Biotechnology Research International http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3 Wed, 23 Apr 2014 15:03:56 +0000 http://www.hindawi.com/journals/btri/2014/293434/ Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L). To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine. Kumar Pranaw, Surender Singh, Debjani Dutta, Surabhi Chaudhuri, Sudershan Ganguly, and Lata Nain Copyright © 2014 Kumar Pranaw et al. All rights reserved. Biocontrol Activity of the Local Strain of Metschnikowia pulcherrima on Different Postharvest Pathogens Thu, 17 Apr 2014 06:37:32 +0000 http://www.hindawi.com/journals/btri/2014/397167/ The strains of the yeast Metschnikowia pulcherrima have strong biocontrol activity against various microorganisms. Biocontrol activity of M. pulcherrima largely depends on its iron immobilizing pigment pulcherrimin. Biocontrol activity of pulcherrimin producing strain, M. pulcherrima UMY15, isolated from local vineyards, was tested on different molds that cause food spoilage. M. pulcherrima UMY15 was a very effective biocontrol agent against Penicillium roqueforti, P. italicum, P. expansum, and Aspergillus oryzae in in-vitro plate tests. However, the inhibitory activity of M. pulcherrima UMY15 was less effective on Fusarium sp. and A. niger species in biocontrol assays. In addition, M. pulcherrima UMY15 strain completely inhibited the germination and mycelia growth of A. oryzae, A. parasiticus, and Fusarium sp. spores on artificial wounds of apples when they coinoculated with M. pulcherrima UMY15. Moreover, when coinoculated, M. pulcherrima UMY15 strain also inhibited the growth of P. roqueforti, P. italicum, P. expansum, A. oryzae, Fusarium sp., and Rhizopus sp. in grape juice, indicating that M. pulcherrima UMY15 can be used as a very effective biocontrol yeast against various species of postharvest pathogens, including   Penicillium, Aspergillus, Fusarium, and Rhizopus. Sezai Türkel, Mihriban Korukluoğlu, and Mümine Yavuz Copyright © 2014 Sezai Türkel et al. All rights reserved. Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases Sun, 06 Apr 2014 11:09:24 +0000 http://www.hindawi.com/journals/btri/2014/970595/ Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger. Christian Bach, William Sherman, Jani Pallis, Prabir Patra, and Hassan Bajwa Copyright © 2014 Christian Bach et al. All rights reserved. Characterization of β-Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose Tue, 01 Apr 2014 12:29:52 +0000 http://www.hindawi.com/journals/btri/2014/317092/ β-Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF) was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein) adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications. Anderson Baraldo Junior, Diogo G. Borges, Paulo W. Tardioli, and Cristiane S. Farinas Copyright © 2014 Anderson Baraldo Junior et al. All rights reserved. Production of Bioactive Compounds by Actinomycetes and Their Antioxidant Properties Wed, 26 Mar 2014 13:37:56 +0000 http://www.hindawi.com/journals/btri/2014/217030/ An actinomycete was isolated from mangrove soil collected from Nellore region of Andhra Pradesh, India, and screened for its ability to produce bioactive compounds. The cultural, morphological, and biochemical characters and 16S rRNA sequencing suggest that the isolated strain is Nocardiopsis alba. The bioactive compounds produced by this strain were purified by column chromatography. The in vitro antioxidant capacity of the isolated compounds (fractions) was estimated and fraction F2 showed very near values to the standard ascorbic acid. The potential fraction obtained by column chromatography was subjected to HPLC for further purification, then this purified fraction F2 was examined by FTIR, NMR, and mass spectroscopy to elucidate its chemical structure. By spectral data, the structure of the isolated compound was predicted as “(Z)-1-((1-hydroxypenta-2,4-dien-1-yl)oxy)anthracene-9,10-dione.” Avilala Janardhan, Arthala Praveen Kumar, Buddolla Viswanath, D. V. R. Saigopal, and Golla Narasimha Copyright © 2014 Avilala Janardhan et al. All rights reserved. Kojic Acid Production from Agro-Industrial By-Products Using Fungi Sun, 23 Mar 2014 07:09:53 +0000 http://www.hindawi.com/journals/btri/2014/642385/ A total of 278 different isolates of filamentous fungi were screened using synthetic medium for respective ability to produce kojic acid. Nineteen, six, and five isolates proved to be low, moderate, and high kojic acid producers, respectively. Levels of kojic acid produced were generally increased when shaking cultivation was used rather than those obtained using static cultivation. A trial for the utilization of 15 agro-industrial wastes or by-products for kojic acid production by the five selected higher kojic acid producer isolates was made. The best by-product medium recorded was molasses for kojic acid. A. flavus numbers 7 and 24 were able to grow and produce kojic acid on only 12 out of 15 wastes or by-products media. The best medium used for kojic acid production by A. flavus number 7 was rice fragments followed by molasses, while the best medium used for kojic acid production by A. flavus number 24 was the molasses followed by orange, pea, and rice fragments. An attempt for production of kojic acid using a 1.5 L laboratory fermentor has been made. Aspergillus flavus number 7 was used and grown on molasses medium; maximum level (53.5 g/L) of kojic acid was obtained after eight days of incubation. Ismael A. El-Kady, Abdel Naser A. Zohri, and Shimaa R. Hamed Copyright © 2014 Ismael A. El-Kady et al. All rights reserved. Expeditious Quantification of Lignocellulolytic Enzymes from Indigenous Wood Rot and Litter Degrading Fungi from Tropical Dry Evergreen Forests of Tamil Nadu Wed, 26 Feb 2014 13:30:32 +0000 http://www.hindawi.com/journals/btri/2014/127848/ In this study thirty wood rotting and litter degrading basidiomycetes were screened for the production of lignocellulolytic enzymes such as, laccase, peroxidase, and cellulase using rapid micro quantification assay. Out of the 30 indigenous isolates Trametes gibbosa was identified to be a potential lignocellulolytic enzyme producer, producing a maximum amount of cellulase ( IU/L) and laccase ( U/L). Moreover, it is the second leading producer of peroxidase enzyme ( U/L). Tricholomopsis sp. a wood rot basidiomycete was found to be the leading lignin decomposer with maximum peroxidase activity ( U/L) and second maximum laccase activity ( U/L). However, its cellulolytic potential was found to be moderate ( U/L). A higher level of lignocellulolytic enzymes was recorded in wood rotting basidiomycetes, whereas very low levels of lignolytic enzymes were found in litter inhabiting basidiomycetes. However, their cellulolytic potential was found to be moderate. Jenefar Sudarson, Shenbhagaraman Ramalingam, Premalatha Kishorekumar, and Kaviyarasan Venkatesan Copyright © 2014 Jenefar Sudarson et al. All rights reserved. In Vitro Flower Induction from Shoots Regenerated from Cultured Axillary Buds of Endangered Medicinal Herb Swertia chirayita H. Karst. Tue, 25 Feb 2014 00:00:00 +0000 http://www.hindawi.com/journals/btri/2014/264690/ In vitro flowering and effective micropropagation protocol were studied in Swertia chirayita, an important medicinal plant using axillary bud explants. The Murashige and Skoog's medium (MS) supplemented with benzyl amino purine (BAP) 1.0  and adenine sulfate 70.0  was found optimum for production of multiple shoots. In the present study, incubation of flowering cultures on BAP supplemented medium (during shoot multiplication) was found necessary for flowering (6 weeks). However, concentrations of auxins-like IBA (0–2.0 mg/L) were ineffective to form reproductive buds. Subculture duration, photoperiod, and carbon source type do have influence on the in vitro flowering. The mature purple flowers were observed when the cultures were maintained in the same medium. This is the very first report that describes in vitro flowering system to overcome problems associated with flower growth and development as well as lay foundation for fruit and seed production in vitro in Swertia chirayita. Vikas Sharma, Barkha Kamal, Nidhi Srivastava, Anoop Kumar Dobriyal, and Vikash Singh Jadon Copyright © 2014 Vikas Sharma et al. All rights reserved. Thermostability of Probiotics and Their α-Galactosidases and the Potential for Bean Products Tue, 18 Feb 2014 15:46:50 +0000 http://www.hindawi.com/journals/btri/2014/472723/ Soybeans and other pulses contain oligosaccharides which may cause intestinal disturbances such as flatulence. This study was undertaken to investigate α-galactosidase-producing probiotics added to frozen foods which can survive warming treatments used in thawing and consumption of the pulses. The maximum α-galactosidase activity (1.26 U/mg protein) was found in Bifidobacterium breve S46. Lactobacillus casei had the highest α-galactosidase thermostability among the various strains, with D values of 35, 29, and 9.3 minutes at 50°C, 55°C, and 60°C, respectively. The enzyme activity was less affected than viable cells by heating. However, the D values of two bacterial enzymes were lower than those of three commercial α-galactosidase-containing products. Freshly grown cells and their enzymes were more stable than the rehydrated cultures and their enzymes. Practical Application. Enzymes and cultures can be added to foods in order to enhance the digestibility of carbohydrates in the gastrointestinal tract. However since many foods are warmed, it is important that the thermostability of the enzymes be assessed. This paper provides data on the stability of α-galactosidase, which could potentially be added to food matrices containing stachyose or raffinose, such as beans. Xiaoli Liu, Claude P. Champagne, Byong H. Lee, Joyce I. Boye, and Michel Casgrain Copyright © 2014 Xiaoli Liu et al. All rights reserved. Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics Tue, 21 Jan 2014 12:36:35 +0000 http://www.hindawi.com/journals/btri/2014/495384/ The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values , , , and were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent and values for starch were 3.4 mg mL−1 and 19.5 IU mg−1 protein, respectively. The optimum temperature and pH for α-amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol−1, respectively. Both enthalpies () and entropies of activation () for denaturation of α-amylase were lower than those reported for other thermostable α-amylases. Romana Tabassum, Shazia Khaliq, Muhammad Ibrahim Rajoka, and Foster Agblevor Copyright © 2014 Romana Tabassum et al. All rights reserved. High Level Ethanol from Sugar Cane Molasses by a New Thermotolerant Saccharomyces cerevisiae Strain in Industrial Scale Sun, 01 Dec 2013 16:03:41 +0000 http://www.hindawi.com/journals/btri/2013/253286/ A new local strain of S. cerevisiae F-514, for ethanol production during hot summer season, using Egyptian sugar cane molasses was applied in Egyptian distillery factory. The inouluum was propagated through 300 L, 3 m3, and 12 m3 fermenters charged with diluted sugar cane molasses containing 4%-5% sugars. The yeast was applied in fermentation vessels 65 m3 working volume to study the varying concentrations of urea, DAP, orthophosphoric acid (OPA), and its combinations as well as magnesium sulfate and inoculum size. The fermenter was allowed to stay for a period of 20 hours to give time for maximum conversion of sugars into ethanol. S. cerevisiae F-514 at molasses sugar level of 18% (w/v), inoculum size of 20% (v/v) cell concentration of /mL, and combinations of urea, diammonium phosphate (DAP), orthophosphoric acid (OPA), and magnesium sulfate at amounts of 20, 10, 5, and 10 kg/65 m3 working volume fermenters, respectively, supported maximum ethanol production (9.8%, v/v), fermentation efficiency (FE) 88.1%, and remaining sugars (RS) 1.22%. The fermentation resulted 13.4 g dry yeast/L contained 34.6% crude protein and 8.2% ash. By selecting higher ethanol yielding yeast strain and optimizing, the fermentation parameters both yield and economics of the fermentation process can be improved. M. Fadel, Abeer A. Keera, Foukia E. Mouafi, and Tarek Kahil Copyright © 2013 M. Fadel et al. All rights reserved. Influence of Cow Bone Particle Size Distribution on the Mechanical Properties of Cow Bone-Reinforced Polyester Composites Thu, 14 Nov 2013 08:40:58 +0000 http://www.hindawi.com/journals/btri/2013/725396/ This work was carried out to investigate the influence of cow bone particle size distribution on the mechanical properties of polyester matrix composites in order to consider the suitability of the materials as biomaterials. Cow bone was procured from an abattoir, washed with water, and sun-dried for 4 weeks after which it was crushed with a sledge hammer and was further pulverized with laboratory ball mill. Sieve size analysis was carried out on the pulverized bone where it was sieved into three different sizes of 75, 106, and 300 m sieve sizes. Composite materials were developed by casting them into tensile and flexural tests moulds using predetermined proportions of 2, 4, 6, and 8%. The samples after curing were striped from the moulds and were allowed to be further cured at room temperature for 3 weeks before tensile and flexural tests were performed on them. Both tensile and flexural strength were highly enhanced by 8 wt% from 75 m while toughness was highly enhanced by 6 and 8 wt% from 300 m. This shows that fine particles lead to improved strength while coarse particles lead to improved toughness. The results show that these materials are structurally compatible and are being developed from animal fibre based particle; it is expected to also aid the compatibility with the surface conditions as biomaterials. Isiaka Oluwole Oladele and Temitope Akinyemi Adewole Copyright © 2013 Isiaka Oluwole Oladele and Temitope Akinyemi Adewole. All rights reserved. Chiral Phosphinate Degradation by the Fusarium Species: Scope and Limitation of the Process Sun, 10 Nov 2013 15:21:23 +0000 http://www.hindawi.com/journals/btri/2013/927361/ Biodegradable capacities of fungal strains of Fusarium oxysporum (DSMZ 2018) and Fusarium culmorum (DSMZ 1094) were tested towards racemic mixture of chiral 2-hydroxy-2-(ethoxyphenylphosphinyl) acetic acid—a compound with two stereogenic centres. The effectiveness of decomposition was dependent on external factors such as temperature and time of the process. Optimal conditions of complete mineralization were established. Both Fusarium species were able to biodegrade every isomer of tested compound at 30°C, but F. culmorum required 10 days and F. oxysporum 11 days to accomplish the process, which was continuously monitored using the 31P NMR technique. Natalia Kmiecik, Magdalena Klimek-Ochab, Małgorzata Brzezińska-Rodak, Paulina Majewska, and Ewa Żymańczyk-Duda Copyright © 2013 Natalia Kmiecik et al. All rights reserved. Bioremediating Oil Spills in Nutrient Poor Ocean Waters Using Fertilized Clay Mineral Flakes: Some Experimental Constraints Sun, 23 Jun 2013 13:18:17 +0000 http://www.hindawi.com/journals/btri/2013/704806/ Much oil spill research has focused on fertilizing hydrocarbon oxidising bacteria, but a primary limitation is the rapid dilution of additives in open waters. A new technique is presented for bioremediation by adding nutrient amendments to the oil spill using thin filmed minerals comprised largely of Fullers Earth clay. Together with adsorbed N and P fertilizers, filming additives, and organoclay, clay flakes can be engineered to float on seawater, attach to the oil, and slowly release contained nutrients. Our laboratory experiments of microbial activity on weathered source oil from the Deepwater Horizon spill in the Gulf of Mexico show fertilized clay treatment significantly enhanced bacterial respiration and consumption of alkanes compared to untreated oil-in-water conditions and reacted faster than straight fertilization. Whereas a major portion (up to 98%) of the alkane content was removed during the 1 month period of experimentation by fertilized clay flake interaction; the reduced concentration of polyaromatic hydrocarbons was not significantly different from the non-clay bearing samples. Such clay flake treatment could offer a way to more effectively apply the fertilizer to the spill in open nutrient poor waters and thus significantly reduce the extent and duration of marine oil spills, but this method is not expected to impact hydrocarbon toxicity. Laurence N. Warr, André Friese, Florian Schwarz, Frieder Schauer, Ralph J. Portier, Laura M. Basirico, and Gregory M. Olson Copyright © 2013 Laurence N. Warr et al. All rights reserved. Production of Pectinolytic Enzymes by the Yeast Wickerhanomyces anomalus Isolated from Citrus Fruits Peels Wed, 17 Apr 2013 09:09:21 +0000 http://www.hindawi.com/journals/btri/2013/435154/ Wickerhamomyces anomalus is pectinolytic yeast isolated from citrus fruits peels in the province of Misiones, Argentine. In the present work, enzymes produced by this yeast strain were characterized, and polygalacturonase physicochemical properties were determined in order to evaluate the application of the supernatant in the maceration of potato tissues. W. anomalus was able to produce PG in liquid medium containing glucose and citrus pectin, whose mode of action was mainly of endo type. The supernatant did not exhibit esterase or lyase activity. No others enzymes, capable of hydrolyzing cell wall polymers, such as cellulases and xylanases, were detected. PG showed maximal activity at pH 4.5 and at temperature range between 40°C and 50°C. It was stable in the pH range from 3.0 to 6.0 and up to 50°C at optimum pH. The enzymatic extract macerated potato tissues efficiently. Volume of single cells increased with the agitation speed. The results observed make the enzymatic extract produced by W. anomalus appropriate for future application in food industry, mainly for the production of fruit nectars or mashed of vegetables such as potato or cassava, of regional interest in the province of Misiones, Argentine. María A. Martos, Emilce R. Zubreski, Oscar A. Garro, and Roque A. Hours Copyright © 2013 María A. Martos et al. All rights reserved. Antioxidant and Hepatoprotective Properties of Tofu (Curdle Soymilk) against Acetaminophen-Induced Liver Damage in Rats Thu, 28 Feb 2013 14:06:23 +0000 http://www.hindawi.com/journals/btri/2013/230142/ The antioxidant and hepatoprotective properties of tofu using acetaminophen to induce liver damage in albino rats were evaluated. Tofus were prepared using calcium chloride, alum, and steep water as coagulants. The polyphenols of tofu were extracted and their antioxidant properties were determined. The weight gain and feed intake of the rats were measured. The analysis of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities and the concentrations of albumin, total protein, cholesterol, and bilirubin were analyzed. The result reveals that the antioxidant property of both soluble and bound polyphenolic extracts was significantly higher in all tofus, but the steep water coagulated tofu was recorded higher. Rats fed with various tofus and acetaminophen had their serum ALP, ALT, AST, and LDH activities; total cholesterol; and bilirubin levels significantly () reduced, and total protein and albumin concentrations increased when compared with basal diet and acetaminophen administered group. Therefore, all tofus curdled with various coagulants could be used to prevent liver damage caused by oxidative stress. Ndatsu Yakubu, Ganiyu Oboh, and Amuzat Aliyu Olalekan Copyright © 2013 Ndatsu Yakubu et al. All rights reserved. Statistical Analysis of Metal Chelating Activity of Centella asiatica and Erythroxylum cuneatum Using Response Surface Methodology Wed, 27 Feb 2013 07:21:14 +0000 http://www.hindawi.com/journals/btri/2013/137851/ The purpose of the study is to evaluate the relationship between the extraction parameters and the metal chelating activity of Centella asiatica (CA) and Erythroxylum cuneatum (EC). The response surface methodology was used to optimize the extraction parameters of methanolic extract of CA and EC with respect to the metal chelating activity. For CA, Run 17 gave optimum chelating activity with IC50 = 0.93 mg/mL at an extraction temperature of 25°C, speed of agitation at 200 rpm, ratio of plant material to solvent at 1 g : 45 mL and extraction time at 1.5 hour. As for EC, Run 13 with 60°C, 200 rpm, 1 g : 35 mL and 1 hour had metal chelating activity at IC50 = 0.3817 mg/mL. Both optimized extracts were further partitioned using a solvent system to evaluate the fraction responsible for the chelating activity of the plants. The hexane fraction of CA showed potential activity with chelating activity at IC50 = 0.090 and the ethyl acetate fraction of EC had IC50 = 0.120 mg/mL. The study showed that the response surface methodology helped to reduce the extraction time, temperature and agitation and subsequently improve the chelating activity of the plants in comparison to the conventional method. R. J. Mohd Salim, M. I. Adenan, A. Amid, M. H. Jauri, and A. S. Sued Copyright © 2013 R. J. Mohd Salim et al. All rights reserved. Production of Enzymes from Agroindustrial Wastes by Biosurfactant-Producing Strains of Bacillus subtilis Tue, 26 Feb 2013 13:42:51 +0000 http://www.hindawi.com/journals/btri/2013/103960/ Bacteria in the genus Bacillus are the source of several enzymes of current industrial interest. Hydrolases, such as amylases, proteases, and lipases, are the main enzymes consumed worldwide and have applications in a wide range of products and industrial processes. Fermentation processes by Bacillus subtilis using cassava wastewater as a substrate are reported in the technical literature; however, the same combination of microorganisms and this culture medium is limited or nonexistent. In this paper, the amylase, protease, and lipase production of ten Bacillus subtilis strains previously identified as biosurfactant producers in cassava wastewater was evaluated. The LB1a and LB5a strains were selected for analysis using a synthetic medium and cassava wastewater and were identified as good enzyme producers, especially of amylases and proteases. In addition, the enzymatic activity results indicate that cassava wastewater was better than the synthetic medium for the induction of these enzymes. Francisco Fábio Cavalcante Barros, Ana Paula Resende Simiqueli, Cristiano José de Andrade, and Gláucia Maria Pastore Copyright © 2013 Francisco Fábio Cavalcante Barros et al. All rights reserved. Family-Specific Degenerate Primer Design: A Tool to Design Consensus Degenerated Oligonucleotides Thu, 21 Feb 2013 13:54:30 +0000 http://www.hindawi.com/journals/btri/2013/383646/ Designing degenerate PCR primers for templates of unknown nucleotide sequence may be a very difficult task. In this paper, we present a new method to design degenerate primers, implemented in family-specific degenerate primer design (FAS-DPD) computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. To assess their efficiency, four different genome collections were used, covering a wide range of genomic lengths: Arenavirus ( nucleotides), Baculovirus ( to  bp), Lactobacillus sp. ( to  bp), and Pseudomonas sp. ( to  bp). In each case, FAS-DPD designed primers were tested computationally to measure specificity. Designed primers for Arenavirus and Baculovirus were tested experimentally. The method presented here is useful for designing degenerate primers on collections of related protein sequences, allowing detection of new family members. Javier Alonso Iserte, Betina Ines Stephan, Sandra Elizabeth Goñi, Cristina Silvia Borio, Pablo Daniel Ghiringhelli, and Mario Enrique Lozano Copyright © 2013 Javier Alonso Iserte et al. All rights reserved. Structural Variations of Human Glucokinase Glu256Lys in MODY2 Condition Using Molecular Dynamics Study Wed, 13 Feb 2013 13:58:12 +0000 http://www.hindawi.com/journals/btri/2013/264793/ Glucokinase (GK) is the predominant hexokinase that acts as glucose sensor and catalyses the formation of Glucose-6-phosphate. The mutations in GK gene influence the affinity for glucose and lead to altered glucose levels in blood causing maturity onset diabetes of the young type 2 (MODY2) condition, which is one of the prominent reasons of type 2 diabetic condition. In view of the importance of mutated GK resulting in hyperglycemic condition, in the present study, molecular dynamics simulations were carried out in intact and 256 E-K mutated GK structures and their energy values and conformational variations were correlated. Energy variations were observed in mutated GK (3500 Kcal/mol) structure with respect to intact GK (5000 Kcal/mol), and it showed increased γ-turns, decreased β-turns, and more helix-helix interactions that affected substrate binding region where its volume increased from 1089.152 Å2 to 1246.353 Å2. Molecular docking study revealed variation in docking scores (intact = −12.199 and mutated = −8.383) and binding mode of glucose in the active site of mutated GK where the involvement of A53, S54, K56, K256, D262 and Q286 has resulted in poor glucose binding which probably explains the loss of catalytic activity and the consequent prevailing of high glucose levels in MODY2 condition. Nanda Kumar Yellapu, Kalpana Kandlapalli, Koteswara Rao Valasani, P. V. G. K. Sarma, and Bhaskar Matcha Copyright © 2013 Nanda Kumar Yellapu et al. All rights reserved. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71 Wed, 13 Feb 2013 09:07:33 +0000 http://www.hindawi.com/journals/btri/2013/431315/ In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle. Nadimpalli Ravi S. Varma, Haryanti Toosa, Hooi Ling Foo, Noorjahan Banu Mohamed Alitheen, Mariana Nor Shamsudin, Ali S. Arbab, Khatijah Yusoff, and Raha Abdul Rahim Copyright © 2013 Nadimpalli Ravi S. Varma et al. All rights reserved. Optimization of Culture Conditions for Some Identified Fungal Species and Stability Profile of -Galactosidase Produced Mon, 28 Jan 2013 10:23:43 +0000 http://www.hindawi.com/journals/btri/2013/920759/ Microbial α-galactosidase preparations have implications in medicine and in the modification of various agricultural products as well. In this paper, four isolated fungal strains such as AL-3, WF-3, WP-4 and CL-4 from rhizospheric soil identified as Penicillium glabrum (AL-3), Trichoderma evansii (WF-3), Lasiodiplodia theobromae (WP-4) and Penicillium flavus (CL-4) based on their morphology and microscopic examinations, are screened for their potential towards α-galactosidases production. The culture conditions have been optimized and supplemented with specific carbon substrates (1%, w/v) by using galactose-containing polysaccharides like guar gum (GG), soya casein (SC) and wheat straw (WS). All strains significantly released galactose from GG, showing maximum production of enzyme at 7th day of incubation in rotary shaker (120 rpm) that is 190.3, 174.5, 93.9 and 28.8 U/mL, respectively, followed by SC and WS. The enzyme activity was stable up to 7days at −20°C, then after it declines. This investigation reveals that AL-3 show optimum enzyme activity in guar gum media, whereas WF-3 exhibited greater enzyme stability. Results indicated that the secretion of proteins, enzyme and the stability of enzyme activity varied not only from one strain to another but also differed in their preferences of utilization of different substrates. A. S. Chauhan, N. Srivastava, H. K. Kehri, and B. Sharma Copyright © 2013 A. S. Chauhan et al. All rights reserved. Bioprocessing of “Hair Waste” by Paecilomyces lilacinus as a Source of a Bleach-Stable, Alkaline, and Thermostable Keratinase with Potential Application as a Laundry Detergent Additive: Characterization and Wash Performance Analysis Mon, 17 Dec 2012 13:15:40 +0000 http://www.hindawi.com/journals/btri/2012/369308/ Paecilomyces lilacinus (Thom) Samson LPS 876, a locally isolated fungal strain, was grown on minimal mineral medium containing “hair waste,” a residue from the hair-saving unhairing process, and produced a protease with keratinolytic activity. This enzyme was biochemically characterized. The optimum reaction conditions, determined with a response surface methodology, were 60°C and pH 6.0. It was remarkably stable in a wide range of pHs and temperatures. Addition of Ca2+, Mg2+, or sorbitol was found to be effective in increasing thermal stability of the protease. PMSF and Hg2+ inhibited the proteolytic activity indicating the presence of a thiol-dependent serine protease. It showed high stability toward surfactants, bleaching agents, and solvents. It was also compatible with commercial detergents (7 mg/mL) such as Ariel, Skip, Drive, and Ace, retaining more than 70% of its proteolytic activity in all detergents after 1 h of incubation at 40°C. Wash performance analysis revealed that this protease could effectively remove blood stains. From these properties, this enzyme may be considered as a potential candidate for future use in biotechnological processes, as well as in the formulation of laundry detergents. Ivana A. Cavello, Roque A. Hours, and Sebastián F. Cavalitto Copyright © 2012 Ivana A. Cavello et al. All rights reserved. Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51 Thu, 13 Dec 2012 15:23:54 +0000 http://www.hindawi.com/journals/btri/2012/135498/ This paper reports the isolation and identification of chitinase-producing Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125 kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer. Secil Berna Kuzu, Hatice Korkmaz Güvenmez, and Aziz Akin Denizci Copyright © 2012 Secil Berna Kuzu et al. All rights reserved. Isolation and Characterization of Some Phytochemicals from Indian Traditional Plants Tue, 11 Dec 2012 10:29:41 +0000 http://www.hindawi.com/journals/btri/2012/549850/ The present study was designed to evaluate relative contribution of different polyphenols (total phenolics, flavonoids, flavonols) and their antioxidants activities in aqueous extracts of different parts of some plants; Argemone mexicana, Datura metel, Calotropis procera, Thevetia peruviana, and Cannabis sativa. The antioxidants (total phenolics, flavonoids, flavones) were determined by chemical methods. The antioxidant capacities of these extracts were evaluated by FRAP assay. The results demonstrated that phenolic content was maximally present in leaves of T. peruviana. This plant exhibited minimum phenolic content in its flower as compared to other plants. The flower of D. metel contained maximum phenolic content. The flavonoids were present in highest quantity in leaves of C. procera while T. peruviana flowers showed maximum flavonoid content. The fruits of C. sativa contained maximum quantity of flavonoid as compared to other plants tested. The flower extract of C. sativa possessed highest FRAP value followed by A. mexicana and fruit of C. procera. The values of ratios of different polyphenolic compounds present in plant extracts indicated that flower of D. metel contained maximum total flavonoids and minimum phenolics. These results suggested that levels of total phenolics, flavonoids and their FRAP indices exhibited specificity to different plants and their parts. Neeharika Srivastava, Aishwarya Singh Chauhan, and Bechan Sharma Copyright © 2012 Neeharika Srivastava et al. All rights reserved. Properties and Therapeutic Application of Bromelain: A Review Mon, 10 Dec 2012 15:17:08 +0000 http://www.hindawi.com/journals/btri/2012/976203/ Bromelain belongs to a group of protein digesting enzymes obtained commercially from the fruit or stem of pineapple. Fruit bromelain and stem bromelainare prepared differently and they contain different enzymatic composition. “Bromelain” refers usually to the “stem bromelain.” Bromelain is a mixture of different thiol endopeptidases and other components like phosphatase, glucosidase, peroxidase, cellulase, escharase, and several protease inhibitors. In vitro and in vivo studies demonstrate that bromelain exhibits various fibrinolytic, antiedematous, antithrombotic, and anti-inflammatory activities. Bromelain is considerably absorbable in the body without losing its proteolytic activity and without producing any major side effects. Bromelain accounts for many therapeutic benefits like the treatment of angina pectoris, bronchitis, sinusitis, surgical trauma, and thrombophlebitis, debridement of wounds, and enhanced absorption of drugs, particularly antibiotics. It also relieves osteoarthritis, diarrhea, and various cardiovascular disorders. Bromelain also possesses some anticancerous activities and promotes apoptotic cell death. This paper reviews the important properties and therapeutic applications of bromelain, along with the possible mode of action. Rajendra Pavan, Sapna Jain, Shraddha, and Ajay Kumar Copyright © 2012 Rajendra Pavan et al. All rights reserved. High-Frequency Regeneration of the Drought-Tolerant Tree Melia volkensii Gurke Using Low-Cost Agrochemical Thidiazuron Tue, 27 Nov 2012 09:15:24 +0000 http://www.hindawi.com/journals/btri/2012/818472/ Melia volkensii Gurke is a drought-tolerant tree native to East Africa’s arid and semiarid lands (ASALs), with vast but underutilized potential for agroforestry and sustainable livelihoods in the ASALs. Its cultivation is limited by difficulties in propagation via conventional means. Full exploitation of the ability of thidiazuron (TDZ) to elicit regeneration in plant tissue cultures, as sole plant growth regulator (PGR), is hampered by high costs. This study tested the effectiveness of a low-cost agrochemical TDZ for in vitro propagation of M. volkensii. Zygotic embryos from mature seeds were cultured on Gamborg’s B5 medium containing 0 to 4 mg/L of agrochemical TDZ from Kingtai Chemicals Co.,Ltd., China. Callus induction frequency was 96.67 to 100%. Significantly large callus fresh mass was produced at 0.05 mg/L TDZ concentration (ANOVA, ). The effect of TDZ on embryogenicity was significant over certain ranges of concentrations (Anova, ). Multiple somatic embryos developed within 14 days of subculture to hormone-free B5 medium. Somatic embryos developed into microshoots which elongated when transferred to MS medium supplemented with 0.1 mg/L 6-benzylaminopurine plus 10% coconut water. The Kingtai-TDZ showed a high potency and suitability for use in M. volkensii tissue culture. Eliud Sagwa Mulanda, Mark Ochieng Adero, Nelson Onzere Amugune, Elijah Akunda, and Jenesio I. Kinyamario Copyright © 2012 Eliud Sagwa Mulanda et al. All rights reserved. Production of Alkaline Cellulase by Fungi Isolated from an Undisturbed Rain Forest of Peru Mon, 19 Nov 2012 11:38:31 +0000 http://www.hindawi.com/journals/btri/2012/934325/ Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL−1) with higher specific productivities (>30 U g−1 h−1). Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry. Karin Vega, Gretty K. Villena, Victor H. Sarmiento, Yvette Ludeña, Nadia Vera, and Marcel Gutiérrez-Correa Copyright © 2012 Karin Vega et al. All rights reserved. In Vitro Propagation of Muña-Muña (Clinopodium odorum (Griseb.) Harley) Thu, 18 Oct 2012 14:19:04 +0000 http://www.hindawi.com/journals/btri/2012/196583/ A micropropagation protocol was developed which may assist in the safeguarding and augmentation of dwindling natural populations of Clinopodium odorum (Griseb.) Harley, a critically and endangered medicinal plant. Factors affecting culture initiation bud sprouting and growth, rooting, and acclimatization were studied, using nodal segments of in vitro germinated seedling as primary explants on six media supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) (0.5–1.5 and 2-Naphthalene acetic acid (NAA) (0.5–1.5). Best results for culture initiation with sustainable multiplication rates (100%) were obtained on WP medium without any growth regulator. WP with the addition of 0.5 : 1 or 0.5 : 1.5) of BAP and NAA promoted a higher elongation; however, the optimum number of nodes were obtained in plantlets grown on 1/2 MS with the addition of 1 : 1.5 of BAP and NAA. Culture of sectioned individual nodes transferred to the media with different rates of BAP and NAA 1/2 MS-9 (1.5 : 1.5), SH-8 (1.5 : 1.0), and 1/2 B5-4 (1.0 : 0.5) media resulted in no proliferated shoots. The in vitro plants were successfully acclimatized garden soil and sand (2 : 1) in the greenhouse, with over 90% survival rate. The in vitro-grown plants could be transferred to ex vitro conditions and the efficacy in supporting ex vitro growth was assessed, with a view to develope longer-term strategies for the transfer and reintroduction into natural habitats. María Soledad Diaz, Lorena Palacio, Ana Cristina Figueroa, and Marta Ester Goleniowski Copyright © 2012 María Soledad Diaz et al. All rights reserved. Recent Advances in the Genetic Transformation of Coffee Wed, 29 Aug 2012 14:27:23 +0000 http://www.hindawi.com/journals/btri/2012/580857/ Coffee is one of the most important plantation crops, grown in about 80 countries across the world. The genus Coffea comprises approximately 100 species of which only two species, that is, Coffea arabica (commonly known as arabica coffee) and Coffea canephora (known as robusta coffee), are commercially cultivated. Genetic improvement of coffee through traditional breeding is slow due to the perennial nature of the plant. Genetic transformation has tremendous potential in developing improved coffee varieties with desired agronomic traits, which are otherwise difficult to achieve through traditional breeding. During the last twenty years, significant progress has been made in coffee biotechnology, particularly in the area of transgenic technology. This paper provides a detailed account of the advances made in the genetic transformation of coffee and their potential applications. M. K. Mishra and A. Slater Copyright © 2012 M. K. Mishra and A. Slater. All rights reserved.