Properties of the endogenous cellulase from Panesthia cribrata saussure and purification of major endo-β-1,4-glucanase components
ABSTRACT The cellulase of Panesthia cribrata consists of at least six endo-β-1,4-glucanase (EC 22.214.171.124) and two β-glucosidase (EC 126.96.36.199) components. The two major endo-β-1,4-glucanase components, named EG1 and EG2, comprise 13% of the soluble protein in the foregut and midgut contents. They were purified by a combination of gel chromatography and ion-exchange FPLC and had Mr of 53,600 and 48,800, respectively. With carboxymethylcellulose (CMC) the Km and Vmax values for EG1 and 9.4 mg/ml and 22.2 mg reducing sugar/min/mg protein; the corresponding values for EG2 were 6.8 and 88.3l. Values for Km, Vmax and kcat were also calculated for activity against cellotetraose and cellopentaose. Neither component hydrolysed cellobiose or cellotriose. The relative activities of EG1 and EG2 against CMC and microcrystalline cellulose Sigmacell Type 20 were 5200:1 and 3500:1. It is proposed that the inefficiency of EG1 and EG2 is compensated for by their secretion in large amounts into the gut. Four minor endo-β-1,4-glucanase components, comprising between 4 and 10% of the endo-β-1,4-glucanase activity were also present but could not be separated from the β-glucosidase components and therefore were not characterised. There were two β-glucosidase components, named GD1 and GD2, which were partially purified and characterised. The Km for GD1, the major component, was 10.6 mM with . Km values were also determined for both components with cellodextrins ranging from cellobiose to cellopentaose. GD1 was competitively inhibited by gluconon-δ-1, 5-lactone (Ki = 0.33 mM) but was unaffected by glucose at physiological concentrations. Neither component was active against crystalline cellulose or CMC.
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ABSTRACT: Abstract Cellulosic ethanol has been identified as a crucial biofuel resource due to its sustainability and abundance of cellulose feedstocks. However, current methods to obtain glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass. Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic material as a source of metabolic glucose. Even though traditional biochemical methods have been used to identify and characterize these enzymes, the advancement of genomic and proteomic research tools are expected to allow new insights into insect digestion of cellulose. This information is highly relevant to the design of improved industrial processes of biofuel production and to identify potential new targets for development of insecticides. This review describes the diverse methodologies used to detect, quantify, purify, clone and express cellulolytic enzymes from insects, as well as their advantages and limitations.Insect Science 05/2010; 17(3):184 - 198. · 1.79 Impact Factor
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ABSTRACT: In the past decade, a century of debate over whether termites produce their own cellulases has been resolved by the application of molecular genetic techniques. Cellulase genes were present in ancient bilaterian animals, and have been passed down to termites and many other invertebrate lineages over several hundred million years. Termites contain multiple endoglucanase gene copies, all of which come from glycosyl hydrolase family 9, but the roles of the different gene copies are not yet clear. Enzyme assays and RNAi experiments indicate that endogenous cellulases play a key role in termite metabolism. The overall contribution of these enzymes in members of the Termitidae (which lack cellulolytic flagellates) appears to be greater than in members of other families. A major shift in the site of expression of endoglucanases and β-glucosidases from the salivary glands to the midgut has occurred in some members of the speciose family Termitidae. Investigations into the roles of different members of the termite colony in digesting cellulose have begun, and have revealed major variations in the level of expression, including differences between different sized workers. In fungus-growers and soil-feeders, endogenous cellulases appear to be of relatively minor importance, but have nonetheless been retained in the genome.10/2010: pages 51-67;
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ABSTRACT: The demand for the usage of natural renewable polymeric material is increasing in order to satisfy the future needs for energy and chemical precursors. Important steps in the hydrolysis of polymeric material and bioconversion can be performed by microorganisms. Over about 150 million years, termites have optimized their intestinal polysaccharide-degrading symbiosis. In the ecosystem of the "termite gut," polysaccharides are degraded from lignocellulose, such as cellulose and hemicelluloses, in 1 day, while lignin is only weakly attacked. The understanding of the principles of cellulose degradation in this natural polymer-degrading ecosystem could be helpful for the improvement of the biotechnological hydrolysis and conversion of cellulose, e.g., in the case of biogas production from natural renewable plant material in biogas plants. This review focuses on the present knowledge of the cellulose degradation in the termite gut.Applied Microbiology and Biotechnology 07/2013; · 3.69 Impact Factor