A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine
TL;DR: This is the first report on the use of Gram’s iodine for the detection of cellulase production by microorganisms using plate assay, which is rapid and efficient and can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi.
Abstract: Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram’s iodine instead of the reagents just mentioned. Gram’s iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram’s iodine for the detection of cellulase production by microorganisms using plate assay.
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TL;DR: The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.
Abstract: Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.
527 citations
Cites background from "A rapid and easy method for the det..."
...Recently, Kasana and colleagues found that Gram’s iodine for plate flooding in place of hexadecyltrimethyl ammonium bromide or Congo red, gave a more rapid and highly discernable result [27]....
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TL;DR: Concept component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny, but there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology.
Abstract: Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.
458 citations
TL;DR: The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article.
Abstract: Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.
247 citations
Cites background from "A rapid and easy method for the det..."
...Recently in a zymogram assay, Gram’s iodine has been used for a fast and easy detection of EG activity, which makes a sharp and distinct zone around the cellulose-producing microbial colonies in a bluish-black background within a short time (3–4 min) (Kasana et al., 2008)....
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TL;DR: The review describes the current knowledge of cellulase production by submerged fermentation and solid state fermentation, properties of cellulasing and cloning and expression of cellulases gene, and the biotechnological aspect of cellul enzyme research and their future prospects are discussed.
Abstract: Cellulose is an abundant natural biopolymer on earth and most dominating Agricultural waste. This cellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. It can be degraded by cellulase produced by cellulolytic bacteria. This enzyme has various industrial applications and now considered as major group of industrial enzyme. The review discusses application of cellulase, classification of cellulase, quantification of cellulase, the types of cellulolytic bacteria and their screening. It describes the current knowledge of cellulase production by submerged fermentation and solid state fermentation, properties of cellulase and cloning and expression of cellulase gene. The biotechnological aspect of cellulase research and their future prospects are also discussed.
228 citations
Cites background from "A rapid and easy method for the det..."
...Kasana et al. 2008 found that Gram's iodine for plate flooding in place of hexadecyltrimethyl ammonium bromide or Congo red, gave a more rapid and highly discernable result [19]....
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...2008 found that Gram's iodine for plate flooding in place of hexadecyltrimethyl ammonium bromide or Congo red, gave a more rapid and highly discernable result [19]....
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TL;DR: This is the first report to describe cacti-associated bacteria from Brazilian semi-arid with plant growth-promoting abilities, isolated based on their ability to grow in medium with reduced water availability.
209 citations
References
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01 Jan 1990
26,241 citations
TL;DR: The present article is an overview of the biotechnological state-of-the-art for cellulases and related enzymes.
1,353 citations
TL;DR: The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity and an increasing number of three-dimensional structures are becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes.
Abstract: Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.
1,327 citations
"A rapid and easy method for the det..." refers background in this paper
...Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants [ 1 ]....
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TL;DR: Structure de la cellulose, sources et production d'enzymes cellulolytiques: regulation, secretion, mutation, mutation et clonage, multiplicite des enzymes.
Abstract: (1985). The Properties of Fungal and Bacterial Cellulases with Comment on their Production and Application. Biotechnology and Genetic Engineering Reviews: Vol. 3, No. 1, pp. 39-110.
356 citations