Studies on isolation, purification and properties of Endoglucanase from the hepatopancreas of Perna viridis

In the attempt to find out catalytic potency and properties of the endoglucanase of green mussel, it could be highlighted that the enzyme is efficient in degrading carboxymethylcellulose to reducing sugars. The immobilized enzyme will find applications in the food industry, paper and pulp industry, wood preservation, alcohol and pharmaceutical industry.The purification method employed i.e. Sephadex G100 chromatography employing affinity and exclusion principles simplify the purification procedure.Addition of Mg2+ and Co2+ at 10mM concentrations enhances endoglucanase activity of green mussel.The immobilized endoglucanase can be used for deinking mixed office waste paper. The endoglucanase if supplemented with exoglucanase and B-glucosidase under appropriate conditions would help in the recycling of paper.

Enzymatic upgrading of eucalypt paper-grade kraft pulp within dissolving pulp production

Dissolving-grade pulps are commonly used for the production of cellulose derivatives and regenerated cellulose. High cellulose content, low content of non-cellulosic material, high brightness, a uniform molecular weight distribution and high cellulose reactivity are the key features that determine the quality of a dissolving pulp. The first part of this work was an optimization study regarding the application of selected enzymes in different stages of a new purification process recently developed in Novozymes for purifying an eucalypt Kraft pulp into dissolving pulp, as an alternative to the pre-hydrolysis kraft (PHK) process. In addition, a viscosity reduction was achieved by cellulase (endoglucanase) treatment in the beginning of the sequence, while the GH11 and GH10 xylanases contributed to boost the brightness of the final pulp. The second part of the work aimed at exploring different auxiliary enzyme activities together with a key xylanase towards further removal of recalcitrant hemicelluloses from a partially bleached Eucalypt Kraft pulp. The resistant fraction (ca. 6% xylan in pulp) was not hydrolysable by the different combinations of enzymes tested. Production of a dissolving pulp was successful when using a cold caustic extraction (CCE) stage in the end of the sequence O-X-DHCE-X-HCE-D-CCE. The application of enzymes improved process efficiency. The main requirements for the production of a dissolving pulp (suitable for viscose making) were fulfilled: 2,7% residual xylan, 92,4% of brightness, a viscosity within the values of a commercial dissolving pulp and increased reactivity.

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

Identification of a putative cellulase gene in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

Nutrition plays an important role in the development of all organisms and in particular that of farmed aquatic species where costs associated with feed can often exceed 60% of total production costs. Crustacean species in addition, have the added metabolic requirement for regular moulting to allow normal growth and this requires large amounts of energy in the form of sugars (glucose). The current study explored the capacity of the giant freshwater prawn to produce endogenous cellulose-degrading enzymes capable of extracting nutrients (simple sugars) from plant sources in formulated feeds used in the prawn aquaculture industry. We identified a putative cellulase cDNA fragment in the target organism of 1576 base pairs in length of non-microbial origin that after protein modelling exhibited a TM-score of 0.916 with a described cellulase reported from another crustacean species. The functional role of cellulase enzymes is to hydrolyse cellulose to glucose and the fragment identified in GFP was highly expressed in the hepatopancreas, the site of primary food digestion and absorption in crustaceans. Hepatopancreatic tissue from Macrobrachium rosenbergii also showed active digestion of cellulose to glucose following an endoglucanase assay. Cellulase gene(s) are present in the genomes of many invertebrate taxa and play an active role in the conversion of cellulose to available energy. Identification and characterization of endogenous cellulase gene(s) in giant freshwater prawn can assist development of the culture industry because the findings confirm that potentially greater levels of low-cost plant-material could be included in artificial formulated diets in the future without necessarily compromising individual growth performance. Ultimately, this development may contribute to more efficient, cost-effective production systems for freshwater prawn culture stocks that meet the animal's basic nutritional requirements and that also support good individual growth rates.

Endoglucanase production with the newly isolated Myceliophtora sp. i-1d3b in a packed bed solid state fermentor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

Purification of particles of subterranean clover red leaf virus using an industrial-grade cellulase

Particles of two isolates of subterranean clover red leaf virus were purified by a method in which infected plant tissue was digested with an industrial-grade cellulase, Celluclast® 2.0 L type X. The yields of virus particles using this enzyme were comparable with those obtained using either of two laboratory-grade cellulases, Cellulase type 1 (Sigma) and Driselase®. However, the specific infectivity or aphid transmissibility of the particles purified using Celluclast® was 10-100 times greater than those of preparations obtained using laboratory-grade cellulases or no enzyme. The main advantage of using Celluclast® is that at present in Australia its cost is only ca. 1% of laboratory-grade cellulases.

An investigation of functional diversity of endogenous cellulase and expression of other digestive enzyme genes in freshwater crayfish (Cherax) species

The project evaluated potential of soluble cellulose as a cheap feed ingredient for major farmed Australian freshwater crayfish species testing their growth performance, digestive enzyme activity and digestive enzyme gene expression patterns. Test animals showed an innate capacity to utilise a range of carbohydrate sources including complex structural polysaccharides. Results suggest that more plant-derived ingredient can be incorporated in formulated low-cost feeds for the culture industry.

Purification and characterization of an extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity

Multiple forms of beta-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One beta-glucosidase was purified 16-fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl beta-D-glucosides and beta-D-linked diglucosides. It was optimally active at pH 5.4, at 65-degrees-C. The apparent K(m) values for p-nitrophenyl beta-D-glucoside (PNPG) and cellobiose were 0.29 and 0.83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited beta-glucosidase competitively. At high (> 1 mm) substrate concentration, beta-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a beta-linked tetramer of glucose. Admixtures of beta-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. Beta-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.

Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei

The potential for performing cellulase-catalyzed reactions on cellulose dissolved in 1-butyl-3-methylimidazolium chloride ([bmim] Cl) has been investigated. We have carried out a systematic study on the irreversible solvent and ionic strength-induced inactivation and unfolding of cellulase from Trichoderma reesei ( E.C.#3.2.1.4). Experiments, varying both cellulase and IL solvent concentrations, have indicated that [bmim] Cl, and several other ILs, as well as dimethylacetamide-LiCl (a well-known solvent system for cellulose), inactivate cellulase under these conditions. Despite cellulase inactivity, results obtained from this study led to valuable insights into the requirements necessary for enzyme activity in IL systems. Enzyme stability was determined during urea, NaCl, and [bmim] Cl-induced denaturation observed through fluorescence spectroscopy. Protein stability of a PEG-supported cellulase in [bmim] Cl solution was investigated and increased stability/activity of the PEG-supported cellulase in both the [bmim] Cl and citrate buffer solutions were detected.

Wood degradation, and cellulase and ligninase production, by Trametes and other wood-inhabiting basidiomycetes from indigenous forests of Zimbabwe

Nine species of Trametes and five other wood inhabiting basidiomycetes, were collected from the indigenous forests of Zimbabwe and analysed for cellulases, ligninases, extracellular phenolases and wood degrading ability for the first time. Cellulase enzyme activities varied widely among the species. After 15 d growth exo-glucanase activity had increased in the majority of species whilst Biter paper activity showed the opposite trend, being greatly reduced in all species on day 15 compared to day IO. Endo-glucanase activity was relatively uniform at both sampling times. The fungi were more active against water soluble cellulose derivatives than filter paper cellulase. In all the fungi tested, cellulose activity on filter paper was significantly less than endo- and exo-glucanase activities. The highest cellulase activity was expressed by Cerrena meyenii (683 U mg(-1)) Phaeotrametes decipiens, Trametes modesta, and T. pocas also expressed relatively high cellulase activity on all types of cellulose tested. All Trametes species tested positive for extracellular phenol oxidases whilst Fomotopsis spragueii and Irpex stereoides tested negative. Ail but one of the Trametes species in the study were able to degrade two different lignin preparations in tests for lignin degradation. T. menziesii was unable to degrade both lignin preparations although it had tested positive for production of extracellular oxidase. The species in this study degraded hardwood to a greater extent than softwood. Eight of them caused more than 80% dry weight loss of wood blocks during 70 d incubation. Those fungi that expressed high cellulase activity also caused high weight loss on wood.