Showing papers in "Enzyme Research in 2014"
TL;DR: The fungal source of laccases and their application in environment protection are described, and several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity.
Abstract: Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection.
332 citations
TL;DR: This review describes briefly the classification of mammalian peroxidases and their role in the human immune system and in host cell damage, and summarizes present knowledge on the mode of action of lactoperoxidase.
Abstract: Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity. Their best known effect is their antimicrobial activity that arouses much interest in in vivo and in vitro applications. In this context, the proper use of lactoperoxidase needs a good understanding of its mode of action, of the factors that favor or limit its activity, and of the features and properties of the active molecules. The first part of this review describes briefly the classification of mammalian peroxidases and their role in the human immune system and in host cell damage. The second part summarizes present knowledge on the mode of action of lactoperoxidase, with special focus on the characteristics to be taken into account for in vitro or in vivo antimicrobial use. The last part looks upon the characteristics of the active molecule produced by lactoperoxidase in the presence of thiocyanate and/or iodide with implication(s) on its antimicrobial activity.
115 citations
TL;DR: The immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate and poly(vinyl alcohol) and PVA, found to be optimally active between 30 and 40°C and more stable than the free enzyme.
Abstract: This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C.
99 citations
TL;DR: Investigation of the affects of varying chromium(VI) levels on the key enzymes of nitrogen metabolism in clusterbean found that the enzyme activity increased with advancement of growth to reach maximum at flowering stage and thereafter decreased at grain filling stage, which was found to be lethal to clusterbean plants.
Abstract: Heavy metals are the intrinsic component of the environment with both essential and nonessential types. Their excessive levels pose a threat to plant growth and yield. Also, some heavy metals are toxic to plants even at very low concentrations. The present investigation (a pot experiment) was conducted to determine the affects of varying chromium(VI) levels (0.0, 0.5, 1.0, 2.0, and 4.0 mg chromium(VI) soil in the form of potassium dichromate) on the key enzymes of nitrogen metabolism in clusterbean. Chromium treatment adversely affect nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate dehydrogenase in various plant organs at different growth stages as specific enzyme activity of these enzymes decreased with an increase in chromium(VI) levels from 0 to 2.0 mg chromium(VI) soil and 4.0 mg chromium(VI) soil was found to be lethal to clusterbean plants. In general, the enzyme activity increased with advancement of growth to reach maximum at flowering stage and thereafter decreased at grain filling stage.
61 citations
TL;DR: This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp.
Abstract: Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production.
50 citations
TL;DR: The study showed that homogenization speed as an independent variable had maximum effect on particle size and zeta potential and internal-external phase volume ratio hadmaximum effect on drug encapsulation.
Abstract: The present study was initiated to understand the effect of PLGA concentration, PVA concentration, internal-external phase ratio, homogenization speed, and homogenization time on mean particle size, zeta potential, and percentage drug encapsulation using fractional factorial design. Using PLGA (50-50) as the carrier, hyaluronidase loaded PLGA nanoparticles were prepared using double emulsion solvent evaporation technique. The particle size was analyzed by dynamic light scattering technique and protein content by Lowry method. The study showed that homogenization speed as an independent variable had maximum effect on particle size and zeta potential. Internal-external phase volume ratio had maximum effect on drug encapsulation. Mean particle size also had high dependency on the combined effect of PVA concentration and phase volume ratio. Using fractional factorial design particle size of <400 nm, zeta potential of <−30 mV, and percentage encapsulation of 15–18% were achieved.
46 citations
TL;DR: Relatively lower but consistent production of laccase for a longer period is likely to be an ecologically important phenomenon under low temperature environment and enhancement in production of enzyme using various supplements will be useful for its use in specific biotechnological applications.
Abstract: Production of laccase by a cold and pH tolerant strain of Penicillium pinophilum has been investigated under different cultural conditions for up to 35 days of incubation. The fungus was originally isolated from a low temperature environment under mountain ecosystem of Indian Himalaya. The estimations were conducted at 3 temperatures (15, 25, and 35°C), a range of pH (3.5–11.5), and in presence of supplements including carbon and nitrogen sources, vitamins, and antibiotics. Optimum production of laccase was recorded at 25°C (optimum temperature for fungal growth) and 7.5 pH. The production of enzyme was recorded maximum on day 28 ( U/L) following a slow decline at day 35 of incubation ( U/L). Fructose and potassium nitrate (0.2%) among nutritional supplements, chloramphenicol (0.1%) among antibiotics, and folic acid (0.1%) among vitamins were found to be the best enhancers for production of laccase. Relatively lower but consistent production of laccase for a longer period is likely to be an ecologically important phenomenon under low temperature environment. Further, enhancement in production of enzyme using various supplements will be useful for its use in specific biotechnological applications.
42 citations
TL;DR: The results indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications, and the results revealed that the optimum values are pH 7.0 and temperature 35°C.
Abstract: Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.
42 citations
TL;DR: Significantly, gas chromatography analysis showed a remarkable biodiesel production yield of 95% by using the LPA complex at 40°C for 6-hours reaction time, whereas the yield was 86% when using free lyophilized lipase.
Abstract: Biodiesel production technology is competitive in terms of low cost and alternative source of energy which should be not only sustainable but also environmentally friendly. Designing of the lipase immobilization for biodiesel production has a remarkable impact and is still challenging. In this work, biodiesel production from soybean oil was enhanced and facilitated by using a novel biocatalyst consisting of commercial lipase (EC 3.1.1.3), silver nanoparticles, and polydopamine. Silver nanoparticles (AgNPs) were synthesized with a size range of 10–20 nm. Polydopamine (PD) was delivered by the self-polymerization of dopamine in 10 mM Tris-HCl pH 8.5 and simultaneously coated the AgNPs to form a PD/AgNPs complex. Lipase was immobilized on the PD/AgNPs complex surface via covalent bonds to form a tailor-made biocatalyst consisting of immobilized lipase/PD/AgNPs complex (LPA). The formation and morphology of each composition were characterized by UV-Vis spectroscopy and scanning electron microscope (SEM). Significantly, gas chromatography analysis showed a remarkable biodiesel production yield of 95% by using the LPA complex at 40°C for 6-hours reaction time, whereas the yield was 86% when using free lyophilized lipase. The LPA complex was apparently reusable after 7 batches and the latter conversion rate of soybean oil was decreased by only 27%.
38 citations
TL;DR: Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer and low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides and no homology to other psychrophilic proteases.
Abstract: Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.
27 citations
TL;DR: This work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797 and afforded increased cellulase production in the order of 3.6 to 9.5 times higher than production using nonoptimised conditions.
Abstract: Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases), which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD) was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L−1) as carbon source was determined to include urea (1.2 g·L−1), yeast extract (1.0 g·L−1), KH2PO4 (6.0 g·L−1), and MgSO4·7H2O (1.2 g·L−1). The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L−1 for FPase, 9,204 U·L−1 for endoglucanase, and 2,395 U·L−1 for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions.
TL;DR: This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes.
Abstract: Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1). This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB), suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata.
TL;DR: The results indicate that the α-amylase reflects their potentiality towards industrial utilization in Streptomyces sp.
Abstract: A partial purification and biochemical characterization of the α-amylase from Streptomyces sp. MSC702 were carried out in this study. The optimum operational conditions for enzyme substrate reaction for amylolytic enzyme activity from the strain were evaluated. The optimum pH, temperature, and incubation period for assaying the enzyme were observed to be 5.0, 55°C, and 30 min, respectively. The extracellular extract was concentrated using ammonium sulfate precipitation. It was stable in the presence of metal ions (5 mM) such as K
TL;DR: The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale, and high accuracy of the polynomial model with significant similarities between the predicted and the observed results.
Abstract: The enormous increase in world population has resulted in generation of million tons of agricultural wastes. Biotechnological process for production of green chemicals, namely, enzymes, provides the best utilization of these otherwise unutilized wastes. The present study elaborates concomitant production of protease and amylase in solid state fermentation (SSF) by a newly isolated Bacillus megaterium B69, using agroindustrial wastes. Two-level statistical model employing Plackett-Burman and response surface methodology was designed for optimization of various physicochemical conditions affecting the production of two enzymes concomitantly. The studies revealed that the new strain concomitantly produced 1242 U/g of protease and 1666.6 U/g of amylase by best utilizing mustard oilseed cake as the substrate at 20% substrate concentration and 45% moisture content after 84 h of incubation. An increase of 2.95- and 2.04-fold from basal media was observed in protease and amylase production, respectively. ANOVA of both the design models showed high accuracy of the polynomial model with significant similarities between the predicted and the observed results. The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale.
TL;DR: The pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source and was able to digest apple and citrus pectins with different degrees of methyl esterification.
Abstract: A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.
TL;DR: Enzymatic antioxidant system is impaired in recurrent aphthous stomatitis patients and seems to play a crucial role in its pathogenesis.
Abstract: Background and Aim. Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae. Despite extensive research, the exact etiology of RAS remains elusive. Recently oxidant-antioxidant imbalance of the body has been implicated in the pathogenesis of recurrent aphthous stomatitis. Thus, the aim of the study was to evaluate the enzymatic antioxidant levels in patients with recurrent aphthous stomatitis. Materials and Methods. The serum levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured in 30 patients with recurrent aphthous stomatitis and compared to the control group, which included 30 healthy subjects. Student’s -test was performed for statistical evaluation. Results. The mean levels of superoxide dismutase (130.2 ± 15.94 U/mL) and glutathione peroxidase (3527.93 ± 488.32 U/L) were found to be significantly lower in study group as compared to control group (211.9 ± 20.93 U/mL, 8860.93 ± 1105.31 U/L, resp.) () while level of catalase in study group was significantly higher when compared to control group (10981.00 ± 1018.07 U/mL versus 9764.00 ± 1621.19 U/mL) (). Conclusion. Enzymatic antioxidant system is impaired in recurrent aphthous stomatitis patients and seems to play a crucial role in its pathogenesis.
TL;DR: This study presents a simple and reproducible purification procedure of G6PD from the camel liver, which displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8.
Abstract: Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.
TL;DR: The purification of an active and efficient enzyme, extracellular poly(3-hydroxybutyrate) (PHB)-depolymerase, suitable for industrial applications is proposed by the application of an easy, fast, and cheap route, skipping the chromatography step.
Abstract: This work proposes the purification of an active and efficient enzyme, extracellular poly(3-hydroxybutyrate) (PHB)-depolymerase, suitable for industrial applications. This is achieved by the application of an easy, fast, and cheap route, skipping the chromatography step. Chromatography with one or two columns is a common step in the purification procedure, which however renders the isolation of the enzyme a time consuming and an expensive process. A strain of the fungus Penicillium pinophilum (ATCC 9644) is used for the isolation of extracellular PHB-depolymerase. The molecular weight of the purified enzyme is about 35 kDa and is estimated by gel electrophoresis (SDS-PAGE, 12% polyacrylamide). The enzymatic activity of the isolated enzyme is determined to be 3.56-fold similar to that found by other researchers that have used chromatography for the isolation. The as-isolated enzyme disintegrates the poly(3-hydroxybutyrate) (PHB) films successfully, as it is demonstrated by the biodegradation test results provided here.
TL;DR: Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield and one of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized, which retained 97% activity in methanol.
Abstract: Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a K m and V max of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol.
TL;DR: Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour, and effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated.
Abstract: A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory.
TL;DR: The glucoamylase production was found to increase from 4.57 U/mL to 12.27 U/mL, a 2.68-fold enhancement, as compared to the unoptimized medium.
Abstract: Glucoamylase is an industrially important enzyme which converts soluble starch into glucose. The media components for the production of glucoamylase from thermophilic fungus Humicola grisea MTCC 352 have been optimized. Eight media components, namely, soluble starch, yeast extract, KH2PO4, K2HPO4, NaCl, CaCl2, MgSO4·7H2O, and Vogel’s trace elements solution, were first screened for their effect on the production of glucoamylase and only four components (soluble starch, yeast extract, K2HPO4, and MgSO4·7H2O) were identified as statistically significant using Plackett-Burman design. It was fitted into a first-order model ( ). Steepest ascent method was performed to identify the location of optimum. Central composite design was employed to determine the optimum values (soluble starch: 28.41 g/L, yeast extract: 9.61 g/L, K2HPO4: 2.42 g/L, and MgSO4·7H2O: 1.91 g/L). The experimental activity of 12.27 U/mL obtained was close to the predicted activity of 12.15. High value (0.9397), low PRESS value (9.47), and AARD values (2.07%) indicate the accuracy of the proposed model. The glucoamylase production was found to increase from 4.57 U/mL to 12.27 U/mL, a 2.68-fold enhancement, as compared to the unoptimized medium.
TL;DR: When quail was exposed orally for 48 h to concentrations of carbamate or organophosphate compounds of 3–200 mg/kg, the percentage inhibition of cholinesterase was in each case larger than that of carboxylesterase and reached statistical significance (P < 0.05) at lower concentrations.
Abstract: The aims of this study were to investigate the presence of different esterase activities in plasma and liver for Japanese quail and to combine determination of both carboxylesterase and cholinesterase as biochemical biomarker in order to identify the effects of carbamate and organophosphate compounds exposure. Carboxylesterase exhibits larger sensitivity to carbamate and organophosphate compounds than to cholinesterase and is present at higher levels. This permitted nature and distribution of carboxylesterase or cholinesterase to be measured. One predominant toxicological form of enzyme level constant in its patterns of motivation and inhibition with cholinesterase was identified in plasma with an apparent Michaelis constant for butyrylthiocholine iodide of 0.394 mM. Carboxylesterase activity in liver was considered by its preferential hydrolysis of the S-phenyl thioacetate. A concentration dependent decrease of carboxylesterase and cholinesterase has demonstrated during in vitro incubation of malathion, parathion, and trichlorfon in the range 0.125–2 mM, while with methomyl was in the range 0.25–4 mM. When quail () was exposed orally for 48 h to concentrations of carbamate or organophosphate compounds of 3–200 mg/kg, the percentage inhibition of cholinesterase was in each case larger than that of carboxylesterase and reached statistical significance () at lower concentrations.
TL;DR: The present results can be a guideline in mutagenesis studies to improve processing by Cel48F.
Abstract: Molecular dynamics (MD) and metadynamics techniques were used to study the cellulase Cel48F-sugar. Cellulase is enzyme that breaks cellulose fibers into small sugar units and is potentially useful in second generation alcohol production. In MD simulations, the overall structure of equilibrated Cel48F did not significantly change along the trajectory, retaining root mean square deviation below 0.15 nm. A set of 15 residues interacting with the sugar chains via hydrogen bonding throughout the simulation was observed. The free energy of dissociation (ΔGdiss.) of the chains in the catalytic tunnel of Cel48F was determined by metadynamics. The ΔGdiss. values of the chains entering and leaving the wild-type Cel48F cavity were 13.9 and 62.1 kcal/mol, respectively. We also mutated the E542 and Q543 to alanine residue and obtained ΔGdiss. of 41.8 and 45.9 kcal/mol, respectively. These mutations were found to facilitate smooth dissociation of the sugar chain across the Cel48F tunnel. At the entry of the Cel48F tunnel, three residues were mutated to alanine: T110, T213, and L274. Contrary to the T110A-Cel48F, the mutants T213-Cel48F and L274-Cel48F prevented the sugar chain from passing across the leaving site. The present results can be a guideline in mutagenesis studies to improve processing by Cel48F.
TL;DR: The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process.
Abstract: The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C. The shift of the spectral centre of mass above 50°C is also apparently cooperative with midpoint transition of 56.3 ± 0.2°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process. Further corroboration comes from differential scanning calorimetry experiments. At protein concentrations ≤0.56 mg·mL−1 the calorimetric transition is reversible and the data were fitted to a non-two-state model and deconvoluted into six transitions, whereas at concentrations greater than 0.56 mg·mL−1 the calorimetric transition is irreversible with an exothermic contribution to the thermogram. The apparent increased linearly with the scan rate according to first order inactivation kinetics. The effect of additives on the calorimetric transition of xylanase is dependent on their nature. The addition of sorbitol transforms reversible transitions into irreversible transitions while stabilizing the protein as the apparent increases linearly with sorbitol concentration. D-Glucono-1,5-lactone, a noncompetitive inhibitor in xylanase kinetics, and soluble xylan change irreversible processes into reversible processes at high protein concentration.
TL;DR: The strain Aspergillus 6V4 was the best amylase producer in the screening assay of starch hydrolysis in petri dishes (ASHPD) and in the assay in submerged fermentation (ASbF).
Abstract: The aim of this study was screening fungi strains, isolated from manipueira (a liquid subproduct obtained from the flour production of Manihot esculenta), for amylases production and investigating production of these enzymes by the strain Aspergillus 6V4. The fungi isolated from manipueira belonged to Ascomycota phylum. The strain Aspergillus 6V4 was the best amylase producer in the screening assay of starch hydrolysis in petri dishes (ASHPD) and in the assay in submerged fermentation (ASbF). The strain Aspergillus 6V4 produced high amylase levels (335 UI/L) using wheat bran infusion as the exclusive substrate and the supplementation of this substrate with peptone decreased the production of this enzyme. The moisture content of 70% was the best condition for the production of Aspergillus 6V4 amylases (385 IU/g) in solid state fermentation (SSF).
TL;DR: Mouse cytotoxic T-lymphocyte antigen-2α, Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor belong to a novel family of cysteINE protease inhibitors (I29), and their inhibitory mechanisms were studied comparatively.
Abstract: Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.
TL;DR: The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA, and should be evaluated in a study with a larger sample size as well as in tissue samples.
Abstract: Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (). Also, there was no correlation between ACE activity and severity () and duration of disease () in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA.
TL;DR: The effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established.
Abstract: The potential of polysaccharide Irvingia gabonensis matrix as enzyme immobilization support was investigated. Lipase of Aspergillus niger F7-02 was immobilized by entrapment using glutaraldehyde as the cross-linking agent and stabilized in ethanolic-formaldehyde solution. The pH and temperature stability and activity yield of the immobilized enzyme were determined. Such parameters as enzyme load, bead size, number of beads, and bead reusability were also optimized. Adequate gel strength to form stabilized beads was achieved at 15.52% (w/v) Irvingia gabonensis powder, 15% (v/v) partially purified lipase, 2.5% (v/v) glutaraldehyde, and 3 : 1 (v/v) ethanolic-formaldehyde solution. There was 3.93-fold purification when the crude enzyme was partially purified in two-step purification using Imarsil and activated charcoal. Optimum lipase activity 75.3 Ug−1 was achieved in 50 mL test solution containing 15 beads of 7 mm bead size. Relative activity 80% was retained at eight repeated cycles. The immobilization process gave activity yield of 59.1% with specific activity of 12.3 Umg−1 and stabilized at optimum pH 4.5 and temperature 55°C. Thus the effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established.