Enzyme Research https://www.hindawi.com The latest articles from Hindawi © 2018 , Hindawi Limited . All rights reserved. The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View Tue, 05 Dec 2017 00:00:00 +0000 http://www.hindawi.com/journals/er/2017/4086845/ Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme’s active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch. Umi Baroroh, Muhammad Yusuf, Saadah Diana Rachman, Safri Ishmayana, Mas Rizky A. A. Syamsunarno, Jutti Levita, and Toto Subroto Copyright © 2017 Umi Baroroh et al. All rights reserved. “In Silico” Characterization of 3-Phytase A and 3-Phytase B from Aspergillus niger Mon, 20 Nov 2017 00:00:00 +0000 http://www.hindawi.com/journals/er/2017/9746191/ Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43% alpha-helix, 12% beta-sheet, and 45% others and 38% alpha-helix, 12% beta-sheet, and 50% others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of −0,304 and −0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84 kDa) and 1QFX (50.78 kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions. Doris C. Niño-Gómez, Claudia M. Rivera-Hoyos, Edwin D. Morales-Álvarez, Edgar A. Reyes-Montaño, Nury E. Vargas-Alejo, Ingrid N. Ramírez-Casallas, Kübra Erkan Türkmen, Homero Sáenz-Suárez, José A. Sáenz-Moreno, Raúl A. Poutou-Piñales, Janneth González-Santos, and Azucena Arévalo-Galvis Copyright © 2017 Doris C. Niño-Gómez et al. All rights reserved. Characterization of Pectinase from Bacillus subtilis Strain Btk 27 and Its Potential Application in Removal of Mucilage from Coffee Beans Mon, 11 Sep 2017 00:00:00 +0000 http://www.hindawi.com/journals/er/2017/7686904/ The demand for enzymes in the global market is projected to rise at a fast pace in recent years. There has been a great increase in industrial applications of pectinase owing to their significant biotechnological uses. For applying enzymes at industrial scale primary it is important to know the features of the enzyme. Thus, this study was undertaken with aims of characterizing the pectinase enzyme from Bacillus subtilis strain Btk27 and proving its potential application in demucilisation of coffee. In this study, the maximum pectinase activity was achieved at pH 7.5 and 50°C. Also, the enzyme activity was found stimulated with Mg2+ and Ca2+ metal ions. Moreover, it was stable on EDTA, Trixton-100, Tween 80, and Tween 20. Since Bacillus subtilis strain Btk27 was stable in most surfactants and inhibitors it could be applicable in various industries whenever pectin degradation is needed. The enzyme m and max values were identified as 1.879 mg/ml and 149.6 U, respectively. The potential application of the enzyme for coffee processing was studied, and it is found that complete removal of mucilage from coffee beans within 24 hours of treatment indicates the potential application in coffee processing. Oliyad Jeilu Oumer and Dawit Abate Copyright © 2017 Oliyad Jeilu Oumer and Dawit Abate. All rights reserved. Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus Wed, 30 Aug 2017 07:09:29 +0000 http://www.hindawi.com/journals/er/2017/6980565/ Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters ( of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile. Dennis J. Díaz-Rincón, Ivonne Duque, Erika Osorio, Alexander Rodríguez-López, Angela Espejo-Mojica, Claudia M. Parra-Giraldo, Raúl A. Poutou-Piñales, Carlos J. Alméciga-Díaz, and Balkys Quevedo-Hidalgo Copyright © 2017 Dennis J. Díaz-Rincón et al. All rights reserved. Inhibition of α-Amylases by Condensed and Hydrolysable Tannins: Focus on Kinetics and Hypoglycemic Actions Sun, 14 May 2017 00:00:00 +0000 http://www.hindawi.com/journals/er/2017/5724902/ The aim of the present study was to compare the in vitro inhibitory effects on the salivary and pancreatic α-amylases and the in vivo hypoglycemic actions of the hydrolysable tannin from Chinese natural gall and the condensed tannin from Acacia mearnsii. The human salivary α-amylase was more strongly inhibited by the hydrolysable than by the condensed tannin, with the concentrations for 50% inhibition (IC50) being 47.0 and 285.4 μM, respectively. The inhibitory capacities of both tannins on the pancreatic α-amylase were also different, with IC50 values being 141.1 μM for the hydrolysable tannin and 248.1 μM for the condensed tannin. The kinetics of the inhibition presented complex patterns in that for both inhibitors more than one molecule can bind simultaneously to either the free enzyme of the substrate-complexed enzyme (parabolic mixed inhibition). Both tannins were able to inhibit the intestinal starch absorption. Inhibition by the hydrolysable tannin was concentration-dependent, with 53% inhibition at the dose of 58.8 μmol/kg and 88% inhibition at the dose of 294 μmol/kg. For the condensed tannin, inhibition was not substantially different for doses between 124.4 μmol/kg (49%) and 620 μmol/kg (57%). It can be concluded that both tannins, but especially the hydrolysable one, could be useful in controlling the postprandial glycemic levels in diabetes. Camila Gabriel Kato, Geferson de Almeida Gonçalves, Rosely Aparecida Peralta, Flavio Augusto Vicente Seixas, Anacharis Babeto de Sá-Nakanishi, Lívia Bracht, Jurandir Fernando Comar, Adelar Bracht, and Rosane Marina Peralta Copyright © 2017 Camila Gabriel Kato et al. All rights reserved. Recycle of Immobilized Endocellulases in Different Conditions for Cellulose Hydrolysis Wed, 29 Mar 2017 10:21:46 +0000 http://www.hindawi.com/journals/er/2017/4362704/ The immobilization of cellulases could be an economical alternative for cost reduction of enzyme application. The derivatives obtained in the immobilization derivatives were evaluated in recycles of paper filter hydrolysis. The immobilization process showed that the enzyme recycles were influenced by the shape (drop or sheet) and type of the mixture. The enzyme was recycled 28 times for sheets and 13 times for drops . The derivative showed the highest stability in the recycle obtaining 0.05 FPU/g, RA of 10%, and FPU Yield of 1.64 times, higher than FPU spent or Net FPU Yield of 5.3 times, saving more active enzymes. The derivative B showed stability in recycles reaching 0.15 FPU/g of derivative, yield of Recovered Activity (RA) of 25%, and FPU Yield of 1.57 times, higher than FPU spent on immobilization or Net PFU Yield of 2.81 times. The latex increased stability and resistance of the drops but did not improve the FPU/gram of derivative. D. F. Silva, A. F. A. Carvalho, T. Y. Shinya, G. S. Mazali, R. D. Herculano, and P. Oliva-Neto Copyright © 2017 D. F. Silva et al. All rights reserved. Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization Tue, 21 Mar 2017 00:00:00 +0000 http://www.hindawi.com/journals/er/2017/5947581/ Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a of 6.87 × 10−5 mM s−1, with an apparent of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges. Edwin D. Morales-Álvarez, Claudia M. Rivera-Hoyos, Ángela M. Cardozo-Bernal, Raúl A. Poutou-Piñales, Aura M. Pedroza-Rodríguez, Dennis J. Díaz-Rincón, Alexander Rodríguez-López, Carlos J. Alméciga-Díaz, and Claudia L. Cuervo-Patiño Copyright © 2017 Edwin D. Morales-Álvarez et al. All rights reserved. Diagnostic Value of Adenosine Deaminase and Its Isoforms in Type II Diabetes Mellitus Mon, 05 Dec 2016 07:15:58 +0000 http://www.hindawi.com/journals/er/2016/9526593/ Background and Aims. In the present study, we have investigated the activity of adenosine deaminase (ADA) as a diagnostic marker in type 2 (or II) diabetes mellitus (T2DM). Design and Methods. The deaminase activity of ADA1 and ADA2 was determined in serum from 33 patients with type 2 (or II) diabetes mellitus and 35 healthy controls. We also determined the proportion of glycated hemoglobin (HbA1c). Results. Our results showed significant differences between total serum ADA (tADA) and ADA2 activities in the diabetic groups with HbA1c < 8 (%) and HbA1c ≥ 8 (%) with respect to the values in healthy individuals (). ADA2 activity in patients with high HbA1c was found to be much higher than that in patients with low HbA1c (). In addition, total ADA activity showed a significant correlation with HbA1c (, ). Conclusions. Total serum ADA activity, specially that due to ADA2, could be useful test for the diagnosis of type 2 (or II) diabetes mellitus. Bagher Larijani, Ramin Heshmat, Mina Ebrahimi-Rad, Shohreh Khatami, Shirin Valadbeigi, and Reza Saghiri Copyright © 2016 Bagher Larijani et al. All rights reserved. Molecular Analysis of CYP21A2 Gene Mutations among Iraqi Patients with Congenital Adrenal Hyperplasia Thu, 29 Sep 2016 13:54:08 +0000 http://www.hindawi.com/journals/er/2016/9040616/ Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries. Ruqayah G. Y. Al-Obaidi, Bassam M. S. Al-Musawi, Munib Ahmed K. Al-Zubaidi, Christian Oberkanins, Stefan Németh, and Yusra G. Y. Al-Obaidi Copyright © 2016 Ruqayah G. Y. Al-Obaidi et al. All rights reserved. Agroindustrial Wastes as Alternative for Lipase Production by Candida viswanathii under Solid-State Cultivation: Purification, Biochemical Properties, and Its Potential for Poultry Fat Hydrolysis Tue, 20 Sep 2016 16:32:08 +0000 http://www.hindawi.com/journals/er/2016/1353497/ The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w) supplemented with 25.0% (w/w) olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w) was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w), cultivation temperature (30°C), and substrate moisture (40%, w/v), lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids. Alex Fernando de Almeida, Kleydiane Braga Dias, Ana Carolina Cerri da Silva, César Rafael Fanchini Terrasan, Sâmia Maria Tauk-Tornisielo, and Eleonora Cano Carmona Copyright © 2016 Alex Fernando de Almeida et al. All rights reserved. Salivary Myeloperoxidase, Assessed by 3,3′-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects Thu, 05 May 2016 10:43:57 +0000 http://www.hindawi.com/journals/er/2016/7517928/ Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects () and gingivitis () and periodontitis patients () were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3′-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects () and higher than in gingivitis patients (). No difference was found between gingivitis and healthy groups (). Significant correlations were observed () between salivary MPO activity and GI (, ), BOP (, ), PD (, ), and CAL (, ) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community. Supaporn Klangprapan, Ponlatham Chaiyarit, Doosadee Hormdee, Amonrujee Kampichai, Tueanjit Khampitak, Jureerut Daduang, Ratree Tavichakorntrakool, Bhinyo Panijpan, and Patcharee Boonsiri Copyright © 2016 Supaporn Klangprapan et al. All rights reserved. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation Tue, 16 Feb 2016 11:33:35 +0000 http://www.hindawi.com/journals/er/2016/3016149/ A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (), quotient energy (), , and were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. Gastón Ezequiel Ortiz, Diego Gabriel Noseda, María Clara Ponce Mora, Matías Nicolás Recupero, Martín Blasco, and Edgardo Albertó Copyright © 2016 Gastón Ezequiel Ortiz et al. 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.