Identification of a fibrinolytic enzyme by <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and its potential as a bacteriolytic enzyme against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg⁻¹. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.     

Structure of the peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on hydrocarbons

The peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on aliphatic hydrocarbons were isolated. They contained muramic acid. glucosamine, alanine, d-glutamic acid and meso-diaminopimelic acid in a molar ratio of about 0.5:0.5:1.6:1.0:1.0 (M. glucidolytica) and 0.8:0.7:1.3:1.0:1.0 (M. lwoffi).The peptidoglycans were lysozyme-resistant. However, when treated with formamide, they could be partially degraded by lysozyme. The fragments were purified and their structure determined. In both strains, the peptide subunits consisted mainly of tripeptides (l-Ala-d-Glu-meso-DAP) and tetrapeptides (l-Ala-d-Glu-meso-DAP-d-Ala), most of them being directly cross-linked. It is concluded that in both strains the primary structures of the peptidoglycans are closely related.     

Magnetite-alginate beads for purification of some starch degrading enzymes

Starch degrading enzymes, viz., β-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. β-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.     

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.     

Medicinal herb extracts inhibit growth of<Emphasis Type="Italic">Rhabditis elongate</Emphasis> (Nematoda:<Emphasis Type="Italic">Rhabditidae</Emphasis>)

We tested the influence of extracts from three medicinal herbs —Salvia miltiorrhiza, Schizandra chinensis, andEugenia caryophyllata — on activity of the nematodeRhabditis elongate. Treatment with f.caryophyllata was most useful, causing the greatest decrease in populations and mobility, but did not have any detrimental effect on the initial growth of the host microorganism,Escherichia coli. For example, when 0.5 g/L of the extract was added to an inoculated liquid culture, we counted 710 nematodes/mL, with a multiplication rate 5 times greater than the initial population. This was in contrast to the control sample, which had a count of 1100 nematodes/mL and a growth ratio of 11. For our field test, nematode mobility in the presence of the extract also decreased, to 6.8 mm/day, compared with 20 mm/day for the control. Likewise, when 1.0 g/L of the extract was added to the soil, the total number of nematodes was reduced to only 30- to 40% of the control population.     

Degradation of tannic acid and purification and characterization of tannase from Enterococcus faecalis

Tannins, present in various foods, feeds and forages, have anti-nutritional activity; however, presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxification of these tannins. The present investigation was carried out to study the degradation profile of tannins by Enterococcus faecalis and to purify tannase. E. faecalis was observed to degrade tannic acid (1.0% in minimal media) to gallic acid, pyrogallol and resorcinol. Tannase from E. faecalis was purified up to 18.7 folds, with a recovery of 41.7%, using ammonium sulphate precipitation, followed by DEAE-cellulose and Sephadex G-150. The 45 kDa protein had an optimum activity at 40 °C and pH 6.0 at substrate concentration of 0.25 mM methyl gallate.     

Evaluation of cyst loss in standard procedural steps for detecting ofGiardia lamblia andCryptosporidium parvum in water

The standard procedure outlined by the United States Environmental Protection Agency (US EPA) in Method 1623 for analyzingGiardia lamblia cysts andCryptosporidium parvum oocysts in water samples consists of filtration, elution, centrifugal concentration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) followed by microscopic examination. In this study, the extent of (oo)cyst loss in each step of this procedure was evaluated by comparing recovery yields in segmented analyses: (i) IMS+IFA, (ii) concentration +IMS+IFA, and (iii) filtration/elution + concentration +IMS+IFA. The complete (oo)cyst recovery by the full procedure was 52–57%. The (oo)cyst loss in the IMS step was only 0–6%, implying that IMS is a fairly reliable method for (oo)cyst purification. Centrifugal concentration of the eluted sample and pellet collection before IMS resulted in a loss of 8–14% of the (oo)cysts. The largest (oo)cyst loss occurred in the elution step, with 68–71% of the total loss. The permeated loss of (oo)cysts was negligible during filtration of the water sample with a 1.0-μm pore polyethersulfone (PES) capsule. These results demonstrated that the largest fraction of (oo)cyst loss in this procedure occurred due to poor elution from the filter matrix. Improvements in the elution methodology are therefore required to enhance the overall recovery yield and the reliability of the detection of these parasitic protozoa.     

Compositional consistency of a heteropolysaccharide-7 produced by Beijerinckia indica

The component sugars of heteropolysaccharide-7 (PS-7) produced by Beijerinckia indica were rhamnose and glucose (1.0:4.8, mol:mol) by gas chromatographic analysis. Galacturonic acid, previously reported as a repeat unit of PS-7, was not found in purified PS-7. The yield of PS-7 varied with physiological conditions, such as concentration of carbon source and initial pH of medium, but the molar ratio of rhamnose to glucose stayed within 1.0 to 4.6-5.1. B. indica utilized glucose and some glucose analogs as carbon sources and produced exopolymers, although there was no direct incorporation of these sugars into PS-7. The molar ratio of rhamnose to glucose in each polymer synthesized from glucose-related sugars showed no significant variation (1.0 to 4.5-4.7)     

Submersed Culture Production of Extracellular Wax Esters by the Marine Bacterium <Emphasis Type="Italic">Fundibacter jadensis</Emphasis>

Fundibacter jadensis strain T9, a marine gram-negative bacterium, was isolated from the intertidal sediment of the German North Sea coast by our group. The cells were able to produce considerable amounts of extracellular wax esters when cultivated with n-alkanes (hexadecane or tetradecane) as a carbon source. The dependence of wax ester production and the composition of the purified wax on different culture conditions (C:N:P ratio and dissolved oxygen tension) were tested. Our results show that wax ester production was not directly growth-linked. The C:N:P ratio had no significant influence on the yield of alkane-free purified wax. The dissolved oxygen tension affected the produced amount of the alkane-free purified wax and the composition of the purified wax; when lower than 2% it decreased the yield of purified wax and led to an altered wax ester composition. Tetradecane as a carbon source enhanced the spectrum of the wax ester composition.     

Production and characterization of an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in low nutrition medium

A novel intracellular bioflocculant (named MBF-W6) produced by Chryseobacterium daeguense W6 cultured in low nutrition medium was investigated in this study. The effects of carbon source, nitrogen source, C/N ratio, initial pH, inoculum size, culture temperature and shaking speed on MBF-W6 production were studied. Chemical analysis showed that the purified MBF-W6 was mainly composed of 32.4% protein, 13.1% polysaccharide and 6.8% nucleic acid. Fourier-transform infrared (FTIR) spectroscopy indicated the presence of carboxyl, hydroxyl, and methoxyl groups. The elemental analysis of purified MBF-W6 revealed that the mass proportion of C, H, O, N and S was 40.92:6.53:44.01:8.53:1.01 (w/w) correspondingly. MBF-W6 had good flocculating rate in Kaolin suspension without any cation addition. The highest flocculating rate of 96.9% was achieved under the optimal conditions (bioflocculant dosage 1.2 mg l⁻¹, pH 5.6 and temperature 15 °C).     

Optimization of process variables in castor oil hydrolysis by Candida rugosa lipase with buffer as dispersion medium

In this study, castor oil is hydrolyzed in presence of Candida rugosa lipase, while in the buffer (aqueous) phase as a dispersion medium. The following conditions were used to optimize the process: speed of agitation, initial pH of buffer phase, temperature, and ratio of buffer phase volume to oil weight. The optimal conditions are 1,100 rpm, pH 6.5, temperature 35°C, and 3:1 buffer phase volume to oil weight ratio. Under these described conditions, the reusability of lipase was tested and it was found that nearly 80% of original hydrolysis percentage was achieved after the first recycle.     

Foam separation of Escherichia Coli with a cationic surfactant

An experimental investigation of the foam separation of E. coli from distilled water suspension using a cationic surface-active agent, ethylhexadecyldimethyl-ammonium bromide (EHDA-Br) is presented. Results are evaluated in terms of total cell count, using a membrane filtration technique. Cell concentrations in the initial suspensions are varied from 5.0 × 10⁵ to 1.0 × 10⁸ cells/ml. Surfactant concentrations in the initial cell suspensions are varied from 0.015 to 0.040 mg./ml., and foaming times are varied from 2 to 20 min. The residual quantity of cells decreases exponentially with foaming time to about 0.02% of the initial quantity after 20 min. The cell enrichment ratio, varying from 10 to 1,000,000, is an inverse power function of the initial surfactant concentration and an exponential function of foaming time. Foaminess decreases with increasing initial cell concentrations, and for an initial surfactant concentration of 0.030 mg./ml., the residual cell concentration is a linear function of the initial cell concentration.     

Variation in the carbon content of two Asplanchna species

The rotifers of the genus Asplanchna were sampled four times during the summer from eight lakes of different types. The mean individual carbon content in the population varied between 0.15–0.66 µg C ind.^-1 (n = 21) for A. priodonta and 1.0–1.6 µg C ind.^-1 (n = 3) for A. herricki. The carbon content and the size of A. priodonta varied considerably between the populations of both different lakes and dates.The carbon level of both Asplanchna species (sample mean 0.2–1% of wet weight) was considerably lower than is generally found for rotifers. Much of the variation of carbon level could be explained by an inverse relationship with wet weight. The high variation in the carbon content of individuals suggests that Asplanch population may adapt their mean body size to fit prevailing environmental conditions.     

Purification and properties of xylanase fromAureobasidium

Summary The yeast-like fungusAureobasidium is a promising source of xylanase (EC 3.2.1.8) with an exceptionally high specific activity. For enzyme production in volumes of several liters, xylose was the preferred carbon source and inducer. Xylanase in clarified cultures was concentrated by reversible adsorption to cation-exchange matrix to 5% of the initial volume, and recovered at nearly 2 million IU/1. Selective conditions permitted 97% recovery of xylanase with a 1.8-fold enrichment in specific activity, to 70% of purity. The predominant xylanase species (20 kDa) was subsequently purified to >99% of homogeneity by gel filtration chromatography. Purified enzyme exhibited an isoelectric point of 8.5, and specific activity of 2100 IU/mg under optimal conditions, determined to be pH 4.5 and 45°C. The activity of purified enzyme was specific for polymeric xylan.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Dept. of Agirculture over other firms or similar products not mentioned.     

Changes in M:G ratios of extracted and residual alginate fractions on boiling with water the dried brown alga Kjellmaniella crassifolia (Laminariales,Phaeophyta)

Kjellmaniella crassifolia, the edible macro-brown alga in Japan contained nearly 27% of alginates of which nearly 7% was extractable from the fronds with boiling water for 6 h and the residual alginates in the frond were almost exhaustively extracted with a dilute alkali at 60 °C for 6 h. The alginates dissolved in all these extracts with both boiling water and dilute alkali were purified by fractionation with MgCl₂ and alcohol.The content of MM blocks in the boiling water-soluble alginate sample increased remarkably during heating for 6 h while that of GG blocks from the same sample decreased. In contrast, MM blocks in the alkali-soluble alginate sample decreased during 6 h heating while GG blocks continued to increase. Since the amounts of MG blocks showed slight fluctation, the M:G ratio of alginates extracted with boiling water increased towards the end of extraction whereas the reverse is true for the alkali-soluble alginates.     

The acidic polysaccharide from Palmaria decipiens (Palmariales,Rhodophyta)

Palmaria decipiens, one of the most abundant red seaweeds of the chilean Antarctic, was collected in King George Island. The hot water extract (26% yield) showed by acid hydrolysis to contain xylose, galactose and traces of glucose. Fractionation with cetrimide gave a soluble neutral xylan and an insoluble fraction. The insoluble fraction afforded an acidic polysaccharide that contained 4.8% of uronic acids, 2.8% of sulfate and 18.9% of protein. Polyacrylamide gel electrophoresis showed that it was homogeneous. The GLC and HPLC analysis of the total acidic hydrolysis products showed that the acidic polysaccharide was composed of the neutral sugars galactose and xylose in the molar ratio 8.2:1.0 and of galacturonic and glucuronic acid in the ratio 1.5:1.0. The second-derivative FT-IR spectrum showed the characteristic amide I, II and III bands of proteins. Alkaline cleavage with 0.1 M NaOH indicated the presence of a glycoprotein with O-glycosidic linkage.Results found in this work suggest that the acidic polysaccharide extracted from Palmaria decipiens is an acidic xylogalactan-protein complex.     

Norms of reaction and diversifying selection

The numbers of progeny produced by comparable numbers of female Drosophila melanogaster of 26 geographic strains on nine different culture media are examined in the context of norms of reaction. Having emphasized that diversifying selection is seldom discussed simultaneously with its seemingly related topic, norms of reaction, I present the following argument: diversifying selection has generally been viewed as involving sub-populations inhabiting separate localities and subject to different patterns of selection, norms of reaction as variation whose weighted average determines the relative fitnesses of different genotypes within individual sub-populations. Should environmental challenges frequently involve life or death (including sterility) outcomes, norms of reaction involving components of fitness engender diversifying selection within local populations (demes).     

Compared cycling in a soil-plant system of pea and barley residue nitrogen

Field experiments were carried out on a temperate soil to determine the decline rate, the stabilization in soil organic matter and the plant uptake of N from ¹⁵N-labelled crop residues. The fate of N from field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) residues was followed in unplanted and planted plots and related to their chemical composition. In the top 10 cm of unplanted plots, inorganic N was immobilized after barley residue incorporation, whereas the inorganic N pool was increased during the initial 30 days after incorporation (DAI) of pea residues. Initial net mineralization of N was highly correlated to the concentrations of soluble C and N and the lignin: N ratio of residues. The contribution of residue-derived N to the inorganic N pool was at its maximum 30 DAI (10–55%) and declined to on average 5% after 3 years of decomposition.Residual organic labelled N in the top 10 cm soil declined rapidly during the initial 86 DAI for all residue types. Leaching of soluble organic materials may have contributed to this decline. At 216 DAI 72, 59 and 45% of the barley, mature pea and green pea residue N, respectively, were present in organic N-forms in the topsoil. During the 1–3 year period, residual organic labelled N from different residues declined at similar rates, mean decay constant: 0.18 yr^-1. After 3 years, 45% of the barley and on average 32% of the pea residue N were present as soil organic N. The proportion of residue N remaining in the soil after 3 years of decomposition was most strongly correlated with the total and soluble N concentrations in the residue. The ratio (% inorganic N derived from residues): (% organic N derived from residues) was used as a measure of the rate residue N stabilization. From initial values of 3–7 the ratios declined to on average 1.9 and 1.6 after 2 and 3 yrs, respectively, indicating that a major part of the residue N was stabilized after 2 years of decomposition. Even though the largest proportion of residue N stabilized after 3 years was found for barley, the largest amount of residue N stabilized was found with incorporation of pea residues, since much more N was incorporated with these residues.In planted plots and after one year of decomposition, 7% of the pea and 5% of the barley residue N were recovered in perennial ryegrass (Lolium perenne L.) shoots. After 2 years the cumulative recovery of residue N in ryegrass shoots and roots was 14% for pea and 15% for barley residue N. The total uptake of non-labelled soil N after 2 years of growth was similar in the two residue treatments, but the amount of soil N taken up in each growth period varied between the treatments, apparently because the soil N immobilized during initial decomposition of residues was remineralized later in the barley than in the pea residue treatment. Balances were established for the amounts of barley and mature pea residue N remaining in the 0–10 cm soil layer and taken up in ryegrass after 2 years of decomposition. About 24% of the barley and 35% of the pea residue N were unaccounted for. Since these apparent losses are comparable to almost twice the amounts of pea and barley residue N taken up by the perennial ryegrass crop, there seems to be a potential for improved crop residue management in order to conserve nutrients in the soil-plant system.     

Multiple stressors on an Antarctic microplankton assemblage: water soluble crude oil and enhanced UVBR level at Ushuaia (Argentina)

Changes in phytoplankton pigment content, in vivo fluorescence as well as in abundance and cell characteristics of phyto- and bacterioplankton were investigated on a field-collected microplankton assemblage from Ushuaia Bay (Southern Argentina). Effects of different experimental treatments were examined: natural and enhanced ultraviolet-B radiation (UVBR: 280–320 nm) exposures, and water soluble fraction (WSF) of crude oil contamination under both UVBR exposures. After a 5-day exposure to experimental treatments in microcosms, significant UVBR-induced deleterious effects were observed with afternoon depression of the photochemical yield followed by night-time recovery. A significant increase in photoprotective pigments (PPCs) was also observed. Due to their smaller size, picophytoplankton cells appeared to be more impacted than nanophytoplankton cells as revealed by their increasing mean cell size and decreasing growth rate implying a perturbation of the cell cycle. On the other hand, the differential response between the two bacterial sub-populations identified (i.e., as sub-populations 1 and 2 according to their cellular characteristics) suggests a higher vulnerability for only one of these sub-populations to UVBR stress. WSF alone was also shown to induce deleterious effects on phytoplankton assemblage. Nevertheless, bacteria were positively affected, and particularly bacterial sub-population 2. The combination of WSF and enhanced UVBR exposure resulted in an exacerbation of these individual effects, demonstrating a synergistic effect of both stresses. Moreover, Cryptomonas sp. were observed to develop only under dual stresses in response to their capacity to switch between phototrophic and mixotrophic states following stressed conditions. In situ studies with natural communities provided a unique tool for determining the short-term biological response of microplankton assemblages exposed to multiple stressors.     

Screening and Characterization of the High-Cellulase-Producing Strain Aspergillus glaucus XC9

Cellulose is a kind of renewable resource that is abundant in nature. It can be degraded by microorganisms such as mildew. A mildew strain with high cellulase activity was isolated from mildewy maize cob and classified as Aspergillus glaucus XC9 by morphological and 18S rRNA gene sequence analyses. We studied the effects of nitrogen source, initial pH, temperature, incubation time, medium composition, and surfactants on cellulase production. Maximal activities of carboxymethylcellulase (6,812 U/g dry koji) and filter paperase (172 U/g dry koji) were obtained in conditions as follows: initial pH, 5.5–6.0; temperature, 30°C; cultivation period, 3–4 days; inoculum ratio, 6% (vol/vol); sugarcane bagasse/wheat bran ratio, 4:6. When bagasse was used as substrate and mixed with wet koji at a 1:1 (wt/wt) ratio, the yield of reducing sugars was 36.4%. The corresponding conversion rate of cellulose to reducing sugars went as high as 81.9%. The results suggest that A. glaucus XC9 is a preferred candidate for cellulase production. Translated from the Journal of Xiamen University (Natural Science), 2005, 44(1) (in Chinese)