Applied Biochemistry and Biotechnology (v.160, #4)

Anaerobic digestion is a biological method used to convert organic wastes into a stable product for land application with reduced environmental impacts. The biogas produced can be used as an alternative renewable energy source. Dry anaerobic digestion [>15% total solid (TS)] has an advantage over wet digestion (<10% TS) because it allows for the use of a smaller volume of reactor and because it reduces wastewater production. In addition, it produces a fertilizer that is easier to transport. Performance of anaerobic digestion of animal manure–switchgrass mixture was evaluated under dry (15% TS) and thermophilic conditions (55 °C). Three different mixtures of animal manure (swine, poultry, and dairy) and switchgrass were digested using batch-operated 1-L reactors. The swine manure test units showed 52.9% volatile solids (VS) removal during the 62-day trial, while dairy and poultry manure test units showed 9.3% and 20.2%, respectively. Over the 62 day digestion, the swine manure test units yielded the highest amount of methane 0.337 L CH4 /g VS, while the dairy and poultry manure test units showed very poor methane yield 0.028 L CH4/g VS and 0.002 L CH4/g VS, respectively. Although dairy and poultry manure performed poorly, they may still have high potential as biomass for dry anaerobic digestion if appropriate designs are developed to prevent significant volatile fatty acid (VFA) accumulation and pH drop.
Keywords: Anaerobic; Digestion; Animal manure; Switchgrass; Biogas; Renewable energy

Biosorption of Cadmium, Lead, and Uranium by Powder of Poplar Leaves and Branches by M. S. Al-Masri; Y. Amin; B. Al-Akel; T. Al-Naama (976-987).
The removal of metal ions from aqueous solutions by biosorption plays an important role in water pollution control. In this study, dried leaves and branches of poplar trees were studied for removing some toxic elements (cadmium, lead, and uranium) from aqueous solutions. The equilibrium experiments were systematically carried out in a batch process, covering various process parameters that include agitation time, adsorbent size and dosage, initial cadmium, lead and uranium concentration, and pH of the aqueous solution. Adsorption behavior was found to follow Freundlich and Langmuir isotherms. The results have shown that both dried leaves and branches can be effectively used for removing uranium, while only branches were found to remove lead and cadmium completely from the aqueous solution. The maximum biosorption capacity of leaves for uranium was found to be 2.3 mg g−1 and 1.7 mg g−1 and 2.1 mg g−1 for lead and cadmium on branches, respectively. In addition, the studied biomass materials were used in removing lead and cadmium from contaminated water and the method was found to be effective.
Keywords: Poplar; Uranium; Cadmium; Lead; Biosorption; Wastewater; Pollution

Recent Advances in Microbial Raw Starch Degrading Enzymes by Haiyan Sun; Pingjuan Zhao; Xiangyang Ge; Yongjun Xia; Zhikui Hao; Jianwen Liu; Ming Peng (988-1003).
Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.
Keywords: Raw starch degrading enzymes; Production; Purification; Properties; Application

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K m and V max as 2.2 × 10−5 M and 30.8 mM min−1, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.
Keywords: Aspergillus flavus ; Glycosaminoglycans; Heparinase; Inducible; Purification; rRNA gene homology

An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60°C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0–11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-l-asparagine (Z-Asp) and l-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.
Keywords: Purification; Characterization; Organic solvent-stable protease; Enzymatic synthesis

A Techno-economic Analysis of Polyhydroxyalkanoate and Hydrogen Production from Syngas Fermentation of Gasified Biomass by DongWon Choi; David C. Chipman; Scott C. Bents; Robert C. Brown (1032-1046).
A techno-economic analysis was conducted to investigate the feasibility of a gasification-based hybrid biorefinery producing both hydrogen gas and polyhydroxyalkanoates (PHA), biodegradable polymer materials that can be an attractive substitute for conventional petrochemical plastics. The biorefinery considered used switchgrass as a feedstock and converted that raw material through thermochemical methods into syngas, a gaseous mixture composed mainly of hydrogen and carbon monoxide. The syngas was then fermented using Rhodospirillum rubrum, a purple non-sulfur bacterium, to produce PHA and to enrich hydrogen in the syngas. Total daily production of the biorefinery was assumed to be 12 Mg of PHA and 50 Mg of hydrogen gas. Grassroots capital for the biorefinery was estimated to be $55 million, with annual operating costs at $6.7 million. With a market value of $2.00/kg assumed for the hydrogen, the cost of producing PHA was determined to be $1.65/kg.
Keywords: Syngas fermentation; Synthesis gas fermentation; Polyhydroxyalkanoate; PHA; Techno-economic analysis; Hydrogen; ASPEN Plus; Rhodosprillum rubrum

The messenger RNA differential display technique was performed to investigate the differences of gene expression in the liver tissues from Meishan and Large White pigs. A fragment of one differentially expressed gene was isolated and sequenced. A complete complementary DNA (cDNA) sequence was obtained using the rapid amplification of cDNA end method. Nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 339 amino acids which have high homology with those of the ER lipid-raft-associated 2 isoform 2 (ERLIN2) of eight species—human (97%), rhesus monkey (97%), rat (96%), horse (97%), cattle (97%), mouse (97%), dog (95%), and red jungle fowl (90%)—so that it can be defined as the swine erlin2 gene. The phylogenetic tree analysis revealed that the swine erlin2 gene has a closer genetic relationship with the erlin2 genes of human and rhesus monkey. The tissue expression profile analysis indicated that the swine erlin2 gene is differentially expressed in detected tissues from Meishan and Large White pigs. Our experiment suggested that the swine erlin2 gene might play an important role in the superabundant fat deposition of Chinese pigs.
Keywords: Pig; erlin2 gene; mRNA differential display; RACE

Extraction of Ascorbate Oxidase from Cucurbita maxima by Continuous Process in Perforated Rotating Disc Contactor Using Aqueous Two-Phase Systems by T. S. Porto; P. P. Marques; C. S. Porto; K. A. Moreira; J. L. Lima-Filho; A. Converti; A. Pessoa; A. L. F. Porto (1057-1064).
The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.
Keywords: Ascorbate oxidase; ATPS; Continuous extraction; Cucurbita maxima ; PRDC

It was found that the complex of cytochrome c (Cyt c) and hydrogen peroxide could significantly catalyze the chemiluminescence (CL) reaction from luminol–hydrogen peroxide, and a sensitive, rapid, and simple CL procedure was proposed for the determination of Cyt c in a flow injection system for the first time. The increment of CL intensity was linear over the concentration of Cyt c ranging from 5 to 700 ng ml−1, with a detection limit of 2 ng ml−1 (3σ). At a flow rate of 2.0 ml min−1, a complete analytical process could be performed in 30 s with a relative standard deviation of less than 4.0%. The proposed method was applied successfully for the assay of Cyt c in pharmaceutical injections and human serum, and the recoveries were from 98.0% to 108.8% and 92.5% to 109.0%. The possible mechanism of Cyt c enhanced CL reaction was also discussed.
Keywords: Cytochrome c ; Luminol; Hydrogen peroxide; Chemiluminescence; Flow injection

A crude cellulase preparation from Aspergillus niger was used to depolymerize chitosan. The depolymerization process was followed by measuring the apparent viscocity and the intrinsic viscosity. The optimum conditions for enzymatic hydrolysis were investigated. On the selected optimum conditions (pH 5.0, temperature 50 °C, and an enzyme to substrate ratio of 1:5), chitosan was hydrolyzed for 1, 4, 8, and 24 h, its viscosity-average molecular weights were 3.49 × 104, 1.18 × 104, 5.83 × 103, and 1.13 × 103, respectively. Compared with chitosan having viscosity-average molecular weight of 5.18 × 105 before enzymatic hydrolysis, the crude cellulase preparation had rather apparent effect on depolymerization of chitosan. Through the comparison of different origin of cellulases, the prepared cellulase has good ability of enzymatic hydrolysis. The reproducibility and reversibility for enzymatic hydrolysis was appraised. The data are of value for the production of low-molecular weight chitosans and chitooligomers of medical and biotechnological interest.
Keywords: Chitosan; Depolymerization; Enzymatic hydrolysis; Molecular weight; Viscometric feature

Fermentation properties under the control of multiple genes of industrial Saccharomyces cerevisiae strain are difficult to alter with traditional methods. Here, we describe efficient and reliable genome shuffling to increase ethanol production through the rapid improvement of stress resistance. The strategy is carried out using yeast sexual and asexual reproduction by itself instead of polyethylene glycol-mediated protoplast fusion. After three rounds of genome shuffling, the best performing strain S3-10 was obtained on the special plate containing a high ethanol concentration. It exhibits substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. The cycle of fermentation of S3-10 was not only shortened, but also, ethanol yield was increased by up to 10.96% compared with the control in very-high-gravity (VHG) fermentations. In total, S3-10 possesses optimized fermentation characteristics, which will be propitious to the development of bioethanol fermentation industry.
Keywords: Ethanol; Genome shuffling; Industrial strain; Saccharomyces cerevisiae

Feasibility Study of Introducing Redox Property by Modification of PMBN Polymer for Biofuel Cell Applications by Eileen Hao Yu; Yoko Himuro; Madoka Takai; Kazuhiko Ishihara (1094-1101).
In this study, the feasibility of introducing redox property to an amphiphilic phospholipid polymer (PMBN) was investigated. The active ester group in the side chain of the polymer was used to react with pyrroloquinoline quinine (PQQ). Redox peaks that corresponded to PQQ redox potentials were observed after the modification. Glucose oxidase was immobilized to the modified polymer. Electrochemical oxidation of glucose was carried out with the polymer electrode. The oxidation current increased with elevating glucose concentration indicating electron transfer established between the electrode and enzyme. It suggests that by modification, PMBN is possible to use for enzyme electrode for bioelectronics.
Keywords: PMBN polymer; PQQ; Redox property; Enzyme immobilization; Glucose oxidation; Enzymatic biofuel cells

Rheology of Dilute Acid Hydrolyzed Corn Stover at High Solids Concentration by M. R. Ehrhardt; T. O. Monz; T. W. Root; R. K. Connelly; C. T. Scott; D. J. Klingenberg (1102-1115).
The rheological properties of acid hydrolyzed corn stover at high solids concentration (20–35 wt.%) were investigated using torque rheometry. These materials are yield stress fluids whose rheological properties can be well represented by the Bingham model. Yield stresses increase with increasing solids concentration and decrease with increasing hydrolysis reaction temperature, acid concentration, and rheometer temperature. Plastic viscosities increase with increasing solids concentration and tend to decrease with increasing reaction temperature and acid concentration. The solids concentration dependence of the yield stress is consistent with that reported for other fibrous systems. The changes in yield stress with reaction conditions are consistent with observed changes in particle size. This study illustrates that torque rheometry can be used effectively to measure rheological properties of concentrated biomass.
Keywords: Biomass; Corn stover; Rheology; Yield stress; Viscosity; Acid hydrolysis

In this study, several nonionic surfactants were tried to improve the enzymatic hydrolysis of ginsenoside Rg3 into Rh2 which was catalyzed at 50 °C and pH 5.0 by a crude glucosidase extracted from Fusarium sp. ECU2042. Among the biocompatible nonionic surfactants, polyethylene glycol 350 monomethyl ether was shown to be the best. After optimizing some influencing factors on the reaction, the conversion of Rg3 (5 g/l) with 10 g/l crude enzyme reached almost 100% in the presence of the nonionic surfactant (7.5%, w/v), which was 25% higher than that in buffer without any surfactant. Furthermore, the enzyme stability was affected faintly by the surfactant.
Keywords: Nonionic surfactant; Fusarium sp. ECU2042; Biohydrolysis; Ginsenoside Rg3 ; Ginsenoside Rh2

Mannanase Transfer into Hexane and Xylene by Liquid–Liquid Extraction by Stepan Shipovskov; Karsten M. Kragh; Brian S. Laursen; Charlotte H. Poulsen; Flemming Besenbacher; Duncan S. Sutherland (1124-1129).
The formation of noncovalent complexes between glycosidase, endo-1,4-β-d-mannanase, and ionic surfactant di(2-ethylhexyl) sodium sulfosuccinate (AOT) was shown to promote protein transfer into organic solvents such as xylene and hexane. It was found that mannanase can be solubilized in hexane and in xylene with concentration at least 2.5 and 2.0 mg/ml, respectively. The catalytic activity of the enzyme in hexane spontaneously increases with the concentration of AOT and is about 10% of the activity in aqueous system. In xylene, a catalytic activity higher than that in bulk aqueous conditions was found for the samples containing 0.1–0.3 mg/ml of mannanase, while for the samples with a higher concentration of enzyme, the activity was hardly detected.
Keywords: Endo-1,4-β-d-mannanase; Liquid–liquid extraction; Hexane; Xylene; Organic solvent; Biocatalysis

The different ionic molecules/compounds were used as a ligand for the immobilization of penicillin G acylase on the highly porous cellulose-based polymeric membrane having buffer flux 1,746 LMH (L m−2 h−1) at 0.5 bar pressure. The immobilized enzyme activity around 250 UApp was obtained with the ligand such as proline, tryptophan, casein acid hydrolysate, and brilliant green. Comparatively, proline showed less IMY% (percentage immobilization yield—58) but higher RTA% (percentage of activity retention—71) and specific activity (145 UApp g−1). However, the crosslinked preparation of brilliant green obtained using glutaraldehyde showed 82 ± 2.7% immobilized enzyme activity after the completion of successive five cycles. In comparison with the free enzyme, the enzyme immobilized on the brilliant green coupled membrane showed around 2.4-fold increase in K m value (47.4 mM) as well as similar optimum pH (7.2) and temperature (40 °C). The immobilized enzyme retained almost 50% activity after 107 days and 50 cycles of operation. Almost 50% decrease in buffer flux after enzyme immobilization was observed. At the end of the 30 cycles, flux pattern shows around 38% decrease in buffer flux however, after 16 cycles of operation flux moves closer towards the steady state.
Keywords: Penicillin G acylase; Immobilization; Proline; Brilliant green; Polymeric membrane

Screening of Food Grade Lipases to be Used in Esterification and Interesterification Reactions of Industrial Interest by Ariela Veloso de Paula; Gisele Fátima Morais Nunes; Josiane de Lourdes Silva; Heizir Ferreira de Castro; Júlio César dos Santos (1146-1156).
Seven food grade commercially available lipases were immobilized by covalent binding on polysiloxane–polyvinyl alcohol (POS-PVA) hybrid composite and screened to mediate reactions of industrial interest. The synthesis of butyl butyrate and the interesterification of tripalmitin with triolein were chosen as model reactions. The highest esterification activity (240.63 μM/g min) was achieved by Candida rugosa lipase, while the highest interesterification yield (31%, in 72 h) was achieved by lipase from Rhizopus oryzae, with the production of about 15 mM of the triglycerides C50 and C52. This lipase also showed a good performance in butyl butyrate synthesis, with an esterification activity of 171.14 μM/g min. The results demonstrated the feasibility of using lipases from C. rugosa for esterification and R. oryzae lipase for both esterification and interesterification reactions.
Keywords: Food grade lipases; Interesterification; Esterification; Immobilization; Lipase

Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant β-Glucuronidase by Goran Robić; Cristiane S. Farinas; Elíbio L. Rech; Everson A. Miranda (1157-1167).
Soybean is one of the plant species with potential to be used as seed-based bioreactor. As part of the downstream processing (DSP) of this technology, extraction is a key step, since it defines the composition of the solution from which the recombinant product will be purified. In the present work, the characteristics of soybean seeds used as a bioreactor were evaluated from a process engineering standpoint through analysis of the influence of pH and ionic strength on the extraction of recombinant β-glucuronidase (rGUS). Concentrations of recombinant protein and native soybean compounds were analyzed and compared with similar data from extraction studies using transgenic corn seeds as bioreactor. Efficient rGUS extraction was obtained at pH of around 5.5 with no addition of salt. Soybean seed extracts had lower levels of co-extracted native compounds, than corn seed extracts, and should be considered as a potential plant bioreactor in terms of DSP.
Keywords: Downstream processing; Extraction; Recombinant protein; Soyben seed; Transgenic plants

Heterologous Expression and Characterization of an Endoglucanase from a Symbiotic Protist of the Lower Termite, Reticulitermes speratus by Nemuri Todaka; Crisanto M. Lopez; Tetsushi Inoue; Kanako Saita; Jun-ichi Maruyama; Manabu Arioka; Katsuhiko Kitamoto; Toshiaki Kudo; Shigeharu Moriya (1168-1178).
RsSymEG, an endoglucanase of glycosyl hydrolase family (GHF) 7 encoded by a transcript isolated from the symbiotic protist of the termite Reticulitermes speratus, is expressed in Aspergillus oryzae. Interestingly, purified RsSymEG1 has a relatively higher specific activity (603 μmol min−1 mg−1 protein) and V max value (769.6 unit/mg protein) than previously reported data for GHF7 endoglucanase of Trichoderma ressei. It also has the same K m value (1.97 mg/ml) with Clostridium cellulolyticum enzymes that contain cellulose binding module, a property indicative of high affinity to substrate, though no cellulose binding module is found within it. Thin-layer chromatography analysis revealed that RsSymEG1 preferentially hydrolyzes the β-1,4-cellulosic linkage of cellodextrins into cellobiose and glucose.
Keywords: Cellulase; Biomass; Termite; Protists

Role of Toll-Like Receptor 3, RIG-I, and MDA5 in the Expression of Mesothelial IL-8 Induced by Viral RNA by Markus Wörnle; Matthias Sauter; Kathrin Kastenmüller; Andrea Ribeiro; Thomas Mussack; Roland Ladurner; Thomas Sitter (1179-1187).
Interleukin-8 (IL-8) is a chemokine that has been shown to be a potent chemoattractant for polymorphonuclear neutrophils from the vascular compartment into the pleural space during infectious pleural effusions. Mesothelial cells express the viral receptors Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Activation of these receptors by viral RNA exemplified by poly (I:C) RNA leads to a time- and dose-dependent increase of mesothelial IL-8 synthesis. To show the specific effect of viral receptors, knockdown experiments with short interfering RNA specific for TLR3, RIG-I and MDA5 were performed. This novel finding of functional expression of these viral sensors on human mesothelial cells may indicate a novel link between viral infections and mesothelial inflammation and indicates a pathophysiologic role of viral receptors in these processes.
Keywords: IL-8; MDA5; Poly (I:C) RNA; RIG-I; Toll-like receptor 3

New Model for Polymerization of Oligomeric Alcohol Dehydrogenases into Nanoaggregates by Abolfazl Barzegar; Ali A. Moosavi-Movahedi; Anahita Kyani; Bahram Goliaei; Shahin Ahmadian; Nader Sheibani (1188-1205).
Polymerization and self-assembly of proteins into nanoaggregates of different sizes and morphologies (nanoensembles or nanofilaments) is a phenomenon that involved problems in various neurodegenerative diseases (medicine) and enzyme instability/inactivity (biotechnology). Thermal polymerization of horse liver alcohol dehydrogenase (dimeric) and yeast alcohol dehydrogenase (tetrameric), as biotechnological ADH representative enzymes, was evaluated for the development of a rational strategy to control aggregation. Constructed ADH nuclei, which grew to larger amorphous nanoaggregates, were prevented via high repulsion strain of the net charge values. Good correlation between the variation in scattering and λ −2 was related to the amorphousness of the nanoaggregated ADHs, shown by electron microscopic images. Scattering corrections revealed that ADH polymerization was related to the quaternary structural changes, including delocalization of subunits without unfolding, i.e. lacking the 3D conformational and/or secondary-ordered structural changes. The results demonstrated that electrostatic repulsion was not only responsible for disaggregation but also caused a delay in the onset of aggregation temperature, decreasing maximum values of aggregation and amounts of precipitation. Together, our results demonstrate and propose a new model of self-assembly for ADH enzymes based on the construction of nuclei, which grow to formless nanoaggregates with minimal changes in the tertiary and secondary conformations.
Keywords: Self-assembly; Nanoensembles; Nucleation; Nanoaggregates; Net charge; Scattering correction; Polymerization; Electron microscopy

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A by Wei-Gang Hu; Scott Jager; Damon Chau; Dave Mah; Les P. Nagata (1206-1216).
In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 105 Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K d value of 10−7 M.
Keywords: Recombinant full-length human antibody; Botulinum neurotoxin A; In vitro immunization; Fab library; 2A self-cleavage linker; Monocistronic expression vector; Mammalian expression

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, the most common neoplasm in untreated HIV-1-infected individuals, and several B cell disorders. KSHV infection goes through lytic and latent phases, and the switch from latency to lytic replication is governed by viral replication and transcription activator (RTA). RTA consists of 691 amino acids, containing an N-terminal DNA-binding and a C-terminal activation domain. In the present study, polyclonal antibody against RTA was generated and evaluated. The C-terminal region of RTA (E482~D691) was expressed in Escherichia coli, purified by affinity chromatography, and utilized to raise polyclonal antibody in BALB/c mice. High-affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:13,500 for ELISA and 1:20,000 for Western blot analysis. The antibody can specifically recognize full-length RTA expressed in both E. coli and mammalian cells. Furthermore, endogenous RTA can be detected with the antibody in TPA-induced BCBL-1 cells under various conditions. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of RTA.
Keywords: Replication and transcription activator; Kaposi’s sarcoma-associated herpesvirus; Polyclonal antibody; C-terminal region

Purification and Characterization of a Low Molecular Weight of β-Mannanase from Penicillium occitanis Pol6 by Monia Blibech; Raoudha Ellouz Ghorbel; Ines Fakhfakh; Patricia Ntarima; Katheleen Piens; Abir Ben Bacha; Semia Ellouz Chaabouni (1227-1240).
The highest β-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified β-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 °C and pH 4.0. It was stable for 30 min at 70 °C and has a broad pH stability (2.0–12.0). ManIII showed K m, V max, and K cat values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s−1 with locust bean gum as substrate, respectively. It was inhibited by mannose with a K I of 0.610−3 mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.
Keywords: β-Mannanase; Penicillium occitanis ; Flour of carob seed; Inhibition; Mannose

Removal of azo dyes from effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are mutagenic and carcinogenic. Pseudomonas aeruginosa grew well in the presence of Remazol Orange (RO) and was able to decolorize and degrade it. In the present study, the decolorization and degradation efficiency using single culture P. aeruginosa with RO and textile wastewaters is studied. The elucidation of decolorization pathway for P. aeruginosa is of special interest. The degradation pathway and the metabolic products formed during the degradation were also predicted with the help of high performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analysis. The data show the cleavage of the azo dye RO to form both methyl metanilic acid and 4-aminobenzoic acid after decolorization and finally to oxidation forms benzoic acid, alkenes, aldehydes, and alkynes. The organism was able to decolorize the dye RO and wastewater effectively to the maximum of 82.4% and 62%, respectively.
Keywords: Pseudomonas aeruginosa ; Decolorization; Biodegradation; Metabolic pathway; Textile wastewater treatment; Growth kinetics

Extracellular lipase production by Staphylococcus epidermidis CMST Pi 2 isolated from the intestine of shrimp Penaeus indicus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tested tuna preparations, defatted fish meat supported the maximum lipase production, and 2.5% concentration of the same was found to be optimum for maximizing the lipase production. The effect of carbon sources on lipase production revealed that glucose aided the higher lipase production than any other tested carbon source and a concentration of 2% glucose registered as optimum to enhance the lipase production. The halotolerancy of S. epidermidis CMST Pi 2 for lipase production indicated that 4% of sodium chloride was optimum to yield maximum lipase. Among the surfactants tested, lipase production was high in Tween 20 added medium when compared to other surfactants, and its optimum concentration recorded was 0.8%. Partial characterization of crude enzyme revealed that pH 7 and 55 °C temperature were optimum for maximum lipase activity.
Keywords: Tuna waste; Lipase; Shrimp gut bacteria; S. epidermidis ; Tween 20