Applied Biochemistry and Biotechnology (v.167, #7)

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.
Keywords: Codon optimization; Phosphoenolpyruvate carboxylase; Gene expression; Oxaloacetate; Malate; Escherichia coli

The Use of PAMAM Dendrimers as a Platform for Laccase Immobilization: Kinetic Characterization of the Enzyme by Franciane Pinheiro Cardoso; Sidney Aquino Neto; Pietro Ciancaglini; Adalgisa R. de Andrade (1854-1864).
The kinetic behavior of the enzyme laccase in solution and immobilized onto carbon platforms using poly(amido amine) (PAMAM) dendrimers has been investigated. The results with the immobilized enzymes have demonstrated that almost ten times more enzyme on the carbon support is required for satisfactory kinetic rates to be achieved. Furthermore, the study as a function of the substrate concentration revealed that the kinetic behavior of the enzyme in solution fits the Michaelis–Menten model. However, when the enzyme is immobilized onto the carbon surface, the catalyzed reaction follows a particular kinetic behavior with apparent positive cooperativity. The highest activity with laccase (in solution or immobilized) is achieved around pH 4.5, and the substrate conversion rate clearly diminishes with rising pH. The optimum temperature lies around 60 °C. The enzyme displays good catalytic activity in a wide range of pH and temperature values. The stability tests evidenced that there is no appreciable reduction in the enzymatic activity after immobilization within the first 30 days. Taking into account both the kinetic and stability tests, one can infer that the use of PAMAM dendrimers seems to be a very attractive approach for the immobilization of enzymes, as well as a feasible and useful methodology for the anchoring of enzymes with potential application in many biotechnological areas.
Keywords: Laccase; PAMAM; Enzyme immobilization; Biofuel cell

Bioligninolysis: Recent Updates for Biotechnological Solution by Rashmi Paliwal; Anand Prabha Rawat; Monica Rawat; J. P. N. Rai (1865-1889).
Bioligninolysis involves living organisms and/or their products in degradation of lignin, which is highly resistant, plant-originated polymer having three-dimensional network of dimethoxylated (syringyl), monomethoxylated (guaiacyl), and non-methoxylated (p-hydroxyphenyl) phenylpropanoid and acetylated units. As a major repository of aromatic chemical structures on earth, lignin bears paramount significance for its removal owing to potential application of bioligninolytic systems in industrial production. Early reports illustrating the discovery and cloning of ligninolytic biocatalysts in fungi was truly a landmark in the field of enzymatic delignification. However, the enzymology for bacterial delignification is hitherto poorly understood. Moreover, the lignin-degrading bacterial genes are still unknown and need further exploration. This review deals with the current knowledge about ligninolytic enzyme families produced by fungi and bacteria, their mechanisms of action, and genetic regulation and reservations, which render them attractive candidates in biotechnological applications.
Keywords: Bioligninolysis; Microbial enzymes; Bacterial genes; Omics technologies

Mushroom Polysaccharides and Lipids Synthesized in Liquid Agitated and Static Cultures. Part II: Study of Volvariella volvacea by Panagiota Diamantopoulou; Seraphim Papanikolaou; Eleni Katsarou; Michael Komaitis; George Aggelis; Antonios Philippoussis (1890-1906).
Volvariella volvacea strains were studied in relation with their ability to produce biomass, lipids and polysaccharides. Firstly, screening of four strains (AMLR 188, 190, 191 and 192) was performed in agar cultures, where the mycelial growth rate of the strains was measured, and in static liquid cultures, where the production of biomass, the biosynthesis of total cellular lipids and the consumption of glucose were monitored. For all strains, biomass production was significant (13–15 g l−1) and total lipid in dry weight (%, w/w) ranged from 3 to 12 %. Afterwards, a detailed kinetic analysis of mycelial biomass, extra- and intra- cellular polysaccharides (EPS, IPS, respectively) as well as lipid production by a V. volvacea selected strain was conducted in submerged static and agitated cultures. Maximum values of 15 g l−1 biomass, ∼1.0 g l−1 EPS and 5.5 g l−1 IPS were recorded. Agitation did not have severe impact on biomass, EPS and IPS production, but it increased total lipid in dry weight quantities. EPS, IPS and lipid in dry weight values decreased with time. Glucose was the major cellular carbohydrate detected. Total fatty acid analysis of cellular lipids was performed for all V. volvacea strains and linoleic acid Δ9,12C18:2 was predominant. Neutral lipids constituted the major fraction of cellular lipids, but their quantity decreased as fermentation proceeded. Phospholipids were the most saturated lipid fraction.
Keywords: Volvariella volvacea ; Biomass; Cellular lipids; Polysaccharides; Submerged cultures

Production of Galanthamine by Leucojum aestivum Shoots Grown in Different Bioreactor Systems by Anika Schumann; Strahil Berkov; Diana Claus; André Gerth; Jaume Bastida; Carles Codina (1907-1920).
The production of galanthamine by shoots of Leucojum aestivum grown in different bioreactor systems (shaking and nonshaking batch culture, temporary immersion system, bubble bioreactor, continuous and discontinuous gassing bioreactor) under different culture conditions was studied. The influence of the nutrient medium, weight of inoculum, and size of bioreactor on both growth and galanthamine production was studied. The maximal yield of galanthamine (19.416 mg) was achieved by cultivating the L. aestivum shoots (10 g of fresh inoculum) in a temporary immersion system in a 1-L bioreactor vessel which was used as an airlift culture vessel, gassing 12 times per day (5 min).
Keywords: Alkaloids; Amaryllidaceae; Airlift bioreactors; Bubble bioreactors; Galanthamine; Leucojum aestivum ; Submerged culture; Shoots; Temporary immersion system

Fractionation of Sugarcane Bagasse Using a Combined Process of Dilute Acid and Ionic Liquid Treatments by Danie Diedericks; Eugéne van Rensburg; Johann F. Görgens (1921-1937).
Biorefineries processing lignocellulose will produce chemicals and fuels from chemical constituents, cellulose, hemicelluloses, and lignin to replace fossil-derived products. Fractionation of sugarcane bagasse into three pure streams of chemical constituents was addressed through dissolution of constituents with the ionic liquids, 1-ethyl-3-methylimidazolium acetate ([EMiM]CH3COO) or 1-butyl-3-methylimidazolium methyl sulfate ([BMiM]MeSO4). Constituents were isolated from the reaction mixture with the anti-solvents acetone (Ā), acetone–water (AW), and sodium hydroxide (NaOH). Delignification was enhanced by NaOH, although resulting in impure product streams. Xylose pre-extraction (75 % w/w) by dilute acid pretreatment, prior to ionic liquid treatment, improved lignin purity after anti-solvent separation. Fractionation efficiency of the combined process was maximized (84 %) by ionic liquid treatment at 125 °C for 120 min, resulting in 80.2 % (w/w) lignin removal and 76.5 % (w/w) lignin recovery. Ionic liquids achieved similar degrees of delignification, although fully digestible cellulose-rich solids were produced only by [EMiM]CH3COO treatment.
Keywords: Lignocellulose fractionation; Ionic liquids; Dilute acid pretreatment; 1-Ethyl-3-methylimidazolium acetate; 1-Butyl-3-methylimidazolium methyl sulfate

A fungal strain isolated from rotten banana and identified as Aspergillus alliaceus was found capable of producing thermostable extracellular β-galactosidase enzyme. Optimum cultural conditions for β-galactosidase production by A. alliaceus were as follows: pH 4.5; temperature, 30 °C; inoculum age, 25 h; and fermentation time, 144 h. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 45 °C, 20 min, and 7.2, respectively, for crude and partially purified enzyme. For immobilized enzyme–substrate reaction, these three variable, temperature, time, and pH were optimized at 50 °C, 40 min, and 7.2, respectively. Glucose was found to inhibit the enzyme activity. The K m values of partially purified and immobilized enzymes were 170 and 210 mM, respectively. Immobilized enzyme retained 43 % of the β-galactosidase activity of partially purified enzyme. There was no significant loss of activity on storage of immobilized beads at 4 °C for 28 days. Immobilized enzyme retained 90 % of the initial activity after being used four times.
Keywords: β-Galactosidase; Fungal; Immobilization; Thermostable; Reusability

Gamma-Cyclodextrin Production Using Cyclodextrin Glycosyltransferase from Bacillus clarkii 7364 by Dan Wu; Sheng Chen; Ning Wang; Jian Chen; Jing Wu (1954-1962).
The production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase) from Bacillus clarkii 7364 was studied. Forty-seven percent (w/w) conversion rate to γ-CD was obtained in the process performed by reacting 5 U per gram of starch CGTase with 15 % (w/v) soluble starch in 0.025 M sodium phosphate–NaOH buffer (pH 12) at 55 °C in the presence of 2 % (w/v) glycyrrhizic acid. Meanwhile, the ratio of γ:β-CD was 89:11, with negligible formation of α-CD. Under these conditions, there is a significant increase in overall production of CDs and a marked change in product selectivity for γ-CD. The possible mechanisms were discussed upon different product profiles with respect to the size and amount of CDs synthesized at different reaction conditions. The approach described here can be easily applied to an enzymatic process for the production of γ-CD on an industrial scale, and such high selectivity, at high conversions, is especially attractive from a commercial perspective.
Keywords: γ-Cyclodextrin; Cyclodextrin glycosyltransferase; Bacillus clarkii 7364; Conversion rate; Selectivity

Optimization of a Natural Medium for Cellulase by a Marine Aspergillus niger Using Response Surface Methodology by Dong-Sheng Xue; Hui-Yin Chen; Dong-Qiang Lin; Yi-Xin Guan; Shan-Jing Yao (1963-1972).
The components of a natural medium were optimized to produce cellulase from a marine Aspergillus niger under solid state fermentation conditions by response surface methodology. Eichhornia crassipes and natural seawater were used as a major substrate and a source of mineral salts, respectively. Mineral salts of natural seawater could increase cellulase production. Raw corn cob and raw rice straw showed a significant positive effect on cellulase production. The optimum natural medium consisted of 76.9 % E. crassipes (w/w), 8.9 % raw corn cob (w/w), 3.5 % raw rice straw (w/w), 10.7 % raw wheat bran (w/w), and natural seawater (2.33 times the weight of the dry substrates). Incubation for 96 h in the natural medium increased the biomass to the maximum. The cellulase production was 17.80 U/g the dry weight of substrates after incubation for 144 h. The natural medium avoided supplying chemicals and pretreating substrates. It is promising for future practical fermentation of environment-friendly producing cellulase.
Keywords: Cellulase; Environment friendly; Response surface methodology; Marine A. niger ; Solid state fermentation

Bioactive Compounds Extracted from Ecklonia cava by Using Enzymatic Hydrolysis Protects High Glucose-Induced Damage in INS-1 Pancreatic β-Cells by Seung-Hong Lee; Mi-Hwa Park; Sun-Joo Park; Jaeil Kim; Yong-Tae Kim; Myung-Cheol Oh; Yoonhwa Jeong; Misook Kim; Ji-Sook Han; You-Jin Jeon (1973-1985).
Pancreatic β-cells are very sensitive to oxidative stress and this might play an important role in β-cell death in diabetes. In the present study, we investigated whether the brown alga Ecklonia cava has protective effects against high glucose-induced damage in INS-1 pancreatic β-cells. For that purpose, we prepared an enzymatic hydrolysate from E. cava (EHE) by using the carbohydrase, Celluclast. High-glucose (30 mM) treatment induced glucotoxicity, whereas EHE prevented cells from high glucose-induced damage then restoring cell viability was significantly increased. Furthermore, lipid peroxidation, intracellular reactive oxygen species (ROS) and nitric oxide (NO) were overproduced as the result of the treatment by high glucose; however, these lipid peroxidation, ROS and NO generations were effectively inhibited by addition of EHE in a dose-dependent manner. Moreover, EHE treatment increased activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in high glucose pretreated INS-1 pancreatic β-cells. EHE slightly reduced the expression of pro-apoptotic protein Bax induced by high glucose but increased the expression of Bcl-2, an anti-apoptotic protein. These findings indicate that EHE might be used as potential nutraceutical agent which will protect the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.
Keywords: Ecklonia cava ; Enzymatic hydrolysate; Pancreatic β-cells; Protective effects; High glucose; Oxidative stress

Chemical Composition, Antioxidant and Anticancer Effects of the Seeds and Leaves of Indigo (Polygonum tinctorium Ait.) Plant by Hong-Gi Jang; Buk-Gu Heo; Yong Seo Park; Jacek Namiesnik; Dinorah Barasch; Elena Katrich; Kann Vearasilp; Simon Trakhtenberg; Shela Gorinstein (1986-2004).
Seeds and leaves of indigo (Polygonum tinctorium Ait.) plant were investigated and compared with another medicinal plant named prolipid for their properties such as chemical composition, antioxidant, and anticancer effects by Fourier transform infrared, three-dimensional fluorescence spectroscopy, and electrospray ionization-MS in negative mode. It was found that polyphenols, flavonoids, and flavanols were significantly higher in prolipid (P < 0.05), following by indigo mature leaves, immature leaves, and seeds. Methanol extract of mature indigo leaves in comparison with the ethyl acetate extract showed higher inhibition of proliferation. The interaction between polyphenol extracts of indigo mature leaves and BSA showed that indigo has a strong ability, as other widely used medicinal plants, to quench the intrinsic fluorescence of BSA by forming complexes. In conclusion, indigo mature leaves were compared with prolipid. High content of bioactive compounds, antioxidant, fluorescence, and antiproliferative properties of indigo justifies the use of this plant as a medicinal plant and a new source of antioxidants.
Keywords: Indigo; Extracts; Bioactive compounds; Antioxidant and anticancer activities

Velogenic Newcastle Disease Virus as an Oncolytic Virotherapeutics: In Vitro Characterization by Rajiv Kumar; Ashok K. Tiwari; Uttara Chaturvedi; G. Ravi Kumar; Aditya P. Sahoo; R. S. Rajmani; Lovleen Saxena; Shikha Saxena; Sangeeta Tiwari; Sudesh Kumar (2005-2022).
Cancer is one of the killer diseases in humans and needs alternate curative measures despite recent improvement in modern treatment modalities. Oncolytic virotherapy seems to be a promising nonconventional way to treat cancers. Newcastle disease virus (NDV), a poultry virus, is nonpathogenic to human and domestic animals and has a long history of being used in oncotherapy research in several preclinical studies. The ability of NDV to successfully infect and destroy cancer cells is dependent on the strain and the pathotype of the virus. Adaptation of viruses to heterologous hosts without losing its replicative and oncolytic potential is prerequisite for use as cancer virotherapeutics. In the present study, velogenic NDV was adapted for replication in HeLa cells, and its cytotoxic potential was evaluated by observing morphological, biochemical, and nuclear landmarks of apoptosis. Our results indicated that the NDV-induced apoptosis in HeLa cells was dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspases activation. Different determinants of apoptosis evaluated in the present study indicated that this strain could be a promising candidate for cancer therapy in future.
Keywords: Adaptation; Apoptosis; HeLa cells; NDV; Oncolytic virotherapy

Ascorbate oxidase (AO), a biologically active macromolecule, was successfully immobilized into a biocompatible sandwich-type composite film for developing the vitamin C (VC) biosensor, and the content of VC in commercial juices was amperometrically determined. The biocompatible and conducting poly(3,4-ethylenedioxythiophene) composite film and highly stable and selective multiwalled carbon nanotubes –Nafion composite film were prepared as inner and outer films of biosensor. AO molecules were immobilized between these two composite films. The as-fabricated biosensor displayed an excellent bioelectrocatalytic performance towards the oxidation of VC, a fast current response, a low working potential, a high sensitivity, a wide linear range, and a low detection limit. Moreover, the working mechanism of the biosensor was proposed, and its kinetics was also discussed. In addition, the specificity, reproducibility, and feasibility of the as-fabricated biosensor were also evaluated. Good results of the VC determination in commercial juices indicated that the as-fabricated biosensor was a potential candidate for the electrochemical determination of VC in agricultural crops. Inner and outer films provided a promising platform for the immobilization of biologically active species.
Keywords: Enzyme Immobilization; Ionic liquid microemulsions; Ascorbate oxidase; Vitamin C; Conducting polymers; Biosensor

Thermal Stabilization of an Endoglucanase by Cyclization by Johan F. T. van Lieshout; Odette N. Pérez Gutiérrez; Wietse Vroom; Antoni Planas; Willem M. de Vos; John van der Oost; Sotirios Koutsopoulos (2039-2053).
An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.
Keywords: Thermophilic enzyme; Circular protein; Enzyme stability; Cyclization; Thermostability

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass by Juan Daniel Rivaldi; Boutros Fouad Sarrouh; Ricardo de Freitas Branco; Ismael Maciel de Mancilha; Silvio Silvério da Silva (2054-2067).
Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30 g g−1) and productivity (0.19 g L−1 h−1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3 g L−1 resulted in high biomass production. The highest biomass concentration (21 g L−1), yield (0.45 g g−1) and productivity (0.31 g L−1 h−1), as well as ribonucleotide production (13.13 mg g−1), were observed at 700 rpm and 0.5 vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.
Keywords: Biodiesel-derived glycerol; Yeasts; Microbial biomass; Ribonucleotides

Improvement in the Purification Process of the Capsular Polysaccharide from Haemophilus influenzae Type b by Using Tangential Ultrafiltration and Diafiltration by Silvia Maria Ferreira Albani; Mateus Ribeiro da Silva; Mickie Takagi; Joaquin Cabrera-Crespo (2068-2075).
Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122∼263 and 294∼480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP ≥ 100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.
Keywords: Haemophilus influenzae serotype b; Cocamidopropyl betaine; Sodium deoxycholate; Tangential ultrafiltration; Enzymatic hydrolysis

Cyproterone Synthesis, Recognition and Controlled Release by Molecularly Imprinted Nanoparticle by Ebadullah Asadi; Saman Azodi-Deilami; Majid Abdouss; Saeedeh Khaghani (2076-2087).
In this study, we used novel synthetic conditions of precipitation polymerization to obtain nanosized cyproterone molecularly imprinted polymers for application in the design of new drug delivery systems. The scanning electron microscopy images and Brunauer–Emmett–Teller analysis showed that molecularly imprinted polymer (MIP) prepared by acetonitrile exhibited particles at the nanoscale with a high degree of monodispersity, specific surface area of 246 m2 g−1, and pore volume of 1.24 cm3 g−1. In addition, drug release, binding properties, and dynamic light scattering of molecularly imprinted polymers were studied. Selectivity of MIPs was evaluated by comparing several substances with similar molecular structures to that of cyproterone. Controlled release of cyproterone from nanoparticles was investigated through in vitro dissolution tests and by measuring the absorbance by HPLC-UV. The pH dissolution media employed in controlled release studies were 1.0 at 37 °C for 5 h and then at pH 6.8 using the pH change method. Results show that MIPs have a better ability to control the cyproterone release in a physiological medium compared to the non molecularly imprinted polymers (NMIPs).
Keywords: Molecular imprinting; Cyproterone; Nanoparticles; Drug release; Recognition

Soaking Pretreatment of Corn Stover for Bioethanol Production Followed by Anaerobic Digestion Process by Zhuang Zuo; Shen Tian; Zebing Chen; Jia Li; Xiushan Yang (2088-2102).
The production of ethanol and methane from corn stover (CS) was investigated in a biorefinery process. Initially, a novel soaking pretreatment (NaOH and aqueous-ammonia) for CS was developed to remove lignin, swell the biomass, and improve enzymatic digestibility. Based on the sugar yield during enzymatic hydrolysis, the optimal pretreatment conditions were 1 % NaOH + 8 % NH4OH, 50°C, 48 h, with a solid-to-liquid ratio 1:10. The results demonstrated that soaking pretreatment removed 63.6 % lignin while reserving most of the carbohydrates. After enzymatic hydrolysis, the yields of glucose and xylose were 78.5 % and 69.3 %, respectively. The simultaneous saccharification and fermentation of pretreated CS using Pichia stipitis resulted in an ethanol concentration of 36.1 g/L, corresponding only to 63.3 % of the theoretical maximum. In order to simplify the process and reduce the capital cost, the liquid fraction of the pretreatment was used to re-soak new CS. For methane production, the re-soaked CS and the residues of SSF were anaerobically digested for 120 days. Fifteen grams CS were converted to 1.9 g of ethanol and 1337.3 mL of methane in the entire process.
Keywords: Corn stover; Pretreatment; Re-soaking; Enzymatic hydrolysis; SSF; Biogas production

Emerging Implications of Nonmammalian Cytosine Deaminases on Cancer Therapeutics by Vinod Kumar Yata; P. Gopinath; Siddhartha Sankar Ghosh (2103-2116).
Nonmammalian cytosine deaminases (CDs) have been investigated for last 30 years in the context of cancer therapy. The therapeutic effect of CD is based on its ability to catalyze the conversion of nontoxic prodrug 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU) by deamination of the number 4 carbon of 5FC. This deamination property of CD has been explored to develop innovative therapeutic approach for treatment of cancer. A general overview is needed for the identification of efficient cytosine deaminases for potential use in cancer therapy. In this review, we have discussed about nonmammalian CDs for a variety of prodrug gene/enzyme therapy applications with several recent examples. Finally, we have provided a prospective on the future aspects of CDs and their applications in cancer therapy.
Keywords: Cytosine deaminases; Chemotherapy; Therapeutic effect; Deamination; Cancer therapy; 5-Fluorocytosine; 5-Fluorouracil

Production and Characterization of Extracellular α-Amylase Produced by Wickerhamia sp. X-Fep by Zahuiti Hernández-Montañez; Margarita Juárez-Montiel; Martha Velázquez-Ávila; Eliseo Cristiani-Urbina; César Hernández-Rodríguez; Lourdes Villa-Tanaca; Griselda Chávez-Camarillo (2117-2129).
A yeast isolate able to produce high levels of extracellular α-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26 S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of α-amylases (EC The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the α-amylase was estimated at 54 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5–6 and 50 °C. This thermostable enzyme was inhibited by EDTA–Na2 and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca2+ and Mg2+ cations, which indicates that the α-amylase is a metalloenzyme. α-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of α-amylase.
Keywords: Amylase; Starch; Wickerhamia sp.; Enzyme production; Metalloenzyme; Yeast