http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2013 , Hindawi Publishing Corporation . All rights reserved. Mon, 13 May 2013 08:13:26 +0000 http://www.hindawi.com/journals/er/2013/897420/ Macrophomina phaseolina was cultivated in complex and simple media for the production of extracellular lipolytic enzymes. Culture supernatants were batch foam fractionated for the recovery of these enzymes, and column design and operation included the use of P 2 frit (porosity 40 to 100 μm), air as sparging gas at variable flow rates, and Triton X-100 added at the beginning or gradually in aliquots. Samples taken at intervals showed the progress of the kinetic and the efficiency parameters. Best results were obtained with the simple medium supernatant by combining the stepwise addition of small amounts of the surfactant with the variation of the air flow rates along the separation. Inert proteins were foamed out first, and the subsequent foamate was enriched in the enzymes, showing estimated activity recovery (R), enrichment ratio (E), and purification factor (P) of 45%, 34.7, and 2.9, respectively. Lipases were present in the enriched foamate. Claudia Schinke and José Carlos Germani Copyright © 2013 Claudia Schinke and José Carlos Germani. All rights reserved. Wed, 24 Apr 2013 17:46:29 +0000 http://www.hindawi.com/journals/er/2013/869062/ Laccase production by a temperature and pH tolerant fungal strain (GBPI-CDF-03) isolated from a glacial site in Indian Himalayan Region (IHR) has been investigated. The fungus developed white cottony mass on potato dextrose agar and revealed thread-like mycelium under microscope. ITS region analysis of fungus showed its 100% similarity with Trametes hirsuta. The fungus tolerated temperature from 4 to 48°C ± 2 (25°C opt.) and pH 3–13 (5–7 opt.). Molecular weight of laccase was determined approximately 45 kDa by native PAGE. Amplification of laccase gene fragment (corresponding to the copper-binding conserved domain) contained 200 bp. The optimum pH for laccase production, at optimum growth temperature, was determined between 5.5 and 7.5. In optimization experiments, fructose and ammonium sulfate were found to be the best carbon and nitrogen sources, respectively, for enhancing the laccase production. Production of laccase was favored by high carbon/nitrogen ratio. Addition of CuSO4 (up to 1.0 mM) induced laccase production up to 2-fold, in case of 0.4 mM concentration. Addition of organic solvents also induced the production of laccase; acetone showed the highest (2-fold) induction. The study has implications in bioprospecting of ecologically resilient microbial strains. Kusum Dhakar and Anita Pandey Copyright © 2013 Kusum Dhakar and Anita Pandey. All rights reserved. Thu, 11 Apr 2013 16:10:58 +0000 http://www.hindawi.com/journals/er/2013/597028/ Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. Juhi Sikarwar, Sanket Kaushik, Mau Sinha, Punit Kaur, Sujata Sharma, and Tej P. Singh Copyright © 2013 Juhi Sikarwar et al. All rights reserved. Sun, 31 Mar 2013 18:16:02 +0000 http://www.hindawi.com/journals/er/2013/803415/ A commercial amylase (amy) was immobilized by adsorption onto Luffa operculata fibers (LOFs). The derivative LOF-amy presented capacity to hydrolyze starch continuously and repeatedly for over three weeks, preserving more than 80% of the initial activity. This system hydrolyzed more than 97% of starch during 5 min, at room temperature. LOF-amy was capable to hydrolyze starch from different sources, such as maize (93.96%), wheat (85.24%), and cassava (79.03%). A semi-industrial scale reactor containing LOF-amy was prepared and showed the same yield of the laboratory-scale system. After five cycles of reuse, the LOF-amy reactor preserved over 80% of the initial amylase activity. Additionally, the LOF-amy was capable to operate as a kitchen grease trap component in a real situation during 30 days, preserving 30% of their initial amylase activity. Ricardo R. Morais, Aline M. Pascoal, Samantha S. Caramori, Flavio M. Lopes, and Kátia F. Fernandes Copyright © 2013 Ricardo R. Morais et al. All rights reserved. Wed, 27 Feb 2013 09:48:08 +0000 http://www.hindawi.com/journals/er/2013/874050/ The purified fish visceral protease enzyme was immobilized by using various concentrations of sodium alginate and calcium chloride to optimize the best concentration for the formation of the beads. Then it was characterized by assaying the optimal pH, temperature, storage stability and reusability. The results on immobilization with sodium alginate and calcium chloride showed that a combination of 2% sodium alginate and 0.3 M calcium chloride weas found to be the optimum concentration for the formation of spherical and stable beads, this gave a maximal entrapped activity of 48.31%, and there was no change in the optimum pH 8.0 and temperature 40°C of protease before and after entrapment. The results on stability and reusability indicated that it was stable at 4°C retaining 100% residual activity after 5 days of storage and 67% loss of activity after ten days of storage and it retained 100% residual activity on the first reuse, 75% residual activity on the second reuse, 25% residual activity on the third use and complete loss in the activity on the fourth reuse. S. Geethanjali and Anitha Subash Copyright © 2013 S. Geethanjali and Anitha Subash. All rights reserved. Thu, 21 Feb 2013 08:15:58 +0000 http://www.hindawi.com/journals/er/2013/784973/ Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) for L-lactate and a higher value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions. Neal J. Dawson, Ryan A. V. Bell, and Kenneth B. Storey Copyright © 2013 Neal J. Dawson et al. All rights reserved. Sat, 29 Dec 2012 11:02:55 +0000 http://www.hindawi.com/journals/er/2012/248983/ Biomass-degrading enzymes are one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels. This work evaluates the effects of operational conditions on biomass-degrading multienzyme production by a selected strain of Aspergillus niger. The fungus was cultivated under solid-state fermentation (SSF) of soybean meal, using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity, and initial substrate moisture content on multienzyme (FPase, endoglucanase, and xylanase) production were evaluated using a statistical design methodology. Highest production of FPase (0.55 IU/g), endoglucanase (35.1 IU/g), and xylanase (47.7 IU/g) was achieved using an initial substrate moisture content of 84%, an inlet air humidity of 70%, and a flow rate of 24 mL/min. The enzymatic complex was then used to hydrolyze a lignocellulosic biomass, releasing 4.4 g/L of glucose after 36 hours of saccharification of 50 g/L pretreated sugar cane bagasse. These results demonstrate the potential application of enzymes produced under SSF, thus contributing to generate the necessary technological advances to increase the efficiency of the use of biomass as a renewable energy source. Gabriela L. Vitcosque, Rafael F. Fonseca, Ursula Fabiola Rodríguez-Zúñiga, Victor Bertucci Neto, Sonia Couri, and Cristiane S. Farinas Copyright © 2012 Gabriela L. Vitcosque et al. All rights reserved. Sun, 23 Dec 2012 07:52:08 +0000 http://www.hindawi.com/journals/er/2012/731427/ Tyrosinase is a ubiquitous enzyme with diverse physiologic roles related to pigment production. Tyrosinase inhibition has been well studied for cosmetic, medicinal, and agricultural purposes. We simulated the docking of tyrosinase and D-(−)-arabinose and found a binding energy of −4.5 kcal/mol for theup-formof D-(−)-arabinose and −4.4 kcal/mol for thedown-form of D-(−)-arabinose. The results of molecular dynamics simulation suggested that D-(−)-arabinose interacts mostly with HIS85, HIS259, and HIS263, which are believed to be in the active site. Our kinetic study showed that D-(−)-arabinose is a reversible, mixed-type inhibitor of tyrosinase (-value ,  M). Measurements of intrinsic fluorescence showed that D-(−)-arabinose induced obvious tertiary changes to tyrosinase (binding constant  M−1, binding number ). This strategy of predicting tyrosinase inhibition based on specific interactions of aldehyde and hydroxyl groups with the enzyme may prove useful for screening potential tyrosinase inhibitors. Hong-Jian Liu, Sunyoung Ji, Yong-Qiang Fan, Li Yan, Jun-Mo Yang, Hai-Meng Zhou, Jinhyuk Lee, and Yu-Long Wang Copyright © 2012 Hong-Jian Liu et al. All rights reserved. Thu, 06 Dec 2012 11:18:58 +0000 http://www.hindawi.com/journals/er/2012/157643/ Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and CoCl2·6H2O were selected based on their positive influence on cellulase production. The composition of the selected components was optimized using Response Surface Methodology (RSM). The optimum conditions are as follows: Avicel: 25.30 g/L, Soybean cake flour: 23.53 g/L, KH2PO4: 4.90 g/L, and CoCl2·6H2O: 0.95 g/L. These conditions are validated experimentally which revealed an enhanced Cellulase activity of 7.8 IU/mL. P. Saravanan, R. Muthuvelayudham, and T. Viruthagiri Copyright © 2012 P. Saravanan et al. All rights reserved. Wed, 05 Dec 2012 13:47:08 +0000 http://www.hindawi.com/journals/er/2012/845465/ Histidine acid phytases (HAPhy) are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA), homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal “RHGXRXP” and C-terminal “HD.” Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 “SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF” was present in 38 protein sequences representing clusters 1 (PhyA) and 2 (PhyB). Cluster 3 (AppA) contains motif 9 “KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP” as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. This in silico analysis might contribute in the classification and future genetic engineering of this most diverse class of phytase. Vinod Kumar, Gopal Singh, A. K. Verma, and Sanjeev Agrawal Copyright © 2012 Vinod Kumar et al. All rights reserved. Wed, 28 Nov 2012 12:13:27 +0000 http://www.hindawi.com/journals/er/2012/793708/ The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulase-producing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g−1). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient () of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement. Camila Florencio, Sonia Couri, and Cristiane Sanchez Farinas Copyright © 2012 Camila Florencio et al. All rights reserved. Sun, 11 Nov 2012 16:06:49 +0000 http://www.hindawi.com/journals/er/2012/987523/ Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v); yeast extract 1.25% (w/v); NaCl 0.45% (w/v) olive oil 0.1% (v/v) with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16) and olive oil with optimal activity (100%) compared to other substrates. Anuradha Balan, Darah Ibrahim, Rashidah Abdul Rahim, and Fatimah Azzahra Ahmad Rashid Copyright © 2012 Anuradha Balan et al. All rights reserved. Wed, 31 Oct 2012 08:10:35 +0000 http://www.hindawi.com/journals/er/2012/459190/ A general mathematical model for a fixed bed immobilized enzyme reactor was developed to simulate the process of diffusion and reaction inside the biocatalyst particle. The modeling and simulation of starch hydrolysis using immobilized α-amylase were used as a model for this study. Corn starch hydrolysis was carried out at a constant pH of 5.5 and temperature of . The substrate flow rate was ranging from 0.2 to 5.0 mL/min, substrate initial concentrations 1 to 100 g/L. α-amylase was immobilized on to calcium alginate hydrogel beads of 2 mm average diameter. In this work Michaelis-Menten kinetics have been considered. The effect of substrate flow rate (i.e., residence time) and initial concentration on intraparticle diffusion have been taken into consideration. The performance of the system is found to be affected by the substrate flow rate and initial concentrations. The reaction is controlled by the reaction rate. The model equation was a nonlinear second order differential equation simulated based on the experimental data for steady state condition. The simulation was achieved numerically using FINITE ELEMENTS in MATLAB software package. The simulated results give satisfactory results for substrate and product concentration profiles within the biocatalyst bead. Ameel M. R. Al-Mayah Copyright © 2012 Ameel M. R. Al-Mayah. All rights reserved. Wed, 17 Oct 2012 16:08:40 +0000 http://www.hindawi.com/journals/er/2012/138634/ A thermophilic fungal strain producing polygalacturonase was isolated after primary screening of 40 different isolates. The fungus was identified as Rhizomucor pusilis by Microbial Type Culture Collection (MTCC), Chandigarh, India. An extracellular polygalacturonase (PGase) from R. pusilis was purified to homogeneity by two chromatographic steps using Sephadex G-200 and Sephacryl S-100. The purified enzyme was a monomer with a molecular weight of 32 kDa. The PGase was optimally active at 55°C and at pH 5.0. It was stable up to 50°C for 120 min of incubation and pH condition between 4.0 and 5.0. The stability of PGase decreases rapidly above 60°C and above pH 5.0. The apparent and values were 0.22 mg/mL and 4.34 U/mL, respectively. It was the first time that a polygalacturonase enzyme was purified in this species. It would be worthwhile to exploit this strain for polygalacturonase production. Polygalacturonase from this strain can be recommended for the commercial production because of its constitutive and less catabolically repressive nature, thermostability, wide range of pH, and lower properties. However, scale-up studies are needed for the better output for commercial production. Mohd. Asif Siddiqui, Veena Pande, and Mohammad Arif Copyright © 2012 Mohd. Asif Siddiqui et al. All rights reserved. Thu, 13 Sep 2012 15:33:14 +0000 http://www.hindawi.com/journals/er/2012/173831/ Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant 𝐾𝑚 and 𝑉max values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([𝑃]∞−[𝑃]𝑡) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[𝑃]∞ versus 1/[𝑆] yielded 𝑘+0 and 𝑘′+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of 𝑘+0 and 𝑘′+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed. Charles O. Nwamba and Ferdinand C. Chilaka Copyright © 2012 Charles O. Nwamba and Ferdinand C. Chilaka. All rights reserved. Thu, 06 Sep 2012 13:59:59 +0000 http://www.hindawi.com/journals/er/2012/921362/ The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase. Siew Ling Hii, Joo Shun Tan, Tau Chuan Ling, and Arbakariya Bin Ariff Copyright © 2012 Siew Ling Hii et al. All rights reserved. Thu, 16 Aug 2012 10:08:14 +0000 http://www.hindawi.com/journals/er/2012/542589/ Hydrolysis of virgin coconut oil (VCO) had been carried out by using an immobilised lipase from Mucor miehei (Lipozyme) in a water-jacketed batch reactor. The kinetic of the hydrolysis was investigated by varying the parameters such as VCO concentration, enzyme loading, water content, and reaction temperature. It was found that VCO exhibited substrate inhibition at the concentration more than 40% (v/v). Lipozyme also achieved the highest production of free fatty acids, 4.56 mM at 1% (w/v) of enzyme loading. The optimum water content for VCO hydrolysis was 7% (v/v). A relatively high content of water was required because water was one of the reactants in the hydrolysis. The progress curve and the temperature profile of the enzymatic hydrolysis also showed that Lipozyme could be used for free fatty acid production at the temperature up to 50°C. However, the highest initial reaction rate and the highest yield of free fatty acid production were at 45 and 40°C, respectively. A 100 hours of initial reaction time has to be compensated in order to obtain the highest yield of free fatty acid production at 40°C. Lee Suan Chua, Meisam Alitabarimansor, Chew Tin Lee, and Ramli Mat Copyright © 2012 Lee Suan Chua et al. All rights reserved. Wed, 08 Aug 2012 13:24:21 +0000 http://www.hindawi.com/journals/er/2012/281384/ The production of a thermostable and highly alkaline pectinase by Bacillus pumilus dcsr1 was optimized in solid-state fermentation (SSF) and the impact of various treatments (chemical, enzymatic, and in combination) on the quality of ramie fibres was investigated. Maximum enzyme titer (348.0±11.8 Ug−1 DBB) in SSF was attained, when a mixture of agro-residues (sesame oilseed cake, wheat bran, and citrus pectin, 1 : 1 : 0.01) was moistened with mineral salt solution (𝑎𝑤 0.92, pH 9.0) at a substrate-to-moistening agent ratio of 1 : 2.5 and inoculated with 25% of 24 h old inoculum, in 144 h at 40°C. Parametric optimization in SSF resulted in 1.7-fold enhancement in the enzyme production as compared to that recorded in unoptimized conditions. A 14.2-fold higher enzyme production was attained in SSF as compared to that in submerged fermentation (SmF). The treatment with the enzyme significantly improved tensile strength and Young’s modulus, reduction in brittleness, redness and yellowness, and increase in the strength and brightness of ramie fibres. Deepak Chand Sharma and T. Satyanarayana Copyright © 2012 Deepak Chand Sharma and T. Satyanarayana. All rights reserved. Sun, 05 Aug 2012 11:46:33 +0000 http://www.hindawi.com/journals/er/2012/421683/ Chitosan is a deacetylated product of chitin produced by chitin deacetylase, an enzyme that hydrolyses acetamido groups of N-acetylglucosamine in chitin. Chitosan is a natural polymer that has great potential in biotechnology and in the biomedical and pharmaceutical industries. Commercially, it is produced from chitin via a harsh thermochemical process that shares most of the disadvantages of a multistep chemical procedure. It is environmentally unsafe and not easily controlled, leading to a broad and heterogeneous range of products. An alternative or complementary procedure exploiting the enzymatic deacetylation of chitin could potentially be employed, especially when a controlled and well-defined process is required. In this study, 20 strains of bacteria were isolated from soil samples collected from different beaches of Chennai, India. Of these 20 bacterial strains, only 2 strains (S3, S14) are potent degrader of chitin and they are also a good producer of the enzyme chitin deacetylase so as to release chitosan. Kuldeep Kaur, Vikrant Dattajirao, Vikas Shrivastava, and Uma Bhardwaj Copyright © 2012 Kuldeep Kaur et al. All rights reserved. Sun, 05 Aug 2012 10:24:24 +0000 http://www.hindawi.com/journals/er/2012/572939/ Catalase (EC 1.11.1.6) oxidizes ethanol to acetaldehyde within the brain and variations in catalase activity may underlie some consequences of ethanol consumption. The goals of this study were to measure catalase activity in subcellular fractions from rat brain and to compare the levels of this enzyme in several important settings. In the first series of studies, levels of catalase were compared between juvenile and adult rats and between the Long-Evans (LE) and Sprague-Dawley (SD) strains. Levels of catalase appear to have achieved the adult level by the preadolescent period defined by postnatal age (P, days) P25–P28, and there were no differences between strains at the developmental stages tested. Thus, variation in catalase activity is unlikely to be responsible for differences in how adolescent and adult rats respond to ethanol. In the second series of studies, periadolescent and adult rats were administered ethanol chronically through an ethanol-containing liquid diet. Diet consumption and blood ethanol concentrations were significantly higher for periadolescent rats. Catalase activities remained unchanged following ethanol consumption, with no significant differences within or between strains. Thus, the brain showed no apparent adaptive changes in levels of catalase, even when faced with the high levels of ethanol consumption characteristic of periadolescent rats. Dennis E. Rhoads, Cherly Contreras, and Salma Fathalla Copyright © 2012 Dennis E. Rhoads et al. All rights reserved. Wed, 18 Jul 2012 11:33:08 +0000 http://www.hindawi.com/journals/er/2012/416062/ In 2008 melamine-contaminated infant formula and dairy products in China led to over 50,000 hospitalizations of children due to renal injuries. In North America during 2007 and in Asia during 2004, melamine-contaminated pet food products resulted in numerous pet deaths due to renal failure. Animal studies have confirmed the potent renal toxicity of melamine combined with cyanuric acid. We showed previously that the solubility of melamine cyanurate is low at physiologic pH and ionic strength, provoking us to speculate how toxic levels of these compounds could be transported through the circulation without crystallizing until passing into the renal filtrate. We hypothesized that melamine might be sequestered by heme proteins, which could interfere with heme enzyme activity. Four heme peroxidase enzymes were selected for study: horseradish peroxidase (HRP), lactoperoxidase (LPO), and cyclooxygenase-1 and -2 (COX-1 and -2). Melamine exhibited noncompetitive inhibition of HRP (𝐾𝑖9.5±0.7mM), and LPO showed a mixed model of inhibition (𝐾𝑖14.5±4.7mM). The inhibition of HRP and LPO was confirmed using a chemiluminescent peroxidase assay. Melamine also exhibited COX-1 inhibition, but inhibition of COX-2 was not detected. Thus, our results demonstrate that melamine inhibits the activity of three heme peroxidases. Pattaraporn Vanachayangkul and William H. Tolleson Copyright © 2012 Pattaraporn Vanachayangkul and William H. Tolleson. All rights reserved. Sun, 08 Jul 2012 19:23:08 +0000 http://www.hindawi.com/journals/er/2012/835731/ Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway. Antón Barreiro-Iglesias and Michael I. Shifman Copyright © 2012 Antón Barreiro-Iglesias and Michael I. Shifman. All rights reserved. Sun, 08 Jul 2012 09:08:57 +0000 http://www.hindawi.com/journals/er/2012/196853/ Aspergillus fumigatus was grown on chopped wheat straw in a solid state fermentation (SSF) process carried out in constant presence of isolated free water inside the fermentation chamber. The system allowed maintaining a constant vapor pressure inside the fermentor throughout the fermentation process. Crude endoglucanase produced by A. fumigatus under such conditions was more thermostable than previously reported enzymes of the same fungal strain which were produced under different conditions and was also more thermostable than a number of other previously reported endoglucanases as well. Various thermostability parameters were calculated for the crude endoglucanase. Half lives (T1/2) of the enzyme were 6930, 866, and 36 min at 60°C, 70°C, and 80°C, respectively. Enthalpies of activation of denaturation (Δ𝐻∗𝐷) were 254.04, 253.96, and 253.88 K J mole−1, at 60°C, 70°C and 80°C, respectively, whereas entropies of activation of denaturation (Δ𝑆∗𝐷) and free energy changes of activation of denaturation (Δ𝐺∗𝐷) were 406.45, 401.01, and 406.07 J mole−1 K−1 and 118.69, 116.41, and 110.53 K J mole−1 at 60°C, 70°C and 80°C, respectively. Abdul A. N. Saqib, Ansa Farooq, Maryam Iqbal, Jalees Ul Hassan, Umar Hayat, and Shahjahan Baig Copyright © 2012 Abdul A. N. Saqib et al. All rights reserved. Wed, 06 Jun 2012 10:30:15 +0000 http://www.hindawi.com/journals/er/2012/272706/ A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding. Jiyong Su and Karl Forchhammer Copyright © 2012 Jiyong Su and Karl Forchhammer. All rights reserved. Mon, 26 Mar 2012 13:19:02 +0000 http://www.hindawi.com/journals/er/2012/317314/ Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation. Ryan A. V. Bell, Neal J. Dawson, and Kenneth B. Storey Copyright © 2012 Ryan A. V. Bell et al. All rights reserved. Mon, 26 Mar 2012 10:35:54 +0000 http://www.hindawi.com/journals/er/2012/192867/ The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms. Su-Fang Wang, Sangho Oh, Yue-Xiu Si, Zhi-Jiang Wang, Hong-Yan Han, Jinhyuk Lee, and Guo-Ying Qian Copyright © 2012 Su-Fang Wang et al. All rights reserved. Sun, 25 Mar 2012 13:39:08 +0000 http://www.hindawi.com/journals/er/2012/375309/ Rhus laccase was isolated and purified from acetone powder obtained from the exudates of Chinese lacquer trees (Rhus vernicifera) from the Jianshi region, Hubei province of China. There are two blue bands appearing on CM-sephadex C-50 chromatography column, and each band corresponding to Rhus laccase 1 and 2, the former being the major constituent, and each had an average molecular weight of approximately 110 kDa. The purified and crude Rhus laccases were immobilized on zirconium chloride in ammonium chloride solution, and the kinetic properties of free and immobilized Rhus laccase, such as activity, molecular weight, optimum pH, and thermostability, were examined. In addition, the behaviors on catalytic oxidation of phenols also were conducted. Rong Lu and Tetsuo Miyakoshi Copyright © 2012 Rong Lu and Tetsuo Miyakoshi. All rights reserved. Sun, 11 Mar 2012 10:44:20 +0000 http://www.hindawi.com/journals/er/2012/980681/ Alane Beatriz Vermelho, Claudiu T. Supuran, and Jose M. Guisan Copyright © 2012 Alane Beatriz Vermelho et al. All rights reserved. Mon, 27 Feb 2012 09:22:28 +0000 http://www.hindawi.com/journals/er/2011/907423/ Claudio Alejandro Pereira, Ariel Mariano Silber, and Elena Gonzalez-Rey Copyright © 2011 Claudio Alejandro Pereira et al. All rights reserved. Mon, 23 Jan 2012 08:31:12 +0000 http://www.hindawi.com/journals/er/2012/329178/ This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150 min incubation time, pH 4.76, and an enzyme/support ratio of 1282 U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone. Roberta Bussamara, Luciane Dall'Agnol, Augusto Schrank, Kátia Flávia Fernandes, and Marilene Henning Vainstein Copyright © 2012 Roberta Bussamara et al. All rights reserved.