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Enzymatic hydrolysis is a method to generate biofuel from biomass, and autohydrolysis is a popular method to pretreat biomass prior to enzymatic hydrolysis. The primary aim of the present study was to determine the role of lignin produced in the autohydrolysis process on the enzymatic hydrolysis of biomass. The HSQC and 31 P NMR analyses confirmed that β-O-4 of lignin was reduced, while β-5, β-β, and S/G-ratio of lignin were increased with intensifying the hydrolysis intensity.The increase in the hydrolysis intensity significantly enhanced the condensed and non-condensed phenolic OH group of lignin. Interestingly, the cellulase enzyme adsorbed more on lignin that had more phenolic content, and its association with lignin reduced its activity for hydrolyzing cellulose microcrystals. Strong negative correlations were observed between the enzymatic hydrolysis yield and the condensed S-OH (r 2  = 0.978) and G-OH (r 2  = 0.961) of lignin generated in the autohydrolysis process., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Baliospermum montanum (Willd.) Muell. Arg, a medicinal plant distributed throughout India from Kashmir to peninsular-Indian region is extensively used to treat jaundice, asthma, and constipation. In the current study, 203 endophytic fungi representing twenty-nine species were isolated from tissues of B. montanum. The colonization and isolation rate of endophytes were higher in stem followed by seed, root, leaf and flower. The phytochemical analysis revealed 70% endophytic isolates showed alkaloids and flavonoids, 13% were positive for phenols, saponins and terpenoids. Further, these endophytes produced remarkable extracellular enzymes such as amylase, cellulase, phosphates, protease and lipase. The most promisive three endophytic fungi were identified by ITS region and secreted metabolites were identified by gas chromatography-mass spectrometry (GC-MS/MS). The GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from flower exhibited nine bioactive compounds namely, 2-Cyclopentenone, 2-(4-chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl and 1-Acetyl-20a-hydroxy-16-methylene. The endophyte, Aspergillus brasiliensis isolated from root and Fusarium oxysporum isolated from seed produced nine and seven bioactive compounds, respectively. Overall, a significant contribution of bioactive compounds was noticed from the diverse endophytic fungi associated with B. montanum and could be explored for development of novel drug with commercial values., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Improved sequencing technologies and the maturation of metagenomic approaches allow the identification of gene variants with potential industrial applications, including cellulases. Cellulase identification from metagenomic environmental surveys is complicated by inconsistent nomenclature and multiple categorization systems. Here, we summarize the current classification and nomenclature systems, with recommendations for improvements to these systems. Addressing the issues described will strengthen the annotation of cellulose-active enzymes from environmental sequence data sets-a rapidly growing resource in environmental and applied microbiology., (Copyright © 2020 American Society for Microbiology.)

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root-microorganism interactions.

Solid-state fermentation (SSF) is the preferred method of enhancing the phenolic content of oats, while scientific optimization for improving specific phenolic compounds is limited. In this study, sequential targeting of phenolic conversion in simultaneous hydrolysis and fermentation (SHF) of oats was investigated. The results revealed that SHF with adding cellulase at 0, 6 and 12 days could increase the total phenolic content by 4.4%, 67.8% and 59.1%, respectively, over that of SSF. The α-amylase and CMCase activity were highly correlated with the soluble and insoluble phenolic contents in SHF (-6 and -12) systems (r > 0.8, p < 0.05). Interestingly, the content of phenolic fraction, such as ferulic acid, was up-regulated, whereas sinapic acid was down-regulated. These results indicated that the phenolic conversion occurred in SHF, resulting in variation in DPPH and ABTS + radical scavenging abilities. This research provided metabolic understanding of the optimization of phenolic compounds to increase the functional ingredient of oats., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

Digital microfluidics (DMF) represents an alternative to the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps. The automation component of DMF have pushed the barriers of this "lab-on-chip" technology. However, integration with external components (i.e., "world-to-chip") interfaces have been a challenge. Two common "world-to-chip" challenges are (1) delivering biological samples to DMF devices and (2) accurately controlling temperatures on device. To address these challenges, this work describes two "world-to-chip" interface features that have been integrated on a DMF platform: a reagent delivery system and a thermal control apparatus. This platform enables a variety of biological or chemical experiments to be conducted on-chip while reducing manual intervention. Specifically, our platform increases reagent volumes available to device reservoirs volume by at least 50-fold eliminating the need to manually refill reservoirs while improving droplet dispensing reproducibility. In addition, we have integrated a closed-loop temperature control system that offers precise temperature control on-chip. To validate our "world-to-chip" interface, we have automated bacterial transformation and enzymatic assay protocols, showing that such a system enhances DMF performance. Overall, we propose that this system will improve biological experimentation which requires fluidic and temperature control integrated on DMF platforms.

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei's Genetic diversity index; h = 0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei's genetic diversity index as h = 0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.

Effective and low-cost inducer is a prerequisite for efficiency improvement in cellulase production by Trichoderma reesei. In this study, spores of Aspergillus niger enriched with cellobiase were embedded in Ca-alginate microcapsules, attaining a sustained cellobiase-release system (named CRMA) which was then used to catalyze the conversion of glucose into glucose-sophorose sugar mixture via transglycosylation. Further, the process and optimal parameters of transglycosylation reaction were studied in a 5 L reactor and the sophorose concentration reached 70.55 g/L at 50 °C, pH 4.0 with CRMA loading of 200 mL after 72 h of reaction. The obtained sugar mixture was subsequently used as both carbon source and inducer in cellulase production by Trichoderma reesei under fed-batch process. The filter paper activity (FPA) reached 90.28 IU/mL at 144 h fermentation time, 1.85-fold higher than that induced by lactose. These results indicate that the newly established strategy for soluble inducer synthesis has the potential for efficiency improvement and cost reduction in cellulase production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The responses of cellulase enzymes of three bacterial isolates and their impacts on cattle manure decomposition were assessed in a greenhouse model in vivo pond ecosystem. Fifty grams of fresh cattle manure was placed in a fastened nylon bag (mesh size ~ 50 μm dia.) and placed in triplicate in a plastic bucket with 10 l of pond water which was hung inside the enclosed polyhouse, semi-closed and open systems for 4 weeks. Samples of manure residue directly from nylon bag and water from manure leached bucket water, water, and soil from the enclosed polyhouse were collected for enzymatic assays, enumeration of aerobic cellulose decomposing and heterotrophic bacteria, and determination of water and soil quality parameters. Responses of cellulases to different temperatures in situ were also elucidated. The values of test bacteria, endoglucanase, exoglucanase and β-glucosidase, and organic carbon were significantly (P ˂ 0.05) higher in the closed system compared to semi-closed or open system. Priming of all the enzymes coupled with the peak of aerobic cellulose decomposing bacteria and heterotrophic bacterial populations occurred on the day 14 or 21 in vivo. Since the peaks of three cellulases of bacterial isolates (KUPH1, KUPH6, and KUPH8) were demonstrated between 35 and 40 °C, and that temperature coincided with temperature of the greenhouse model, this temperature range appeared to favor the growth of cellulose decomposing bacterial populations and involved cellulase enzymes.

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, 'Candidatus Reconcilibacillus cellulovorans', possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the 'Ca. Reconcilibacillus cellulovorans' multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.

Microbial fish pathogens are prevalent in aquaculture. Control of bacterial fish pathogens is important and bio control of pathogenic bacteria is a novel field of study. The aim of this study was to evaluate the antagonistic activity of bacteria isolated from Anabas testudineus against potent fish pathogens. The cellular components/preparations and filtered cell free culture supernatants were effective against six fish pathogens. Altogether 110 strains were isolated from fish proximal and distal intestine, out of which 10 strains were selected through well diffusion method. From them a strain HGA4C having prominent antimicrobial activity was selected as candidate probiotic strain. The whole-cell product, heat-killed whole-cell product and the filtered broth were exhibited bactericidal activity against the tested pathogens. Among them cell free culture supernatant showed maximum inhibition. In addition, isolated candidate probiotic bacterium was capable of producing extracellular enzymes important for the digestion of food ingredients and was effectively grown in fish mucus obtained from Oreochromis niloticus. The strain tolerated gradient of bile juice secreted by the host and effectively produced biofilm. Analysis of 16S rDNA sequence revealed that isolated strain HGA4C was Paenibacillus polymyxa (MF457398.1). Furthermore intraperitoneal injection of the bacterium did not induce any pathological anomalies or mortalities in Oreochromis niloticus and disclosed the safety of this bacterium as a candidate probiotic in aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Industrial production of cellulase by filamentous fungi is largely dependent on cellulose, which serves as a natural inducer of cellulase expression. However, insoluble cellulose is unfavorable to submerged fermentation and thus limits the production level of cellulase. The possibility of cellulase production under non-inducing conditions is explored in Penicillium oxalicum by overexpressing two chimeric transcription factors. The chimeric transcription factors contain the DNA binding domain of cellulase transcriptional activator ClrB linked to the C-terminal sequences of XlnR A871V , a constitutively active mutant of hemicellulase transcriptional activator. The obtained recombinant mutants exhibited dramatically improved basal production of cellulase, which was not observed with the overexpression of intact ClrB. When cultivated in a complex cellulosic medium, one of these mutants, OE-CX C -S-1, displayed a 7.3-fold increase in cellulase production (2.8 U mL -1 ) relative to the parent strain. The results demonstrate that the dependence of cellulase synthesis on cellulose could be reduced by the overexpression of artificially designed chimeric transcription factors, and offers a potential strategy to engineer fungal strains for improving cellulase production., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals., Results: Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24-62%) and β-glucosidase (22-76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5-4.5 g l -1 ., Conclusion: Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

Lentinula edodes has the potential to degrade woody and nonwoody lignocellulosic biomass. However, the mechanism of lignocellulose degradation by L. edodes is unclear. The aim of this work is to explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L. edodes. For that, we compared the secretomes of L. edodes grown on microcrystalline cellulose, cellulose with lignosulfonate and glucose. Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate, 230 proteins were identified. Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-β-1,4-glucanase, α-galactosidase, polygalacturonase and glucoamylase in both cellulosic secretomes. In contrast, enzymes involved in lignin degradation were most abundant in glucose culture, with laccase 1 being the predominant protein (13.13%). When the cellulose and cellulose with lignosulfonate secretomes were compared, the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures, which was confirmed by enzyme activity assays. In addition, qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased (by 32.2- to 1166.7-fold) when L. edodes was grown in cellulose with lignosulfonate medium., Biological Significance: In this article, the secretomes of L. edodes grown on three different carbon sources were compared. The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation, which is helpful to further explore the mechanism of biomass bioconversion by L. edodes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10 -8 U per assay, being 10 3 -10 4 -fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications.

Two specific, sensitive, and precise stability-indicating chromatographic methods were developed, optimized, and validated for the determination of Azintamide (AZ) in the presence of its degradation product. The first method was TLC combined with the densitometric determination of the separated bands. Separation was achieved using silica gel 60 F254 TLC plates and chloroform-acetone-glacial acetic acid (7.5 + 2.1 + 0.4, v/v/v) as the developing system. Good correlations were obtained between the integrated peak area of the studied drug and its corresponding concentrations in the linearity range. The second method used HPLC with UV diode-array detection, in which the proposed method was applied for the quantitative determination of AZ in the presence of its acidic degradation product and the quantitative determination of the acid-induced degradation product of AZ (AZ Deg) using pentoxifylline as the internal standard. The proposed components were separated on a reversed-phase C18 analytical column using acetonitrile-water (50 + 50, v/v). The flow rate was maintained at 0.55 mL/min and the detection wavelength was 260 nm. Linear regressions were obtained in the range of 1-30 and 0.3-16 μg/mL for AZ and AZ Deg, respectively. Different parameters affecting the suggested methods were optimized for maximum separation of the cited components. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and successfully applied for the determination of AZ in its pure powder form and in its pharmaceutical formulation. Both methods were also statistically compared with the reported method with no significant difference in performance observed.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A liquid crystal (LC)-based sensor, which is capable of monitoring enzymatic activity at the aqueous/LC interface and detecting cellulase and cysteine (Cys), was herein reported. When functionalized with a surfactant, dodecyl β-d-glucopyranoside, the 4-cyano-4'-pentylbiphenyl (5CB) displays a dark-to-bright transition in the optical appearance for cellulase. We attribute this change to the orientational transition of LCs, as a result of enzymatic hydrolysis between cellulase and surfactant. Furthermore, by adding cellulase and Cu(2+), our surfactant-LCs system performs an interesting ability to detect Cys, even though Cys could not interact with surfactant or LC directly. Alternatively, through the strong binding between Cys and Cu(2+), cellulase was able to hydrolyze surfactant in the presence of Cu(2+), leading to the transition of LCs from dark to bright. The detection limit of the LC sensor was around 1×10(-5)mg/mL and 82.5μM for cellulase and Cys, respectively. The LC-based sensor may contribute to the development of low-cost, expedient, and label-free detection for cellulase and Cys and the design strategy may also provide a novel way for detecting multiple analytes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Knowledge regarding the enzymatic machinery of fungi is decisive to understand their ecological role. The species of the genus Geastrum are known to grow extremely slowly in pure culture, which makes it difficult to evaluate physiological parameters such as enzyme activity. Qualitative assays were performed on isolates of four species of this genus, showing evidence of laccase, cellulase, pectinase, amylase and lipase activity and suggesting that a wide range of carbon sources can be exploited by these species. For the first time in this genus, quantitative assays verified manganese peroxidase activity (up to 0.6mU/g) in 30-day old cultures, as well as laccase, β-glycosidase and β-xylosidase activities., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

endo-1,4-β-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside. Graphical Abstract Principle of the CELLG5 assay.

Pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB was evaluated under submerged (SmF) and solid state (SSF) culture conditions. Number of bacteria was 2.7 times higher in SmF as compared with SSF. Enzymes produced under SmF were xylanase, LiP, MnP and laccase. In SSF xylanase and laccase were detected. CMCase, FPase and β-glucosidase were not detected. Delignification was highest in SmF with 19.94% and 10.43% removal of hemicelluloses and lignin, respectively. FTIR analysis suggested the degradation of lignin/hemicellulose component. SEM analysis showed pores were three times bigger in SmF as compared with raw bagasse. Maximum CR dye was absorbed by treated SmF bagasse. Enzymatic saccharification increased by 3.7 times after SmF treatment in comparison to raw bagasse. Pretreatment of bagasse by Pandoraea sp. ISTKB was more efficient under SmF than SSF. High negative correlation between saccharification vs lignin/hemicelluloses content justified the need for pretreatment of lignocellulosic waste before saccharification., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

Identification of microorganisms for the production of carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. To this end, dye-polysaccharide interactions which provide a visual indication of polymer hydrolysis (clear zones or halos) have been used for decades. For the detection of extracellular cellulase or xylanase activity many laboratories use Gram's iodine as the chromogenic dye, as it is a more rapid initial screening method compared to the use of other dyes. Here, we compared Gram's iodine and Congo red as indicators of polysaccharide hydrolysis. We attempted to detect cellulase activity using carboxymethylcellulose, and xylanase activity using birchwood xylan, in fourteen uncharacterized bacteria isolated from wood chips. Our results indicate that Gram's iodine may lead to identification of false positives in a typical screening protocol and that Congo red allows for avoidance of such pitfall. Congo red allowed detection of cellulase activity from live microbial colonies but not Gram's iodine. To confirm this, detection of enzymatic activity was also assessed using cell-free enzyme preparations. Congo red was found to be reliable in detecting cellulase activity with isolated enzymes preparations. Under the same conditions, neither of these dyes detected xylanase activity, despite independent evidence of xylanase activity for one of the preparations. We detected xylanase activity for this particular enzyme preparation using a coloured derivative of xylan (Remazol Brillant Blue R-xylan adduct) that respond to xylan hydrolysis. Our results suggest that methods that rely on interactions between a dye (Congo red or Gram's iodine) and a polymeric substrate (carboxymethylcellulose or birchwood xylan) for indirect detection of hydrolysis may require the use of relevant controls and independent confirmation of enzymatic activities., (Copyright © 2014. Published by Elsevier Inc.)

Xylanase gene xynB, cellulase genes egxA and bgl4 were subjected to codon optimisation using two opposing strategies. One was designated the 'one amino acid-one codon' approach, which employs only the codon most used by humans for each amino acid. The other one is referred to as the "humanised" codon usage method, which selects synonymous codons for each amino acid according to the human codon usage table to mimic patterns used in humans. Protein expression levels in mammalian cell lines from each sequence were measured using fluorescence-activated cell sorting, western blotting and enzymatic activity assay. The results indicate that compared with the humanised codon usage method, the relatively simple 'one amino acid-one codon' approach could enhance heterologous protein expression in mammalian cells without apparent drawbacks.

There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The diversity of marine-derived Penicillium from Korea was investigated using morphological and multigene phylogenetic approaches, analyzing sequences of the internal transcribed spacer region, β-tubulin gene, and RNA polymerase subunit II gene. In addition, the biological activity of all isolated strains was evaluated. We tested for the extracellular enzyme activity of alginase, endoglucanase, and β-glucosidase, and antifungal activity against two plant pathogens (Colletotrichum acutatum and Fusarium oxysporum). A total of 184 strains of 36 Penicillium species were isolated, with 27 species being identified. The most common species were Penicillium polonicum (19.6 %), P. rubens (11.4 %), P. chrysogenum (11.4 %), and P. crustosum (10.9 %). The diversity of Penicillium strains isolated from soil (foreshore soil and sand) and marine macroorganisms was higher than the diversity of strains isolated from seawater. While many of the isolated strains showed alginase and β-glucosidase activity, no endoglucanase activity was found. More than half the strains (50.5 %) showed antifungal activity against at least one of the plant pathogens tested. Compared with other strains in this study, P. citrinum (strain SFC20140101-M662) showed high antifungal activity against both plant pathogens. The results reported here expand our knowledge of marine-derived Penicillium diversity. The relatively high proportion of strains that showed antifungal and enzyme activity demonstrates that marine-derived Penicillium have great potential to be used in the production of natural bioactive products for pharmaceutical and/or industrial use.

In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity.

A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end d-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free d-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion.

A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein-nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F(1)-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10-15 h, depending mainly on the substrate surface to be used.

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.

Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.

Opuntia ficus-indica Mill. (forage cactus) is farmed with relative success in the semi-arid region of the Brazilian northeast for commercial purposes, particularly as forage and food. Endophytic microorganisms are those that can be isolated inside plant tissues and can be a new source to production of enzymes with different potentialities. The objective of this study was to describe the richness of endophytic fungi from O. ficus-indica and to detect the capacity of these species to produce extracellular hydrolytic enzymes. Forty-four endophytic fungi species were isolated. Among them, the most commonly found were Cladosporium cladosporioides (20.43%) and C. sphaerospermum (15.99%). Acremonium terricola, Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata are being reported for the first time as endophytic fungi for Brazil. The majority of isolated fungi exhibited enzymatic potential. Aspergillus japonicus and P. glandicola presented pectinolytic activity. Xylaria sp. was the most important among the other 14 species with positive cellulase activity. All 24 isolates analysed were xylanase-positive. Protease was best produced by isolate PF103. The results indicate that there is a significant richness of endophytic fungi in O. ficus-indica, and that these isolates indicate promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases and proteases.

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay "Congo Red Analysis of Cellulose Concentration," or "CRACC." CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

A cellulose-degrading composite microbial system containing a mixture of microbes was previously shown to demonstrate a high straw-degrading capacity. To estimate its potential utilization as an inoculant to accelerate straw biodegradation after returning straw to the field, two cellulose-degrading composite microbial systems named ADS3 and WSD5 were inoculated into wheat straw-amended soil in the laboratory. The microbial survival of the inoculant was confirmed by a denaturing gradient gel electrophoresis (DGGE) analysis, whereas the enhancement of straw degradation in soil was assessed by measuring the mineralization of the soil organic matter and the soil cellulase activity. The results indicated that most of the DGGE bands from ADS3 were detected after inoculation into straw-amended autoclaved soil, yet only certain bands from ADS3 and WSD5 were detected after inoculation into straw-amended non-autoclaved soil during five weeks of incubation; some bands were detected during the first two weeks after inoculation, and then disappeared in later stages. Organic matter mineralization was significantly higher in the soil inoculants ADS3 and WSD5 than in the uninoculated controls during the first week, yet the enhanced degradation did not persist during the subsequent incubation. Similar to the increase in soil organic matter, the cellulase activity also increased during the first week in the ADS3 and WSD5 treatments, yet decreased during the remainder of the incubation period. Thus, it was concluded that, although the survival and performance of the two inoculants did not persist in the soil, a significant enhancement of degradation was present during the early stage of incubation.

The natural biodiversity that is found in tropical areas offers countless biotechnological opportunities; especially if we take in account that many biomolecules from several microorganisms have supported for many years, different industrial applications in areas such as pharmacology, agro-industry, bioprocess, environmental technology, and bioconversion. In order to find new lignocellulolytic enzymes and evaluate bamboo fibers as substrate, Schizophyllum commune a fungus with broad distribution was isolated and grown during 15 days in liquid culture medium containing 1% lignocellulosic fibers from bamboo, banana stem, and sugarcane bagasse. The enzymatic activity of xylanase, mannanase, polygalacturonase, CMCase, FPase, and avicelase were evaluated. Sugarcane bagasse and banana stem showed to induce higher hollocellulase activity when compared with bamboo as the main carbon source. The physical mechanism that the fungus uses to degrade bamboo was observed not only in fibers naturally infected but also in healthy fibers that were treated and untreated with enzyme solution. SEM analysis showed the structural disruption and invasion of the vascular bundles, parenchyma cells, and parenchymatous tissues as a consequence of the presence of this fungus and the catalytic action of its enzymes into the plant tissue.

This study was focused on investigating the role of the initial residue community, i.e. microorganisms and enzymes from the epiphytic and endophytic compartments, in soil decomposition processes. Aerial and underground parts (leaves and roots) of maize (Zea mays L.) plants were γ-irradiated, surface-sterilized with sodium hypochlorite (NaOCl)/ethanol or non-sterilized (controls), while the outer surface morphology of maize leaves and roots was examined by scanning electron microscopy (SEM). Non-sterilized and sterilized maize leaves and roots were incubated in soil to study carbon (C) mineralization kinetics and enzyme dynamics (L-leucine aminopeptidase, CBH-1, xylanase, cellulase and laccase). SEM results showed that initial microbial colonization was more pronounced on non-sterilized leaf and root surfaces than on sterilized samples. The hypochlorite treatment removed a part of the soluble components of leaves by washing and no specific effect of any type of colonizing microorganisms was observed on C mineralization. In contrast, γ irradiation and hypochlorite treatments did not affect root chemical characteristics and the quantitative effect of initial residue-colonizing microorganisms on C mineralization was demonstrated. The variations in C mineralization and enzyme dynamics between non-sterilized and sterilized roots suggested that activities of epiphytic and endogenic microorganisms were of the same order of magnitude., (Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for fungal attachment was employed for production of cellulase by Chaetomium crispatum and xylanase by Gliocladium viride. The design allowed comparison of production between CCFs with hydrophobic surface (PMMA-CCF), hydrophilic glass surface (GS-CCF) and 500-ml Erlenmeyer flask (EF). Compared with the EF, endo-β-1,4-glucanase and FPase (filter paper degradation) activities increased from 0.044 to 0.156 and from 0.008 to 0.021 IU/ml, respectively, in the PMMA-CCF, while growth of C. crispatum was higher by at most 1.38-fold compared with the other vessels. Xylanase production in the EF was at most 5.08-fold higher and growth of G. viride was at most 1.52-fold higher compared with the other vessels. Temporal pattern of biofilm development based on two-channel fluorescence detection of extracellular polymeric substances (EPSs) and whole cells in a confocal laser scanning microscope demonstrated increase by 100% in biovolume, 25% in thickness and 62.5% both in substratum coverage and total spreading of C. crispatum biofilm in PMMA-CCF over 6 days. Biovolume of G. viride biofilm in GS-CCF increased by 150% over 4 days while that in PMMA-CCF enhanced by 200% over 2 days. Biofilm thickness in PMMA-CCF was 44% higher compared with GS-CCF and increased by 175% over 2 days. Substratum coverage was 38% higher in GS-CCF compared with PMMA-CCF. Thus, reactor surface area and property, shear forces and biofilm formation influenced enzyme production.

A new enzymatic activity detection assay based on colour change of the semi-reflective films is presented. The method is based on the preparation of multilayered thin films of controlled thickness obtained by sequential deposition of cellulose nanocrystals and xyloglucan. The hydrolysis of the films leads to a decrease in layer thickness that enables to detect enzyme activity, to the naked eye, from the resulting colour changes in a span of few minutes. The method allows direct, fast, highly sensitive, and easy-to-use characterization of enzymatic activities., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with high transparency, surrounded by well-developed forests and located at San Carlos de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we characterized yeast distribution and diversity along a south-to-north transect and established a relationship between the ability to produce photoprotective compounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of yeast at different collection points. Subsurface water samples were filtered for yeast isolation. Total yeast counts ranged between 22 and 141 CFU L(-1) , and the highest values corresponded to the most impacted sites. Littoral sites had a low proportion of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably a result of the high transparency of the water and the increased UV exposure. The yeast community from NH Lake showed a high species richness and a uniform distribution of taxa between pelagic and border collection points. Yeasts were identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and Cryptococcus victoriae were the most frequently found species, representing 14.4% and 13.6% of the total yeast isolates, respectively. Most of the yeast isolates demonstrated at least one extracellular enzymatic activity (mainly cellulase and lipase activities), which suggested that these microorganisms are metabolically active in the lake., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A poly(dimethylsiloxane) (PDMS) viscometer was developed to analyze the activity of endo-β-1,4-glucanases (EC3.2.1.4) on hydrolyzing sodium carboxymethylcellulose (NaCMC). In the viscometer, a PDMS pump was used as the vacuum source to drive the fluid flow in the PDMS microchannel. The numerical simulation by COMSOL suggested that the vacuum level of the PDMS pump increased during the viscosity measurement. The increasing vacuum level of the PDMS pump allowed more stable fluid flow than the constant vacuum of the conventional vacuum pump. By monitoring the fluids traveling in the microchannel, the viscosity of the endoglucanase-NaCMC solution was derived at different reaction times. The non-Newtonian behavior of the NaCMC solution was studied to ensure that the viscosity measurement was performed in the Newtonian range. A protocol was established to convert the viscometric activity to the activity of standard international units with Almin's method. The PDMS viscometer required only 20 μL sample for the endoglucanase assay and the results were in good agreement with the results from Ubbelohde viscometers., (This journal is © The Royal Society of Chemistry 2011)