Enzyme Research http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility Sun, 31 Jan 2016 08:52:50 +0000 http://www.hindawi.com/journals/er/2016/5098985/ Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. Madison A. Smith, Jesica Gonzalez, Anjum Hussain, Rachel N. Oldfield, Kathryn A. Johnston, and Karlo M. Lopez Copyright © 2016 Madison A. Smith et al. All rights reserved. Characterization of a Hyperthermostable Alkaline Lipase from Bacillus sonorensis 4R Thu, 21 Jan 2016 08:57:37 +0000 http://www.hindawi.com/journals/er/2016/4170684/ Hyperthermostable alkaline lipase from Bacillus sonorensis 4R was purified and characterized. The enzyme production was carried out at 80°C and 9.0 pH in glucose-tween inorganic salt broth under static conditions for 96 h. Lipase was purified by anion exchange chromatography by 12.15 fold with a yield of 1.98%. The molecular weight of lipase was found to be 21.87 KDa by SDS-PAGE. The enzyme activity was optimal at 80°C with of 150 min and at 90°C, 100°C, 110°C, and 120°C; the respective values were 121.59 min, 90.01 min, 70.01 min, and 50 min. The enzyme was highly activated by Mg and values at 80°C were increased from 150 min to 180 min when magnesium and mannitol were added in combination. The activation energy calculated from Arrhenius plot was 31.102 KJ/mol. At 80–120°C, values of and were in the range of 28.16–27.83 KJ/mol and 102.79 KJ/mol to 111.66 KJ/mol, respectively. Lipase activity was highest at 9.0 pH and stable for 2 hours at this pH at 80°C. Pretreatment of lipase with MgSO4 and CaSO4 stimulated enzyme activity by 249.94% and 30.2%, respectively. The enzyme activity was greatly reduced by CoCl2, CdCl2, HgCl2, CuCl2, Pb(NO3)2, PMSF, orlistat, oleic acid, iodine, EDTA, and urea. Hemlata Bhosale, Uzma Shaheen, and Tukaram Kadam Copyright © 2016 Hemlata Bhosale et al. All rights reserved. Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324 Tue, 19 Jan 2016 12:38:43 +0000 http://www.hindawi.com/journals/er/2016/4379403/ A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability () of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS). Preety Vatsyayan and Pranab Goswami Copyright © 2016 Preety Vatsyayan and Pranab Goswami. All rights reserved. Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry Sun, 03 Jan 2016 13:08:46 +0000 http://www.hindawi.com/journals/er/2016/9034364/ Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v), respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v), 4% (v/v), and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5%) or the sole crude enzyme (8.9%). It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards. Deyaa M. Abol Fotouh, Reda A. Bayoumi, and Mohamed A. Hassan Copyright © 2016 Deyaa M. Abol Fotouh et al. All rights reserved. Immunomodulatory Effects of Chitotriosidase Enzyme Sun, 03 Jan 2016 09:04:49 +0000 http://www.hindawi.com/journals/er/2016/2682680/ Chitotriosidase enzyme (EC: is the major active chitinase in the human body. It is produced mainly by activated macrophages, in which its expression is regulated by multiple intrinsic and extrinsic signals. Chitotriosidase was confirmed as essential element in the innate immunity against chitin containing organisms such as fungi and protozoa; however, its immunomodulatory effects extend far beyond innate immunity. In the current review, we will try to explore the expanding spectrum of immunological roles played by chitotriosidase enzyme in human health and disease and will discuss its up-to-date clinical value. Mohamed A. Elmonem, Lambertus P. van den Heuvel, and Elena N. Levtchenko Copyright © 2016 Mohamed A. Elmonem et al. All rights reserved. Modulation of Aromatase by Phytoestrogens Mon, 21 Dec 2015 14:34:47 +0000 http://www.hindawi.com/journals/er/2015/594656/ The aromatase enzyme catalyzes the conversion of androgens to estrogens in many human tissues. Estrogens are known to stimulate cellular proliferation associated with certain cancers and protect against adverse symptoms during the peri- and postmenopausal intervals. Phytoestrogens are a group of plant derived naturally occurring compounds that have chemical structures similar to estrogen. Since phytoestrogens are known to be constituents of animal/human food sources, these compounds have received increased research attention. Phytoestrogens may contribute to decreased cancer risk by the inhibition of aromatase enzyme activity and CYP19 gene expression in human tissues. This review covers (a) the aromatase enzyme (historical descriptions on function, activity, and gene characteristics), (b) phytoestrogens in their classifications and applications to human health, and (c) a chronological coverage of aromatase activity modulated by phytoestrogens from the early 1980s to 2015. In general, phytoestrogens act as aromatase inhibitors by (a) decreasing aromatase gene expression, (b) inhibiting the aromatase enzyme itself, or (c) in some cases acting at both levels of regulation. The findings presented herein are consistent with estrogen’s impact on health and phytoestrogen’s potential as anticancer treatments, but well-controlled, large-scale studies are warranted to determine the effectiveness of phytoestrogens on breast cancer and age-related diseases. Edwin D. Lephart Copyright © 2015 Edwin D. Lephart. All rights reserved. Actinomycetes: A Source of Lignocellulolytic Enzymes Thu, 17 Dec 2015 14:21:00 +0000 http://www.hindawi.com/journals/er/2015/279381/ Lignocellulose is the most abundant biomass on earth. Agricultural, forest, and agroindustrial activities generate tons of lignocellulosic wastes annually, which present readily procurable, economically affordable, and renewable feedstock for various lignocelluloses based applications. Lignocelluloses are the focus of present decade researchers globally, in an attempt to develop technologies based on natural biomass for reducing dependence on expensive and exhaustible substrates. Lignocellulolytic enzymes, that is, cellulases, hemicellulases, and lignolytic enzymes, play very important role in the processing of lignocelluloses which is prerequisite for their utilization in various processes. These enzymes are obtained from microorganisms distributed in both prokaryotic and eukaryotic domains including bacteria, fungi, and actinomycetes. Actinomycetes are an attractive microbial group for production of lignocellulose degrading enzymes. Various studies have evaluated the lignocellulose degrading ability of actinomycetes, which can be potentially implemented in the production of different value added products. This paper is an overview of the diversity of cellulolytic, hemicellulolytic, and lignolytic actinomycetes along with brief discussion of their hydrolytic enzyme systems involved in biomass modification. Anita Saini, Neeraj K. Aggarwal, Anuja Sharma, and Anita Yadav Copyright © 2015 Anita Saini et al. All rights reserved. Estimation of Inhibitory Effect against Tyrosinase Activity through Homology Modeling and Molecular Docking Tue, 15 Dec 2015 14:24:24 +0000 http://www.hindawi.com/journals/er/2015/262364/ Tyrosinase is a key enzyme in melanogenesis. Generally, mushroom tyrosinase from A. bisporus had been used as a model in skin-whitening agent tests employed in the cosmetic industry. The recently obtained crystal structure of bacterial tyrosinase from B. megaterium has high similarity (33.5%) to the human enzyme and thus it was used as a template for constructing of the human model. Binding of tyrosinase to a series of its inhibitors was simulated by automated docking calculations. Docking and MD simulation results suggested that N81, N260, H263, and M280 are involved in the binding of inhibitors to mushroom tyrosinase. E195 and H208 are important residues in bacterial tyrosinase, while E230, S245, N249, H252, V262, and S265 bind to inhibitors and are important in forming pi interaction in human tyrosinase. Daungkamon Nokinsee, Lalida Shank, Vannajan Sanghiran Lee, and Piyarat Nimmanpipug Copyright © 2015 Daungkamon Nokinsee et al. 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.