Enzyme Research http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Chitinases from Bacteria to Human: Properties, Applications, and Future Perspectives Thu, 19 Nov 2015 12:14:46 +0000 http://www.hindawi.com/journals/er/2015/791907/ Chitin is the second most plenteous polysaccharide in nature after cellulose, present in cell walls of several fungi, exoskeletons of insects, and crustacean shells. Chitin does not accumulate in the environment due to presence of bacterial chitinases, despite its abundance. These enzymes are able to degrade chitin present in the cell walls of fungi as well as the exoskeletons of insect. They have shown being the potential agents for biological control of the plant diseases caused by various pathogenic fungi and insect pests and thus can be used as an alternative to chemical pesticides. There has been steady increase in demand of chitin derivatives, obtained by action of chitinases on chitin polymer for various industrial, clinical, and pharmaceutical purposes. Hence, this review focuses on properties and applications of chitinases starting from bacteria, followed by fungi, insects, plants, and vertebrates. Designing of chitinase by applying directed laboratory evolution and rational approaches for improved catalytic activity for cost-effective field applications has also been explored. Abhishek Singh Rathore and Rinkoo D. Gupta Copyright © 2015 Abhishek Singh Rathore and Rinkoo D. Gupta. All rights reserved. Extracellular Polyhydroxyalkanoate Depolymerase by Acidovorax sp. DP5 Tue, 17 Nov 2015 10:51:29 +0000 http://www.hindawi.com/journals/er/2015/212159/ Bacteria capable of degrading polyhydroxyalkanoates (PHA) by secreting extracellular depolymerase enzymes were isolated from water and soil samples collected from various environments in Malaysia. A total of 8 potential degraders exhibited clear zones on poly(3-hydroxybutyrate) [P(3HB)] based agar, indicating the presence of extracellular PHA depolymerase. Among the isolates, DP5 exhibited the largest clearing zone with a degradation index of 6.0. The highest degradation activity of P(3HB) was also observed with depolymerase enzyme of DP5 in mineral salt medium containing P(3HB). Based on biochemical characterization and 16S rRNA cloning and sequencing, isolate DP5 was found to belong to the genus Acidovorax and subsequently named as Acidovorax sp. DP5. The highest extracellular depolymerase enzyme activity was achieved when 0.25% (w/v) of P(3HB) and 1 g/L of urea were used as carbon and nitrogen source, respectively, in the culture media. The most suitable assay condition of the depolymerase enzyme in response to pH and temperature was tested. The depolymerase produced by strain Acidovorax sp. DP5 showed high percentage of degradation with P(3HB) films in an alkaline condition with pH 9 and at a temperature of 40°C. S. Vigneswari, T. S. Lee, Kesaven Bhubalan, and A. A. Amirul Copyright © 2015 S. Vigneswari et al. All rights reserved. Determining the IC50 Values for Vorozole and Letrozole, on a Series of Human Liver Cytochrome P450s, to Help Determine the Binding Site of Vorozole in the Liver Mon, 09 Nov 2015 09:35:22 +0000 http://www.hindawi.com/journals/er/2015/321820/ Vorozole and letrozole are third-generation aromatase (cytochrome P450 19A1) inhibitors. [11C]-Vorozole can be used as a radiotracer for aromatase in living animals but when administered by IV, it collects in the liver. Pretreatment with letrozole does not affect the binding of vorozole in the liver. In search of finding the protein responsible for the accumulation of vorozole in the liver, fluorometric high-throughput screening assays were used to test the inhibitory capability of vorozole and letrozole on a series of liver cytochrome P450s (CYP1A1, CYP1A2, CYP2A6, and CYP3A4). It was determined that vorozole is a potent inhibitor of CYP1A1 (IC50 = 0.469 μM) and a moderate inhibitor of CYP2A6 and CYP3A4 (IC50 = 24.4 and 98.1 μM, resp.). Letrozole is only a moderate inhibitor of CYP1A1 and CYP2A6 (IC50 = 69.8 and 106 μM) and a very weak inhibitor of CYP3A4 (<10% inhibition at 1 mM). Since CYP3A4 makes up the majority of the CYP content found in the human liver, and vorozole inhibits it moderately well but letrozole does not, CYP3A4 is a good candidate for the protein that [11C]-vorozole is binding to in the liver. Lendelle Raymond, Nikita Rayani, Grace Polson, Kylie Sikorski, Ailin Lian, and Melissa A. VanAlstine-Parris Copyright © 2015 Lendelle Raymond et al. All rights reserved. Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases Mon, 26 Oct 2015 11:55:04 +0000 http://www.hindawi.com/journals/er/2015/806240/ This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry. Michele Dutra Rosolen, Adriano Gennari, Giandra Volpato, and Claucia Fernanda Volken de Souza Copyright © 2015 Michele Dutra Rosolen et al. All rights reserved. Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity Thu, 22 Oct 2015 06:32:29 +0000 http://www.hindawi.com/journals/er/2015/370705/ Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte’s AChE exhibited for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type ( value, 3.6 mM) which negatively influenced both the and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead. Vivek Kumar Gupta, Rajnish Pal, Nikhat Jamal Siddiqi, and Bechan Sharma Copyright © 2015 Vivek Kumar Gupta et al. All rights reserved. Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase Wed, 21 Oct 2015 14:25:59 +0000 http://www.hindawi.com/journals/er/2015/404607/ Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate . In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity. Paola R. Beassoni, Lucas A. Gallarato, Cristhian Boetsch, Mónica N. Garrido, and Angela T. Lisa Copyright © 2015 Paola R. Beassoni et al. All rights reserved. Improved Enzyme Catalytic Characteristics upon Glutaraldehyde Cross-Linking of Alginate Entrapped Xylanase Isolated from Aspergillus flavus MTCC 9390 Wed, 12 Aug 2015 14:21:20 +0000 http://www.hindawi.com/journals/er/2015/210784/ Purified fungal xylanase was entrapped in alginate beads. Its further cross-linking using glutaraldehyde resulted in large enzyme aggregates which may function as both a catalyst and a support material for numerous substrate molecules. Enzyme cross-linking presented a negative impact on enzyme leaching during repeated washings and recovery of enzyme activity was substantial after twelve cycles of usage. The entrapment followed by cross-linking doubled the total bound activity and also greatly improved the enzyme stability at extreme chemical environment. The wide pH stability, better thermo- and storage stability, lowered Km value, and protection from some metal ions are salient achievements of present immobilization. The study shows the efficacy, durability, and sustainability of immobilized catalytic system which could be efficiently used for various juice processing operations. Bharat Bhushan, Ajay Pal, and Veena Jain Copyright © 2015 Bharat Bhushan et al. All rights reserved. Effect of Diffusion on Discoloration of Congo Red by Alginate Entrapped Turnip (Brassica rapa) Peroxidase Thu, 05 Feb 2015 08:46:10 +0000 http://www.hindawi.com/journals/er/2015/575618/ Enzymatic discoloration of the diazo dye, Congo red (CR), by immobilized plant peroxidase from turnip “Brassica rapa” is investigated. Partially purified turnip peroxidase (TP) was immobilized by entrapment in spherical particles of calcium alginate and was assayed for the discoloration of aqueous CR solution. Experimental data revealed that pH, reaction time, temperature, colorant, and H2O2 concentration play a significant role in dye degradation. Maximum CR removal was found at pH 2.0, constant temperature of 40°C in the presence of 10 mM H2O2, and 180 mg/L of CR. More than 94% of CR was removed by alginate immobilized TP after 1 h of incubation in a batch process under optimal conditions. About 74% removal efficiency was retained after four recycles. Diffusional limitations in alginate beads such as effectiveness factor η, Thiele modulus , and effective diffusion coefficients (De) of Congo red were predicted assuming a first-order biodegradation kinetic. Results showed that intraparticle diffusion resistance has a significant effect on the CR biodegradation rate. Afaf Ahmedi, Mahmoud Abouseoud, Amrane Abdeltif, and Couvert Annabelle Copyright © 2015 Afaf Ahmedi et al. All rights reserved. An Efficient and Improved Methodology for the Screening of Industrially Valuable Xylano-Pectino-Cellulolytic Microbes Mon, 26 Jan 2015 06:43:34 +0000 http://www.hindawi.com/journals/er/2015/725281/ Xylano-pectino-cellulolytic enzymes are valuable enzymes of the industrial sector. In our earlier study, we have reported a novel and cost effective methodology for the qualitative screening of cellulase-free xylano-pectinolytic microorganisms by replacing the commercial, highly expensive substrates with agricultural residues, but the microorganisms with xylanolytic, pectinolytic, cellulolytic, xylano-pectinolytic, xylano-cellulolytic, pectino-cellulolytic, and xylano-pectino-cellulolytic potential were obtained. The probability of getting the desired combination was low, so efforts were made to further improve this cost effective methodology for obtaining the high yield of the microbes capable of producing desired combination of enzymes. By inclusion of multiple enrichment steps in sequence, using only practically low cost substrates and without any nutrient media till primary screening stage, this improved novel protocol for screening gave only the desired microorganisms with xylano-pectino-cellulolytic activity. Using this rapid, efficient, cost effective, and improved methodology, microbes with required combination of enzymes can be obtained and the probability of getting the desired microorganisms is cent percent. This is the first report presenting the methodology for the isolation of xylano-pectino-cellulolytic positive microorganisms at low cost and consuming less time. Avtar Singh, Amanjot Kaur, Anita Dua, and Ritu Mahajan Copyright © 2015 Avtar Singh et al. All rights reserved. Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus Mon, 19 Jan 2015 10:07:03 +0000 http://www.hindawi.com/journals/er/2015/837842/ DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX ( and γ), and components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions. Wendy Ribble, Shawn D. Kane, and James M. Bullard Copyright © 2015 Wendy Ribble et al. All rights reserved. Chloride Activated Halophilic α-Amylase from Marinobacter sp. EMB8: Production Optimization and Nanoimmobilization for Efficient Starch Hydrolysis Sun, 18 Jan 2015 11:26:21 +0000 http://www.hindawi.com/journals/er/2015/859485/ Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Present work encompasses production optimization and nanoimmobilization of an α-amylase from moderately halophilic Marinobacter sp. EMB8. Media ingredients and culture conditions were optimized by “one-at-a-time approach.” Starch was found to be the best carbon source at 5% (w/v) concentration. Glucose acted as catabolic repressor for amylase production. Salt proved critical for amylase production and maximum production was attained at 5% (w/v) NaCl. Optimization of various culture parameters resulted in 48.0 IU/mL amylase production, a 12-fold increase over that of unoptimized condition (4.0 IU/mL). α-Amylase was immobilized on 3-aminopropyl functionalized silica nanoparticles using glutaraldehyde as cross-linking agent. Optimization of various parameters resulted in 96% immobilization efficiency. Starch hydrolyzing efficiency of immobilized enzyme was comparatively better. Immobilized α-amylase retained 75% of its activity after 5th cycle of repeated use. Sumit Kumar and S. K. Khare Copyright © 2015 Sumit Kumar and S. K. Khare. All rights reserved. Immobilization of Papain on Chitin and Chitosan and Recycling of Soluble Enzyme for Deflocculation of Saccharomyces cerevisiae from Bioethanol Distilleries Thu, 01 Jan 2015 09:34:47 +0000 http://www.hindawi.com/journals/er/2015/573721/ Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1–10% w·v−1), polyethyleneimine (0.5% v·v−1), and tripolyphosphate (1–10% w·v−1) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L−1. Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes. Douglas Fernandes Silva, Henrique Rosa, Ana Flavia Azevedo Carvalho, and Pedro Oliva-Neto Copyright © 2015 Douglas Fernandes Silva et al. All rights reserved. Immobilization of Aspergillus niger F7-02 Lipase in Polysaccharide Hydrogel Beads of Irvingia gabonensis Matrix Wed, 31 Dec 2014 13:49:04 +0000 http://www.hindawi.com/journals/er/2014/967056/ The potential of polysaccharide Irvingia gabonensis matrix as enzyme immobilization support was investigated. Lipase of Aspergillus niger F7-02 was immobilized by entrapment using glutaraldehyde as the cross-linking agent and stabilized in ethanolic-formaldehyde solution. The pH and temperature stability and activity yield of the immobilized enzyme were determined. Such parameters as enzyme load, bead size, number of beads, and bead reusability were also optimized. Adequate gel strength to form stabilized beads was achieved at 15.52% (w/v) Irvingia gabonensis powder, 15% (v/v) partially purified lipase, 2.5% (v/v) glutaraldehyde, and 3 : 1 (v/v) ethanolic-formaldehyde solution. There was 3.93-fold purification when the crude enzyme was partially purified in two-step purification using Imarsil and activated charcoal. Optimum lipase activity 75.3 Ug−1 was achieved in 50 mL test solution containing 15 beads of 7 mm bead size. Relative activity 80% was retained at eight repeated cycles. The immobilization process gave activity yield of 59.1% with specific activity of 12.3 Umg−1 and stabilized at optimum pH 4.5 and temperature 55°C. Thus the effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established. Safaradeen Olateju Kareem, Olayinka Quadri Adio, and Michael Bamitale Osho Copyright © 2014 Safaradeen Olateju Kareem et al. All rights reserved. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation Wed, 31 Dec 2014 09:41:53 +0000 http://www.hindawi.com/journals/er/2014/353915/ A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking. Danielle Biscaro Pedrolli and Eleonora Cano Carmona Copyright © 2014 Danielle Biscaro Pedrolli and Eleonora Cano Carmona. All rights reserved. Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver Thu, 25 Dec 2014 00:10:08 +0000 http://www.hindawi.com/journals/er/2014/714054/ Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver. Mahmoud A. Ibrahim, Abdel-Hady M. Ghazy, Ahmed M. H. Salem, Mohamed A. Ghazy, and Mohamed M. Abdel-Monsef Copyright © 2014 Mahmoud A. Ibrahim et al. All rights reserved. Assessment of Serum Enzymatic Antioxidant Levels in Patients with Recurrent Aphthous Stomatitis: A Case Control Study Wed, 10 Dec 2014 06:40:38 +0000 http://www.hindawi.com/journals/er/2014/340819/ Background and Aim. Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae. Despite extensive research, the exact etiology of RAS remains elusive. Recently oxidant-antioxidant imbalance of the body has been implicated in the pathogenesis of recurrent aphthous stomatitis. Thus, the aim of the study was to evaluate the enzymatic antioxidant levels in patients with recurrent aphthous stomatitis. Materials and Methods. The serum levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured in 30 patients with recurrent aphthous stomatitis and compared to the control group, which included 30 healthy subjects. Student’s -test was performed for statistical evaluation. Results. The mean levels of superoxide dismutase (130.2 ± 15.94 U/mL) and glutathione peroxidase (3527.93 ± 488.32 U/L) were found to be significantly lower in study group as compared to control group (211.9 ± 20.93 U/mL, 8860.93 ± 1105.31 U/L, resp.) () while level of catalase in study group was significantly higher when compared to control group (10981.00 ± 1018.07 U/mL versus 9764.00 ± 1621.19 U/mL) (). Conclusion. Enzymatic antioxidant system is impaired in recurrent aphthous stomatitis patients and seems to play a crucial role in its pathogenesis. Ishita Gupta, Arvind Shetti, Vaishali Keluskar, and Anjana Bagewadi Copyright © 2014 Ishita Gupta et al. All rights reserved. A Fractional Factorial Design to Study the Effect of Process Variables on the Preparation of Hyaluronidase Loaded PLGA Nanoparticles Wed, 10 Dec 2014 00:10:26 +0000 http://www.hindawi.com/journals/er/2014/162962/ The present study was initiated to understand the effect of PLGA concentration, PVA concentration, internal-external phase ratio, homogenization speed, and homogenization time on mean particle size, zeta potential, and percentage drug encapsulation using fractional factorial design. Using PLGA (50-50) as the carrier, hyaluronidase loaded PLGA nanoparticles were prepared using double emulsion solvent evaporation technique. The particle size was analyzed by dynamic light scattering technique and protein content by Lowry method. The study showed that homogenization speed as an independent variable had maximum effect on particle size and zeta potential. Internal-external phase volume ratio had maximum effect on drug encapsulation. Mean particle size also had high dependency on the combined effect of PVA concentration and phase volume ratio. Using fractional factorial design particle size of <400 nm, zeta potential of <−30 mV, and percentage encapsulation of 15–18% were achieved. K. Narayanan, V. M. Subrahmanyam, and J. Venkata Rao Copyright © 2014 K. Narayanan et al. All rights reserved. Contemporaneous Production of Amylase and Protease through CCD Response Surface Methodology by Newly Isolated Bacillus megaterium Strain B69 Wed, 12 Nov 2014 08:45:23 +0000 http://www.hindawi.com/journals/er/2014/601046/ The enormous increase in world population has resulted in generation of million tons of agricultural wastes. Biotechnological process for production of green chemicals, namely, enzymes, provides the best utilization of these otherwise unutilized wastes. The present study elaborates concomitant production of protease and amylase in solid state fermentation (SSF) by a newly isolated Bacillus megaterium B69, using agroindustrial wastes. Two-level statistical model employing Plackett-Burman and response surface methodology was designed for optimization of various physicochemical conditions affecting the production of two enzymes concomitantly. The studies revealed that the new strain concomitantly produced 1242 U/g of protease and 1666.6 U/g of amylase by best utilizing mustard oilseed cake as the substrate at 20% substrate concentration and 45% moisture content after 84 h of incubation. An increase of 2.95- and 2.04-fold from basal media was observed in protease and amylase production, respectively. ANOVA of both the design models showed high accuracy of the polynomial model with significant similarities between the predicted and the observed results. The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale. Rajshree Saxena and Rajni Singh Copyright © 2014 Rajshree Saxena and Rajni Singh. All rights reserved. Partial Purification and Characterization of a Heat Stable α-Amylase from a Thermophilic Actinobacteria, Streptomyces sp. MSC702 Wed, 08 Oct 2014 00:00:00 +0000 http://www.hindawi.com/journals/er/2014/106363/ A partial purification and biochemical characterization of the α-amylase from Streptomyces sp. MSC702 were carried out in this study. The optimum operational conditions for enzyme substrate reaction for amylolytic enzyme activity from the strain were evaluated. The optimum pH, temperature, and incubation period for assaying the enzyme were observed to be 5.0, 55°C, and 30 min, respectively. The extracellular extract was concentrated using ammonium sulfate precipitation. It was stable in the presence of metal ions (5 mM) such as K+, Co2+, and Mo2+, whereas Pb2+, Mn2+, Mg2+, Cu2+, Zn2+, Ba2+, Ca2+, Hg2+, Sn2+, Cr3+, Al3+, Ag+, and Fe2+ were found to have inhibitory effects. The enzyme activity was also unstable in the presence of 1% Triton X-100, 1% Tween 80, 5 mM sodium lauryl sulphate, 1% glycerol, 5 mM EDTA, and 5 mM denaturant urea. At temperature 60°C and pH 5.0, the enzyme stability was maximum. α-amylase retained 100% and 34.18% stability for 1 h and 4 h, respectively, at 60°C (pH 7.0). The enzyme exhibited a half-life of 195 min at 60°C temperature. The analysis of kinetic showed that the enzyme has of 2.4 mg/mL and of 21853.0 μmol/min/mg for soluble potato starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization. Renu Singh, Vijay Kumar, and Vishal Kapoor Copyright © 2014 Renu Singh et al. All rights reserved. Angiotensin Converting Enzyme Activity in Alopecia Areata Wed, 01 Oct 2014 09:44:48 +0000 http://www.hindawi.com/journals/er/2014/694148/ Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (). Also, there was no correlation between ACE activity and severity () and duration of disease () in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. Mohammad Reza Namazi, Armaghan Ashraf, Farhad Handjani, Ebrahim Eftekhar, and Amir Kalafi Copyright © 2014 Mohammad Reza Namazi et al. All rights reserved. A Simple Route for Purifying Extracellular Poly(3-hydroxybutyrate)-depolymerase from Penicillium pinophilum Tue, 23 Sep 2014 08:43:53 +0000 http://www.hindawi.com/journals/er/2014/159809/ This work proposes the purification of an active and efficient enzyme, extracellular poly(3-hydroxybutyrate) (PHB)-depolymerase, suitable for industrial applications. This is achieved by the application of an easy, fast, and cheap route, skipping the chromatography step. Chromatography with one or two columns is a common step in the purification procedure, which however renders the isolation of the enzyme a time consuming and an expensive process. A strain of the fungus Penicillium pinophilum (ATCC 9644) is used for the isolation of extracellular PHB-depolymerase. The molecular weight of the purified enzyme is about 35 kDa and is estimated by gel electrophoresis (SDS-PAGE, 12% polyacrylamide). The enzymatic activity of the isolated enzyme is determined to be 3.56-fold similar to that found by other researchers that have used chromatography for the isolation. The as-isolated enzyme disintegrates the poly(3-hydroxybutyrate) (PHB) films successfully, as it is demonstrated by the biodegradation test results provided here. Elpiniki Panagiotidou, Constantinos Konidaris, Apostolos Baklavaridis, Ioannis Zuburtikudis, Dimitris Achilias, and Paraskevi Mitlianga Copyright © 2014 Elpiniki Panagiotidou et al. All rights reserved. Mode of Action of Lactoperoxidase as Related to Its Antimicrobial Activity: A Review Tue, 16 Sep 2014 09:03:49 +0000 http://www.hindawi.com/journals/er/2014/517164/ Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity. Their best known effect is their antimicrobial activity that arouses much interest in in vivo and in vitro applications. In this context, the proper use of lactoperoxidase needs a good understanding of its mode of action, of the factors that favor or limit its activity, and of the features and properties of the active molecules. The first part of this review describes briefly the classification of mammalian peroxidases and their role in the human immune system and in host cell damage. The second part summarizes present knowledge on the mode of action of lactoperoxidase, with special focus on the characteristics to be taken into account for in vitro or in vivo antimicrobial use. The last part looks upon the characteristics of the active molecule produced by lactoperoxidase in the presence of thiocyanate and/or iodide with implication(s) on its antimicrobial activity. F. Bafort, O. Parisi, J.-P. Perraudin, and M. H. Jijakli Copyright © 2014 F. Bafort et al. All rights reserved. Immobilization of Lipase on Silver Nanoparticles via Adhesive Polydopamine for Biodiesel Production Wed, 10 Sep 2014 07:52:41 +0000 http://www.hindawi.com/journals/er/2014/389739/ Biodiesel production technology is competitive in terms of low cost and alternative source of energy which should be not only sustainable but also environmentally friendly. Designing of the lipase immobilization for biodiesel production has a remarkable impact and is still challenging. In this work, biodiesel production from soybean oil was enhanced and facilitated by using a novel biocatalyst consisting of commercial lipase (EC, silver nanoparticles, and polydopamine. Silver nanoparticles (AgNPs) were synthesized with a size range of 10–20 nm. Polydopamine (PD) was delivered by the self-polymerization of dopamine in 10 mM Tris-HCl pH 8.5 and simultaneously coated the AgNPs to form a PD/AgNPs complex. Lipase was immobilized on the PD/AgNPs complex surface via covalent bonds to form a tailor-made biocatalyst consisting of immobilized lipase/PD/AgNPs complex (LPA). The formation and morphology of each composition were characterized by UV-Vis spectroscopy and scanning electron microscope (SEM). Significantly, gas chromatography analysis showed a remarkable biodiesel production yield of 95% by using the LPA complex at 40°C for 6-hours reaction time, whereas the yield was 86% when using free lyophilized lipase. The LPA complex was apparently reusable after 7 batches and the latter conversion rate of soybean oil was decreased by only 27%. Kanchana Dumri and Dau Hung Anh Copyright © 2014 Kanchana Dumri and Dau Hung Anh. All rights reserved. Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study Sun, 07 Sep 2014 08:11:01 +0000 http://www.hindawi.com/journals/er/2014/708676/ The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C. The shift of the spectral centre of mass above 50°C is also apparently cooperative with midpoint transition of 56.3 ± 0.2°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process. Further corroboration comes from differential scanning calorimetry experiments. At protein concentrations ≤0.56 mg·mL−1 the calorimetric transition is reversible and the data were fitted to a non-two-state model and deconvoluted into six transitions, whereas at concentrations greater than 0.56 mg·mL−1 the calorimetric transition is irreversible with an exothermic contribution to the thermogram. The apparent increased linearly with the scan rate according to first order inactivation kinetics. The effect of additives on the calorimetric transition of xylanase is dependent on their nature. The addition of sorbitol transforms reversible transitions into irreversible transitions while stabilizing the protein as the apparent increases linearly with sorbitol concentration. D-Glucono-1,5-lactone, a noncompetitive inhibitor in xylanase kinetics, and soluble xylan change irreversible processes into reversible processes at high protein concentration. Gloria López and Pilar Estrada Copyright © 2014 Gloria López and Pilar Estrada. All rights reserved. Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom Thu, 14 Aug 2014 13:11:05 +0000 http://www.hindawi.com/journals/er/2014/120739/ Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications. Kamal Uddin Zaidi, Ayesha S. Ali, and Sharique A. Ali Copyright © 2014 Kamal Uddin Zaidi et al. All rights reserved. Sequential Statistical Optimization of Media Components for the Production of Glucoamylase by Thermophilic Fungus Humicola grisea MTCC 352 Wed, 09 Jul 2014 13:25:28 +0000 http://www.hindawi.com/journals/er/2014/317940/ Glucoamylase is an industrially important enzyme which converts soluble starch into glucose. The media components for the production of glucoamylase from thermophilic fungus Humicola grisea MTCC 352 have been optimized. Eight media components, namely, soluble starch, yeast extract, KH2PO4, K2HPO4, NaCl, CaCl2, MgSO4·7H2O, and Vogel’s trace elements solution, were first screened for their effect on the production of glucoamylase and only four components (soluble starch, yeast extract, K2HPO4, and MgSO4·7H2O) were identified as statistically significant using Plackett-Burman design. It was fitted into a first-order model (). Steepest ascent method was performed to identify the location of optimum. Central composite design was employed to determine the optimum values (soluble starch: 28.41 g/L, yeast extract: 9.61 g/L, K2HPO4: 2.42 g/L, and MgSO4·7H2O: 1.91 g/L). The experimental activity of 12.27 U/mL obtained was close to the predicted activity of 12.15. High value (0.9397), low PRESS value (9.47), and AARD values (2.07%) indicate the accuracy of the proposed model. The glucoamylase production was found to increase from 4.57 U/mL to 12.27 U/mL, a 2.68-fold enhancement, as compared to the unoptimized medium. Vinayagam Ramesh and Vytla Ramachandra Murty Copyright © 2014 Vinayagam Ramesh and Vytla Ramachandra Murty. All rights reserved. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives Thu, 03 Jul 2014 07:34:57 +0000 http://www.hindawi.com/journals/er/2014/109303/ A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory. Nilesh P. Nirmal and R. Seeta Laxman Copyright © 2014 Nilesh P. Nirmal and R. Seeta Laxman. All rights reserved. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 Mon, 30 Jun 2014 11:02:06 +0000 http://www.hindawi.com/journals/er/2014/197938/ Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. Norsyuhada Alias, Mu’adz Ahmad Mazian, Abu Bakar Salleh, Mahiran Basri, and Raja Noor Zaliha Raja Abd. Rahman Copyright © 2014 Norsyuhada Alias et al. All rights reserved. Optimisation of Cellulase Production by Penicillium funiculosum in a Stirred Tank Bioreactor Using Multivariate Response Surface Analysis Wed, 25 Jun 2014 15:29:11 +0000 http://www.hindawi.com/journals/er/2014/703291/ Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases), which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD) was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L−1) as carbon source was determined to include urea (1.2 g·L−1), yeast extract (1.0 g·L−1), KH2PO4 (6.0 g·L−1), and MgSO4·7H2O (1.2 g·L−1). The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L−1 for FPase, 9,204 U·L−1 for endoglucanase, and 2,395 U·L−1 for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions. Marcelle Lins de Albuquerque de Carvalho, Daniele Fernandes Carvalho, Edelvio de Barros Gomes, Roberto Nobuyuki Maeda, Lidia Maria Melo Santa Anna, Aline Machado de Castro, and Nei Pereira Jr. Copyright © 2014 Marcelle Lins de Albuquerque de Carvalho et al. All rights reserved. Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors Thu, 19 Jun 2014 12:11:54 +0000 http://www.hindawi.com/journals/er/2014/848937/ Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed. Bui T. T. Nga, Yuki Takeshita, Misa Yamamoto, and Yoshimi Yamamoto Copyright © 2014 Bui T. T. Nga et al. All rights reserved.