Enzyme Research http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. A cDNA Cloning of a Novel Alpha-Class Tyrosinase of Pinctada fucata: Its Expression Analysis and Characterization of the Expressed Protein Thu, 10 Apr 2014 12:30:02 +0000 http://www.hindawi.com/journals/er/2014/780549/ 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. Ryousuke Takgi and Tomoyuki Miyashita Copyright © 2014 Ryousuke Takgi and Tomoyuki Miyashita. All rights reserved. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads Thu, 10 Apr 2014 10:23:47 +0000 http://www.hindawi.com/journals/er/2014/738739/ 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. Bárbara M. Bonine, Patricia Peres Polizelli, and Gustavo O. Bonilla-Rodriguez Copyright © 2014 Bárbara M. Bonine et al. All rights reserved. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165 Thu, 03 Apr 2014 07:02:32 +0000 http://www.hindawi.com/journals/er/2014/494682/ 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 and of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. Deepthy Alex, Anju Shainu, Ashok Pandey, and Rajeev K. Sukumaran Copyright © 2014 Deepthy Alex et al. All rights reserved. Effect of Chromium(VI) Toxicity on Enzymes of Nitrogen Metabolism in Clusterbean (Cyamopsis tetragonoloba L.) Tue, 18 Mar 2014 07:58:56 +0000 http://www.hindawi.com/journals/er/2014/784036/ 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. Punesh Sangwan, Vinod Kumar, and U. N. Joshi Copyright © 2014 Punesh Sangwan et al. All rights reserved. Prolonged Laccase Production by a Cold and pH Tolerant Strain of Penicillium pinophilum (MCC 1049) Isolated from a Low Temperature Environment Sun, 09 Mar 2014 13:37:29 +0000 http://www.hindawi.com/journals/er/2014/120708/ 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. Kusum Dhakar, Rahul Jain, Sushma Tamta, and Anita Pandey Copyright © 2014 Kusum Dhakar et al. All rights reserved. Aspergillus 6V4, a Strain Isolated from Manipueira, Produces High Amylases Levels by Using Wheat Bran as a Substrate Sun, 02 Mar 2014 11:20:28 +0000 http://www.hindawi.com/journals/er/2014/725651/ 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). Jessyca dos Reis Celestino, Ana Caroline Duarte, Cláudia Maria de Melo Silva, Hellen Holanda Sena, Maria do Perpétuo Socorro Borges Carriço Ferreira, Neila Hiraishi Mallmann, Natacha Pinheiro Costa Lima, Chanderlei de Castro Tavares, Rodrigo Otávio Silva de Souza, Érica Simplício Souza, and João Vicente Braga Souza Copyright © 2014 Jessyca dos Reis Celestino et al. All rights reserved. A Method for Fast Assessment of OP/CB Exposure in the Japanese Quail (Coturnix coturnix japonica) Using Combined Esterases Enzyme Activity as Biomarkers Thu, 09 Jan 2014 11:58:11 +0000 http://www.hindawi.com/journals/er/2014/812302/ 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. Kasim Sakran Abass Copyright © 2014 Kasim Sakran Abass. All rights reserved. Stability of a Lipase Extracted from Seeds of Pachira aquatica in Commercial Detergents and Application Tests in Poultry Wastewater Pretreatment and Fat Particle Hydrolysis Mon, 23 Dec 2013 09:36:48 +0000 http://www.hindawi.com/journals/er/2013/324061/ A protein extract containing a plant lipase from oleaginous seeds of Pachira aquatica was tested using soybean oil, wastewater from a poultry processing plant, and beef fat particles as substrate. The hydrolysis experiments were carried out at a temperature of 40°C, an incubation time of 90 minutes, and pH 8.0-9.0. The enzyme had the best stability at pH 9.0 and showed good stability in the alkaline range. It was found that P. aquatica lipase was stable in the presence of some commercial laundry detergent formulations, and it retained full activity up to 0.35% in hydrogen peroxide, despite losing activity at higher concentrations. Concerning wastewater, the lipase increased free fatty acids release by 7.4 times and promoted the hydrolysis of approximately 10% of the fats, suggesting that it could be included in a pretreatment stage, especially for vegetable oil degradation. Patrícia Peres Polizelli, Fernanda Dell Antonio Facchini, and Gustavo Orlando Bonilla-Rodriguez Copyright © 2013 Patrícia Peres Polizelli et al. All rights reserved. Modelling and Optimization Studies on a Novel Lipase Production by Staphylococcus arlettae through Submerged Fermentation Thu, 19 Dec 2013 07:51:18 +0000 http://www.hindawi.com/journals/er/2013/353954/ Microbial enzymes from extremophilic regions such as hot spring serve as an important source of various stable and valuable industrial enzymes. The present paper encompasses the modeling and optimization approach for production of halophilic, solvent, tolerant, and alkaline lipase from Staphylococcus arlettae through response surface methodology integrated nature inspired genetic algorithm. Response surface model based on central composite design has been developed by considering the individual and interaction effects of fermentation conditions on lipase production through submerged fermentation. The validated input space of response surface model (with value of 96.6%) has been utilized for optimization through genetic algorithm. An optimum lipase yield of 6.5 U/mL has been obtained using binary coded genetic algorithm predicted conditions of 9.39% inoculum with the oil concentration of 10.285% in 2.99 hrs using pH of 7.32 at 38.8°C. This outcome could contribute to introducing this extremophilic lipase (halophilic, solvent, and tolerant) to industrial biotechnology sector and will be a probable choice for different food, detergent, chemical, and pharmaceutical industries. The present work also demonstrated the feasibility of statistical design tools integration with computational tools for optimization of fermentation conditions for maximum lipase production. Mamta Chauhan, Rajinder Singh Chauhan, and Vijay Kumar Garlapati Copyright © 2013 Mamta Chauhan et al. All rights reserved. A Highly Thermostable Xylanase from Stenotrophomonas maltophilia: Purification and Partial Characterization Sat, 14 Dec 2013 12:31:00 +0000 http://www.hindawi.com/journals/er/2013/429305/ Seven xylanolytic bacterial strains were isolated from saw-dust dump soil. The bacterial strain X6 was selected on the basis of the highest xylanase activity with no cellulase contamination. It was identified as Stenotrophomonas maltophilia by biochemical tests and 16S rRNA gene sequencing approach. Xylanase production studies by S. maltophilia on different commercial xylans and agro-industrial residues suggested that wheat bran was the best carbon source for xylanase production (26.4 ± 0.6 IU/mL). The studies with inorganic and organic nitrogen sources suggested yeast extract as the best support for xylanase production (25 ± 0.6 IU/mL). Maximum xylanase production was observed at initial medium pH = 8.0 (23.8 ± 0.4 IU/mL) with production at pH = 7.0 and pH = 9.0 being almost comparable. Xylanase produced by S. maltophilia was purified to homogeneity using ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The final purification was 5.43-fold with recovery of 19.18%. The molecular weight of the purified xylanase protein was ~142 kDa. Both crude and purified xylanase had good stability at pH = 9.0 and 80°C with activity retention greater than 90% after 30 min incubation. The enzyme stability at high temperature and alkaline pH make it potentially effective for industrial applications. Abhay Raj, Sharad Kumar, and Sudheer Kumar Singh Copyright © 2013 Abhay Raj et al. All rights reserved. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism Wed, 23 Oct 2013 18:42:01 +0000 http://www.hindawi.com/journals/er/2013/461374/ The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. Ali Shahriari, Neal J. Dawson, Ryan A. V. Bell, and Kenneth B. Storey Copyright © 2013 Ali Shahriari et al. All rights reserved. Purification and Properties of Polygalacturonase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI-756 on Solid-State Fermentation Thu, 12 Sep 2013 11:30:20 +0000 http://www.hindawi.com/journals/er/2013/438645/ Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries, textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by thermophilic fungus Thermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45°C for 4 days. PG obtained was purified by gel filtration and ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80% of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity, whereas at pH 10.0 it remained around 44%. The optimum temperature was 60–65°C. The enzyme was completely stable when incubated for 1 hour at 50°C. At 55°C and 60°C, the activity decreased 55% and 90%, respectively. The apparent molecular weight was 29.3 kDa, of 1.58 mg/mL and of 1553.1 μmol/min/mg. The presence of Zn+2, Mn+2, and Hg+2 inhibited 59%, 77%, and 100% of enzyme activity, respectively. The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme. Eduardo da Silva Martins, Rodrigo Simões Ribeiro Leite, Roberto da Silva, and Eleni Gomes Copyright © 2013 Eduardo da Silva Martins et al. All rights reserved. Purification and Characterization of Phenylalanine Ammonia Lyase from Trichosporon cutaneum Thu, 12 Sep 2013 10:10:15 +0000 http://www.hindawi.com/journals/er/2013/670702/ Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (: μmol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8–8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe,  mM) and positive allostery (Tyr,  mM, Hill coefficient ) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme’s ability to act on both substrates. Andrea Goldson-Barnaby and Christine H. Scaman Copyright © 2013 Andrea Goldson-Barnaby and Christine H. Scaman. All rights reserved. Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol Thu, 22 Aug 2013 08:31:55 +0000 http://www.hindawi.com/journals/er/2013/240219/ The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) ( IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems. Carla Eliana Todero Ritter, Marli Camassola, Denise Zampieri, Mauricio Moura Silveira, and Aldo José Pinheiro Dillon Copyright © 2013 Carla Eliana Todero Ritter et al. All rights reserved. An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues Wed, 10 Jul 2013 11:37:07 +0000 http://www.hindawi.com/journals/er/2013/287343/ Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at C/pH 4. rEG A retained 100% of activity when incubated at 45 and C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as  mg/mL,  mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol. Eveline Queiroz de Pinho Tavares, Marciano Regis Rubini, Thiago Machado Mello-de-Sousa, Gilvan Caetano Duarte, Fabrícia Paula de Faria, Edivaldo Ximenes Ferreira Filho, Cynthia Maria Kyaw, Ildinete Silva-Pereira, and Marcio Jose Poças-Fonseca Copyright © 2013 Eveline Queiroz de Pinho Tavares et al. All rights reserved. Solvent-Free Synthesis of Flavour Esters through Immobilized Lipase Mediated Transesterification Thu, 30 May 2013 10:50:52 +0000 http://www.hindawi.com/journals/er/2013/367410/ The synthesis of methyl butyrate and octyl acetate through immobilized Rhizopus oryzae NRRL 3562 lipase mediated transesterification was studied under solvent-free conditions. The effect of different transesterification variables, namely, molarity of alcohol, reaction time, temperature, agitation, addition of water, and enzyme amount on molar conversion (%) was investigated. A maximum molar conversion of 70.42% and 92.35% was obtained in a reaction time of 14 and 12 h with the transesterification variables of 0.6 M methanol in vinyl butyrate and 2 M octanol in vinyl acetate using 80 U and 60 U immobilized lipase with the agitation speed of 200 rpm and 0.2% water addition at 32°C and 36°C for methyl butyrate and octyl acetate, respectively. The immobilized enzyme has retained good relative activity (more than 95%) up to five and six recycles for methyl butyrate and octyl acetate, respectively. Hence, the present investigation makes a great impingement in natural flavour industry by introducing products synthesized under solvent-free conditions to the flavour market. Vijay Kumar Garlapati and Rintu Banerjee Copyright © 2013 Vijay Kumar Garlapati and Rintu Banerjee. All rights reserved. Immobilization and Biochemical Properties of the Enantioselective Recombinant NStcI Esterase of Aspergillus nidulans Mon, 27 May 2013 11:56:58 +0000 http://www.hindawi.com/journals/er/2013/928913/ The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1 h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72 h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, of 0.07, and 120 h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures. Carolina Peña-Montes, María Elena Mondragón-Tintor, José Augusto Castro-Rodríguez, Ismael Bustos-Jaimes, Arturo Navarro-Ocaña, and Amelia Farrés Copyright © 2013 Carolina Peña-Montes et al. All rights reserved. Recovery of Extracellular Lipolytic Enzymes from Macrophomina phaseolina by Foam Fractionation with Air 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. Laccase Production from a Temperature and pH Tolerant Fungal Strain of Trametes hirsuta (MTCC 11397) 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. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii 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. Immobilization of -Amylase onto Luffa operculata Fibers 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. Optimization and Immobilization of Purified Labeo rohita Visceral Protease by Entrapment Method 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. Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans 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. Production of Biomass-Degrading Multienzyme Complexes under Solid-State Fermentation of Soybean Meal Using a Bioreactor 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. The Effect of D-(−)-arabinose on Tyrosinase: An Integrated Study Using Computational Simulation and Inhibition Kinetics 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. Application of Statistical Design for the Production of Cellulase by Trichoderma reesei Using Mango Peel 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. In Silico Characterization of Histidine Acid Phytase Sequences 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. Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains 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. Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra 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. Simulation of Enzyme Catalysis in Calcium Alginate Beads 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.