of soil isolates of Nocardia asteroides

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, α-glucosidase and β-glucosidase. In addition, β-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-β-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in >95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-glucoronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region. This revised version was published online in June 2006 with corrections to the Cover Date.     

β-Mannanolytic system of Aureobasidium pullulans

A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-d-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase. Received: 24 June 1996 / Accepted: 26 September 1996     

The influence of the host on expression of intestinal microbial enzyme activities involved in metabolism of foreign compounds

The activities of four enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) in selected intestinal bacteria ( Escherichia coli, Clostridium sp., Streptococcus sp., Bacteroides sp. and Lactobacillus salivarius ) were measured after growth in vitro and in vivo . The five strains differed in their activites with Clostridium sp. being the most active for β-gjucosidase, β-glucuronidase and nitroreductase, and E. coli the most active producer of nitrate reductase. Enzyme activity in vivo tended to be higher than in vitro but there were instances where the comparative activities were reversed.     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

Simultaneous production of high activities of thermostable endoglucanase and β-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus

The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and β-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l⁻¹ Solka Floc as the carbon source and 9 g l⁻¹ soymeal as the organic nitrogen source yielded 1130 nkat ml⁻¹ endoglucanase and 116 nkat ml⁻¹β-glucosidase activities after 264 h as shake cultures. In addition, good levels of β-xylanase (3479 nkat ml⁻¹) and low levels of filter-paper cellulase, β-xylosidase, α-l-arabinofuranosidase, β-mannanase, β-mannosidase, α-galactosidase and β-galactosidase were detected. Batch fermentation in a 5-l laboratory fermentor using the optimised medium allowed the production of 940 nkat ml⁻¹ endoglucanase and 102 nkat ml⁻¹β-glucosidase in 192 h. Endoglucanase and β-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 °C. Endoglucanase and β-glucosidase showed good stability at pH values 4–8 and 4–7, respectively, after a prolonged incubation (48 h at 50 °C). Endoglucanase had half-lives of 98 h at 70 °C and 4.1 h at 75 °C, while β-glucosidase had half-lives of 23.5 h at 70 °C and 1.7 h at 75 °C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h. Received: 12 November 1999 / Accepted: 14 November 1999     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Synthesis of β-mercaptoethyl-glycosides by enzymatic reverse hydrolysis and transglycosylation

Summary The synthetic potential ofAlmond β-glucosidase andJack bean α-mannosidase in the presence of high amounts of β-mercaptoethanol as glycosyl acceptor for the synthesis of β-mercaptoethyl-glycosides was studied. The regioselectivity, O-glycosylation and/or S-glycosylation, and the stereoselectivity were analyzed with the reverse hydrolysis and the transglycosylation methods. With both enzymes, high yields of condensation are obtained without the use of chemical protective groups.     

Glycosidases and Cerebellar Ontogenesis in the Rat

Abstract: Five glysosidases (α- and β-D-mannosidase, β-D-galactosidase, β- N -acetyl-glucosaminidase, and α-L-fucosidase) were studied during the postnatal development of the rat cerebellum. Each glycosidase has a particular developmental pattern. Transient decreases in the specific activities of β-mannosidase, β-D-galactosidase, and β-D- N -acetyl-glucosaminidase could be correlated with the phase of massive cell multiplication. A possible more specific role for α-D-mannosidase is discussed.     

Enzymatic studies of the extracellular mucilage of two aquatic hyphomycetes,Lemonniera aquatica andMycocentrospora filiformis

The effect of three carbohydrate-digesting enzymes, β-glucuronidase, lyticase and α-mannosidase and three proteolytic enzymes, α-chymotrypsin, papain and pronase E, on the strength of conidial attachment ofLemonniera aquatica andMycoentrospora filiformis was determined using the LH_Fowler cell Adhesion Measurement Module. Carbohydrate-digesting enzyme treatments showed significant differences in number of attached and detached, conidia versus control samples; little or no effect was observed for the proteolytic enzymes. Scanning and transmission electron microscopy showed different degrees of mucilage digestion by the carbohydrate-digesting enzymes on the germ hyphae, hyphae subtending appressoria, and appressoria of the two species. The loss of mucilage integrity and decrease in mucilage thickness were more pronounced on the hyphal sheaths than on the appressorial sheaths. Lyticase caused the most severe damage to the mucilage and cytoplasm of both fungi, particularlyL. aquatica. β-Glucuronidase and α-mannosidase exhibited more effective mucilage digestion onM. filiformis than onL. aquatica. Results indicate that the mucilage of the two species is mainly polysaccharide, containing more β-1,3-glucans than β-glucuronide and α-mannosyl residues. Variability of mucilage composition exists between these species and also between different structures of the same fungus.     

Effects of dietary fructan on cecal enzyme activities in rats

In this study, we have attempted to determine the effects of dietary fructose polymers (fructan), high molecular-weight β-(2,6)-linked levan, and low-molecular-weight β-(2,1)-linked inulin, on two intestinal enzymes (β-glucuronidase and β-glucosidase). As a preliminary experiment, when intestinal microflora were cultured in anaerobic media harboring levan or its oligosaccharides, bacterial cell growth was observed in the levanoligosaccharide-supplemented media, but not in the levan-supplemented media, indicating that levan’s size is important for the utilization by intestinal bacteria of levan as an energy source. In our animal study, the intake of a levan-rich diet was determined to significantly attenuate the activity of the harmful enzyme β-glucuronidase, but did not affect the activity of β-glucosidase.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Glucosidase Activity in Acanthamoeba (Mayorella) palestinensis. The Effect of Glucose and Natural Glucosides on α- and β-Glucosidases

SYNOPSIS. Acanthamoeba ( Mayorella ) palestinensis produces high basal levels of α- and β -glucosidases, the latter being much more active than the former. Glucose, an essential growth substance, has a dual effect on the glucosidase activity. Growth concentrations (1%) of glucose inhibit, while low levels elevate the activity of both enzymes. Natural α-glucosides support growth in the same manner as glucose and raise the activity of both enzymes to the same extent. β -glucosides, on the other hand, are weak growth substrates, but stronger inducers, especially for β -glucosidase activity. The role of the glucosidases in the over-all metabolism of the ameba is discussed.