Ethanol-insoluble Material Research Articles

Formulation and effectiveness of solid soap preparations from citronella grass (Cymbopogon nardus L.) essential oil extract against Listeria monocytogenes

Background: The components of essential oils, such as citronellol, citronellal, and geraniol have been reported to possess the ability to inhibit bacteria. Furthermore, Listeria monocytogenes is a bacteria that often affects humans and its infections can cause meningitis and bacteremia. In pregnant women, Listeria monocytogenes can cause flu-like syndrome with complications of miscarriage, infant death, and meningitis in infants. Objective: This study evaluated the suitability of solid soap preparations from Citronella grass essential oil, based on its physical properties, such as organoleptic tests, as well as assessments of chemical tests and the effectiveness of the sample produced against bacteria. Method: The soap-making process was initiated by mixing the fat fraction (stearic acid and coconut oil) with an alkali, namely 30% NaOH. The soap stock was then added to the other ingredients and stirred until it became homogeneous for testing. Results: The test showed a pH value, water content, total fat, ethanol insoluble material, free fatty acids, chloride content, and unsaponificable fat of 10.8, 14.26, 74.84, 2.4, 0.26, 0.48, and 85.8, respectively. Furthermore, the sample showed a high level of inhibition against Listeria monocytogenes. Conclusion: Evaluation of citronella essential oil extract soap met the requirements, except for its unsaponifiable fat.

Pengaruh Penambahan Sodium Lauryl Sulfat terhadap Karakteristik Sabun Padat pada Mata Kuliah Praktikum Analitik Proses

Solid soap as a result from process analysis practicum can be produced using with hot process method using raw material of palm oil and a strong base in the form of NaOH. The aim of this research is that apart from fostering student inquiry regarding the soap making process, they are also able to analyze the parameters of solid soap over a certain period based on variations in the amount of added surfactant. The methods used in practicum activities are dividing practicum groups, making solid soap, and collecting practicum reports. In the activity of making solid soap, the concentration of sodium lauryl sulfate (SLS) was varied for each group, 0, 1, 2 and 3% w/w respectively. The parameters measured in this research were the pH value, water content, ability to form foam, free fatty acid content and ethanol insoluble material in solid soap. The pH characterization of the results of the saponification process shows that soap at all variations in SLS concentration is predominantly alkaline. The characterization carried out showed that the levels of free fatty acids and ethanol insoluble substances met SNI 3532:2016 standards. Water content parameters do not meet these standards because the measurements were carried out during practical activities and without the process of holding the soap product. From the results of observing these parameters, in general it can be concluded that the solid soap products resulting from the practical work have the best quality under the condition of adding SLS with a concentration of 1%.

Formulation and evaluation liquid hand soap of celery leaf (Apium graviolens) extract

Washing hands with soap is one of the new habits during the COVID-19 pandemic, which is very important to continue to apply. Therefore, alternative natural ingredients are needed to manufacture liquid hand soap that is safe to use in the long term. Celery leaves contain flavonoids, saponins, tannins, essential oils, choline, lipase, and alkaloids. The content of these secondary metabolites has antimicrobial activity. This study aims to formulate liquid hand soap preparations from celery leaf extract following SNI 2588.2.2017. This research is experimental laboratory research that formulates soap preparations with concentrations of 5%, 10%, and 15% and is followed by an evaluation of the preparations based on the referenced SNI quality requirements. This study resulted in an extract yield of 37.12% with organoleptic test results in a thick liquid dosage form, characteristic odor of celery, and green color. In the evaluation test of preparations based on SNI 2588:2017, the results obtained that liquid hand soap preparations meet the requirements. PH for FI, FII, and FIII respectively 9.4, 9.5, and 9.3. In the total active ingredient test, the FI 34.6% was obtained; FII 32.2% and FIII 32.6%. Then in the ethanol insoluble material test, the FI results were 0.094%, FII 0.086%, and FIII 0.112%. Free alkali test obtained FI 0.044%, FII 0.0472%, and FIII 0.0488%. Then the total plate number test was obtained in FI, FII, and FIII preparations; namely, there was no colony growth in samples with dilutions of 10-1, 10-2, and 10-3. The formulation of liquid hand soap meets the quality requirements of SNI 2588.2.2017.

Effect of VCO and Castor Oil Concentrations on Physicochemical Properties of Patikan Kebo <i>(Euphorbia hirta) </i>Liquid Handsoap

This study aimed to investigate the impact of VCO and castor oil compositions on the physicochemical properties of liquid soap from patikan kebo (Euphorbia hirta) extract and compare it with the marketed liquid hand-soap. The liquid hand-soap was manufactured by adding patikan kebo extract with VCO and castor oil as fatty acid sources. The concentration of VCO and castor oil were varied in 5 formula with ratio 1 : 0, 3 : 1, 1 : 1, 1 : 3, and 0 : 1 of 200 mL of soap perspectively. Several tests such as organoleptic inspection, homogeneity, density, viscosity, foam volume and stability, pH, insoluble materials, free fatty acids, total active ingredients, and total plate number, were performed to determine the physicochemical properties of prepared handsoap. The results of the five formulas were known to the greater VCO the higher the viscosity and percent height of foam, the greater the castor oil the higher the value of specific gravity The five liquid soap formulas produced meet the requirements of SNI 2588-2017. The second liquid soap formula with a ratio of VCO and castor oil 3: 1 was declared to be the best product with free fatty acid 0.78%, pH 8.31, total active ingredient 24.3%, ethanol insoluble material 0.29%, specific gravity 1.09 g/mL, foam high stability 82.85%, and no colonies in testing the Total Plate Figures. It can be concluded that variations in the concentration of VCO and castor oil may affect the physicochemical properties of liquid soap for hand washing extracted from Patikan kebo.

Improved efficiency of brewer’s spent grain arabinoxylans by ultrasound-assisted extraction

Arabinoxylan (AX) rich extracts from brewer’s spent grain (BSG) were produced by the application of ultrasound-assisted extraction (UAE) and conventional alkaline extraction (AKE). UAE and AKE were optimised for the production of the highest yield of ethanol insoluble material using response surface methodology (RSM). The efficiency of UAE was established by the significant reduction of time (7h to 25min) and energy when compared to AKE, to recover similar amounts of AX (60%) from BSG, leading to the production of starch-free AX-rich extracts.

Utilization of some plant polysaccharides for improving yoghurt consistency

Six plant polysaccharides (PS) were extracted from mature okra fruits (Hibiscus esculents), whole plant Jew’s-mallow (Corchorus olitorius) and taro corm (Arum colocasia). Three of these products were isolated as ethanol insoluble materials (PS1-type products), while the other three were attained as ethanol soluble substance (PS2-type products). Different concentrations (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) of extracted PS1 and PS2-types, were used in the manufacture of yoghurt mainly for stabilizing of yoghurt texture. The resultant yoghurt was analysed organoleptically (appearance, body and texture and flavour) and chemically after production as well as during cold storage (0, 3, 5 and 7 days) at 5±1°C. Results showed that yoghourt with good appearance, body and texture and flavour could be produced by using PS extracted from taro at concentration 0.3%, okra at concentration 0.1% and Jew’s-mallow at concentration 0.1%.

Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [(14)C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of d-[U-14C]glucose or d-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and α-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of d-[U-14C]glucose or d-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.

Application of CP-MAS and liquid-like solid-state NMR experiments for the study of the ripening-associated cell wall changes in tomato

13C and 1H NMR spectra of an ethanol insoluble material (EIM) prepared from the pericarp of mature-green (MG) and red-ripe (RR) tomato fruits were acquired in ‘liquid-like’ and cross-polarisation with dipolar decoupling and magic angle spinning (CPMAS) conditions using the same triple resonance probe. Such a strategy allowed acquisitions of various NMR experiments aimed at detecting compositional differences as well as distinguishing differences in molecular mobility for various constituent polysaccharides related with the two ripening stages. Increase of the proton dipolar decoupling power levels from 3 to 50–55 kHz during single pulse 13C acquisition, led to more intense signals for pectic and hemicellulosic polysaccharides. This behaviour was interpreted as reflecting motional restrictions of these polysaccharides inside the porous cell wall network. Measurements of the proton rotating frame relaxation times T 1ρ in the ‘liquid-like’ conditions and of the proton transverse relaxation times T 2 from CPMAS spectra, revealed changes in mobilities for some pectic polysaccharides in relation with ripening, particularly for the H1 and H5 protons of α-1,5 arabinan (Ara) side chains of rhamnogalacturonans. These data are discussed in relation with known pectic modifications occurring during ripening and associated with the tomato fruit softening.

イチジク果実の細胞壁成分の品種間差異と成熟過程における変化

To elucidate the varietal differences in cell wall composition of fig fruits at harvest, the uronic acid and neutral sugar contents in the syconia of Caprifig 'VC-180' (Ficus carica L. var. intermedia Shinn×F. carica L. var. sylvestris Shinn), San Pedro type 'King' and 'San Pedro White' (F. carica L. var. intermedia Shinn) and common figs 'Masui Dauphine' and 'Houraishi' (F. carica L. var. hortensis Shinn) were analyzed. In addition, the changes in cell wall composition of ripening 'Masui Dauphine' fruits were investi-gated, comparing uronic acid and neutral sugar contents in the syconia exhibiting 10, 50, and 100% colored peel. Cell wall polysaccharides were sequentially extracted from etha-nol insoluble materials of pistillate flowers with water, hot-water, EDTA, KOH (hemicellulose fraction), and H2SO4 (cellulose fraction) solutions.1. In the water fractions, the varietal difference in cell wall composition was very small. The total sugar contents, including uronic acid and neutral sugar, in the hot-water, EDTA, hemicellulose, and cellulose fractions were significantly highest in the syconia of 'VC-180'. Except for 'VC-180', the difference in neutral sugar contents in the cellulose fractions was largest among other cultivars.2. The uronic acid contents per unit weight in the water fractions, extracted from 'Masui Dauphine' syconia during ripening, became higher in the order of 50, 100, and 10% colored peel, but there was little difference in the neutral sugar contents in the same fractions. In the hot-water and EDTA fractions, the uronic acid contents per unit weight decreased with color development ; therefore, the total sugar contents from pectic substances likewise decreased. In the hemicellulose and cellulose fractions, both the uro-nic acid and neutral sugar contents per unit weight decreased with color development.3. The cell wall composition per fruit of 'Masui Dauphine' was different from that per unit weight, because the ripening fruits increased in weight. As the percentage of color-ing in the peel increased, the uronic acid contents per fruit increased markedly in the wa-ter fractions and decreased in the hot-water and EDTA fractions ; the neutral sugar con-tents per fruit increased in the water and hot-water fractions. However, there were no differences in the total sugars per fruit derived from the pectic substances or in the uro-nic acid, neutral sugar, and total sugars per fruit derived from the hemicellulose and cel-lulose fractions during the ripening process.

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction: (fertilization/acrosome reaction/sea urchin/sperm/N-linked oligosaccharides).

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH4 , preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.

Acetaldehyde Stimulation of Net Gluconeogenic Carbon Movement from Applied Malic Acid in Tomato Fruit Pericarp Tissue

Applied acetaldehyde is known to lead to sugar accumulation in fruit including tomatoes (Lycopersicon esculentum) (O Paz, HW Janes, BA Prevost, C Frenkel [1982] J Food Sci 47: 270-274) presumably due to stimulation of gluconeogenesis. This conjecture was examined using tomato fruit pericarp discs as a test system and applied i-[U-(14)C]malic acid as the source for gluconeogenic carbon mobilization. The label from malate was recovered in respiratory CO(2), in other organic acids, in ethanol insoluble material, and an appreciable amount in the ethanol soluble sugar fraction. In Rutgers tomatoes, the label recovery in the sugar fraction and an attendant label reduction in the organic acids fraction intensified with fruit ripening. In both Rutgers and in the nonripening tomato rin, these processes were markedly stimulated by 4000 ppm acetaldehyde. The onset of label apportioning from malic acids to sugars coincided with decreased levels of fructose-2,6-biphosphate, the gluconeogenesis inhibitor. In acetaldehyde-treated tissues, with enhanced label mobilization, this decline reached one-half to one third of the initial fructose-2,6-biphosphate levels. Application of 30 micromolar fructose-2,6-biphosphate or 2,5-anhydro-d-mannitol in turn led to a precipitous reduction in the label flow to sugars presumably due to inhibition of fructose-1,6-biphosphatase by the compounds. We conclude that malic and perhaps other organic acids are carbon sources for gluconeogenesis occurring normally in ripening tomatoes. The process is stimulated by acetaldehyde apparently by attenuating the fructose-2,6-biphosphate levels. The mode of the acetaldehyde regulation of fructose-2,6-biphosphate metabolism awaits clarification.

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5′-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-14C-glucose or UDP-14C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl2 was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl2 the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10μM UDP-glucose in the presence of 3 mM MnCl2 was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 μM UDP-xylose in the presence of 3 mM MnCl2, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-14C-glucose, and fractions enriched in Golgi apparatus, both (1→3)- and (1→4)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (1→3)-glucosyl. With the substrate, UDP-14C-xylose, mostly (1→4)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.

Submergence of Rice. II. Adverse Effects of Low CO2 Concentrations

Submergence of rice in water at low CO2 concentrations was studied in phytotron experiments using plants in the 3rd to 4th leaf stage. Cultivars known to differ in tolerance to complete submergence were adversely affected by the same mechanisms but to a different degree. Submergence for 4-12 days either reduced dry weight production of the whole plant by 6 to 10 fold or even resulted in a loss of dry weight. Nevertheless, the emerging leaf elongated, and both ethanol insoluble material and protein content increased with time. These increases were associated with translocation of dry matter and nitrogen from expanded to expanding leaves. Submergence also reduced concentrations of soluble sugars and starch in all plant parts by 4 to 12 fold. In contrast, concentrations of potassium and free amino acids in shoots were either the same or, in the case of the emerging leaf, higher than in plants which were not submerged. These results indicate (i) these solutes were not limiting growth and (ii) the tissues retained their semipermeability to these solutes during submergence. Insufficient capacity of root metabolism in submerged plants was indicated by low rates of respiration, which persisted in the presence of glucose, and by a low ability to consume ethanol. A model is presented on the adverse effects of submergence of rice which considers possible interactions between CO2, low O2 and high ethylene concentrations.

Extraction and characterisation of extracellular polymers in digested sewage sludge

AbstractFive different bacterial extracellular polymer extraction methods were compared on digested sewage sludge. A washing step was necessary to remove and eliminate the slime from the sludge sample, in order to distinguish it from the material more tightly bound to the sludge solids. An ion‐exchange resin method was the most effective and least disruptive of all treatments tested, based on the relatively high extracellular polymer yield and the small amount of cell disruption demonstrated by the fractionation and characterisation of the extracted material.The extracellular polymer extracted contained protein and polysaccharide in a ratio of approximately 3:1, and contributed a relatively small proportion of the total solids in digested sludge. This was comparatively less than is extracted in activated sludge. The ion‐exchange resin method yielded 1% by weight of polysaccharide and 3% of protein, whereas ethanol‐insoluble material accounted for 3.5% of the solids in digested sludge.

Stimulation of extracellular polysaccharide synthesis in oat protoplasts by the host-specific phytotoxin victorin

The host-specific phytotoxin victorin (HV-toxin) stimulates mesophyll protoplasts of susceptible but not of resistant oat (Avena sativa L.) to produce an amorphous, ethanol-insoluble extracellular material which stains with Calcofluor white and aniline blue. Over a 24-h period incorporation of [(14)C]glucose into ethanol-insoluble products is maximally stimulated by 60 pg victorin/ml, whereas at 6 ng/ml initial rates of incorporation are higher but the protoplasts collapse. The extracellular material produced in response to victorin is solubilized by cold 4.4 N NaOH and by commercial laminarinase and pectinase. Incorporation of [(14)C]glucose into cellulose (material resistant to Updegraff's acetic-nitric acid reagent) is stimulated as much as incorporation into other wall polysaccharides, but cellulose constitutes less than 15% of the total victorin-stimulated incorporation. Synthesis of ethanol-insoluble material that can be digested by pronase, i.e. protein, is inhibited by victorin above 60 pg victorin/ml. Formation of extracellular polysaccharide is stimulated at concentrations of victorin which cause almost complete inhibition of protein synthesis, indicating that de-novo protein synthesis is not involved. Preincubation of protoplasts with inhibitors of RNA or protein synthesis prevents both extracellular polysaccharide synthesis and cell death in response to victorin. Although previous studies have indicated a link between calcium and the action of victorin, several compounds which interact with calcium do not influence this response to victorin.

The Effects of Pectin and Plant Root Surface Carbohydrates on Encystment and Development of Phytophthora cinnamomi Zoospores

Citrus pectin induces highly synchronous differentiation in populations of Phytophthora cinnamomi zoospores in the absence of a host plant. This action is independent of the presence of Ca2+ in the zoospore suspension medium. Water-soluble, ethanol-insoluble material present on the surface of the plant roots also induces differentiation of P. cinnamomi zoospore populations with the same time course as pectin. It is proposed that this material provides the natural recognition signal which induces P. cinnamomi zoospores to differentiate, and that pectin is sufficiently similar in structure for it to mimic its action.

Pectic substances in developing and ripening plum fruits

AbstractThe fresh weight of plum fruits increased throughout the growing season whilst the weight of ethanol insoluble material increased to a peak in the penultimate sample and then declined. Pectin content paralleled the change in weight of the insoluble residue. There were no significant changes in the proportional composition of monosaccharides in pectin or the total solids. Pectic substances were examined by gel permeation chromatography and high voltage electrophoresis. There was no evidence for extensive degradation of the pectic substances, but changes in the electrophoretic mobility were consistent with de‐esterification during ripening.

Removal of extracellular material techniques and pitfalls

Extracellular material—specifically capsular material—has been strongly implicated in the flocculation of activated sludge and other mixed cultures. This study examines the steps involved to extract and collect this material, and suggests one suitable method which evolved after a study of numerous alternatives. Five steps are needed to obtain extracellular capsular material from the mixed culture which produced it. They are biomass concentration and removal of slime (washing); stripping of the capsule from the cell; precipitation of the stripped material from solution; collection of the precipitate; and an optional purification step. In the study reported herein, it was found that washing was essential to eliminate slime, as well as any dissolved salts which could contaminate the final product. Physical stripping by blending followed by centrifugation was more convenient than chemical stripping using K 2HPO 4; high-speed centrifugation alone did not result in the capsule being stripped from the cell. Precipitation was accomplished by adding the centrate to ethanol and refrigerating overnight. The precipitate was collected by filtering through tared glass-fibre filter paper. Further studies were done to assess whether blending would cause cell disruption (hence contamination by intracellular material), what level of repeatibility could be expected, and whether a single-pass blending step would remove all of the capsule. DNA measurements of the extract and of lysed cells showed that no significant disruption had been caused by blending. Duplicate tests indicated that repeatability of approx. 5% or better could be expected. Successive extractions showed that significant quantities of material (as much as 50% of the first extraction) could be removed by two further blending steps, but it is also likely that these extracts would contain higher amounts of intracellular material. Extracellular material is frequently referred to as “biopolymer”. However, because this material, having been extracted by methods as described above, has been shown to include organic monomers as well as inorganic components, an alternative description is suggested. This uses the precipitating agent (generally an alcohol) as the basis, and in the present study the term “ethanol insoluble material (eim)” has been used.

Association of UDPG Transferase Activity with Cell Walls of Achlya Ambisexualis

Fungal cell wall growth has been postulated to involve the cooperative action of both wall-hydrolyzing and wall-synthesizing enzymes (Park and Robinson, 1966; Gooday and Trinci, 1980). Investigations of cell wall morphogenesis in Achlya have dealt primarily with hydrolytic events, particularly those mediated by endo-l,4-D-glucanases (Thomas and Mullins, 1967, 1969; Hill and Mullins, 1979, 1980). Much less attention has been devoted to wall-synthesizing enzymes in Achlya and other cellulosic fungi because the activity of glucan-synthesizing enzymes is difficult to demonstrate in vitro, and the low level of activity makes characterization of the products uncertain. In a previous investigation, uridinediphosphoglucose (UDPG) transferase (EC 2.1.4.12) was demonstrated at low activity in association with subcellular particles that also exhibited cellulase activity (Hill and Mullins, 1980). Since these particles were similar to apical vesicles this association suggests that UDPG transferase in Achlya may be involved in cell wall synthesis. In the present paper, activity of UDPG transferase is demonstrated in cell walls of Achlya, and the solubility properties of the in vitro reaction products are described. Achlya ambisexualis Raper strain E87 was cultured for 48 h as described previously (Mullins, 1973). UDPG tranferase activity was assayed by a modification of the techniques of Ray et al. (1969) and Shore and Maclachlan (1975). The reaction mixture contained (mM): UDPG, 0.24; cellobiose, 5; MgCl2, 11; dithiothreitol (DTT), 1.7; pH 5.8 sodium phosphate buffer, 67; and incubation was 20 min at 23 C. The UDPG was glucose-uL-14C (250 utCi/Aumole, New England Nuclear) and as used gave 7.3 x 10-8 moles with 119 nCi of radioactivity. Products were recovered by centrifugation of 70% ethanol-insoluble material after addition of 30 mg of powdered Whatman cellulose as a carrier. To determine the distribution of transferase activity between wall and protoplasm fractions, mycelia were homogenized by ultrasonication at 5-10 C in a buffered homogenizing solution containing 20% w/w sucrose, 10 mM DTT, and 20 mM tris HCl buffer, pH 7.6. Hyphal fragments were sedimented by centrifugation, then resuspended, and the entire process repeated three times. The supernatant fractions were pooled and the final sediment, when examined by phase contrast microscopy, consisted of wall fragments without apparent cytoplasmic contamination. UDPG transferase activity was assayed in each fraction and protein was estimated with the Branford (1976) Bio-Rad assay. Results are displayed in TABLE I. Although less transferase activity was found in the wall fraction than 851