Simultaneous microwave-assisted phosphotungstic acid catalysis for rapid improvements on the accessibility and reactivity of Kraft-based dissolving pulp.

Improving the cellulose accessibility and reactivity in an efficient and convenient way has become the focused issue in the field of dissolving pulp manufacturing. We herein demonstrate a simple yet efficient strategy, namely a simultaneous microwave (MW)-assisted phosphotungstic acid (PTA) catalysis (MW-PTAsim). The MW-PTAsim treatment was efficient to improve Fock reactivity from 49.1 % to 85.8 % and decrease viscosity from 561 to 360 mL/g within 10 min, which was superior to the single MW treatment and the sequential MW-PTAseq treatment. Besides, the MW-PTAsim treated fiber had rougher and more fibrillated surfaces with an enhanced fiber accessibility, showing increased specific surface area (SSA) from 1.43 to 6.31 m2/g, mean pore diameter (MPD) from 6.92 to 11.20 nm and water retention value (WRV) from 101 % to 172 %. These positive enhancements are mainly due to a synergy that MW-enhanced rotation of PTA mediums was served as "spinning cutters" to attack the fibers, plus MW-accelerated PTA transfer and catalytic hydrolysis further improved the fiber accessibility. Moreover, PTA also demonstrates a high reusability and chemical stability. This process offers an effective and sustainable alternative for manufacturing a premium dissolving pulp.

Impact of feed supplement with alfa-amylase and beta-glucanase on ingestive-related biomarkers registered with real-time sensors.

The aim of this study was to investigate the impact of feed supplements with alfa-amylase and beta-glucanase (Optipartum C+ 200) on ingestive-related behaviour biomarkers registered with real-time sensors: rumination behaviours and reticulorumen parameters (pH and temperature). Cows (n=20) in the treatment group (TG) were fed with Optipartum C+ 200 (Enzymes feed supplement: Alfa-Amylase 57 Units; Beta-Glucanase 107 Units) from 21 days before calving until 30 days after calving with a feeding rate of 200 g/cow/day. Cows (n=22) in the control group (CG) were fed a feed ration without feed supplement. Measurements started from 6 days before calving and continued until 21 days after calving. The following indicators were registered: with the RumiWatch System: Rumination time; Eating time; Drinking time; Rumination chews; Eating chews; Drinking gulps; Bolus; Chews per minute; Chews per bolus. With the SmaXtec system: the temperature, pH of the contents of the cows' reticulorumens, and cows' walking activity. According to our results, feed supplementation with alfa-amylase and beta-glucanase (Optipartum C+ 200) in the TG group resulted in increases in the following parameters: 9% rumination time and eating time, 19% drinking time, 11% rumination chews, 16% eating chews, 13% number of boluses per rumination, 5% chews per minute and 16% chews per bolus. The rumination time showed a strong, positive relation with rumination chews and bolus indicators in both groups (TG and CG) (p⟨0.001); while the rumination time in both groups of cows showed an opposite direction and was negatively related to eating time and eating chews (p⟨0.05). We found a 1.28 % lower reticulorumen pH and a 0.64 % lower reticulorumen temperature in cows fed with the supplement compared with cows in the control group. Cows in TG were 8.80% more active than those in the CG group. For improvement of ingestive-related behaviour we suggest adding a feed supplement with alfa-amylase and beta-glucanase (Optipartum C+ 200).

In vitro activities of cellulase and ceftazidime, alone and in combination against Pseudomonas aeruginosa biofilms.

BACKGROUND: Biofilms are a main pathogenicity feature of Pseudomonas aeruginosa and has a significant role in antibiotic resistance and persistent infections in humans. We investigated the in vitro activities of antibiotic ceftazidime and enzyme cellulase, either alone or in combination against biofilms of P. aeruginosa. RESULTS: Both ceftazidime and cellulase significantly decreased biofilm formation in all strains in a dose-dependent manner. Combination of enzyme at concentrations of 1.25, 2.5, 5, and 10 U/mL tested with 1/16× MIC of antibiotic led to a significant reduction in biofilm biomass. Cellulase showed a significant detachment effect on biofilms at three concentrations of 10 U/mL, 5 U/mL, and 2.5 U/mL. The MIC, MBC, and MBEC values of ceftazidime were 2 to 4 µg/mL, 4 to 8 µg/mL, and 2048 to 8192 µg/mL. When combined with cellulase, the MBECs of antibiotic showed a significant decrease from 32- to 128-fold. CONCLUSIONS: Combination of the ceftazidime and the cellulase had significant anti-biofilm effects, including inhibition of biofilm formation and biofilm eradication in P. aeruginosa. These data suggest that glycoside hydrolase therapy as a novel strategy has the potential to enhance the efficacy of antibiotics and helps to resolve biofilm-associated wound infections caused by this pathogen.

Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes.

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa.

Endoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett-Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 µg.

Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis.

Tuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

Characteristics of a recombinant Fusarium verticillioides cutinase and its effects on enzymatic hydrolysis of rice straw.

The current study heterologously expressed a cutinase from Fusarium verticillioides by Pichia pastoris and investigated its properties and effects on the hydrolysis of rice straw. The optimal pH and temperature for F. verticillioides cutinase were 8.0 and 50 °C, respectively. F. verticillioides cutinase had poor thermal stability and could be inhibited by some metal ions, inhibitors, and detergents (5 mM), including Ni2+, Zn2+, Cu2+, Ca2+, Mn2+, sodium dodecyl sulfate, EDTA, and Tween-20. F. verticillioides cutinase could tolerate 15% methanol and dimethyl sulfoxide but was significantly repressed by 15% ethanol and acetone with 48% and 63% residual activity, respectively. F. verticillioides cutinase could degrade the cuticle of rice straw with palmitic acid and stearic acid as the main products. However, the dissolving sugars released from the rice straw treated with F. verticillioides cutinase were significantly reduced by 29.2 µg/mL compared with the control (107.9 µg/mL). Similarly, the reducing sugars produced from the cellulase hydrolysis of rice straw pretreated with F. verticillioides cutinase were reduced by 63.5 µg/mL relative to the control (253.6 µg/mL). Scanning electron microscopy results showed that numerous tuberculate or warty protrusions were present nearly everywhere on the surface of rice straw treated with F. verticillioides cutinase, and some protrusions even covered and blocked the stomata of the rice straw surface. Current limited data indicate that F. verticillioides cutinase might not be an appropriate choice for improving the utilization of agricultural straws.

Synergy between endo/exo-glucanases and expansin enhances enzyme adsorption and cellulose conversion.

Effective binding between cellulases and cellulose is essential for enzymatic hydrolysis of lignocellulose. Expansin can loosen the cellulose structure and can enhance the efficiency of cellulase. However, possible synergy between cellulases and expansin is not clear. In this work, the real-time adsorption of exoglucanases (Cel7A) or endoglucanases (Cel7B) with Bacillus subtilis expansin (BsEXLX1) and the enzymatic hydrolysis of cellulose were followed using quartz crystal microbalance with dissipation (QCM-D). Initial adsorption rate, adsorption capacity, and pseudo-steady-state rate of cellulose hydrolysis by Cel7A/Cel7B increased in the presence of BsEXLX1. When injecting Cel7A or Cel7B together with BsEXLX1 at a mass ratio of 1:1, the hydrolysis rate was almost 5 times the rate for Cel7A or Cel7B alone at 25 °C. These results increase our understanding of the real-time synergism between cellulases and expansin on cellulose, as well as the impact of their synergy on the enzymatic hydrolysis of cellulose.

Effects of dietary exogenous fibrolytic enzymes on ruminal fermentation characteristics of beef steers fed high- and low-quality growing diets1.

The effects of dietary pretreatment with fibrolytic enzyme-based cocktail were evaluated in 2 studies: (1) in vitro true digestibility; and (2) intake, digestibility, feeding behavior, and ruminal fermentation of beef steers fed growing diets. For the in vitro assessment, the ruminal inoculum was collected from 2 steers (BW = 543 ± 45 kg; 4-h after feeding; growing diets) and enzymes included or not (Trichoderma reesei fermentation extract; 0.75 µL/g of substrate DM). Within in vitro batches (n = 4), 12 substrates were incubated and in vitro true nutrient digestibility was evaluated. For study 2, 5 ruminally cannulated beef steers (BW = 520 ± 30 kg) were used in a 5 × 4 unbalanced Latin square using a 2 × 2 factorial arrangement of treatments: (a) diet quality (high = HQ; and low = LQ) and (b) enzyme inclusion (0 or 0.75 mL/kg of diet DM). Steers were fed ad libitum during four 21-d periods consisting of 14-d of adaptation and 7-d of collections. An enzyme × substrate was observed (P < 0.01), in which DM, OM, and NDF disappearance of sorghum grain increased with enzymes addition. Addition of enzymes increased (P < 0.01) ADF digestibility for all substrates. No diet quality × enzyme (P ≥ 0.18) was observed for intake variables in study 2. Enzyme-fed steers increased (P ≤ 0.05) intake of DM, digestible DM, NDF, and ADF compared with steers not fed fibrolytic enzymes. Addition of enzyme did not affect (P ≥ 0.28) apparent total tract digestibility of beef steers. Steers fed HQ diets consumed more (P ≤ 0.04) DM, digestible DM and OM, and less (P ≤ 0.03) total and digestible fiber than steers fed LQ diets. Ruminal pH average decreased (P = 0.01) for steers fed HQ or enzyme-fed diets compared with other treatments. A tendency (P = 0.06) toward improved total VFA was observed on enzyme-fed steers with HQ diets, but not for LQ diets. The molar proportion of ruminal propionate increased (P = 0.01) when steers were fed enzyme. Steers fed HQ diets had greater (P < 0.01) propionate and valerate molar proportions, lower (P < 0.01) acetate and acetate:propionate ratio than steers fed LQ diets. In vitro methane and total gas production were not affected (P ≥ 0.50) by dietary treatments. Fibrolytic enzymes positively affected digestion of multiple roughage sources commonly fed to cattle and might have additional benefit when used on unprocessed sorghum grain. Fibrolytic enzymes in beef cattle growing diets stimulated intake and generated positive impacts on ruminal fermentation.

Encapsulation of graphene quantum dot-crosslinked chitosan by carboxymethylcellulose hydrogel beads as a pH-responsive bio-nanocomposite for the oral delivery agent.

Oral delivery most commonly used due to the non-invasive nature and the fact that avoids patient pain and discomfort in compression with the intravenous administration. Herein, the obtained graphene quantum dots (GQDs) from citric acid were employed as a cross-linker for chitosan (CS). Sodium salicylate (SS) as a model drug was loaded in the prepared graphene quantum dots-crosslinked chitosan hybrid bio-nanocomposite beads (CS-GQD). SS-loaded CS-GQD (CS-GQD/SS) was protected with pH-sensitive biopolymeric carboxymethylcellulose (CMC) hydrogel beads. The CMC encapsulated CS-GQD/SS bio-nanocomposite hydrogel beads (CS-GQD/SS@CMC) were characterized using FT-IR, PL and SEM analysis. For determination of surficial charge of the carrier, pH point of zero charges (pHpzc) was measured. In-vitro drug delivery tests were carried out in simulating the gastrointestinal tract conditions for proving the efficiency of the prepared beads as a controlled oral drug delivery. The synergistic effects of CMC and CS enhanced the stability of drug dosing for a long time with controlling the drug releases in the gastrointestinal tract conditions. The MTT test confirmed that the bio-nanocomposite beads have low toxicity against human colon adenocarcinoma HT29 cells. The obtained results showed that the prepared novel CS-GQD/SS@CMC could potentially be used as a safe carrier for oral drug delivery.

The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin.

BACKGROUND: Trichoderma fungi live in the soil rhizosphere and are beneficial for plant growth and pathogen resistance. Several species and strains are currently used worldwide in co-cultivation with crops as a biocontrol alternative to chemical pesticides even though little is known about the exact mechanisms of the beneficial interaction. We earlier found alamethicin, a peptide antibiotic secreted by Trichoderma, to efficiently permeabilise cultured tobacco cells. However, pre-treatment with Trichoderma cellulase made the cells resistant to subsequent alamethicin, suggesting a potential mechanism for plant tolerance to Trichoderma, needed for mutualistic symbiosis. RESULTS: We here investigated intact sterile-grown Arabidopsis thaliana seedlings germinated in water or growth medium. These could be permeabilised by alamethicin but not if pretreated with cellulase. By following the fluorescence from the membrane-impermeable DNA-binding probe propidium iodide, we found alamethicin to mainly permeabilise root tips, especially the apical meristem and epidermis cells, but not the root cap and basal meristem cells nor cortex cells. Alamethicin permeabilisation and cellulase-induced resistance were confirmed by developing a quantitative in situ assay based on NADP-isocitrate dehydrogenase accessibility. The combined assays also showed that hyperosmotic treatment after the cellulase pretreatment abolished the induced cellulase resistance. CONCLUSION: We here conclude the presence of cell-specific alamethicin permeabilisation, and cellulase-induced resistance to it, in root tip apical meristem and epidermis of the model organism A. thaliana. We suggest that contact between the plasma membrane and the cell wall is needed for the resistance to remain. Our results indicate a potential mode for the plant to avoid negative effects of alamethicin on plant growth and localises the point of potential damage and response. The results also open up for identification of plant genetic components essential for beneficial effects from Trichoderma on plants.

The impact of direct-fed microbials and enzymes on the health and performance of dairy cows with emphasis on colostrum quality and serum immunoglobulin concentrations in calves.

Thirty-six cows were blocked by calving date and randomly assigned to one of three treatments. Cows were on treatments 3 weeks prepartum through 8 weeks post-partum. Treatments were as follows: (i) no direct-fed microbial (DFM) or cellulase and amylase enzymes (C), (ii) 45.4 g/day of DFM (D) or (iii) 45.4 g/day of DFM and 18.2 g/day of enzyme (DE). Total mixed ration fed and refused were measured daily to determine dry matter intake (DMI). Blood samples were taken three times weekly and analysed for ß-hydroxybutyrate, glucose and non-esterified fatty acids. Body weight (BW) was measured weekly. Colostrum was weighed and analysed for IgA and IgG concentration. Calves were fed 4 L of colostrum within 2 hr of birth. Calf blood samples were taken at 0 and 24 hr for analysis of IgA and IgG concentrations and apparent efficiency of absorption. Milk yield was measured daily and samples collected weekly. Initial BW was different among treatments with D being lesser than C or DE treatments. Body weight, weight gain, efficiency of gain, DMI and blood parameters were unaffected. Treatment did not affect colostrum yield. Ash percentage of colostrum tended to increase with D and DE, while IgA and total solids yield decreased with D. Colostrum fat yield was decreased in D and DE. Treatments did not impact BW, serum IgA and IgG concentrations or apparent efficiency of absorption of calves. Post-partum BW, DMI, blood parameters, milk production and composition were unaffected by treatment. However, cows on D gained more BW and tended to have greater efficiency of gain compared to those on DE, but were similar to C. Somatic cell scores were greatest for D. Results indicate that DFM and enzyme supplementation did not improve health and performance of dairy cattle during the pre- and post-partum periods under conditions of this study.

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens.

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® fresh™, FreshKon® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection.

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments.

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes-Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI-BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI-BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis.

Effect of Enzymatic pre-treatment of microalgae extracts on their anti-tumor activity.

BACKGROUND: There is an increasing need to find natural bioactive compounds for pharmaceutical applications, because they have less harmful side effects compared to their chemical alternatives. Microalgae (MA) have been identified as a promising source for these bioactive compounds, and this work aimed to evaluate the anti-proliferative effects of semi-purified protein extracted from MA against several tumor cell lines. METHODS: Tested samples comprised MA cell extracts treated with cellulase and lysozyme, prior to extraction. The effect of dialysis, required to remove unnecessary small molecules, was also tested. The anti-cancer efficacies of the dialyzed and undialyzed extracts were determined by measuring cell viability after treating four human cancer cell lines, specifically A549 (human lung carcinoma), MCF-7 (human breast adenocarcinoma), MDA MB-435 (human melanoma), and LNCap (human prostate cancer cells derived from a metastatic site in the lymph node). This was compared to the effects of the agents on the human BPH-1 cell line (benign human prostate epithelial cells). The t-test was used to statistically analyze the results and determine the significance. RESULTS: Against LNCap and A549 cells, the performance of cellulase-treated extracts was better (with p-values < 0.05, as compared to the control) than that of lysozyme-treated preparations (with p-values mainly > 0.05, as compared to the control); however, they had similar effects against the other two tumor cell lines (with p-values mainly < 0.05, as compared to the control). Moreover, based on their effect on BPH-1 cells, extracts from lysozyme-treated MA cells were determined to be safer against the benign prostate hyperplasia cells, BPH-1 (with p-values mainly > 0.05, as compared to the control). After dialysis, the performance of MA extracts from lysozyme-treated cells was enhanced significantly (with p-values dropping to < 0.05, as compared to the control). CONCLUSIONS: The results of this work provide important information and could provide the foundation for further research to incorporate MA constituents into pharmaceutical anti-cancer therapeutic formulations.

Enhanced lipid recovery from Nannochloropsis microalgae by treatment with optimized cell wall degrading enzyme mixtures.

A statistical mixture design approach was used to investigate the effects of cell wall degrading enzymes on the recovery of lipids from Nannochloropsis sp. A preliminary screening of potentially suitable enzyme preparations, including lysozyme, cellulase and different types of hemicellulases, was carried out. The most effective preparations were then taken as basic components for the formulation of enzyme mixtures. Optimized ternary mixtures consisting of cellulase and two hemicellulases were obtained which allowed the recovery of up to 37.2g of lipids per 100g of dry biomass. SEM and TEM images of the enzymatically treated microalga revealed extensive cell damage, with degradation of the cell wall and release of intracellular material. Overall, the results obtained demonstrate that the mixture design method can be used to prepare cell wall degrading enzyme cocktails that can significantly improve the recovery of lipids or other valuable components from microalgae.

Nutrient value of spray field forages fed to pigs and the use of feed enzymes to enhance nutrient digestibility.

This study determined the DE, ME, apparent total tract digestibility (ATTD) of N, and N retention of spray field forages (Bermuda grass, forage sorghum, and sweet sorghum) fed to pigs and the effects of the supplemental feed enzymes on energy and N utilization. A basal diet was formulated with 96% corn and 4% amino acids, minerals, and vitamins. Test diets contained 85% basal diet + 15% Bermuda grass, forage sorghum, or sweet sorghum. Allzyme SSF (Alltech, Nicholasville, KY) was used as a feed enzyme, which was composed of cellulase, glucanase, xylanase, phytase, and protease. The basal diet and test diets were evaluated by using 4 sets of 2 × 2 Latin square designs consisting of 2 pigs and 2 periods with a total of 32 barrows (38.7 ± 7.9 kg). Each period (10-d adjustment and 4-d collection) had 2 Latin squares. The 2 treatments were levels of enzyme supplementation (0 or 200 mg/kg). Pigs received experimental diets twice daily (0700 and 1700 h) at a fixed amount based on BW of pigs (0.09 × BW0.75 kg). On d 10, chromic oxide (0.5%) was added to the diets at 1700 h as an external marker. Fecal and urine samples were collected during 4 consecutive days. The basal diet contained 3,850 kcal DE/kg, 3,769 kcal ME/kg, 86.06% ATTD of N, and 71.10% N retention and was not affected by enzyme supplementation. Bermuda grass contained 893 kcal DE/kg, 845 kcal ME/kg, -16.50% ATTD of N, and -37.49% N retention and tended to be improved by enzyme supplementation to 1,211 kcal DE/kg (P = 0.098), 1,185 kcal ME/kg (P = 0.081), and -10.54% N retention (P = 0.076). The ATTD of N of Bermuda grass increased (P < 0.05) to 0.08% by enzyme supplementation. The forage sorghum contained 1,520 kcal DE/kg, 1,511 kcal ME/kg, -0.72% ATTD of N, and -16.99% N retention. The sweet sorghum contained 1,086 kcal DE/kg, 1,061 kcal ME/kg, -75.47% ATTD of N, and -49.22% N retention. Enzyme supplementation did not improve energy digestibility of forage sorghum and sweet sorghum. Nitrogen in these forages was poorly utilized. In conclusion, spray field forages including Bermuda grass, forage sorghum, and sweet sorghum can partly be utilized in pig feed to provide energy, although N is rather poorly digested. Feed enzymes could enhance both energy and N utilization in Bermuda grass but not sorghum.

High consistency cellulase treatment of hardwood prehydrolysis kraft based dissolving pulp.

For enzymatic treatment of dissolving pulp, there is a need to improve the process to facilitate its commercialization. For this purpose, the high consistency cellulase treatment was conducted based on the hypothesis that a high cellulose concentration would favor the interactions of cellulase and cellulose, thus improves the cellulase efficiency while decreasing the water usage. The results showed that compared with a low consistency of 3%, the high consistency of 20% led to 24% increases of cellulase adsorption ratio. As a result, the viscosity decrease and Fock reactivity increase at consistency of 20% were enhanced from 510 mL/g and 70.3% to 471 mL/g and 77.6%, respectively, compared with low consistency of 3% at 24h. The results on other properties such as alpha cellulose, alkali solubility and molecular weight distribution also supported the conclusion that a high consistency of cellulase treatment was more effective than a low pulp consistency process.