Applied Biochemistry and Biotechnology (v.157, #1)
High-Level Expression, Purification, and In Vitro Refolding of Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) by Dongming Wang; Linmei Shi (1-9).
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. In this paper, we report the expression, purification, and preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) without posttranslational modifications, which may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant nonfusion sTRAIL protein, the encoding region for sTRAIL was cloned between KpnI and BamHI in pET32a. The Trx (thioredoxin)/sTRAIL fusion proteins were expressed in the form of inclusion bodies in Escherichia coli host strain BL21 (DE3). The expression level was more than 35% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2 M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >98% were obtained. Trx/sTRAIL L proteins were digested by enterokinase to both Trx and sTRAIL fragments, which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rsTRAIL (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the recombinant sTRAIL may have cancer therapeutic applications.
Keywords: sTRAIL; Fusion protein; Inclusion bodies; Protein expression; Refolding; Protein purification
Phytodegradation Potential of Erythrina crista-galli L., Fabaceae, in Petroleum-Contaminated Soil by Vanessa de Farias; Leila Teresinha Maranho; Eliane Carvalho de Vasconcelos; Marco Aurélio da Silva Carvalho Filho; Luiz Gustavo Lacerda; Jayme Augusto Menegassi Azevedo; Ashok Pandey; Carlos Ricardo Soccol (10-22).
This work aimed at investigating both the tolerance and the phytodegradation potential of Erythrina crista-galli L. in petroleum-contaminated soil. It consisted in analyzing E. crista-galli germination, surviving, growth, and development when cultivated at different contaminant concentrations and pollutant degradation rates. This specimen was selected because it presented a special behavior among others also exposed to petroleum in an accident that occurred in the Araucaria region (south of Brazil), resulting in a four-million-liter oil spill. The experiment was carried out in a greenhouse containing non-contaminated soil (NCS), vegetated contaminated soil (VCS), and non-vegetated contaminated soil (NVCS) at the following petroleum concentrations: 25 g kg−1 (VCS-25), 50 g kg−1 (VCS-50), and 75 g kg−1 (VCS-75). After 60 days, the soil samples were analyzed by gas chromatography. Germination was more and more evident as higher petroleum concentrations were observed. The surviving rates of groups NCS, VCS-25, VCS-50, and VCS-75 were 64%, 70%, 61%, and 96%, respectively. The VCS group growth was reduced when compared to the control group (NCS). The individuals exposed to petroleum pollution presented differences in the anatomic structure of their roots when compared to the NCS group. It was observed that the petroleum degradation rate was higher for VCS group than for NVCS. E. crista-galli is potentially recommended for petroleum-contaminated soils because of its positive association in the presence of contamination.
Keywords: Phytodegradation; Petroleum; Erythrina ; Root; Fabaceae
Purification and Characterization of Thermostable Chitinase from Bacillus licheniformis SK-1 by Sanya Kudan; Rath Pichyangkura (23-35).
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K m and V max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.
Keywords: Bacillus licheniformis SK-1; Characterization; Chitinase; N-bromosuccinamide; Purification
Isolation and Identification of a Newly Isolated Alternaria sp. ND-16 and Characterization of Xylanase by Yin Li; Zhiqiang Liu; Fengjie Cui; Lifeng Ping; Chongyan Qiu; Geng Li; Lijiao Yan (36-49).
Alternaria sp. ND-16, a bacterium isolated from soil sample, was identified as a strain of Alternaria mali based on the morphology and comparison of internal transcribed spacer rDNA gene sequence studies. Furthermore, it is demonstrated that this strain has xylanase activity, and the activity can be optimized under suitable growing conditions where wheat bran and urea are the primary sources of carbon and nitrogen. Partially purified xylanase from Alternaria sp. ND-16 is shown to have an optimal pH of 6.0 and optimal temperature of 50 °C, making this enzyme potentially suitable for industrial applications. It is also demonstrated that Na+ and Mn2+ show strong inhibition of the xylanase while K+, Li+, Fe2+, Cu2+, and Zn2+ have no significant effect on the activity.
Keywords: Alternaria sp. ND-16; Isolation; Identification; Production; Xylanase
New Thermostable Amylase from Bacillus cohnii US147 with a Broad pH Applicability by Raoudha Ellouz Ghorbel; Sameh Maktouf; Ezedine Ben Massoud; Samir Bejar; Semia Ellouz Chaabouni (50-60).
A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed K m and V max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO3, 1 mM H2O2, 1 mM Zn+2, and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca 2+ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.
Keywords: Bacillus cohnii ; Amylase; Acid and basic pH activity; Purification; Characterization
Kinetic and Stoichiometric Parameters in the Production of Carotenoids by Sporidiobolus salmonicolor (CBS 2636) in Synthetic and Agroindustrial Media by Eunice Valduga; Alexsandra Valério; Helen Treichel; Agenor Furigo Júnior; Marco Di Luccio (61-69).
With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h−1, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P x) of 0.08 g L−1 h−1 and maximum total carotenoid productivity (P car) of 14.2 μg L−1 h−1. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.
Keywords: Sporidiobolus salmonicolor ; Synthetic media; Agroindustrial media; Carotenoids; Kinetic parameters; Stoichiometric parameters
Survival of Freeze-dried Leuconostoc mesenteroides and Lactobacillus plantarum Related to Their Cellular Fatty Acids Composition during Storage by Ibourahema Coulibaly; Anastasie Yao Amenan; Georges Lognay; Marie Laure Fauconnier; Philippe Thonart (70-84).
Lactic acid bacteria strains Lactobacillus plantarum CWBI-B534 and Leuconostoc ssp. mesenteroïdes (L. mesenteroïdes) Kenya MRog2 were produced in bioreactor, concentrated, with or without cryoprotectants. In general, viable population did not change significantly after freeze-drying (p > 0.05). In most cases, viable population for cells added with cryoprotectants was significantly lower than those without (p < 0.05). Cellular fatty acids (CFAs) from the two strains in this study were analyzed before and after freeze-drying. Six CFAs were identified, namely, palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic (C18:3) acids were identified. Four of them, C16:0, C16:1, C18:0, and C18:1, make up more than 94% or 93% of the fatty acids in L. mesenteroides and L. plantarum, respectively, with another one, namely, C18:3, making a smaller (on average 5–6%, respectively) contribution. The C18:2 contributed very small percentages (on average ≤ 1%) to the total in each strain. C16:0 had the highest proportion at most points relative to other fatty acids. Moisture content and water activity (a w) increased significantly during the storage period. It was observed that C16:1/C16:0, C18:0/C16:0 and C18:1/C16:0 ratios for freeze-dried L. mesenteroides or L. plantarum, with or without cryoprotectants, did not change significantly during the storage period. According to the packaging mode and storage temperatures, C18:2/C16:0 and C18:3/C16:0 ratios for freeze-dried L. mesenteroides and L. plantarum with or without cryoprotectants decreased as the storage time increased. However, a higher C18:2/C16:0 or C18:3/C16:0 ratio for L. mesenteroides and L. plantarum was noted in the freeze-dried powder held at 4 °C or under vacuum and in dark than at 20 °C or in the presence of oxygen and light.
Keywords: Viability; Oxidative stress; Heat stress; Freeze-drying; Cellular fatty acids; Lactic acid bacteria
Isolation and Biomass Production of a Saccharomyces cerevisiae Strain Binding Copper and Zinc Ions by Aurore Stroobants; Jean-Marc Delroisse; Franck Delvigne; Julien Delva; Daniel Portetelle; Micheline Vandenbol (85-97).
Copper and zinc are essential trace elements participating in many physiological functions, notably immunity and protection against oxidative stress. Yeasts and the yeast Saccharomyces cerevisiae, in particular, possess in their genome tandem repeats of the CUP1 gene coding for a protein (a metallothionein) capable of capturing and binding toxic elements such as copper ions. The number of copies of this gene in a cell determines its physiological level of resistance to these ions. This paper describes the selection, characterization, and production of a new copper-resistant yeast strain that can bind large quantities of copper and zinc. This approach should lead to increasing the bioavailability of these trace elements and hence to reducing their emission into the environment.
Keywords: Selection; Metallothionein; Probiotic; Prebiotic; Yeast; CUP1 ; Bioreactor
Synthesis of Cephalexin in Aqueous Medium with Carrier-bound and Carrier-free Penicillin Acylase Biocatalysts by Andrés Illanes; Lorena Wilson; Carolina Aguirre (98-110).
The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.
Keywords: Penicillin acylase; Enzyme immobilization; Cephalexin; Cross-linked enzyme aggregates; Multipoint covalent attachment
Structural Genomics on Membrane Proteins by Vikash Kumar Dubey (111-112).