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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #8)
In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Inflorescence Explants and Peroxidase Involvement in Morphogenesis of Populus euphratica Olivier by Yan Zhou; Ziyang Gao; Shumin Gao; Fangfang Sun; Pengjun Cheng; Fenglan Li (pp. 2067-2078).
The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0 mg L−1 6-benzylaminopurine (BA) and 0.5 mg L−1 α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5 mg L−1 BA and 0.2 mg L−1 NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5 mg L−1 BA and 0.2 mg L−1 NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0 mg L−1 indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5 mg L−1 IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8 days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.
Keywords: Inflorescence; Plant regeneration in vitro; POD activities; Populus euphratica Olivier
Biosorption of Heavy Metal Ions (Cu2+, Mn2+, Zn2+, and Fe3+) from Aqueous Solutions Using Activated Sludge: Comparison of Aerobic Activated Sludge with Anaerobic Activated Sludge by Yunhai Wu; Jianxin Zhou; Yajun Wen; Li Jiang; Yunying Wu (pp. 2079-2093).
The potential of using two different kinds of air drying of activated sludge (aerobic activated sludge and anaerobic activated sludge) for the removal of Cu2+, Mn2+, Zn2+, and Fe3+ from aqueous solutions was assessed. Results indicated that the maximum biosorption occurred at pH = 5.0 for Cu2+, Zn2+, and Mn2+ and pH = 3.0 for Fe3+. The kinetic parameters of biosorption data were found to be best fitted to the second-order equation. Also, it was found that the best dosage for biosorption was 0.2 g for both aerobic activated sludge and anaerobic activated sludge. The experimental results were fitted well to the Langmuir, Freundlich, and Dubinin–Radushkevich (D-R) isotherms. The maximum biosorption capacities of Cu2+, Mn2+, Zn2+, and Fe3+ for aerobic activated sludge were 65.789, 44.843, 64.935, and 75.756 mg/g, respectively, while they were 59.880, 49.020, 62.500, and 69.444 mg/g for anaerobic activated sludge, respectively. The mean free energy values evaluated from the D-R model indicated that the biosorptions of studied heavy metal ions onto activated sludge were taken place by chemical interaction. The results of this study provided valuable information on the biosorption of heavy metals by activated sludge that may contribute in wastewater treatment.
Keywords: Biosorption; Activated sludge; Metal ions; Kinetic parameters; Wastewater treatment
Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain That Co-Ferments Glucose and Xylose: II. Influence of pH and Acetic Acid on Ethanol Production by Akinori Matsushika; Shigeki Sawayama (pp. 2094-2104).
The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0–6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150–200 mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.
Keywords: Recombinant Saccharomyces cerevisiae ; Xylose; Glucose; Ethanol; Co-fermentation; pH; Acetic acid
Immobilization of Lipase from Grey Mullet by Alberta N. A. Aryee; Benjamin K. Simpson (pp. 2105-2122).
Grey mullet (Mugil cephalus) lipase was isolated using para-aminobenzamidine agarose and immobilized on octyl Sepharose CL-4B (o-Sep). Immobilized grey mullet lipase (GMLi) had a 10 °C higher optimum temperature compared to the free enzyme and showed remarkable thermal stability. GMLi was most active within the pH range of 8.0–9.5 with an optimum at 8.5. Immobilization also enhanced the storage stability and reusability of the enzyme with minimal changes in efficiency during repeated batches. GMLi showed variable stabilities in various organic solvents. A signal in the amide I absorption region of the FTIR spectrum of GMLi was attributed to the protein layer on o-Sep. The surface morphology of o-Sep was visualized on a Zeiss stereomicroscope as globular-shaped beads.
Keywords: Grey mullet; Delipidation; Lipase; Adsorption; Immobilization; Characterization
Effects of Ion Beams Pretreatment on Damage of UV-B Radiation on Seedlings of Winter Wheat (Triticum aestivum L.) by Shuaipeng Zhao; Qunce Huang; Pengming Yang; Jiajia Zhang; Hongru Jia; Zhen Jiao (pp. 2123-2135).
The seeds of winter wheat were pretreated with three different doses of low-energy N+ beams, and its seedlings were subjected to UV-B irradiation (10.08 kJ m−2 day−1) at three-leaves stage. The growth characteristic of seeds, the oxidative damage to membrane system induced by UV-B radiation, and the alleviating effects of N+ beams pretreatment to radiation damage were investigated. The results showed that the germination rate and seedling rate, respectively, increased 14.09 ± 1.03 and 13.91 ± 1.21 % compared with control (CK) at the dose of 4.0 × 1016 ions/cm2. When seedlings were exposed to UV-B radiation, the pretreatment method under the dose of 4.0 × 1016 ions/cm2 made the activity of peroxidase and superoxide dismutase increasing, the content of chlorophyll enhancing, but the content of malondialdehyde reducing significantly compared with that of the single UV-B radiation. Whereas, the activity of catalase irradiated by UV-B improved notably under the pretreatment dose of 8.0 × 1016 ions/cm2. In addition, after being irradiated with UV-B, the content of soluble protein and glutathione whose seeds were pretreated by the dose of 6.0 × 1016 ions/cm2 were higher than that of the single UV-B radiation. It was suggested that the suitable dose of low-energy ion beams pretreatment to wheat seeds could change its physiological characteristics at seedlings stage to alleviate the damage effects from UV-B radiation.
Keywords: Ion beams; UV-B radiation; Peroxidase (POD); Superoxide dismutase (SOD); Catalase (CAT); Malondialdehyde (MDA); Glutathione (GSH); Chlorophyll
Comparative Evaluation of Pumice Stone as an Alternative Immobilization Material for 1,3-Propanediol Production from Waste Glycerol by Immobilized Klebsiella pneumoniae by Cagdas Gonen; Mine Gungormusler; Nuri Azbar (pp. 2136-2147).
In this study, pumice stone (PS), which is a vastly available material in Turkey, was evaluated as an alternative immobilization material in comparison to other commercially available immobilization materials such as glass beads and polyurethane foam. All immobilized bioreactors resulted in much better 1,3-propanediol production from waste glycerol in comparison to the suspended cell culture bioreactor. It was also demonstrated that the locally available PS material is as good as the commercially available immobilization material. The maximum volumetric productivity (8.5 g L−1 h−1) was obtained by the PS material, which is 220 % higher than the suspended cell system. Furthermore, the immobilized bioreactor system was much more robust against cell washout even at very low hydraulic retention time values.
Keywords: 1,3-Propanediol; Immobilization; Klebsiella pneumoniae ; Glycerol; Biopolymer; Biodiesel
Novel Technology Development through Thermal Drying of Encapsulated Kluyveromyces marxianus in Micro- and Nano-tubular Cellulose in Lactose Fermentation and Its Evaluation for Food Production by Harris Papapostolou; Yiannis Servetas; Loulouda A. Bosnea; Maria Kanellaki; Athanasios A. Koutinas (pp. 2148-2159).
A novel technology development based on the production of a low-cost starter culture for ripening of cheeses and baking is reported in the present study. The starter culture comprises thermally dried cells of Kluyveromyces marxianus encapsulated in micro- and nano-tubular cellulose. For production of a low-cost and effective biocatalyst, whey was used as raw material for biomass production and thermal drying methods (convective, conventional, and vacuum) were applied and evaluated at drying temperatures ranging from 35 to 60 °C. The effect of drying temperature of biocatalysts on fermentability of lactose and whey was evaluated. Storage stability and suitability of biocatalysts as a commercial starter cultures was also assessed and evaluated. All thermally dried biocatalysts were found to be active in lactose and whey fermentation. In all cases, there was sugar conversion ranging from 92 to 100 %, ethanol concentration of up to 1.47 % (v/v), and lactic acid concentrations ranged from 4.1 to 5.5 g/l. However, convective drying of the encapsulated cells of K. marxianus in micro- and nano-tubular cellulose was faster and a more effective drying method while drying at 42 °C appear to be the best drying temperature in terms of cell activity, ethanol, and lactic acid formation. Storage of the biocatalysts for 3 months at 4 °C proved maintenance of its activity even though fermentation times increased by 50–100 % compared with the fresh dried ones.
Keywords: Encapsulation; K. marxianus ; Starter culture; Thermal drying
Dark Fermentative Hydrogen Production from Neutralized Acid Hydrolysates of Conifer Pulp by Marika E. Nissilä; Ya-Chieh Li; Shu-Yi Wu; Jaakko A. Puhakka (pp. 2160-2169).
Concentrated acid hydrolysis of cellulosic material results in high dissolution yields. In this study, the neutralization step of concentrated acid hydrolysate of conifer pulp was optimized. Dry conifer pulp hydrolysis with 55 % H2SO4 at 45 °C for 2 h resulted in total sugar yields of 22.3–26.2 g/L. The neutralization step was optimized for solid Ca(OH)2, liquid Ca(OH)2 or solid CaO, mixing time, and water supplementation. The highest hydrogen yield of 1.75 mol H2/mol glucose was obtained with liquid Ca(OH)2, while the use of solid Ca(OH)2 or CaO inhibited hydrogen fermentation. Liquid Ca(OH)2 removed sulfate to below 30 mg SO4 2−/L. Further optimization of the neutralization conditions resulted in the yield of 2.26 mol H2/mol glucose.
Keywords: Biohydrogen; Dark fermentation; Acid hydrolysis; Neutralization; Conifer pulp
Siderophore-Mediated Iron Uptake Promotes Yeast–Bacterial Symbiosis by RM. Murugappan; M. Karthikeyan; A. Aravinth; MR. Alamelu (pp. 2170-2183).
In the present study, siderophore produced by the marine yeast Aureobasidium pullulans was characterized as hydroxamate by chemical and bioassays. The hydroxamate assignment was supported by the appearance of peaks at 1,647.21–1,625.99 cm−1 and at 1,435.04 cm−1 in the infrared spectrum. The purified siderophore exhibited specific growth-promoting activity under iron-limited conditions for siderophore auxotrophic probiotic bacteria. Cross-utilization of siderophore indicates a symbiotic relationship between the yeast A. pullulans and the selected probiotic bacterial strains. Statistical optimization of medium components for improved siderophore production in A. pullulans was depicted by response surface methodology. The shift in UV–Vis spectroscopy indicates the photoreactive property and subsequent oxidative cleavage of purified siderophore on exposure to sunlight.
Keywords: Aureobasidium pullulans ; Siderophore; Symbiosis; Response surface methodology
Targeted Gene Delivery via PEI Complexed with an Antibody by JooYoun Bae; Masayasu Mie; Eiry Kobatake (pp. 2184-2190).
Stable carriers are required for gene delivery. The use of polyethyleneimine (PEI) has been researched extensively; however, it is not suitable for targeted gene delivery to specific cells. To provide a targeting ability of the specific antibody to PEI, two repeats of Fc-binding domain of protein G (C2) were utilized. The constructed protein containing C2 could bind to a specific antibody and form a larger complex with plasmid DNA/PEI complex. The specific antibody to cell surface protein was bound to the complex through the use of C2 domain, and it was added to cells. As the result, the efficient expression of delivered reporter gene could be realized.
Keywords: Polyethyleneimine; Antibody-binding domain; Target gene delivery; Fusion protein; Transfection efficiency; Gene carrier
Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C by Natesan Balasubramanian; Duarte Toubarro; Mário Teixeira; Nelson Simõs (pp. 2191-2204).
Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76 %, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62 kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by β-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50 °C and pH 7.0, respectively. The purified CMcellulase was stable with about 60 % activity in broad pH ranges from 5 to 10 and temperature of 40 to 60 °C. However, purified CMcellulase was stable at about 70 % at 70 °C and also stable overall at 78 % for surfactants. CMcellulase activity was inhibited by ions such as HgCl2, followed by CuSo4, FeCl2, and MnCl2, while CoCl2 activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.
Keywords: Bacillus mycoides S122C; CMcellulase; Enzyme activity; Enzyme stability; Inhibition; Purification
Bioleaching of Fly Ash by the Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 by Ashok Bankar; Mark Winey; Divya Prakash; Ameeta Ravi Kumar; Suresh Gosavi; Balu Kapadnis; Smita Zinjarde (pp. 2205-2217).
Fly ash collected from an Indian thermal power plant was characterised by scanning electron microscope (SEM)-energy dispersive spectrometer, X-ray diffraction and energy dispersive X-ray fluorescence analysis. The effect of fly ash on the growth and morphology of a metal-tolerant tropical marine yeast, Yarrowia lipolytica NCIM 3589, was studied. The growth of the yeast was unaffected by the presence 0.1, 0.2 or 0.3 % fly ash although the surface-to-volume ratio decreased. The yeast formed biofilms on immobilized fly ash as evidenced by SEM observations. The organism produced citric acid and additional extracellular proteins in the presence of fly ash. Leaching of metals from fly ash by Y. lipolytica was compared with chemical leaching by citric acid. Yeast cells were most effective in leaching Cu (59.41 %) although other metals (Zn, Ni, Cu and Cr) were also extracted. Transmission electron microscope images showed the deposition of metals at the cell wall, cell membrane and in the cytoplasm. This paper thus reports a potential application of Y. lipolytica for removal of different metals from solid waste material (fly ash).
Keywords: Fly ash; Metal extraction; SEM-EDS; Yarrowia lipolytica ; Accumulation
Expression and Characterization of a GH39 β-Xylosidase II from Caulobacter crescentus by Juliana Moço Corrêa; Luciana Graciano; Josielle Abrahão; Eduardo Alexandre Loth; Rinaldo Ferreira Gandra; Marina Kimiko Kadowaki; Caroline Henn; Rita de Cássia Garcia Simão (pp. 2218-2229).
In the present work, the gene xynB2, encoding a β-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 β-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5–7.5 for 24 h and thermotolerance up to 50 °C. The K M and V Max values were 9.3 ± 0.45 mM and 402 ± 19 μmol min−1 for ρ-nitrophenyl-β-d-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family β-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.
Keywords: Gene cloning; Caulobacter crescentus ; β-xylosidase; GH39; Sugarcane bagasse; Aquatic bacterium
Growth Characteristics of Human Adipose-Derived Stem Cells During Long Time Culture Regulated by Cyclin A and Cyclin D1 by Lili Jiang; Tianqing Liu; Kedong Song (pp. 2230-2244).
Abundant and less passaged cells are highly expected in clinical application since repeated subculture reduces stem cell characteristics. Long time culture of stem cells without passage is therefore needed. The growth and cell viability of human adipose-derived stem cells (hADSCs) were investigated by live/dead staining, cck-8 kits, and hemocytometer every day in 30 days of culture. The stem cell characteristics of hADSCs at the beginning and the end of culture were detected by flow cytometry and histochemical staining. hADSCs can be cultured up to the 30th day in one passage while maintaining high level cell viability and their stem cell characteristics. In addition, the cells displayed two plateau phases and three logarithmic phases during 1 month of culture. Increasing expression of cyclin A at protein level resulted in an increase in the percentage of hADSCs in the S and G2/M phases, while decreasing protein level of cyclin D1 induced a decline in the proportion of hADSCs in the G0/G1 phase, regulating cells to move into rapid proliferation. This study demonstrates that a great quantity of hADSCs can be obtained in vitro by prolonging the culture time of each passage. And cyclin A and cyclin D1 affect the distribution of cell cycle and regulate the growth of hADSCs.
Keywords: Human adipose-derived stem cells; Growth curves; Cell cycle; Cyclin A; Cyclin D1
Antioxidative and DNA Protective Effects of Bacillomycin D-Like Lipopeptides Produced by B38 Strain by Olfa Tabbene; Dorra Gharbi; Imen Ben Slimene; Salem Elkahoui; Mohamed Najib Alfeddy; Pascal Cosette; Maria Luisa Mangoni; Thierry Jouenne; Ferid Limam (pp. 2245-2256).
In the present study, we evaluated the antioxidant and the scavenging ability of C14, C15 and C16 bacillomycin D-like lipopeptides produced by B38 strain. They all displayed strong reducing power activity, hydroxyl and superoxide anion radicals scavenging activities and inhibition of lipid peroxidation. In addition, they were found to protect plasmid DNA damage from hydroxyl radical oxidation. Data suggested that their antioxidant potency can be attributed to the hydrophobic and aromatic side-chain groups of their amino acids as well as to the aliphatic chain of their beta amino fatty acids. Note that the hydrocarbon chain length did not interfere with the antioxidant power. Overall, such bacillomycin D lipopeptides which exhibit antioxidant and radical scavenging activities may be useful for cosmetic, therapeutic or pharmaceutical purposes in order to delay or prevent oxidative deterioration of manufactured products.
Keywords: Antioxidant compounds; Antifungal activity; Lipopeptide; Free radicals; Reactive oxygen species
Purification of a Lectin from M. rubra Leaves Using Immobilized Metal Ion Affinity Chromatography and Its Characterization by Thavamani Sureshkumar; Sulochana Priya (pp. 2257-2267).
Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, d-galactosamine and N-acetyl-d-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4–11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe2+, Ca2+, Zn2+, Ni2+, Mn2+, Na+ and K+, HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3+ and Al3+) and Cu2+ did not affect the activity. In the presence of Mg2+ and Hg2+, only 25 % of HA activity remained.
Keywords: Lectin; Haemagglutination; N-acetyl-d-galactosamine
Bioprocess Design for the Microbial Production of Natural Phenolic Compounds by Debaryomyces hansenii by Belén Max; Francisco Tugores; Sandra Cortés-Diéguez; José M. Domínguez (pp. 2268-2284).
Debaryomyces hansenii NRRL Y-7426 metabolised ferulic acid into different phenolic compounds using a factorial design where glucose concentration (in the range of 1–20 g/L), peptone concentration (2–20 g/L) and yeast extract concentration (0.2–10 g/L) were the independent variables. The interrelationship between dependent and operational variables was well fitted (R 2 > 0.95) to models including linear, interaction and quadratic terms. Depending on the glucose and nitrogen concentrations, which redirected the metabolism, the major degradation products were 1,226.2 mg 4-vinyl guaiacol/L after 72 h (molar yield of 86.0 %), 1,077.8 mg vanillic acid/L after 360 h (molar yield of 91.1 %) or 1,682.6 mg acetovanillone/L after 408 h (molar yield of 98.8 %) in fermentations carried out with 2,000 mg ferulic acid/L. Other metabolites such as vanillin, vanillyl alcohol or 4-ethylguaiacol were present in lower amounts.
Keywords: 4-Vinyl guaiacol; Vanillic acid; Acetovanillone; Ferulic acid; Debaryomyces hansenii
Activity and Synergistic Antimicrobial Activity Between Diketopiperazines Against Bacteria In Vitro by S. Nishanth Kumar; J. V. Siji; Bala Nambisan; C. Mohandas (pp. 2285-2296).
The aim of the present study was to determine the synergistic effects of diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp. on the growth of bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic antibacterial activities of the combination of diketopiperazines against pathogenic bacteria were assessed using the checkerboard assay and time–kill methods. The results of the present study showed that the combination effects of diketopiperazines were predominately synergistic (FIC index <0.5). Furthermore, time–kill study showed that the growth of the tested bacteria was completely attenuated with 4–12 h of treatment with 50:50 ratios of diketopiperazines. These results suggest that the combination of diketopiperazines may be microbiologically beneficial. The three diketopiperazines are nontoxic to normal human cell line (L231 lung epithelial) up to 200 m μg/ml. The in vitro synergistic activity of cyclo-(l-Pro-l-Leu), cyclo-(d-Pro-l-Leu), and cyclo-(d-Pro-l-Tyr) against bacteria is reported here for the first time. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (diketopiperazines).
Keywords: Cyclic dipeptides; Synergy; Checkerboard assay; Time–kill assay
Synthesis of Duloxetine Intermediate (S)-3-Chloro-1-(2-thienyl)-1-propanol with Liquid-Core Immobilized Candida pseudotropicalis 104 by Ou Zhimin; Sun Xingyuan; Shi Hanbing; Bi Hongxia (pp. 2297-2308).
(S) -3-Chloro-1-(2-thienyl)-1-propanol was synthesized by the asymmetric reduction of 3-chloro-1-(2-thienyl)propanone with liquid-core immobilized Candida pseudotropicalis 104. The optimum time was 28 h for the re-cultivation of immobilized cells. The optimum film solvent for the liquid-core capsule was 0.3 % chitosan (M w 1.0 × 105). Conversion decreased with the increase of the liquid-core capsule diameter and with the addition of more substrates at the same reduction time. The immobilized cells show good reduction ability in a potassium phosphate buffer (pH 6.6~7.2). The material outside the spread speed of immobilized cells was not restricted when the shaking speed was higher than 160 r/min. Liquid-core immobilized cells can be reused 11 times. Compared with the batch reduction, the continuous reduction of 3-chloro-1-(2-thienyl)propanone in the membrane reactor with liquid-core immobilized cells as catalyst can relieve the inhibition from a high-concentration substrate. Conversion and enantiometric excess of (S)-3-chloro-1-(2-thienyl)-1-propanol reached 100 % and >99 % in a continuous reduction of 12 g/L 3-chloro-1-(2-thienyl)propanone for 10 days.
Keywords: (S)-3-Chloro-1-(2-thienyl)-1-propanol; Immobilized cells; Liquid-core capsule; Candida pseudotropicalis 104
Genotypic Variability Among Soybean Genotypes Under NaCl Stress and Proteome Analysis of Salt-Tolerant Genotype by Khalid Rehman Hakeem; Faheema Khan; Ruby Chandna; Tariq Omer Siddiqui; Muhammad Iqbal (pp. 2309-2329).
The present investigation was conducted to evaluate salt tolerance in ten genotypes of soybean (Glycine max L.). Twelve-day-old seedlings, grown hydroponically, were treated with 0, 25, 50, 75, 100, 125 and 150 mM NaCl for 10 days. Growth, lipid peroxidation and antioxidant enzyme activities were evaluated. Growth, measured in terms of length, fresh weight and dry weight of plants, was drastically reduced in Pusa-24 while there was little effect of NaCl treatment on Pusa-37 genotype of soybean. High level of lipid peroxidation was observed in Pusa-24 as indicated by increased level of malondialdehyde. Activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were maximum in Pusa-37 where 9-, 1-, 5- and 6-fold increase over control were observed, respectively. The results suggested that Pusa-24 and Pusa-37 are salt-sensitive and salt-tolerant genotype of soybean, respectively, and antioxidant defence system is involved in conferring the sensitiveness and tolerance in these genotypes. Salt-tolerant genotype Pusa-37, was further analysed by 2-dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the present study, 173 protein spots were identified. Of these, 40 proteins were responsive to salinity in that they were either up- or downregulated. This study could help us in identifying the possible regulatory switches (gene/s) controlling novel proteins of the salt-tolerant genotype of the crop plants and their possible role in defence mechanism.
Keywords: Lipid peroxidation; Oxidative stress; Proline; Proteomics; Salt tolerance; Soybean; 2D electrophoresis
Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain by Raida Jallouli; Fatma Khrouf; Ahmed Fendri; Tahar Mechichi; Youssef Gargouri; Sofiane Bezzine (pp. 2330-2343).
An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH2-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca2+ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.
Keywords: Fusarium solani ; Fungus; (Phospho)lipase; Purification; Orlistat
A Physiologically Regulated Multidomain Cystatin of Wheat Shows Stage-Dependent Immunity Against Karnal Bunt (Tilletia indica) by Shalini Purwar; Shanthy Sundaram; Praveen Verma; Shaili Srivastava; Anil Kumar (pp. 2344-2357).
To identify novel components of basal resistance against the Tellitia indica of wheat, breeding for disease resistance was carried out on resistant and susceptible genotype of Karnal Bunt. The different members of wheat cystatin gene families were cloned, and their role in triggering differential resistance through co-expression was analyzed in our lab. The multidomain wheat cystatin (WCM) is a proteinase inhibitor characterized by cloning the gene from susceptible (WH542) and resistant genotype (HD 29). A WCM cDNA was isolated from both genotypes and sequenced. The WCM had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited similarity to the cystatin domain. The expression level was significantly (P > 0.001) higher in resistant compared to susceptible genotype at all the physiological stages of wheat spikes. In order to characterize the biochemical properties of WCM, the coding sequence was expressed in Escherichia coli using pET expression vector. The recombinant WCM was purified from soluble fraction of the cell extract by using affinity chromatography. WCM, with 23 KDa molecular mass, showed cysteine proteinase inhibitory activity against papain (Ki 3.039 × 10−7 M) as determined by using BAPNA as substrate. Furthermore, it was able to arrest the fungal mycelial growth of T. indica. Hyphae growth was inhibited, and morphological changes such as swelling and fragmentation of the fungus were observed. Overall, these observations suggest an endogenous high expression of cystatin, possibly associated with the resistance of wheat against Karnal bunt.
Keywords: Wheat; Tilletia indica ; Phytocystatin; Antifungal protein
Regulation of Enzymatic Activity by Deamidation and Their Subsequent Repair by Protein l-isoaspartyl Methyl Transferase by Trina Dutta; Shakri Banerjee; Dhananjay Soren; Sagar Lahiri; Shinjinee Sengupta; Juhi Augusta Rasquinha; Anil K. Ghosh (pp. 2358-2375).
The present study explored both spontaneous and stress-induced deamidation in acid trehalase and endo-xylanase. An alteration in optimum pH by 1.5 units and optimum temperature by 20 °C accelerated the process of deamidation with a rise in isoaspartate formation and ammonia loss. Spontaneous deamidation during an enzyme-substrate reaction at physiological conditions resulted in accretion of isoaspartyl residues within the enzymes which gradually impaired their catalytic efficacy. Deamidation appeared to be more pronounced in endo-xylanase owing to its secondary structure conformation and high asparagine content. The active sites, Ala 549 in acid trehalase and His184 and Trp188 in endo-xylanase contributed to the loss of enzyme activity as they were flanking the deamidation-susceptible Asn residues. Protein l-isoaspartyl methyl transferase seemed to have a repairing capability, which enabled the heat-damaged enzymes to regain their partial activity as evident from there rise in K cat/K m. Endo-xylanase could regain 38.1 % of its biological activity while a lesser 17.5 % reactivation was obtained in acid trehalase. A unique protein l-isoaspartyl methyl transferase recognition site, Asn 151 was also identified in acid trehalase. A mass increment of the tryptic peptides of repaired enzyme due to methylation catalyzed by protein l-isoaspartyl methyl transferase substantiated the repair hypothesis.
Keywords: Deamidation; Ammonia release; Isoaspartate; PIMT; Acid trehalase; Endo-xylanase
Effect of Synergistic Inducement on the Production of Laccase by a Novel Shiraia bambusicola Strain GZ11K2 by Wen Du; Chunlong Sun; Jianping Yu; Jiandong Liang; Zongqi Liang; Yanfeng Han; Xiao Zou (pp. 2376-2386).
In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as laccase secretion indicator. A novel laccase-producing strain was isolated and identified as Shiraia bambusicola Henn. strain GZ11K2 according to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. In further investigation, the production of laccase by S. bambusicola GZ11K2 was greatly enhanced by the nontoxic inducers of copper sulfate and rhodamine B. Copper and rhodamine B were added into the cultivation medium at 24 and 12 h, respectively, and the maximum laccase production was obtained. Under the induction of 2.0 mM copper sulfate and 35 μM rhodamine B, an increment of about 80 times of laccase activity compared with that in the inducer-free medium and about 20 times compared with that in the single copper-supplemented medium was observed. Compared with other species, S. bambusicola GZ11K2 exhibits better laccase-producing characteristics with an activity of 16,400 U/L after 108 h, suggesting its potential ability for industrial application.
Keywords: Shiraia bambusicola ; Laccase; Inducers; Copper; Rhodamine B
Efficient Non-sterilized Fermentation of Biomass-Derived Xylose to Lactic Acid by a Thermotolerant Bacillus coagulans NL01 by Jia Ouyang; Cong Cai; Hai Chen; Ting Jiang; Zhaojuan Zheng (pp. 2387-2397).
Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstock. Its efficient utilization is regarded as a technical barrier to the commercial production of bulk chemicals from lignocellulosic biomass. This work aimed at evaluating the lactic acid production from the biomass-derived xylose using non-sterilized fermentation by Bacillus coagulans NL01. A maximum lactic acid concentration of about 75 g/L was achieved from xylose of 100 g/L after 72 h batch fermentation. Acetic acid and levulinic acid were identified as important inhibitors in xylose fermentation, which markedly reduced lactic acid productivity at 15 and 1.0 g/L, respectively. But low concentrations of formic acid (<2 g/L) exerted a stimulating effect on the lactic acid production. When prehydrolysate containing total 25.45 g/L monosaccharide was fermented with B. coagulans NL01, the same preference for glucose, xylose, and arabinose was observed and18.2 g/L lactic acid was obtained after 48 h fermentation. These results proved that B. coagulans NL01 was potentially well-suited for producing lactic acid from underutilized xylose-rich prehydrolysates.
Keywords: Bacillus coagulans ; Lactic acid; Xylose; Non-sterilized fermentation; Biomass