Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts

Aims: The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. Methods and Results: A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4‐β‐glucosidase activity, but a strain dependent β‐glucanase activity was observed. Some Brettanomyces species did show 1,4‐β‐glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Conclusions: Pronounced exo‐β‐glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. Significance and Impact of the Study: The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo‐β‐glucanase activity. Higher activities can be found in Brettanomyces species with β‐glucosidase activity.     

Genetic and molecular analysis of tissue-culture-derived Ac elements

Summary Our previous experiments on maize (Zea mays L.) plants regenerated from tissue culture revealed genetic activity characteristic of the transposable element Activator (Ac) in the progeny of 2–3% of the plants tested, despite the lack of Ac activity in the progenitor plants. The objective of the present study was to determine whether the presence of Ac activity in tissue-culture-derived plants was associated with changes in the number or structure of Ac-homologous DNA sequences. Families segregating for Ac activity were obtained by crossing plants heterozygous for Ac activity onto Ac-responsive tester plants. A DNA probe derived from a previously isolated Ac sequence was used to examine the Ac-homologous sequences within individual progeny seedlings of segregating families and noncultured control materials. All plants tested had six or more Ac-homologous DNA sequences, regardless of whether Ac activity was present. In the segregating progeny of one tissue-culturederived plant, a 30-kb Ac-homologous SstI restriction fragment and a 10-kb Ac-homologous BglII restriction fragment were found to cosegregate with Ac activity. We propose that these fragments contained a previously silent Ac sequence that had been activated during tissue culture. Although one or more Ac sequences were often hypomethylated at internal PvuII and HpaII sites in plants with Ac activity, hypomethylation was not a prerequisite for activity. Reduced methylation at these sites may have been a result rather than a cause of Ac activity.     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.     

Correlative evidence for peroxidase involvement in disease resistance against <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight of tomato

Eighteen tomato genotypes, with varying degree of response to Alternaria leaf blight disease (ALBD) were used to assess the possible involvement of protease and peroxidase activities in disease response. Pre-infectional protease activity varied noticeably in tested genotypes. Highest pre-infectional protease activity was observed in susceptible genotype CLN-2123. Post-infectional protease activity level was generally lower when compared with pre-infectional level in all genotypes with exception of unchanged level in Tibrido. There was no correlation between post-infectional protease activity and percent disease index (%DI). In contrast, pre- and post-infectional leaf peroxidase activities showed a significant (p < 0.01) negative correlation with %DI. Genotypes with higher pre-infectional peroxidase activity performed better on exposure to Alternaria alternata infection and accumulate enhanced peroxidase activity. Tibrido accumulated highest peroxidase activity while level was lowest in 1621P, which showed highest ALBD incidence. Moreover, genotypes with better resistance to A. alternata infection maintained higher post-infectional peroxidase activity. In resistant (Tibrido) and all moderately resistant genotypes, leaf peroxidase activity raised after inoculation when compared with the pre-inoculation level. I summary, higher pre- and post-infectional peroxidase activity was found to be associated with Alternaria leaf blight resistance. The peroxidase activity can be used as a biochemical tool in marker-assisted screening of tomato germplasm for Alternaria leaf blight resistance.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

Inducible Formation of Urease in Canavalia ensiformis

Increase in urease activity in leaves of Canavdlia ensifomis has been demonstrated. The activity of excised leaves increased by about 100 percent when 1.5 × 10^?1M urea was added externally as inducer. Glycine-1-¹⁴C was used to investigate whether the increase in activity was dependent on de novo protein synthesis. The Incorporation of labeled amino acid into urease was twofold higher in induced samples than in non-induced ones. This indicates that the increase in activity is connected with de nova protein synthesis. The once increased activity was always followed by a rapid decrease. The urease activity was lost constantly with time after the external addition of ammonia in vivo. The inhibitory action of ammonia on urease fa vitro was eliminated by dialysis. Accordingly it may be concluded that the loss of activity was dependent on the product repression by ammonia.     

NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

Differences of urease activity and expression of associated genes according to gastric topography

Background: We hypothesize that pH difference between acid‐secreting corpus and non‐secreting antrum might influence the activity of H. pylori’s urease and/or related genes. We therefore measured urease activity and the expression of amiE whose encoded protein that hydrolyzes short‐chain amides to produce ammonia. Materials and Methods: Fifty‐four patients were recruited into this study. Each gastroscopic biopsy specimen collected from the antrum and body of each patient was immediately used to measure urease activity using serial changes of urease activity (ammonia levels) during 60 minutes. Probe specific for amiE was labeled with a biotin nick‐translation kit and was used to detect expression of these genes (mRNA) in fresh‐frozen gastroscopic biopsy specimens using fluorescent in situ hybridization (FISH). Results: Urease activity at 60 minutes from the gastric antrum and body of all patients infected with H. pylori was 399.5 ± 490.5 and 837.9 ± 1038.9 μg/dL, respectively (p = .004). Urease activity in the antrum was correlated with H. pylori density. Urease activity or H. pylori density in the antrum was significantly correlated with chronic active inflammation; in contrast, this correlation was not found in the gastric body. The expression level of amiE was 1.5 times higher (p < .05) in the gastric body compared with the antrum. Conclusion: Topographically, the urease activity in body was much higher than in antrum. The expression level of amiE was higher in the gastric body compared with the antrum.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Serum malondialdehyde levels,myeloperoxidase and catalase activities in patients with nephrotic syndrome

Abstract

Objectives

Some studies have indicated the pathophysiological importance of reactive oxygen species (ROS) in patients with nephrotic syndrome. Myeloperoxidase (MPO) is a leukocyte-derived enzyme-generating ROS that has been proposed to exert a wide array of pro-atherogenic effects throughout all stages of the atherosclerotic process. The aim of this study was to investigate the serum malondialdehyde (MDA) levels, MPO and catalase activities in patients with adult nephrotic syndrome.

Patients and Methods

Twenty-four patients with nephrotic syndrome and 24 healthy controls were enrolled. Serum MPO activity, catalase activity, and MDA levels were assessed.

Results

Serum MPO activity and MDA levels were signi?cantly higher in patients with nephrotic syndrome than controls (both, P < 0.001), while catalase activity was signi?cantly lower (P < 0.001). Serum catalase activity was found to be significantly correlated with MPO activity (r = ?0.417, P = 0.003) and MDA levels (r = ?0.532, P = 0.007). The serum MDA levels were also found to be significantly correlated with MPO activity (r = 0.419, P = 0.003).

Conclusions

We concluded that serum MPO activity and oxidative stress were increased and that serum catalase activity was decreased in patients with adult nephrotic syndrome. In addition, these results indicate that increased MPO activity is associated with an oxidant–antioxidant imbalance that may contribute to atherosclerosis in patients with adult nephrotic syndrome.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73

An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.     

Differential Phytate Utilization in <Emphasis Type="Italic">Candida</Emphasis> species

The present study was undertaken to evaluate and characterize the phytase activity in different Candida species. A total of 113 Candida isolates representing eight species were examined for phytase activity by an agar plate assay using the calcium salt of phytic acid as the sole phosphorus source. A phytase-positive phenotype was identified by the formation of a clear halo around a fungal colony. Cell-bound differential phytase activity was observed in Candida isolates at inter- and intra-species levels. Although phytase activity was not affected by the supplementation of external phosphate in C. albicans, C. dubliniensis, C. glabrata, and C. kefyr, elevated phytase activity was evident in C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis in phosphate-free medium. Further characterization showed that, in general, relatively higher phytase activity was observed at more acidic pHs, and the phytase activity increased with incubation temperature, reaching a maximum at 55 or 65°C. Taken together, the findings demonstrated, for the first time, differential phytase activities in different Candida species. Phytase activity may be a contributing factor to fungal survival and proliferation within the human gastrointestinal tract, where nutrients are usually scarce.     

Non-destructive measurement of acid phosphatase activity in the rhizosphere using nitrocellulose membranes and image analysis

We developed a method using nitrocellulose membranes and image analysis to localise and quantify acid phosphatase activity in the rhizosphere of two plant species, one with cluster roots (Dryandra sessilis (Knight) Domin) and another with ectomycorrhizal roots (Pinus taeda L.). Membranes were placed in contact with roots and then treated with a solution of x, α-naphthyl phosphate and Fast Red TR. Acid phosphatase activity was visualised as a red imprint on the membrane. We quantified acid phosphatase activity by image analysis of scanned imprints. The method was used to estimate the spatial distribution of acid phosphatase activity within particular root classes (lateral roots, mycorrhizal roots, root clusters). Over 95% of the acid phosphatase activity of the root system of D. sessilis was associated with cluster roots, and between 20 and 32% of the root surface active. About 26 % of the acid phosphatase activity of the root system of P. taeda was associated with mycorrhizal roots and unsuberised white root tips and less than 10% of the root surface was active, irrespective of root type. This non-destructive method can be used for rapid, semi-quantitative assessment of acid phosphatase activity in the laboratory and in situ. This revised version was published online in August 2006 with corrections to the Cover Date.     

Seasonal variation in antifungal,antibacterial and acetylcholinesterase activity in seven South African seaweeds

Seven seaweeds were collected from the intertidal zone at Rocky Bay on the east coast of South Africa. The species were Caulerpa racemosa var. laetevirens, Codium capitatum, Halimeda cuneata, Ulva fasciata, Amphiroa bowerbankii, Amphiroa ephedraea and Dictyota humifusa. Six bimonthly collections were made within a few days of the new moon to correspond with spring tide. Methanol extracts were tested for antifungal, antibacterial and acetylcholinesterase (AChE) inhibitory activity. No seasonal variation was observed in antifungal activity, with D. humifusa extracts being the most active. The seaweed extracts inhibited the growth of the Gram-positive bacteria, with Bacillus subtilis being more susceptible than Staphylococcus aureus. Dictyota humifusa was the only seaweed able to inhibit the Gram-negative Escherichia coli. Seasonal variation in antibacterial activity was observed, with the extracts generally having no activity in summer and having antibacterial activity in late winter (July collection) and early spring (September and November collections). Dictyota humifusa was the most effective seaweed species, having antibacterial activity throughout the year. All the extracts tested had AChE inhibitory activity, with no seasonal variation in the levels of activity. Dictyota humifusa extracts were the most effective at inhibiting AChE activity.     

In vivo Characterization of Nitrate Reductase Activity in Cotton

The in vivo nitrate reductase activity in leaf tissue of cotton (Gossypium hirsutum L.) was characterized. Enzymatic activity was linear with time up to 60 min. The assay for nitrate reductase activity was optimized in leaf slices 400 μm wide incubated in an anaerobic system at 30°C, in a 0.02 M KNO₃ medium at pH 7.0 with 1 % propanol. In vivo activity was highest in recently matured leaves at the top of the plant. Both light and nitrate enhanced in vivo enzymatic activity. The activity was highest after 9 hours in the light and then decreased steadily for several more hours even in the presence of light. The nitrate reductase activity was more strongly correlated to the levels of NO₃-N in the culture solution than to the NO₃-N level in the tissue. The utility of this technique in nitrate reductase assay in a tissue containing large amounts of phenolic compounds is discussed.     

Optical Resolution and Biological Activities of α-Ionylideneacetic Acid.

Nuclease activity associated with cells and protoplasts was analyzed by agarose gel electrophoresis. Datura innoxia protoplasts were found to possess a high exonuclease activity. On the other hand, Datura innoxia cells had an endonuclease activity, but no apparent exonuclease. The exonucleases from the protoplasts were active at pH 5 and 6, but not at pH 9. Endonuclease activity from the cells was also inhibited at pH 9. Cultured cells of Daucus carota, Glycine max, Pisum sativum and Vicia hajastana had endonuclease activity, but did not exhibit exonuclease activity. Nicotiana suaveolens cells had both types of nuclease activity. On the other hand, cells from cereals such as Triticum monococcum, Oryza sativa, and Zea mays had active exonuclease activity.     

Characterization of gastrointestinal chitinase in the lizard Sceloporus undulatus garmani (Reptilia: Phrynosomatidae)

Most studies on chitinase activity in lizards have been concerned with Palaearctic (European) and Laurasian (Middle Eastern and Asian) taxa. Several genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta, have been shown to possess chitinolytic activity. To date, only one New World lizard, Anolis carolinensis, has been reported to exhibit chitinolytic activity. In the present study, chitinase activity was characterized in a second New World taxon, Sceloporus undulatus garmani, a New World, phrynosomatid lizard. Chitinolytic activity was measured by incubating tissue extracts with a radioactive chitin substrate, acetyl-[H³]chitin and determining acid soluble radioactivity as an estimate for chitin hydrolysis. Chitinolytic activity was present in stomach, small intestine and pancreas extracts, with the stomach and pancreas having the highest specific activities. Chitinolytic activity was higher at pH 4.5 than at pH 7.5. The stomach chitinase is immunologically similar to the gastric chitinase previously described for rainbow trout. Western blot analysis showed anti-chitinase cross-reactivity in the extracts of the stomach, but no cross-reactivity in the pancreatic or intestinal extracts, suggesting different isoforms of chitinase. There was no detected lysozyme activity (less than 0.01 mg/ml lysozyme) present in the extracts of the stomach, small intestine and pancreas. The localization of chitinolytic activity in S. u. garmani is in agreement with earlier reptilian reports on the distribution of chitinase.