Determination of the solubilizing activity of a cellulase complex with dyed substrates

When the solubilizing activity of a microbial cellulase complex (e.g.,Trichoderma viride) is determined with conventional methods based on formation of reducing sugars, the results depend on the concentration ratio of cellobiose and glucose in the reaction mixture and thus on the β-glucosidase present and on the type of measurement of reducing sugars. The use of dyed substrates is one way to avoid this problem. The release of coloured compounds from a dyed substrate is proportional to the solubilization.     

Effect of microbial transglutaminase on dyeing properties of natural dyes on wool fabric

The dyeing properties of three natural dyes – curcumin, gardenia yellow and lac dye – on wool fabric after treatment with microbial transglutaminase (MTGase) have been investigated. After 120 min of MTGase treatment, compared with the fabric only pretreated with chemical and protease, the colour strength of curcumin, gardenia yellow and lac dye increased from 8±0.13, 7.5±0.10 and 22±0.12 to about 12.8±0.20, 11.7±0.20 and 27.0±0.41, respectively. The values of wash fastness for dyed wool fabrics increased from 2 to 4 after MTGase treatment, but the light fastness was not obviously improved. By comparing with mordant dyeing, although the colour strength was poorer, MTGase after-treatment did not cause colour shade changes during dyeing and the wash fastness of dyed wool fabric was similar to that of the pre-mordanted samples.     

Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels

A simple, highly sensitive zymogram technique for detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl-D-glucurono-D-xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Antibacterial colorants: characterization of prodiginines and their applications on textile materials

A strain of Vibrio sp. isolated from marine sediments produced large quantities of bright red pigments that could be used to dye many fibers including wool, nylon, acrylics, and silk. Characterization of the pigments by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) revealed three prodiginine-like structures with nonpolar characteristics and low molecular mass. UV-visible spectra of the major constituent in methanol solution showed absorbance at lambda max 530 nm wavelength. The accurate mass result showed that the main isolated product has a molecular mass of m/z 323.1997. Further analysis using mass fragmentation (MS/MS), 1H NMR, COSY, HMQC NMR and DEPT confirmed the detailed structure of the pigment with an elementary composition of C20H25N3O. Fabrics dyed with the microbial prodiginines demonstrated antibacterial activity.     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

Gymnoascus arxii's potential in deteriorating woollen textiles dyed with natural and synthetic dyes

So far, very little is known about microbiological deterioration of dyed woollen textiles. In this paper, the influence of the Gymnoascus arxii fungus on woollen textiles dyed with natural and synthetic dyes was studied. What is more, it was analysed whether the enrichment of the culture medium with additional nutrients has any impact on the deterioration of dyed woollen fabrics caused by a strongly keratinolytic strain. The study was carried out by means of a pure culture method over three different time periods, i.e. 1, 2 and 4 weeks. Within a week, the pure Gymnoascus arxii strain led to a severe deterioration in the mechanical strength of the examined woollen textiles, with the raw fabric being the most severely damaged. After the two-week incubation period, only the fabrics coloured in yellow, i.e. the fabric dyed with natural dye weld, and the synthetic yellow textile as well as the textile dyed with natural dye indigo survived, exclusively on the enriched medium. Solely the weld dyed textile withstood the four-week culture on the nutrient-enriched medium. The conducted studies demonstrated a strong influence of Gymnoascus arxii on dyed fabrics leading to their irreversible destruction. It has been also shown that the presence of nutrients in the substrate that are readily available to microorganism may hinder the development of the Gymnoascus arxii strain and thus, prevent textile deterioration.     

An insoluble chromolytic substrate for the determination of proteolytic activity

A new insoluble chromolytic substrate for the determination of proteolytic activity was prepared by immobilization of dyed casein into the structure of polyacrylamide gel. The prepared substrate could detect approximately 0.1 microgram of trypsin per ml. The spontaneous leakage of dyed casein molecules in water solutions was negligible.     

Insoluble chromolytic substrates for the determination of proteolytic activity

New insoluble chromolytic substrates for the determination of proteolytic activity were prepared by incorporation of dyed proteins into the structure of calcium sulphate dihydrate. The prepared substrates could detect approximately 0.25–1.0 μg of trypsin per ml. The spontaneous leakage of dyed substances in water solutions was negligible.     

Dyed birds achieve higher social status than controls in Harris' sparrows

The status-signalling hypothesis has been proposed to explain colour variation among individuals in flocking birds. Its fundamental assumption is that colour may affect the determination of an individual's social rank in a flock. Here I show for winter Harris' sparrows (Zonotrichia querula) that birds dyed to resemble adults dominate control birds within experimental flocks of young males and young females. The domination of controls by the dyed birds was achieved by a two-step process in both experiments: immediately after the two groups were combined, the controls avoided the dyed birds; then, shortly thereafter, the dyed birds began actively to displace the control birds.     

Determination of proteolytic activity with magnetic dye-stained gelatine

A procedure for the determination of proteolytic activity with dyed magnetic gelatine as an insoluble chromolytic substrate is described. The magnetic nature of the substrate enables magnetic separation of unhydrolysed substrate from the hydrolysed dyed peptide fragments. Such type of substrates could enable the development of new automated protease assays based on the principle of Flow Injection Analysis (FIA).     

Characterization of Bioactive Actinomycetes Isolated from Kadolkele Mangrove Sediments,Sri Lanka

Exploring untapped microbial potentials in previously uncharted environments has become crucial in discovering novel secondary metabolites and enzymes for biotechnological applications. Among prokaryotes, actinomycetes are well recognized for producing a vast range of secondary metabolites and extracellular enzymes. In the present study, we have used surface sediments from ‘Kadolkele’ mangrove ecosystem located in the Negombo lagoon area, Sri Lanka, to isolate actinomycetes with bioactive potentials. A total of six actinomycetes were isolated on modified-starch casein agar and characterized. The isolates were evaluated for their antibacterial activity against four selected bacterial strains and to produce extracellular enzymes: cellulase, amylase, protease, and lipase. Three out of the six isolates exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus cereus, but not against Listeria monocytogenes. Five strains could produce extracellular cellulase, while all six isolates exhibited amylase activity. Only three of the six isolates were positive for protease and lipase assays separately. Ac-1, Ac-2, and Ac-9, identified as Streptomyces spp. with the 16S rRNA gene sequencing, were used for pigment extraction using four different solvents. Acetone-extracted crude pigments of Ac-1and Ac-2 were further used in well-diffusion assays, and growth inhibition of test bacteria was observed only with the crude pigment extract of Ac-2. Further, six different commercially available fabrics were dyed with crude pigments of Ac-1. The dyed fabrics retained the yellow color after acid, alkaline, and cold-water treatments suggesting the potential of the Ac-1 pigment to be used in biotechnological applications.     

Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

Mode of action of cellulases on dyed cotton with a reactive dye

Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.     

Novel media for detection of microbial producers of cellulase and xylanase

Abstract Agar nutrient media containing 0.2% soluble hydroxyethylcellulose covalently dyed with Ostazin Brilliant Red H-3B or soluble beechwood 4-O-methyl- d -glucurono- d -xylan dyed with Remazol Brilliant Blue R were used for sensitive detection of microorganisms producing and secreting into the surrounding medium endo-1,4-β-glucanase and/or endo-1,4-β-xylanase. Pale clearing zones formed around the colonies grown on such media indicated the production of corresponding polysaccharide-hydrolases.     

Amino Acid Regions of Family 45 Endoglucanases Involved in Cotton Defibrillation and in Resistance to Anionic Surfactants and Oxidizing Agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Modification of wool surface by liposomes for dyeing with weld

In this research work, wool surface has been modified by liposome to investigate its effects on dyeing with weld, a yellow natural dye. To do this, samples were first treated with aluminium sulphate and afterward with different concentrations of liposomes at various temperatures for 30?minutes and, finally, dyed with weld at 75, 85, and 95°C for 30, 45, and 60?minutes. K/S values of fabric samples were calculated and washing, light and rub fastness properties of the samples were indicated. The results proposed that the sample treated with 1% liposomes and dyed at 75°C for 60?min has the highest K/S value. The central composite design (CCD) used for the experimental plan with three variables on the results of color strength and statistical analysis confirms the optimum conditions obtained by the experimental results. It was also found that washing, light, wet, and dry rub fastness properties of samples dyed with weld, including liposomes, have not significantly changed. The results of water drop absorption indicated that the hydrophobicity is higher for the samples pretreated with liposomes. The SEM picture of wool sample treated with mordant and liposomes and finally dyed with weld shows a coated layer on the fiber surface.     

用伊文思蓝染色法检测植物整体叶片的细胞活性

本文利用伊丈思蓝（evans blue）作为细胞活性染料,检测了胁迫处理后3种草本和2种灌木植物叶片的细胞活性。结果显示,不同类型的胁迫（温度、PEG、Pb、Cd、NaCl和NaHSO3）均造成了叶片细胞一定程度的伤害。随着各种胁迫的加剧,叶片染色面积和染色后提取液吸光度都呈现梯度增加的趋势。研究分别利用染色面积和其提取液吸光度表达叶片细胞的活性,提出了一种更安全、更准确的植物整体叶片细胞活性检测方法。     

Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.