Studies on Mucor Lipases

Lipases produced by Mucor lipolyticus Aac-0102 were separated into three different fractions (F-1, F-2 and F-3) by CM-Sephadex column chromatography.

Molecular weights of them were estimated to be higher in the order of F-1, F-2, and F-3 by gel filtration on a Sephadex G-100 column. F-1 and F-2 could hydrolyze water soluble substrate such as Tweens. F-3 showed a strong hydrolytic activity toward triglycerides but the activity toward Tweens was almost negligible.

Sodium lauryl sulfate inhibited the olive oil hydrolyzing activity of F-3. However, Tween hydrolyzing activity of F-2 was not affected with it. These results suggested that they are different with each other with respect to their molecular weights, substrate specificities and sensitivities to some inhibitors.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Effect of triton X-100 on alkaline lipase production by Pseudomonas pseudoalcaligenes F-111

Summary An extracellular alkaline lipase was evaluated from Pseudomonas pseudoalcaligenes F-111, which was isolated from soil using a rhodamine B agar plate with Na₂CO₃. For enzyme production, olive oil, soymeal, triton X-100 and sodium ion were found to be essential. The addition of triton X-100 increased the alkaline lipase by 50- fold. The effect of additives as well as the development of suitable culture conditions for lipase production is discussed.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

Glycine Activating Enzyme in the Posterior Silkgland of Silkworm

1. Aminoacyl tRNA synthetase was extracted from the silkgland of silkworm (Bombyxmori Linné) and fractionated on a DEAE-cellulose column. Activities were estimated by ATP-PP_i exchange reaction as well as glycyl tRNA formation.

2. Two peaks, A and B, having ATP-PP_i exchange activity were found in the separated fractions, respectively. There was also observed a marked difference between the both peaks with respect to the pH optimum and activity dependence on MgCl₂ concentration.

3. Peak A showed no activity of glycyl tRNA formation. Only a part of peak B coincided with the activity of glycyl tRNA formation. The activities of both the ATP-PP_i exchange reaction and glycyl tRNA formation were found to be dependent on MgCl₂ concentration, and the optimum concentration was different between two peaks.

4. It also seemed to exist two peaks of activities, a and b, in glycyl tRNA formation which could be separated with a DEAE-cellulose column.     

Compositions of microbial communities associated with oil and water in a mesothermic oil field

Samples of produced water and oil obtained from the Enermark field (near Medicine Hat, Alberta, Canada) were separated into oil and aqueous phases first gravitationally and then through centrifugation at 20°C in an atmosphere of 90% N₂ and 10% CO₂. Biomass that remained associated with oil after gravitational separation (1×g) was dislodged by centrifugation at 25,000×g. DNA was isolated from the aqueous and oil-associated biomass fractions and subjected to polymerase chain reaction amplification with primers targeting bacterial and archaeal 16S rRNA genes. DNA pyrosequencing and bioinformatics tools were used to characterize the resulting 16S rRNA gene amplicons. The oil-associated microbial community was less diverse than that of the aqueous phase and had consistently higher representation of hydrogenotrophs (methanogens of the genera Methanolobus and Methanobacterium and acetogens of the genus Acetobacterium), indicating the oil phase to be a primary source of hydrogen. Many known hydrocarbon degraders were also found to be oil-attached, e.g. representatives of the gammaproteobacterial genus Thalassolituus, the actinobacterial genus Rhodococcus and the alphaproteobacterial genera Sphingomonas, Brevundimonas and Stappia. In contrast, all eight representatives of genera of the Deltaproteobacteria identified were found to be associated with the aqueous phase, likely because their preferred growth substrates are mostly water-soluble. Hence, oil attachment was seen for genera acting on substrates found primarily in the oil phase.     

Different effects on cell proliferation and migration abilities of endothelial cells by LPA1 and LPA3 in mammary tumor FM3A cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA₁ and LPA₃ may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.     

Amylostatins,Other Amylase Inhibitors Produced by Streptomyces diastaticus subsp. Amylostaticus No. 2476

Amylase inhibitors (amylostatins) other than those reported as S-AI were found in the culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476. They were separated grossly into F-1a, F-1b and F-2 fractions by column chromatography on Dowex 50W × 4 (NH₄⁺) and by preparative high performance liquid chromatography. Each fraction was further separated by preparative paper partition chromatography (PPC). Fractions obtained by PPC had different inhibitory activities against various amylases. On the other hand, acid hydrolysis of each active inhibitory fraction produced amylostatin X′ (C₁₃H₂₁NO₇) and/or amylostatin XG (C₁₉H₃₃NO₁₃). The diversities and common features of these amylostatins are discussed.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

Different cytotoxic and clastogenic effects of epigallocatechin gallate in various cell-culture media due to variable rates of its oxidation in the culture medium

Positive genotoxicity results are often observed using mammalian cells in culture with agents that are not in vivo genotoxins. We here illustrate one possible explanation: interaction of test chemicals with the cell-culture media used. We find that the toxicity and clastogenicity of epigallocatechin gallate (EGCG) to Chinese Hamster ovary (CHO) cells is affected by the culture medium used and appears largely or entirely due to variable rates of formation of hydrogen peroxide (H₂O₂) by chemical reactions of EGCG with the culture media. Catalase decreased EGCG toxicity substantially. Of seven different types of commonly used media evaluated, F-10 and F-12 nutrient mixtures were the least prone to produce this artefact. Although it generated H₂O₂ in the culture media, ascorbate was not toxic to CHO cells because the H₂O₂ levels achieved were insufficient to kill these cells. Thus, the culture medium, the cell type and the presence or absence of catalase (e.g. its variable amounts in S9 fractions) must be taken into account in in vitro genotoxicity testing.     

The Release of Carbohydrate Rich Component from Ovomucin Gel during Storage

The fractionation pattern of OMG₀, ovomucin gel(B) in fresh egg white, by density gradient column electrophoresis showed two peaks. Each peak was shown to migrate as a single component, with a mobility of either the fast or slow moving component of ovomucin gel(B). Each peak was named as F-component and S-component.

Carbohydrate and sulfate contents of F-component were much higher than these of S-component. The carbohydrate content of F-component and S-component was found to be about 50 and 15 percents of dry matter, respectively. Serine and threonine contents in F-component were much higher than those in S-component.

The fractionation pattern of OMG₂₀, ovomucin gel(B) in egg white stored for 20 days at 30°C, by density gradient electrophoresis showed only one peak which corresponded to S-component, and that of OMS₂₀, ovomucin sol (B) in egg white stored for 20 days at 30°C, showed two peaks which corresponded to F- and S-component.

Ability of F-component to interact with lysozyme was greater than that of S-component.     

Separation of a Water-Soluble Adjuvant (MAF3) from Delipidated Cells of Mycobacterium tuberculosis Strain Aoyama B by Hydrogenolysis and Gel Filtration

A water-extract from hydrogenolyzed cells of Mycobacterium tuberculosis strain Aoyama B was separated into four portions (F-1 to F-4 fractions) by gel filtration with a Sephadex G-100 column. The third peak (called MAF3) eluted from the column was the most adjuvant-active fraction. The molecular weight determined by gel filtration was around 16 000 daltons. MAF3 consisted of heteropolymer(s) composed of approximately 76 to 79% neutral sugars (Ara, Gal, Man, and Glc) and 19% mucopeptide (MurN, GlcN, Glu, Ala, Dpm, Gly, Asp, Thr, Ser, Leu, Lys, Arg, His, Pro, Tyr, and Phe). The adjuvanticities of MAF3 and other fractions in water-in-oil emulsion were estimated by the enhancing effect on immune response to egg albumin (EA) in guinea pigs. MAF3 stimulated the production of humoral antibodies, particularly IgG2 antibody specific to the antigen, and induced delayed type hypersensitivity against EA in the skin and cornea of antigen-primed guinea pigs. These adjuvanticities of MAF3 were similar to the characteristics of mycobacterial cell wall in Freund's complete adjuvant.     

Chemical Composition,Toxicity and Repellency of Inula graveolens Essential Oils from Roots and Aerial Parts against Stored-Product Beetle Tribolium castaneum (Herbst)

In this work, essential oils extracted from roots and aerial parts of Inula graveolens by hydrodistillation and their fractions obtained by chromatographic simplification were first investigated for their chemical composition by GC/MS and then evaluated for the first time for their repellency and contact toxicity properties against Tribolium castaneumadults. Twenty-eight compounds were identified in roots essential oil (REO), which accounted for 97.9 % of the total oil composition, with modhephen-8-β-ol (24.7 %), cis-arteannuic alcohol (14.8 %), neryl isovalerate (10.6 %) and thymol isobutyrate (8.5 %) as major constituents. Twenty-two compounds were found in the essential oil from aerial parts (APEO), which accounted for 93.9 % of the total oil, with borneol (28.8 %), caryophylla-4(14),8(15)-dien-6-ol (11.5 %), caryophyllene oxide (10.9 %), τ-cadinol (10.5 %) and bornyl acetate (9.4 %) as main compounds.REO and APEO displayed stronger repellency after 2 h of exposure (80.0 and 90.0 %, respectively) against T. castaneum at the concentration of 0.12 μL/cm². After fractionation, fractions R₄ and R₅ exhibited greater effects (83.3 % and 93.3 %, respectively) than the roots essential oil. Furthermore, the fractions AP₂ and AP₃ showed higher repellency (93.3 and 96.6 %, respectively) than the aerial parts oil. The LD₅₀ values of oils from roots and aerial parts topically applied were 7.44 % and 4.88 %, respectively. Results from contact toxicity assay showed that fraction R₄ was more effective than the roots oil with LD₅₀ value of 6.65 %. These results suggests that essential oils of roots and aerial parts from I. graveolens may be explored as potential natural repellent and contact insecticides against T. castaneum in stored products.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites

Eighteen indigenous fungal isolates has been successfully isolated from samples of used motor oil, top five centimetres of soil and drainage water contaminated with used motor oil. All of the pure fungal isolates obtained were identified, characterized and subjected to preliminary screening by evaluating the average growth rate of each fungal isolates on minimal media containing 1% (v/v) used motor oil. Trichoderma asperellum strain TUB F-1067 (SA4), Trichoderma asperellum strain Tr48 (SA5), Trichoderma asperellum strain TUB F-756 (SA6), Penicillium species (P1), and Aspergillus species (P9) were further selected for their hydrocarbon biodegradation potential. Among these five fungal isolates selected, P1 strain presented a significant degree of degradation by degrading almost all of the n-alkanes (n-C-₁₅ to n-C-₂₃ range) present in the used motor oil, thus of greater potential in degrading the aliphatic hydrocarbon compounds of used motor oil. The authors would like to certify that the work have not been sent/considered to be published in other journals.     

Chemical Composition and Insecticidal Activity of Crithmum Maritimum L. Essential Oil against Stored‐Product Beetle Tribolium Castaneum

Several plant essential oils have been used against diverse insect pests since, unlike conventional pesticides, they pose almost no risk to humans and the environment. For this reason, the essential oil (EO) isolated from the fresh leaves of Crithmum maritimum L. and its fractions (F₁–F₅) obtained by chromatographic simplification were investigated for their chemical profile, as well as for their toxicity and repellency effects against Tribolium castaneum (Herbst ) adults. The analysis by GC/MS allowed the identification of 92.8–99.1 % of the compositions of the total oil (EO) and of its fractions (F₁–F₅). The EO and its fractions F₃–F₅ were characterized by the presence of a high amount of phenylpropanoids (94.4, 94.8, 93.6, and 88.7 %, respectively): in all the samples, dill apiole was the most abundant component (EO: 94.1 %, F₃: 94.6 %, F₄: 93.4 %, and F₅: 83.3 %). In addition, the repellency assay results showed that the volatile fraction F₅ and the complete EO exhibited a higher repellency towards T. castaneum (97 % and 93 %, respectively) after 2 h of exposure at the dose of 0.04 μL/cm². The median lethal dose of the topical application of the EO was 9 %. Furthermore, the fraction F₁ possessed interesting contact toxicity against T. castaneum (80 % of mortality) at the concentration of 10 %. These results suggested that the essential oil of C. maritimum leaves might be used as an alternative to synthetic insecticides in order to prevent insects from damaging the stored products.     

Effects of weed abundance and frequency of hand weeding on the establishment of transplanted Imperata cylindrica

Imperata cylindrica is an important component of grasslands in several Asian countries. Its vigorous rhizome elongation enables the plant to quickly cover bare ground, making it a candidate for revegetation projects. In these projects, initial and subsequent management practices often differ. Initial management strategies are important for encouraging rapid cover by the target species. We tested the effects of initial hand weeding on I. cylindrica cover and weed growth over 2 years. Two types of soil (A and B) were scattered on a recently constructed river embankment at a depth of 10 cm. Both soils A and B are nutrient-poor and high (> 7.0) pH. Plots were hand-weeded once or twice a year (W₁ and W₂ treatments, respectively), or mowed but not weeded (M treatment) with four replications. Plug plants of I. cylindrica were introduced. I. cylindrica became dominant in soil A with the W₂ treatment, with 50% cover in year 2. Conversely, I. cylindrica failed to establish in soil B regardless of treatment, due to dominance of the weed Sorghum halepense. Soil B included creeping rhizomes of S. halepense, and vigorous growth from rhizomes is likely a major reason for its predominance. S. halepense seedlings germinated in both soils, but were successfully eradicated when weeded at less than 1 month of age. Thus, for successful revegetation, land managers should use soil that does not contain creeping rhizomes of S. halepense. If rhizomes are not present, hand weeding twice a year for 2 years is sufficient for establishment of I. cylindrica.