Showing papers by "Novozymes published in 2007"

Progress and challenges in enzyme development for biomass utilization.

Abstract: Enzymes play a critical role in the conversion of lignocellulosic waste into fuels and chemicals, but the high cost of these enzymes presents a significant barrier to commercialization. In the simplest terms, the cost is a function of the large amount of enzyme protein required to break down polymeric sugars in cellulose and hemicellulose to fermentable monomers. In the past 6 years, significant effort has been expended to reduce the cost by focusing on improving the efficiency of known enzymes, identification of new, more active enzymes, creating enzyme mixes optimized for selected pretreated substrates, and minimization of enzyme production costs. Here we describe advances in enzyme technology for use in the production of biofuels and the challenges that remain.

Phospholipases and their industrial applications.

Abstract: Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions—white biotechnology is a strong alternative.

Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools

Abstract: A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools.

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

Abstract: In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.

Evaluation of Minimal Trichoderma reesei Cellulase Mixtures on Differently Pretreated Barley Straw Substrates

Abstract: The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-β-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.

Cradle-to-gate environmental assessment of enzyme products produced industrially in denmark by novozymes A/S

Abstract: Enzymes are biological catalysts with an enormous capacity to increase the speed of a huge variety biochemical reactions. Industrially produced enzymes are used in a broad variety of sectors to increase quality, speed and yield of processes, and reduce energy consumption and use of hazardous chemicals. The present paper provides a methodological framework for analysing environmental impacts of enzyme products and environmental data for five characteristic enzyme products. Life cycle assessment is used as an analytical tool and modelling of enzyme production is facilitated in SimaPro 6.0 software. Detailed data on enzyme production are derived from Novozymes’ production facilities in Denmark. Data on ingredients are derived from the literature, publicly available databases and from Novozymes’ suppliers. Cradle-to-gate environmental data for five representative enzyme products produced by Novozymes in Denmark have been determined, and a basis for further assessments of more of Novozymes’ enzyme products has been established. Environmental impacts induced by producing the considered enzyme products vary by a factor 10 or more depending on the impact category considered. Contributions to global warming range, for example, between one and ten kg CO2-equivalents per kg final product. The variation is explained by differences in fermentation time, formulation type, production yield and strength of the final product. The main sources of environmental impact are usually fermentation processes due to electricity and ingredient consumption. Enzyme production has been the subject of significant optimisation during the past decades by implementation of e.g. gene modified production strains, and the provided environmental data are only representative to enzyme products produced by Novozymes at the present optimisation stage. Novozymes produces more than 600 enzyme products for use in a variety of sectors, and the established framework for environmental assessment will be used for assessing more products in order to provide a broad basis for environmental comparison of enzyme assisted technologies and conventional technologies.

The HvNAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis

Abstract: Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence. The gene transcript was isolated by differential display from barley leaves infected with the biotrophic powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The full-length cDNA clone was obtained using 5′-RACE and termed HvNAC6, due to its high similarity to the rice homologue, OsNAC6. Gene silencing of HvNAC6 during Bgh inoculation compromises penetration resistance in barley epidermal cells towards virulent Bgh. Complementing the effect of HvNAC6 gene silencing, transient overexpression of HvNAC6 increases the occurrence of penetration resistant cells towards Bgh attack. Quantitative RT-PCR shows the early and transient induction of HvNAC6 in barley epidermis upon Bgh infection. Additionally, our results show that the Arabidopsis HvNAC6 homologue ATAF1 is also induced by Bgh and the ataf1-1 mutant line shows decreased penetration resistance to this non-host pathogen. Collectively, these data suggest a conserved role of HvNAC6 and ATAF1 in the regulation of penetration resistance in monocots and dicots, respectively.

A new kinetic model for heterogeneous (or spatially confined) enzymatic catalysis : Contributions from the fractal and jamming (overcrowding) effects

Abstract: Heterogeneous catalysis involves catalysts and reactants separated in different phases. In these systems, the interaction between the reactant and catalyst can be quite different from its homogeneous counterpart, because of the peculiarity of the diffusion and molecular collision processes constricted in spaces with dimension less than three. The fractal theory, developed for mathematic, physical, chemical, and biological processes with inherent irregularity and complexity, can be applied to heterogeneous catalysis. To better understand the heterogeneous enzymatic reactions, a fractal Michaelis kinetics was reformulated, after applying the general fractal formalism to the classical model of homogeneous enzymatic reactions. A kinetic “jamming” effect caused by the overcrowding of enzyme/substrate in confined space was also studied. The new kinetic model was applied to the hydrolysis of cellulose by cellobiohydrolase, a representative heterogeneous biocatalytic system highly fractal due to the strong surface adsorption of the enzyme onto the insoluble substrate as well as to the one-dimensional “processive” enzymatic mechanism. The usefulness of the model for the study and application of other enzymatic reactions was discussed.

Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant "Minimal" Enzyme Cocktail Containing beta-Xylosidase and Novel endo-1,4-beta-Xylanase and alpha-L-Arabinofuranosidase Activities

Abstract: This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were approximately 14, approximately 18, and approximately 27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei.

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw

Abstract: In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L® from Trichoderma reesei, and a β-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, ∼48% (g g−1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of ∼39% (g g−1). Addition of extra enzyme (Celluclast 1.5 L®+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32–39 g L−1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.

Methods of increasing secretion of polypeptides having biological activity

Abstract: The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis

Abstract: Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichiastipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability.

Abstract: Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results.

Chemical respiratory allergy: opportunities for hazard identification and characterisation. The report and recommendations of ECVAM workshop 60.

Abstract: Kimber, Ian Agius, Raymond Basketter, David A Corsini, Emanuela Cullinan, Paul Dearman, Rebecca J Gimenez-Arnau, Elena Greenwell, Leona Hartung, Thomas Kuper, Frieke Maestrelli, Piero Roggen, Erwin Rovida, Costanza European Centre for the Validation of Alternative Methods Consensus Development Conference England Alternatives to laboratory animals : ATLA Altern Lab Anim. 2007 May;35(2):243-65.

Anoxybacillus rupiensis sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Abstract: Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127T = NBIMCC 8387T). The 16S rRNA gene sequence data of a strain R270T have been deposited in the EMBL databases under the accession number AJ879076.

Skin sensitisation and epidermal disposition: the relevance of epidermal disposition for sensitisation hazard identification and risk assessment. The report and recommendations of ECVAM workshop 59.

Abstract: This is the 59th report of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). The main goal of ECVAM, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which have scientific relevance and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops, each addressing a specific topic, and at which selected groups of independent international experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward. A workshop on Skin Sensitisation and Epidermal Disposition was held at ECVAM (Ispra, Italy) on 30–31 January 2006, under the chairmanship of David Basketter. The current status of approaches used to measure the disposition of chemicals in skin compartments was reviewed, with particular emphasis on proposing recommendations on how best to use such information to reduce, refine and/or eliminate the need for animal testing, according to the Three Rs principle. The key focus was the relevance of information on epidermal disposition, and how best to integrate such information into non-animal testing strategies for skin sensitisation.

Evaluation of Different Biomass Materials as Feedstock for Fermentable Sugar Production

Abstract: Saline crops and autoclaved municipal organic solid wastes were evaluated for their potential to be used as feedstock for fermentable sugar production through dilute acid pretreatment and enzymatic hydrolysis. The saline crops included two woods, athel (Tamarix aphylla L) and eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose tall wheatgrass (Agropyron elongatum), and creeping wild rye (Leymus triticoides). Each of the biomass materials was first treated with dilute sulfuric acid under selected conditions (acid concentration=1.4% (w/w), temperature=165°C, and time=8 min) and then treated with the enzymes (cellulases and β-glucosidase). The chemical composition (cellulose, hemicellulose, and lignin contents) of each biomass material and the yield of total and different types of sugars after the acid and enzyme treatment were determined. The results showed that among the saline crops evaluated, the two grasses (creeping wild rye and Jose tall wheatgrass) had the highest glucose yield (87% of total cellulose hydrolyzed) and fastest reaction rate during the enzyme treatment. The autoclaved municipal organic solid wastes showed reasonable glucose yield (64%). Of the two wood species evaluated, Athel has higher glucose yield (60% conversion of cellulose) than eucalyptus (38% conversion of cellulose).

Modified messenger rna stabilizing sequences for expressing genes in bacterial cells

Abstract: The present invention relates to methods of producing a polypeptide having biological activity in a bacterial cell, comprising: (a) cultivating a bacterial host cell in a medium conducive for production of the polypeptide, wherein the bacterial host cell comprises a nucleic acid construct comprising a promoter region operably linked to a polynucleotide sequence encoding the polypeptide and a modified mRNA processing/stabilizing sequence located downstream of the promoter region and upstream of the ribosome binding site of the polynucleotide sequence encoding the polypeptide, wherein the modified mRNA processing/stabilizing sequence promotes higher expression of the polynucleotide sequence compared to an unmodified mRNA processing/stabilizing sequence; and (b) isolating the polypeptide having biological activity from the cultivation medium. The present invention also relates to such modified mRNA processing/stabilizing sequences, nucleic acid constructs, and bacterial host cells and to methods of obtaining such bacterial host cells.

Enzyme granules for animal feed

Abstract: The present invention is related to feed granules comprising a feed enzyme and a zinc salt of an organic acid.

Environmental assessment of Ronozyme ® P5000 CT phytase as an alternative to inorganic phosphate supplementation to pig feed used in intensive pig production

Abstract: Goal, Scope and Background. Ronozyme® P5000 CT is an industrially produced enzyme product (phytase) which is able to degrade naturally occurring phytate in animal feed and release the phytate's content of phosphorus for pig's growth. Ronozyme P5000 CT (hereafter called Ronozyme Phytase) can be used as an alternative to inorganic phosphorus supplementation to feed and the study addresses the environmental implications of substituting inorganic phosphorus with Ronozyme Phytase in intensive pig production in Denmark. Methods. Life cycle assessment is used as an analytical tool, and modelling of the two considered systems is facilitated in SimaPro 6.0 software. The study addresses changes induced by switching from the one alternative to the other, and all significant processes influenced by the change are included in the study. Results and Conclusions. Application of Ronozyme Phytase in intensive pig production is justified by major advantages in terms of avoided contributions to global warming, acidification, photochemical ozone formation and particularly nutrient enrichment and by significant energy savings and particularly phosphate savings. A single trade-off in terms of agricultural land use for enzyme production is small and unimportant unless use of agricultural land is given very large relative weight. Recommendations and Perspectives. Hundreds of enzyme products are commercially available on the market today, each with a range of different applications. There are several indications that enzymes like Ronozyme Phytase can play an important role in a transition to a more sustainable society, and more focus should be addressed to the evolving enzyme technology in environmental research.

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry.

Abstract: Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. β-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few β-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover β-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and β-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subject...

Adsorption of amylase enzyme on ultrafiltration membranes.

Abstract: A method to measure the static adsorption on membrane surfaces has been developed and described. The static adsorption of amylase-F has been measured on two different ultrafiltration membranes, both with a cutoff value of 10 kDa (a PES membrane and the ETNA10PP membrane, which is a surface-modified PVDF membrane). The adsorption follows the Langmuir adsorption theory. Thus, the static adsorption consists of monolayer coverage and is expressed both as a permeability drop and an adsorption resistance. From the adsorption isotherms, the maximum static permeability drops and the maximum static adsorption resistances are determined. The maximum static permeability drop for the hydrophobic PES membrane is 75%, and the maximum static adsorption resistance is 0.014 m2·h·bar/L. The maximum static permeability drop for the hydrophilic surface-modified PVDF membrane (ETNA10PP) is 23%, and the maximum static adsorption resistance is 0.0046 m2·h·bar/L. The difference in maximum static adsorption, by a factor of around...