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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.157, #3)

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus by Bo Xiao; Qingwang Li; Bo Feng; Zengsheng Han; Dawei Gao; Rui Zhao; Jian Li; Kun Li; Xubo Zhi; Hai Yang; Zhibin Liu (pp. 357-366).

For producing a large quantity of human nerve growth factor beta (hNGF-β), the recombinant adenovirus containing an expression cassette of hNGF-β complementary DNA gene was constructed and then instilled directly into goats’ mammary glands. The recombinant hNGF-β was detected in the milk. The maximal expression level of recombinant hNGF-β in the milk reached 196.8 mg/l. The biological activity of the recombinant hNGF-β, compared with the commercial hNGF-β, was tested using PC12 cells. The results demonstrate that instilling recombinant adenovirus directly into the mammary gland of goat is an efficient approach to producing a large quantity of hNGF-β.

Keywords: Adenovirus; Goat; Human nerve growth factor beta; Mammary gland; Milk

Biodegradation of Fluoranthene by Basidiomycetes Fungal Isolate Pleurotus Ostreatus HP-1 by Hardik Patel; Akshaya Gupte; Shilpa Gupte (pp. 367-376).

The biodegradation of fluoranthene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), was investigated in submerged culture using the wood decaying fungus isolated from forest locality in Gujarat, India. The basidiomycete fungal isolate was found to have an ability to grow on sabaroud dextrose agar containing 50 mgl⁻¹ of each naphthalene, anthracene, acenaphthene, benzo (a) anthracene, pyrene, flouranthene, carbazole, and biphenyl. The involvement of extracellular fungal peroxidases such as manganese peroxidase (MnP) and laccase (Phenol oxidase) in the degradation of fluoranthene was studied. On the eighth day of incubation 54.09% of 70 mg l⁻¹ fluoranthene was removed. There after no PAHs removal was observed till the 20th day of the incubation period. The isolate was identified as Pleurotus ostreatus by 18S rRNA, 5.8S rRNA, and partial 28S rRNA gene sequencing. To the best of our knowledge this is the first time Pleurotus ostreatus have been reported to degrade such a high concentration of fluoranthene within much lower time period of incubation. Depletion in the residual fluoranthene in the culture medium was determined by HPLC. Attempts were made to identify the degradation product in the culture medium with the help of FT-IR, NMR, and HPTLC analysis. In the present study positive correlation between fluoranthene degradation and the ligninolytic enzyme (MnP and laccase) production is observed, thus this isolate can play an effective role for bioremediation of PAHs contaminated sites.

Keywords: Basidiomycete; Degradation; Fluoranthene; Ligninolytic enzymes; Wood decaying fungus; Pleurotus ostreatus

Heterologous Expression of Legumin Gene in E. coli Isolated from cDNA Clones of Immature Seeds of Pigeonpea (Cajanus cajan L.) by Subodh Gupta; Avinash Mishra; A. K. Verma; Ruchi Pandey; Laxmi Chand (pp. 377-394).

Proteins are one of the targets for improving the nutritional quality, and attempts are being made through manipulation of its native gene(s). Pigeonpea (Cajanus cajan L.) is one of the nutritionally important legumes of tropical and subtropical regions of the world, and studies of the structure of seed storage proteins and their interactions have been limited by the difficulty of isolating single-protein subunits in large amounts from a complex mixture of the seed endosperm. One way to overcome this problem is the expression of seed storage protein-encoded gene(s) in heterologous systems that have additional advantages wherein specific gene modifications can be made and the new gene constructs can quickly be expressed. Legumin protein was extracted from pigeonpea seeds of different developmental stages (5th to 25th day after flowering [DAF]) and characterized. The legumin gene (leg) of size 1.482 kb was screened, using the deoxygenin-labeled legumin probe, from the complementary deoxyribonucleic acid (cDNA) library, constructed from 18-day-old (DAF) immature seeds of pigeonpea and sequenced (accession no. AF3555403). The legumin gene was further characterized by DNA blotting, and its probable secondary structure was predicted using online ExPASy server. Significant Protein Data Bank (PDB) alignment of the deduced legumin protein by BLASTP was observed with proglycinin of soybean. Comparative 3D structural homology was predicted by Cn3D software, and the legumin protein showed the 3D structure alignment and interaction homology with proglycinin chain 1FXZA (PDB no. 1FXZ). The legumin gene was subcloned in vector pET-24a driven by the bacterial promoter, and its expression was detected in Escherichia coli by immunoblotting using polyclonal antibodies, raised against the purified legumin protein.

Keywords: Heterologous expression; Globulin; Legumin; Pigeonpea; Seed storage protein

Characterization of Unique and Differentially Expressed Proteins in Anthracnose-Tolerant Florida Hybrid Bunch Grapes by Hemanth K. N. Vasanthaiah; Ramesh Katam; Sheikh M. Basha (pp. 395-406).

Anthracnose is a major disease in Florida hybrid bunch grapes, caused by a fungus viz. Elsinoe ampelina. Florida hybrid bunch grapes are grown in southeastern USA for their superior wine characteristics. However, the effect of anthracnose on grape productivity and wine quality is a major concern to grape growers. Our research is aimed at determining biochemical basis of anthracnose tolerance in Florida hybrid bunch grape. Leaf samples were collected from the plants infected with E. ampelina at different periods and analyzed for differential protein expression using high throughput two-dimensional gel electrophoresis. Among the 32 differentially expressed leaf proteins, two were uniquely expressed in tolerant genotypes in response to E. ampelina infection. These proteins were identified as mitochondrial adenosine triphosphate synthase and glutamine synthetase, which are known to play a major role in carbohydrate metabolism and defense. Several proteins including ribulose 1-5 bisphosphate-carboxylase involved in photosynthesis were found to be suppressed in susceptible genotypes compared to tolerant genotypes following E. ampelina infection. The results indicate that the anthracnose-tolerant genotypes have the ability to up-regulate and induce new proteins upon infection to defend the invasion of the pathogen as well as maintain the normal regulatory processes.

Keywords: Grape; Anthracnose; Differential protein expression; Mitochondrial ATP synthase; Glutamine synthetase; Ribulose 1-5 bisphosphate-carboxylase

Production of Anti-Methicillin-Resistant Staphylococcus Activity from Bacillus subtilis sp. Strain B38 Newly Isolated from Soil by Olfa Tabbene; Imen Ben Slimene; Faten Bouabdallah; Maria-Luisa Mangoni; Maria-Camino Urdaci; Ferid Limam (pp. 407-419).

B38 bacterial strain, isolated from Tunisian soil showed a strong antimicrobial activity. Based on biochemical characterization and 16S rDNA sequence analysis, B38 strain was identified as Bacillus subtilis. Cell culture supernatant showed antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several Gram-positive and Gram-negative bacteria. Antifungal activity against phytopathogenic fungi was also observed. Antibacterial activity production started at early exponential growth phase, and maximum activity was reached at the stationary phase. This antibacterial activity was neither affected by proteases, lipase, and organic solvents, nor by surfactants. It was stable over a wide pH range and still active after autoclaving at 121 °C during 20 min. Thin layer chromatography followed by bioautography assay allowed the detection of four active spots with R _f values of 0.30, 0.47, 0.70, and 0.82. The single spot with R _f 0.30 showed antifungal activity, whereas the spots with R _f values of 0.47, 0.70, and 0.82 exhibited antibacterial activity.

Keywords: Antibacterial activity; Bacillus subtilis ; Multidrug-resistant pathogenic bacteria; TLC; Bioautography; Peptides antibiotics

Biological Activities of Thermo-tolerant Microbes from Fermented Rice Bran as an Alternative Microbial Feed Additive by J. H. Koh; H. J. Suh (pp. 420-430).

To evaluate the commercial potential of new microbial feed additive, Issatchenkia orientalis Y266 and Bacillus subtilis B266 from commercial fermented rice bran were tested for their tolerance or resistance to pH, bile, oxgall, and temperature. It was found that the strains grew very well up to pH 3.0 and resistant to relatively high concentrations of bile salt and oxgall. I. orientalis and B. subtilis are extremely tolerant in range of 70–90°C in solid medium. B. subtilis B266 also has excellent tolerant property up to 90°C in liquid medium. The health indexes (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the fermented rice bran with these strains (0.25% addition to diet) in comparison with the Avilamycin (20 mg/kg diet)-fed group (p < 0.05). The fermented rice bran-fed group showed a better microbial flora in the small intestines. Accordingly, it would appear that the fermented rice bran with these strains may be a potential candidate for an alternative microbial feed additive.

Keywords: Bacillus sp.; Issatchenkia orientalis ; DFMs; Bile acid; Thermo tolerance

The Methane Monooxygenase Intrinsic Activity of Kinds of Methanotrophs by Yingxin Zhang; Jiaying Xin; Linlin Chen; Chungu Xia (pp. 431-441).

Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-β-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1–5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs.

Keywords: Methanotroph; Methane monooxygenase; Poly-β-hydroxybutyrate; Reducing equivalent

The In Vitro Anti-pathogenic Activity of Immunoglobulin Concentrates Extracted from Ovine Blood by Kyoung-Sik Han; Mike Boland; Harjinder Singh; Paul J. Moughan (pp. 442-452).

An immunoglobulin-rich fraction has been prepared from ovine blood in our laboratory. We have investigated its antibacterial activity and binding activity to pathogenic whole cell antigens, lipopolysaccharide (LPS) and staphylococcal enterotoxin B. Ovine immunoglobulin concentrate (OIC) comprised about 73 ± 2% of IgG and 11 ± 1% of IgM on a protein basis. It inhibited the growth of all 13 strains of pathogens tested, but the inhibitory activity varied according to bacterial strain. The inhibitory activity of OIC was attributed to the high contents of undenatured immunoglobulin present because its inhibitory activity was destroyed by pepsin digestion and heat treatment (65°C for 30 min). OIC bound to all the Gram-positive and Gram-negative pathogens, regardless of cell wall structure. The highest magnitude of crossreactivity to whole cell antigens was against Staphylococcus epidermidis and Shigella soneii strains (p < 0.001). The binding activity of OIC to LPS obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium was assessed by enzyme-linked immunosorbent assay and lymphoblast K-562 proliferation assay. OIC bound to LPS with a binding activity that was dependent on OIC concentration and saturable, showing typical hyperbolic curves. For toxin-binding activity, an OIC concentration-dependent trend like that for LPS-binding activity was also observed. This preliminary evidence suggests that the OIC used in this study could be a promising supplement for protecting against pathogenic bacteria.

Keywords: Ovine immunoglobulin concentrate; Inhibitory activity; Crossreactivity; Lipopolysaccharide (LPS); Staphylococcal enterotoxin B (SEB)

Pilot-Scale Fermentation of Aqueous-Ammonia-Soaked Switchgrass by Asli Isci; Jennifer N. Himmelsbach; John Strohl; Anthony L. Pometto III; D. Raj Raman; Robert P. Anex (pp. 453-462).

Aqueous-ammonia-steeped switchgrass was subject to simultaneous saccharification and fermentation (SSF) in two pilot-scale bioreactors (50- and 350-L working volume). Switchgrass was pretreated by soaking in ammonium hydroxide (30%) with solid to liquid ratio of 5 L ammonium hydroxide per kilogram dry switchgrass for 5 days in 75-L steeping vessels without agitation at ambient temperatures (15 to 33 °C). SSF of the pretreated biomass was carried out using Saccharomyces cerevisiae (D₅A) at approximately 2% glucan and 77 filter paper units per gram cellulose enzyme loading (Spezyme CP). The 50-L fermentation was carried out aseptically, whereas the 350-L fermentation was semiaseptic. The percentage of maximum theoretical ethanol yields achieved was 73% in the 50-L reactor and 52–74% in the 350-L reactor due to the difference in asepsis. The 350-L fermentation was contaminated by acid-producing bacteria (lactic and acetic acid concentrations approaching 10 g/L), and this resulted in lower ethanol production. Despite this problem, the pilot-scale SSF of aqueous-ammonia-pretreated switchgrass has shown promising results similar to laboratory-scale experiments. This work demonstrates challenges in pilot-scale fermentations with material handling, aseptic conditions, and bacterial contamination for cellulosic fermentations to biofuels.

Keywords: Switchgrass; Pilot scale; Aqueous ammonia soaking; Ethanol production; Simultaneous saccharification and fermentation (SSF)

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of Rhodotorula aurantiaca (NCIM 3425) by Atul Kumar; Sanjay Singh; Pankaj Poddar; Asmita Prabhune; Archana Pundle (pp. 463-472).

Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.

Keywords: Penicillin V acylase; Rhodotorula aurantiaca ; Intracellular enzyme; Permeabilization; Atomic force microscopy

Strain Construction for Ethanol Production from Dilute-Acid Lignocellulosic Hydrolysate by Fei Yan; Fali Bai; Shen Tian; Jinxin Zhang; Zuoyang Zhang; Xiushan Yang (pp. 473-482).

In order to construct a strain that converts sugar mixture and resist/metabolize inhibitors in lignocellulosic dilute-acid hydrolysate, the biotechnology of inactive intergeneric fusion between Saccharomyces cerevisiae and Pachysolen tannophilis was performed. Fusant 1 was successfully obtained as a hybrid strain, which was screened out by xylose and mixed sugar (xylose and glucose) fermentation. This strain showed good abilities of ethanol production, ethanol tolerance, and resistance to the toxic inhibitors presenting in the hydrolysate. The maximum volumetric yield of ethanol and yield of xylitol in mixed sugar was 9.52 g/l and 0.44 g/g, respectively. The results indicated that the constructed strain Fusant 1 was a good producer for ethanol and xylitol from lignocellulosic dilute-acid hydrolysate.

Keywords: Inactive intergeneric fusion; Lignocellulosic dilute-acid hydrolysate; Inhibitors; Ethanol fermentation

Catalytic and Thermodynamic Characterization of Endoglucanase (CMCase) from Aspergillus oryzae cmc-1 by Muhammad Rizwan Javed; Muhammad Hamid Rashid; Habibullah Nadeem; Muhammad Riaz; Raheela Perveen (pp. 483-497).

Monomeric extracellular endoglucanase (25 kDa) of transgenic koji (Aspergillus oryzae cmc-1) produced under submerged growth condition (7.5 U mg⁻¹ protein) was purified to homogeneity level by ammonium sulfate precipitation and various column chromatography on fast protein liquid chromatography system. Activation energy for carboxymethylcellulose (CMC) hydrolysis was 3.32 kJ mol⁻¹ at optimum temperature (55 °C), and its temperature quotient (Q ₁₀) was 1.0. The enzyme was stable over a pH range of 4.1–5.3 and gave maximum activity at pH 4.4. V _max for CMC hydrolysis was 854 U mg⁻¹ protein and K _m was 20 mg CMC ml⁻¹. The turnover (k _cat) was 356 s⁻¹. The pK _a1 and pK _a2 of ionisable groups of active site controlling V _max were 3.9 and 6.25, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: ΔH* = 0.59 kJ mol⁻¹, ΔG* = 64.57 kJ mol⁻¹ and ΔS* = −195.05 J mol⁻¹ K⁻¹, respectively. Activation energy for irreversible inactivation ‘E _a(d)’ of the endoglucanase was 378 kJ mol⁻¹, whereas enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) of activation at 44 °C were 375.36 kJ mol⁻¹, 111.36 kJ mol⁻¹ and 833.06 J mol⁻¹ K⁻¹, respectively.

Keywords: CMCase; Activation energy; Enthalpy; Entropy; Gibbs free energy; Thermostability

Characterization of Thermo-stable Endoinulinase from a New Strain Bacillus Smithii T7 by Wei Gao; Yongming Bao; Yang Liu; Xiuli Zhang; Jingyun Wang; Lijia An (pp. 498-506).

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.

Keywords: Inulin; Inulinase; Bacillus smithii ; Enzyme characterization; Thermo-stable enzyme; Optimization

Biomass Productivities in Wild Type and Pigment Mutant of Cyclotella sp. (Diatom) by Michael H. Huesemann; Tom S. Hausmann; Richard Bartha; M. Aksoy; Joseph C. Weissman; John R. Benemann (pp. 507-526).

Microalgae are expected to play a significant role in greenhouse gas mitigation because they can utilize CO₂ from power plant flue gases directly while producing a variety of renewable carbon-neutral biofuels. In order for such a microalgal climate change mitigation strategy to become economically feasible, it will be necessary to significantly improve biomass productivities. One approach to achieve this objective is to reduce, via mutagenesis, the number of light-harvesting pigments, which, according to theory, should significantly improve the light utilization efficiency, primarily by increasing the light intensity at which photosynthesis saturates (I _s). Employing chemical (ethylmethylsulfonate) and UV mutagenesis of a wild-type strain of the diatom Cyclotella, approximately 10,000 pigment mutants were generated, and two of the most promising ones (CM1 and CM1-1) were subjected to further testing in both laboratory cultures and outdoor ponds. Measurements of photosynthetic oxygen production rates as a function of light intensity (i.e., P–I curves) of samples taken from laboratory batch cultures during the exponential and linear growth phase indicated that the light intensity at which photosynthesis saturates (I _s) was two to three times greater in the pigment mutant CM1-1 than in the wild type, i.e., 355–443 versus 116–169 μmol/m² s, respectively. While theory, i.e., the Bush equation, predicts that such a significant gain in I _s should increase light utilization efficiencies and thus biomass productivities, particularly at high light intensities, no improvements in biomass productivities were observed in either semi-continuous laboratory cultures or outdoor ponds. In fact, the maximum biomass productivity in semi-continuous laboratory culture was always greater in the wild type than in the mutant, namely 883 versus 725 mg/L day, respectively, at low light intensity (200 μmol/m² s) and 1,229 versus 1,043 mg/L day, respectively, at high light intensity (1,000 μmol/m² s). Similarly, the biomass productivities measured in outdoor ponds were significantly lower for the mutant than for the wild type. Given that these mutants have not been completely characterized in these initial studies, the exact reasons for their poor performance are not known. Most likely, it is possible that the mutation procedure affected other photosynthetic or metabolic processes. This hypothesis was partially validated by the observation that the pigment mutant had a longer lag period following inoculation, a lower maximum specific growth rate, and poorer stability than the wild type.

Keywords: Biomass productivity; Antenna mutants; Bush equation; Saturating light intensity; Light utilization efficiency; Semi-continuous culture; Outdoor ponds

PVA-Hydrogel Entrapped Candida Guilliermondii for Xylitol Production from Sugarcane Hemicellulose Hydrolysate by Mário A. A. da Cunha; Attilio Converti; Júlio C. Santos; Sylvia T. S. Ferreira; Silvio S. da Silva (pp. 527-537).

Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing–thawing method. Entrapment experiments were planned according to a 2³ full factorial design, using the PVA concentration (80, 100, and 120 g L⁻¹), the freezing temperature (−10, −15, and −20 °C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 °C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L⁻¹, a xylitol yield on consumed xylose of 0.49 g g⁻¹ and a xylitol volumetric productivity of 0.24 g L⁻¹ h⁻¹ were obtained.

Keywords: C. guilliermondii ; Immobilization; PVA-hydrogel; Sugarcane bagasse; Hydrolysate

Polyethylenimine-Based Transfection Method as a Simple and Effective Way to Produce Recombinant Lentiviral Vectors by Jorge R. Toledo; Yanet Prieto; Nayrobis Oramas; Oliberto Sánchez (pp. 538-544).

HIV-1-derived lentiviral vectors (LvV) are within the most attractive gene delivery vehicles in the context of both dividing and quiescent cells. LvV is currently produced by the conventional calcium phosphate precipitation method. Nevertheless, this procedure is highly susceptible to variations in pH and impurities, which lead to inconsistencies in LvV production. Here, we present a simple and robust procedure for LvV production using branched 25 kDa polyethylenimine, with a transfection efficiency of over 90% and viral titer yields of about 1 × 10⁷ infective lentiviral particles per milliliter. The procedure outlined is simple, consistent, and as inexpensive as the CaPO₄-based method.

Keywords: Lentiviral vector; Polyethylenimine; Transfection

HMG-CoA Reductase Inhibitor Regulates Endothelial Progenitor Function Through the Phosphatidylinositol 3′-Kinase/AKT Signal Transduction Pathway by Xiaohong Li; Biao Xu (pp. 545-553).

HMG-CoA reductase inhibitor (statins) are known to have pleiotropic effects. We examined the effect and mechanism of simvastatin on peripheral endothelial progenitor cells (EPCs). Rats were divided into simvastatin group and the control group after cardiac infarction operation. Simvastatin treatment significantly increased the number of peripheral blood CD34+ CD133+ cells, and serum concentration of vascular endothelial growth factor (VEGF) and AKT was markedly increased in vivo. In cultured EPC, simvastatin increased the concentrations of VEGF, AKT and eNOS. Western blots analysis showed that simvastatin increased the phosphorylation of eNOS and FKHRL1, which can be blocked by the PI3K/AKT pathway blocker LY294002 . Our study demonstrated that simvastatin increases the mobilization of EPCs after cardiac infarction. In in vitro study, simvastatin increases the phosphorylation of eNOS and of FKHRL1 through the PI3K/AKT signaling pathway.

Keywords: Simvastatin; Endothelial progenitor cells; VEGF; AKT; NOS

Functional Expression of Bacillus anthracis Protective Antigen in E. coli by Fatemeh Vahedi; Mojtaba Sankian; Mahmoud Mahmoudi (pp. 554-561).

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.

Keywords: Bacillus anthracis ; Protective antigen (PA); Expression; Immunogenicity

Construction, Purification, and Characterization of Anti-BAFF scFv–Fc Fusion Antibody Expressed in CHO/dhfr⁻ Cells by Meng Cao; Peng Cao; Huaijiang Yan; Wuguang Lu; Fang Ren; Yunlong Hu; Shuangquan Zhang (pp. 562-574).

Elevated levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, we have constructed a vector for the expression of a novel compact antibody composed of anti-BAFF single-chain antibody fragment (scFv) and the Fc region (the hinge region, CH2, and CH3 domains) of human IgG1 in Chinese hamster ovary cells. The scFv–Fc fusion protein, showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges, was affinity-purified on protein A Sepharose from culture supernatant. The scFv–Fc antibody was demonstrated to retain high binding affinity to antigen and prolonged clearance time in blood and to possess some human IgG crystallizable fragment effector functions such as protein A binding and antibody-dependent cellular cytotoxicity. These results suggest that this recombinant antibody may have therapeutic applications in the therapy of autoimmune disorders mediated by BAFF.

Keywords: BAFF; CHO cells; Recombinant antibody; scFv–Fc; Stable expression

Purification and Characterization of Cell Suspensions Peroxidase from Cotton (Gossypium hirsutum L.) by Tanoh Hilaire Kouakou; Edmond Ahipo Dué; N’guessan Eugène Jean Parfait Kouadio; Sébastien Niamké; Yatty Justin Kouadio; Jean-Michel Mérillon (pp. 575-592).

Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 °C and was relatively stable at 20–30 °C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and β-mercaptoethanol. Their activities were highly enhanced by Al³⁺, Fe³⁺, Ca²⁺, and Ni²⁺, but they were moderately inhibited by Mn²⁺ and K⁺. The enzyme lost 50% to 62% of its activity in the presence of Zn²⁺ and Hg²⁺.

Keywords: Gossypium hirsutum L.; Cell suspension; Peroxidase; Purification; Characterization

Silk-Fiber Immobilized Lipase-Catalyzed Hydrolysis of Emulsified Sunflower Oil by Sushovan Chatterjee; Lepakshi Barbora; Swaranjit Singh Cameotra; Pinakeswar Mahanta; Pranab Goswami (pp. 593-600).

Lipase was immobilized in silk fibers through glutaraldehyde cross-linking to a maximum loading of 59 U/g silk-fiber and the immobilized lipase was utilized for the hydrolysis of sunflower oil (Helianthus annuus). The hydrolytic activity of the lipase, which was poor in biphasic oil in water system, was increased significantly when the sunflower oil was emulsified in aqueous medium. The hydrolytic activities of the immobilized lipase were 48.73 ± 1.26 U, 36.11 ± 0.96 U, and nil when the substrate sunflower oil was used as emulsion created by a rhamnolipid biosurfactant, Triton X100, and ultrasonication, respectively. Although the efficiency of the immobilized lipase was less than 12% than the corresponding free lipase, the immobilized lipase could be reused for the biosurfactant-mediated hydrolysis of sunflower oil up to third cycle of the reaction. The yield of the fatty acids in the second, third, and fourth cycles were 49.45%, 22.91%, and 5.09%, respectively, of the yield obtained in the first cycle.

Keywords: Sunflower oil; Lipase; Immobilization; Fatty acid; Biosurfactant; Silk fiber

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.157, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.157, #3)