Enzyme Research http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Improved Enzyme Catalytic Characteristics upon Glutaraldehyde Cross-Linking of Alginate Entrapped Xylanase Isolated from Aspergillus flavus MTCC 9390 Wed, 12 Aug 2015 14:21:20 +0000 http://www.hindawi.com/journals/er/2015/210784/ Purified fungal xylanase was entrapped in alginate beads. Its further cross-linking using glutaraldehyde resulted in large enzyme aggregates which may function as both a catalyst and a support material for numerous substrate molecules. Enzyme cross-linking presented a negative impact on enzyme leaching during repeated washings and recovery of enzyme activity was substantial after twelve cycles of usage. The entrapment followed by cross-linking doubled the total bound activity and also greatly improved the enzyme stability at extreme chemical environment. The wide pH stability, better thermo- and storage stability, lowered Km value, and protection from some metal ions are salient achievements of present immobilization. The study shows the efficacy, durability, and sustainability of immobilized catalytic system which could be efficiently used for various juice processing operations. Bharat Bhushan, Ajay Pal, and Veena Jain Copyright © 2015 Bharat Bhushan et al. All rights reserved. Effect of Diffusion on Discoloration of Congo Red by Alginate Entrapped Turnip (Brassica rapa) Peroxidase Thu, 05 Feb 2015 08:46:10 +0000 http://www.hindawi.com/journals/er/2015/575618/ Enzymatic discoloration of the diazo dye, Congo red (CR), by immobilized plant peroxidase from turnip “Brassica rapa” is investigated. Partially purified turnip peroxidase (TP) was immobilized by entrapment in spherical particles of calcium alginate and was assayed for the discoloration of aqueous CR solution. Experimental data revealed that pH, reaction time, temperature, colorant, and H2O2 concentration play a significant role in dye degradation. Maximum CR removal was found at pH 2.0, constant temperature of 40°C in the presence of 10 mM H2O2, and 180 mg/L of CR. More than 94% of CR was removed by alginate immobilized TP after 1 h of incubation in a batch process under optimal conditions. About 74% removal efficiency was retained after four recycles. Diffusional limitations in alginate beads such as effectiveness factor η, Thiele modulus , and effective diffusion coefficients (De) of Congo red were predicted assuming a first-order biodegradation kinetic. Results showed that intraparticle diffusion resistance has a significant effect on the CR biodegradation rate. Afaf Ahmedi, Mahmoud Abouseoud, Amrane Abdeltif, and Couvert Annabelle Copyright © 2015 Afaf Ahmedi et al. All rights reserved. An Efficient and Improved Methodology for the Screening of Industrially Valuable Xylano-Pectino-Cellulolytic Microbes Mon, 26 Jan 2015 06:43:34 +0000 http://www.hindawi.com/journals/er/2015/725281/ Xylano-pectino-cellulolytic enzymes are valuable enzymes of the industrial sector. In our earlier study, we have reported a novel and cost effective methodology for the qualitative screening of cellulase-free xylano-pectinolytic microorganisms by replacing the commercial, highly expensive substrates with agricultural residues, but the microorganisms with xylanolytic, pectinolytic, cellulolytic, xylano-pectinolytic, xylano-cellulolytic, pectino-cellulolytic, and xylano-pectino-cellulolytic potential were obtained. The probability of getting the desired combination was low, so efforts were made to further improve this cost effective methodology for obtaining the high yield of the microbes capable of producing desired combination of enzymes. By inclusion of multiple enrichment steps in sequence, using only practically low cost substrates and without any nutrient media till primary screening stage, this improved novel protocol for screening gave only the desired microorganisms with xylano-pectino-cellulolytic activity. Using this rapid, efficient, cost effective, and improved methodology, microbes with required combination of enzymes can be obtained and the probability of getting the desired microorganisms is cent percent. This is the first report presenting the methodology for the isolation of xylano-pectino-cellulolytic positive microorganisms at low cost and consuming less time. Avtar Singh, Amanjot Kaur, Anita Dua, and Ritu Mahajan Copyright © 2015 Avtar Singh et al. All rights reserved. Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus Mon, 19 Jan 2015 10:07:03 +0000 http://www.hindawi.com/journals/er/2015/837842/ DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX ( and γ), and components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions. Wendy Ribble, Shawn D. Kane, and James M. Bullard Copyright © 2015 Wendy Ribble et al. All rights reserved. Chloride Activated Halophilic α-Amylase from Marinobacter sp. EMB8: Production Optimization and Nanoimmobilization for Efficient Starch Hydrolysis Sun, 18 Jan 2015 11:26:21 +0000 http://www.hindawi.com/journals/er/2015/859485/ Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Present work encompasses production optimization and nanoimmobilization of an α-amylase from moderately halophilic Marinobacter sp. EMB8. Media ingredients and culture conditions were optimized by “one-at-a-time approach.” Starch was found to be the best carbon source at 5% (w/v) concentration. Glucose acted as catabolic repressor for amylase production. Salt proved critical for amylase production and maximum production was attained at 5% (w/v) NaCl. Optimization of various culture parameters resulted in 48.0 IU/mL amylase production, a 12-fold increase over that of unoptimized condition (4.0 IU/mL). α-Amylase was immobilized on 3-aminopropyl functionalized silica nanoparticles using glutaraldehyde as cross-linking agent. Optimization of various parameters resulted in 96% immobilization efficiency. Starch hydrolyzing efficiency of immobilized enzyme was comparatively better. Immobilized α-amylase retained 75% of its activity after 5th cycle of repeated use. Sumit Kumar and S. K. Khare Copyright © 2015 Sumit Kumar and S. K. Khare. All rights reserved. Immobilization of Papain on Chitin and Chitosan and Recycling of Soluble Enzyme for Deflocculation of Saccharomyces cerevisiae from Bioethanol Distilleries Thu, 01 Jan 2015 09:34:47 +0000 http://www.hindawi.com/journals/er/2015/573721/ Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1–10% w·v−1), polyethyleneimine (0.5% v·v−1), and tripolyphosphate (1–10% w·v−1) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L−1. Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes. Douglas Fernandes Silva, Henrique Rosa, Ana Flavia Azevedo Carvalho, and Pedro Oliva-Neto Copyright © 2015 Douglas Fernandes Silva et al. All rights reserved. Immobilization of Aspergillus niger F7-02 Lipase in Polysaccharide Hydrogel Beads of Irvingia gabonensis Matrix Wed, 31 Dec 2014 13:49:04 +0000 http://www.hindawi.com/journals/er/2014/967056/ The potential of polysaccharide Irvingia gabonensis matrix as enzyme immobilization support was investigated. Lipase of Aspergillus niger F7-02 was immobilized by entrapment using glutaraldehyde as the cross-linking agent and stabilized in ethanolic-formaldehyde solution. The pH and temperature stability and activity yield of the immobilized enzyme were determined. Such parameters as enzyme load, bead size, number of beads, and bead reusability were also optimized. Adequate gel strength to form stabilized beads was achieved at 15.52% (w/v) Irvingia gabonensis powder, 15% (v/v) partially purified lipase, 2.5% (v/v) glutaraldehyde, and 3 : 1 (v/v) ethanolic-formaldehyde solution. There was 3.93-fold purification when the crude enzyme was partially purified in two-step purification using Imarsil and activated charcoal. Optimum lipase activity 75.3 Ug−1 was achieved in 50 mL test solution containing 15 beads of 7 mm bead size. Relative activity 80% was retained at eight repeated cycles. The immobilization process gave activity yield of 59.1% with specific activity of 12.3 Umg−1 and stabilized at optimum pH 4.5 and temperature 55°C. Thus the effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established. Safaradeen Olateju Kareem, Olayinka Quadri Adio, and Michael Bamitale Osho Copyright © 2014 Safaradeen Olateju Kareem et al. All rights reserved. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation Wed, 31 Dec 2014 09:41:53 +0000 http://www.hindawi.com/journals/er/2014/353915/ A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking. Danielle Biscaro Pedrolli and Eleonora Cano Carmona Copyright © 2014 Danielle Biscaro Pedrolli and Eleonora Cano Carmona. All rights reserved. Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver Thu, 25 Dec 2014 00:10:08 +0000 http://www.hindawi.com/journals/er/2014/714054/ Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver. Mahmoud A. Ibrahim, Abdel-Hady M. Ghazy, Ahmed M. H. Salem, Mohamed A. Ghazy, and Mohamed M. Abdel-Monsef Copyright © 2014 Mahmoud A. Ibrahim et al. All rights reserved. Assessment of Serum Enzymatic Antioxidant Levels in Patients with Recurrent Aphthous Stomatitis: A Case Control Study Wed, 10 Dec 2014 06:40:38 +0000 http://www.hindawi.com/journals/er/2014/340819/ Background and Aim. Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae. Despite extensive research, the exact etiology of RAS remains elusive. Recently oxidant-antioxidant imbalance of the body has been implicated in the pathogenesis of recurrent aphthous stomatitis. Thus, the aim of the study was to evaluate the enzymatic antioxidant levels in patients with recurrent aphthous stomatitis. Materials and Methods. The serum levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured in 30 patients with recurrent aphthous stomatitis and compared to the control group, which included 30 healthy subjects. Student’s -test was performed for statistical evaluation. Results. The mean levels of superoxide dismutase (130.2 ± 15.94 U/mL) and glutathione peroxidase (3527.93 ± 488.32 U/L) were found to be significantly lower in study group as compared to control group (211.9 ± 20.93 U/mL, 8860.93 ± 1105.31 U/L, resp.) () while level of catalase in study group was significantly higher when compared to control group (10981.00 ± 1018.07 U/mL versus 9764.00 ± 1621.19 U/mL) (). Conclusion. Enzymatic antioxidant system is impaired in recurrent aphthous stomatitis patients and seems to play a crucial role in its pathogenesis. Ishita Gupta, Arvind Shetti, Vaishali Keluskar, and Anjana Bagewadi Copyright © 2014 Ishita Gupta et al. All rights reserved. A Fractional Factorial Design to Study the Effect of Process Variables on the Preparation of Hyaluronidase Loaded PLGA Nanoparticles Wed, 10 Dec 2014 00:10:26 +0000 http://www.hindawi.com/journals/er/2014/162962/ The present study was initiated to understand the effect of PLGA concentration, PVA concentration, internal-external phase ratio, homogenization speed, and homogenization time on mean particle size, zeta potential, and percentage drug encapsulation using fractional factorial design. Using PLGA (50-50) as the carrier, hyaluronidase loaded PLGA nanoparticles were prepared using double emulsion solvent evaporation technique. The particle size was analyzed by dynamic light scattering technique and protein content by Lowry method. The study showed that homogenization speed as an independent variable had maximum effect on particle size and zeta potential. Internal-external phase volume ratio had maximum effect on drug encapsulation. Mean particle size also had high dependency on the combined effect of PVA concentration and phase volume ratio. Using fractional factorial design particle size of <400 nm, zeta potential of <−30 mV, and percentage encapsulation of 15–18% were achieved. K. Narayanan, V. M. Subrahmanyam, and J. Venkata Rao Copyright © 2014 K. Narayanan et al. All rights reserved. Contemporaneous Production of Amylase and Protease through CCD Response Surface Methodology by Newly Isolated Bacillus megaterium Strain B69 Wed, 12 Nov 2014 08:45:23 +0000 http://www.hindawi.com/journals/er/2014/601046/ The enormous increase in world population has resulted in generation of million tons of agricultural wastes. Biotechnological process for production of green chemicals, namely, enzymes, provides the best utilization of these otherwise unutilized wastes. The present study elaborates concomitant production of protease and amylase in solid state fermentation (SSF) by a newly isolated Bacillus megaterium B69, using agroindustrial wastes. Two-level statistical model employing Plackett-Burman and response surface methodology was designed for optimization of various physicochemical conditions affecting the production of two enzymes concomitantly. The studies revealed that the new strain concomitantly produced 1242 U/g of protease and 1666.6 U/g of amylase by best utilizing mustard oilseed cake as the substrate at 20% substrate concentration and 45% moisture content after 84 h of incubation. An increase of 2.95- and 2.04-fold from basal media was observed in protease and amylase production, respectively. ANOVA of both the design models showed high accuracy of the polynomial model with significant similarities between the predicted and the observed results. The model stood accurate at the bench level validation, suggesting that the design model could be used for multienzyme production at mass scale. Rajshree Saxena and Rajni Singh Copyright © 2014 Rajshree Saxena and Rajni Singh. All rights reserved. Partial Purification and Characterization of a Heat Stable α-Amylase from a Thermophilic Actinobacteria, Streptomyces sp. MSC702 Wed, 08 Oct 2014 00:00:00 +0000 http://www.hindawi.com/journals/er/2014/106363/ A partial purification and biochemical characterization of the α-amylase from Streptomyces sp. MSC702 were carried out in this study. The optimum operational conditions for enzyme substrate reaction for amylolytic enzyme activity from the strain were evaluated. The optimum pH, temperature, and incubation period for assaying the enzyme were observed to be 5.0, 55°C, and 30 min, respectively. The extracellular extract was concentrated using ammonium sulfate precipitation. It was stable in the presence of metal ions (5 mM) such as K+, Co2+, and Mo2+, whereas Pb2+, Mn2+, Mg2+, Cu2+, Zn2+, Ba2+, Ca2+, Hg2+, Sn2+, Cr3+, Al3+, Ag+, and Fe2+ were found to have inhibitory effects. The enzyme activity was also unstable in the presence of 1% Triton X-100, 1% Tween 80, 5 mM sodium lauryl sulphate, 1% glycerol, 5 mM EDTA, and 5 mM denaturant urea. At temperature 60°C and pH 5.0, the enzyme stability was maximum. α-amylase retained 100% and 34.18% stability for 1 h and 4 h, respectively, at 60°C (pH 7.0). The enzyme exhibited a half-life of 195 min at 60°C temperature. The analysis of kinetic showed that the enzyme has of 2.4 mg/mL and of 21853.0 μmol/min/mg for soluble potato starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization. Renu Singh, Vijay Kumar, and Vishal Kapoor Copyright © 2014 Renu Singh et al. All rights reserved. Angiotensin Converting Enzyme Activity in Alopecia Areata Wed, 01 Oct 2014 09:44:48 +0000 http://www.hindawi.com/journals/er/2014/694148/ Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (). Also, there was no correlation between ACE activity and severity () and duration of disease () in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. Mohammad Reza Namazi, Armaghan Ashraf, Farhad Handjani, Ebrahim Eftekhar, and Amir Kalafi Copyright © 2014 Mohammad Reza Namazi et al. All rights reserved. A Simple Route for Purifying Extracellular Poly(3-hydroxybutyrate)-depolymerase from Penicillium pinophilum Tue, 23 Sep 2014 08:43:53 +0000 http://www.hindawi.com/journals/er/2014/159809/ This work proposes the purification of an active and efficient enzyme, extracellular poly(3-hydroxybutyrate) (PHB)-depolymerase, suitable for industrial applications. This is achieved by the application of an easy, fast, and cheap route, skipping the chromatography step. Chromatography with one or two columns is a common step in the purification procedure, which however renders the isolation of the enzyme a time consuming and an expensive process. A strain of the fungus Penicillium pinophilum (ATCC 9644) is used for the isolation of extracellular PHB-depolymerase. The molecular weight of the purified enzyme is about 35 kDa and is estimated by gel electrophoresis (SDS-PAGE, 12% polyacrylamide). The enzymatic activity of the isolated enzyme is determined to be 3.56-fold similar to that found by other researchers that have used chromatography for the isolation. The as-isolated enzyme disintegrates the poly(3-hydroxybutyrate) (PHB) films successfully, as it is demonstrated by the biodegradation test results provided here. Elpiniki Panagiotidou, Constantinos Konidaris, Apostolos Baklavaridis, Ioannis Zuburtikudis, Dimitris Achilias, and Paraskevi Mitlianga Copyright © 2014 Elpiniki Panagiotidou et al. All rights reserved. Mode of Action of Lactoperoxidase as Related to Its Antimicrobial Activity: A Review Tue, 16 Sep 2014 09:03:49 +0000 http://www.hindawi.com/journals/er/2014/517164/ Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity. Their best known effect is their antimicrobial activity that arouses much interest in in vivo and in vitro applications. In this context, the proper use of lactoperoxidase needs a good understanding of its mode of action, of the factors that favor or limit its activity, and of the features and properties of the active molecules. The first part of this review describes briefly the classification of mammalian peroxidases and their role in the human immune system and in host cell damage. The second part summarizes present knowledge on the mode of action of lactoperoxidase, with special focus on the characteristics to be taken into account for in vitro or in vivo antimicrobial use. The last part looks upon the characteristics of the active molecule produced by lactoperoxidase in the presence of thiocyanate and/or iodide with implication(s) on its antimicrobial activity. F. Bafort, O. Parisi, J.-P. Perraudin, and M. H. Jijakli Copyright © 2014 F. Bafort et al. All rights reserved. Immobilization of Lipase on Silver Nanoparticles via Adhesive Polydopamine for Biodiesel Production Wed, 10 Sep 2014 07:52:41 +0000 http://www.hindawi.com/journals/er/2014/389739/ Biodiesel production technology is competitive in terms of low cost and alternative source of energy which should be not only sustainable but also environmentally friendly. Designing of the lipase immobilization for biodiesel production has a remarkable impact and is still challenging. In this work, biodiesel production from soybean oil was enhanced and facilitated by using a novel biocatalyst consisting of commercial lipase (EC 3.1.1.3), silver nanoparticles, and polydopamine. Silver nanoparticles (AgNPs) were synthesized with a size range of 10–20 nm. Polydopamine (PD) was delivered by the self-polymerization of dopamine in 10 mM Tris-HCl pH 8.5 and simultaneously coated the AgNPs to form a PD/AgNPs complex. Lipase was immobilized on the PD/AgNPs complex surface via covalent bonds to form a tailor-made biocatalyst consisting of immobilized lipase/PD/AgNPs complex (LPA). The formation and morphology of each composition were characterized by UV-Vis spectroscopy and scanning electron microscope (SEM). Significantly, gas chromatography analysis showed a remarkable biodiesel production yield of 95% by using the LPA complex at 40°C for 6-hours reaction time, whereas the yield was 86% when using free lyophilized lipase. The LPA complex was apparently reusable after 7 batches and the latter conversion rate of soybean oil was decreased by only 27%. Kanchana Dumri and Dau Hung Anh Copyright © 2014 Kanchana Dumri and Dau Hung Anh. All rights reserved. Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study Sun, 07 Sep 2014 08:11:01 +0000 http://www.hindawi.com/journals/er/2014/708676/ The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C. The shift of the spectral centre of mass above 50°C is also apparently cooperative with midpoint transition of 56.3 ± 0.2°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process. Further corroboration comes from differential scanning calorimetry experiments. At protein concentrations ≤0.56 mg·mL−1 the calorimetric transition is reversible and the data were fitted to a non-two-state model and deconvoluted into six transitions, whereas at concentrations greater than 0.56 mg·mL−1 the calorimetric transition is irreversible with an exothermic contribution to the thermogram. The apparent increased linearly with the scan rate according to first order inactivation kinetics. The effect of additives on the calorimetric transition of xylanase is dependent on their nature. The addition of sorbitol transforms reversible transitions into irreversible transitions while stabilizing the protein as the apparent increases linearly with sorbitol concentration. D-Glucono-1,5-lactone, a noncompetitive inhibitor in xylanase kinetics, and soluble xylan change irreversible processes into reversible processes at high protein concentration. Gloria López and Pilar Estrada Copyright © 2014 Gloria López and Pilar Estrada. All rights reserved. Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom Thu, 14 Aug 2014 13:11:05 +0000 http://www.hindawi.com/journals/er/2014/120739/ Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications. Kamal Uddin Zaidi, Ayesha S. Ali, and Sharique A. Ali Copyright © 2014 Kamal Uddin Zaidi et al. All rights reserved. Sequential Statistical Optimization of Media Components for the Production of Glucoamylase by Thermophilic Fungus Humicola grisea MTCC 352 Wed, 09 Jul 2014 13:25:28 +0000 http://www.hindawi.com/journals/er/2014/317940/ Glucoamylase is an industrially important enzyme which converts soluble starch into glucose. The media components for the production of glucoamylase from thermophilic fungus Humicola grisea MTCC 352 have been optimized. Eight media components, namely, soluble starch, yeast extract, KH2PO4, K2HPO4, NaCl, CaCl2, MgSO4·7H2O, and Vogel’s trace elements solution, were first screened for their effect on the production of glucoamylase and only four components (soluble starch, yeast extract, K2HPO4, and MgSO4·7H2O) were identified as statistically significant using Plackett-Burman design. It was fitted into a first-order model (). Steepest ascent method was performed to identify the location of optimum. Central composite design was employed to determine the optimum values (soluble starch: 28.41 g/L, yeast extract: 9.61 g/L, K2HPO4: 2.42 g/L, and MgSO4·7H2O: 1.91 g/L). The experimental activity of 12.27 U/mL obtained was close to the predicted activity of 12.15. High value (0.9397), low PRESS value (9.47), and AARD values (2.07%) indicate the accuracy of the proposed model. The glucoamylase production was found to increase from 4.57 U/mL to 12.27 U/mL, a 2.68-fold enhancement, as compared to the unoptimized medium. Vinayagam Ramesh and Vytla Ramachandra Murty Copyright © 2014 Vinayagam Ramesh and Vytla Ramachandra Murty. All rights reserved. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives Thu, 03 Jul 2014 07:34:57 +0000 http://www.hindawi.com/journals/er/2014/109303/ A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory. Nilesh P. Nirmal and R. Seeta Laxman Copyright © 2014 Nilesh P. Nirmal and R. Seeta Laxman. All rights reserved. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 Mon, 30 Jun 2014 11:02:06 +0000 http://www.hindawi.com/journals/er/2014/197938/ Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. Norsyuhada Alias, Mu’adz Ahmad Mazian, Abu Bakar Salleh, Mahiran Basri, and Raja Noor Zaliha Raja Abd. Rahman Copyright © 2014 Norsyuhada Alias et al. All rights reserved. Optimisation of Cellulase Production by Penicillium funiculosum in a Stirred Tank Bioreactor Using Multivariate Response Surface Analysis Wed, 25 Jun 2014 15:29:11 +0000 http://www.hindawi.com/journals/er/2014/703291/ Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases), which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD) was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L−1) as carbon source was determined to include urea (1.2 g·L−1), yeast extract (1.0 g·L−1), KH2PO4 (6.0 g·L−1), and MgSO4·7H2O (1.2 g·L−1). The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L−1 for FPase, 9,204 U·L−1 for endoglucanase, and 2,395 U·L−1 for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions. Marcelle Lins de Albuquerque de Carvalho, Daniele Fernandes Carvalho, Edelvio de Barros Gomes, Roberto Nobuyuki Maeda, Lidia Maria Melo Santa Anna, Aline Machado de Castro, and Nei Pereira Jr. Copyright © 2014 Marcelle Lins de Albuquerque de Carvalho et al. All rights reserved. Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors Thu, 19 Jun 2014 12:11:54 +0000 http://www.hindawi.com/journals/er/2014/848937/ Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed. Bui T. T. Nga, Yuki Takeshita, Misa Yamamoto, and Yoshimi Yamamoto Copyright © 2014 Bui T. T. Nga et al. All rights reserved. Molecular Dynamics and Metadynamics Simulations of the Cellulase Cel48F Wed, 21 May 2014 00:00:00 +0000 http://www.hindawi.com/journals/er/2014/692738/ Molecular dynamics (MD) and metadynamics techniques were used to study the cellulase Cel48F-sugar. Cellulase is enzyme that breaks cellulose fibers into small sugar units and is potentially useful in second generation alcohol production. In MD simulations, the overall structure of equilibrated Cel48F did not significantly change along the trajectory, retaining root mean square deviation below 0.15 nm. A set of 15 residues interacting with the sugar chains via hydrogen bonding throughout the simulation was observed. The free energy of dissociation (ΔGdiss.) of the chains in the catalytic tunnel of Cel48F was determined by metadynamics. The ΔGdiss. values of the chains entering and leaving the wild-type Cel48F cavity were 13.9 and 62.1 kcal/mol, respectively. We also mutated the E542 and Q543 to alanine residue and obtained ΔGdiss. of 41.8 and 45.9 kcal/mol, respectively. These mutations were found to facilitate smooth dissociation of the sugar chain across the Cel48F tunnel. At the entry of the Cel48F tunnel, three residues were mutated to alanine: T110, T213, and L274. Contrary to the T110A-Cel48F, the mutants T213-Cel48F and L274-Cel48F prevented the sugar chain from passing across the leaving site. The present results can be a guideline in mutagenesis studies to improve processing by Cel48F. Osmair Vital de Oliveira Copyright © 2014 Osmair Vital de Oliveira. All rights reserved. Fungal Laccases and Their Applications in Bioremediation Thu, 15 May 2014 00:00:00 +0000 http://www.hindawi.com/journals/er/2014/163242/ Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection. Buddolla Viswanath, Bandi Rajesh, Avilala Janardhan, Arthala Praveen Kumar, and Golla Narasimha Copyright © 2014 Buddolla Viswanath et al. All rights reserved. Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology Mon, 12 May 2014 12:14:42 +0000 http://www.hindawi.com/journals/er/2014/764898/ Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production. K. Pandiyan, Rameshwar Tiwari, Surender Singh, Pawan K. S. Nain, Sarika Rana, Anju Arora, Shashi B. Singh, and Lata Nain Copyright © 2014 K. Pandiyan et al. All rights reserved. A cDNA Cloning of a Novel Alpha-Class Tyrosinase of Pinctada fucata: Its Expression Analysis and Characterization of the Expressed Protein Thu, 10 Apr 2014 12:30:02 +0000 http://www.hindawi.com/journals/er/2014/780549/ Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1). This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB), suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata. Ryousuke Takgi and Tomoyuki Miyashita Copyright © 2014 Ryousuke Takgi and Tomoyuki Miyashita. All rights reserved. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads Thu, 10 Apr 2014 10:23:47 +0000 http://www.hindawi.com/journals/er/2014/738739/ This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C. Bárbara M. Bonine, Patricia Peres Polizelli, and Gustavo O. Bonilla-Rodriguez Copyright © 2014 Bárbara M. Bonine et al. All rights reserved. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165 Thu, 03 Apr 2014 07:02:32 +0000 http://www.hindawi.com/journals/er/2014/494682/ Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a and of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. Deepthy Alex, Anju Shainu, Ashok Pandey, and Rajeev K. Sukumaran Copyright © 2014 Deepthy Alex et al. All rights reserved.