Applied Biochemistry and Biotechnology (v.159, #1)
Mobilization and Biodegradation of 2-Methylnaphthalene by Amphiphilic Polyurethane Nano-Particle by Young-Bum Kim; Ju-Young Kim; Eun-ki Kim (1-10).
Amphiphilic polyurethane (APU) nano-particle enhanced the mobilization of 2-methylnaphthalene (2-MNPT) in soil. Significant increase in the solubility of 2-MNPT was achieved. The molar solubilization ratio was 0.4 (mole 2-MNPT/mole APU). Simple precipitation of APU particle by 2 N CaCl2 recovered 95% of APU particle and 92% of 2-MNPT simultaneously. Also, 2-MNPT, which was entrapped inside the APU particle, was directly degraded by Acinetobacter sp. as same efficiency as without APU particle. These results showed the potentials of APU particle in the mobilization and biodegradation of hydrophobic compounds from soil.
Keywords: APU particle; 2-Methylnaphthalene; Soil washing; Polymeric surfactant; Biodegradation
Degradation of Phytates in Distillers’ Grains and Corn Gluten Feed by Aspergillus niger Phytase by H. Noureddini; J. Dang (11-23).
Distillers’ dried grains with solubles (DDGS) and corn gluten feed (CGF) are major coproducts of ethanol production from corn dry grind and wet milling facilities, respectively. These coproducts contain important nutrients and high levels of phytates. The phytates in these products cannot be digested by nonruminant animals; consequently, large quantities of phytate phosphorus (P) are deposited into the soil with the animal wastes which potentially could cause P pollution in soil and underground water resources. To reduce phytates in DDGS and CGF, a phytase from Aspergillus niger, PhyA, was investigated regarding its capability to catalyze the hydrolysis of phytates in light steep water (LSW) and whole stillage (WS). LSW and WS streams are the intermediate streams in the production of CGF and DDGS, respectively, and contribute to most of the P in these streams. Enzyme loadings with activity of 0.1, 1, 2, and 4 FTU/g substrate and temperatures of 35 and 45 °C were investigated regarding their influences on the degree of hydrolysis. The analysis of the hydrolyzate suggested to a sequentially degradation of phytates to lower order myo-inositol phosphate isomers. Approximately 90% phytate P of LSW and 66% phytate P of WS were released, suggesting myo-inositol monophosphate as the end product. The maximum amount of released P was 4.52 ± 0.03 mg/g LSW and 0.86 ± 0.01 mg/g WS.
Keywords: Distillers’ grains; Light steep water; Whole stillage; Aspergillus niger phytase; Hydrolysis; Phytates; Myo-insotitol phosphate isomers; Inorganic phosphate
Endoxanthanase, a Novel β-d-Glucanase Hydrolyzing Backbone Linkage of Intact Xanthan from Newly Isolated Microbacterium sp. XT11 by Bing Li; Jiqiang Guo; Wenfu Chen; Xiaoyi Chen; Ligang Chen; Zhiwen Liu; Xianzhen Li (24-32).
A novel endoxanthanase was produced and isolated from the culture of Microbacterium sp. XT11 growing on xanthan. Optimum pH and temperature for the enzyme activity were 6.0 and 35–40 °C, respectively. The endoxanthanase cleaves the backbone endo-β-1,4-glucosidic linkage of the xanthan molecule, which is specific to the intact xanthan. However, the lyase-treated xanthan or carboxymethyl cellulose could not be cleaved by endoxanthanase.
Keywords: Xanthan; Endoxanthanase; Xanthan degradation; β-d-glucanase; Microbacterium
Isolation, Purification and Characterization of a Surfactants-, Laundry Detergents- and Organic Solvents-Resistant Alkaline Protease from Bacillus sp. HR-08 by Fatemeh Moradian; Khosro Khajeh; Hossein Naderi-Manesh; Majid Sadeghizadeh (33-45).
Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited maximum activity at pH 10.0 and 60 °C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 °C in the presence of 10 mM CaCl2. Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min at 60 and 70 °C, respectively. This enzyme had good stability in the presence of H2O2, nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration (50%, v/v) of DMF and DMSO.
Keywords: Alkaline protease; 16S rDNA; Phylogenetic tree; Thermostability; Purification; Organic solvent; Detergent
Yarrowia lipolytica Growth Under Increased Air Pressure: Influence on Enzyme Production by Marlene Lopes; Nelma Gomes; Manuel Mota; Isabel Belo (46-53).
Improvement of microbial cell cultures oxygenation can be achieved by the increase of total air pressure, which increases oxygen solubility in the medium. In this work, a pressurized bioreactor was used for Yarrowia lipolytica batch cultivation under increased air pressure from 1 to 6 bar. Cell growth was strongly enhanced by the pressure rise. Fivefold and 3.4-fold increases in the biomass production and in specific growth rate, respectively, were observed under 6 bar. The increase of oxygen availability caused the induction of the antioxidant enzyme superoxide dismutase, which indicates that the defensive mechanisms of the cells against oxidative stress were effective and cells could cope with increased pressure. The pregrowth of Y. lipolytica under increased pressure conditions did not affect the lipase production ability of the cells. Moreover, the extracellular lipase activity increased 96% using a 5-bar air pressure instead of air at 1-bar pressure during the enzyme production phase. Thus, air pressure increase in bioreactors is an effective mean of cell mass and enzyme productivity enhancement in bioprocess based in Y. lipolytica cultures.
Keywords: Yarrowia lipolytica ; Lipase; Oxidative stress; Oxygen; Pressure
Application of Quartz Crystal Nanobalance for Simultaneous Determination of Vanillylmandelic and Homovanillic Acids by a Net Analyte Signal-Based Method by Maryam Shojaei; Abdolreza Mirmohseni; Yadollah Omidi; Maryam Farbodi (54-64).
Homovanillic acid (HVA) and vanillylmandelic acid (VMA) were selectively determined by quartz crystal nanobalance sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including HVA, VMA, and some common and structurally similar urine compounds. The selection of the optimal time range involved the calculation of the NAS regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of predicted error sum of squares value was carried out by applying a moving window strategy. Based on the obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. Several figures of merit like selectivity, sensitivity, analytical sensitivity, and limit of detection were calculated for both compounds. The results showed that the method was successfully applied for the determination of VMA and HVA.
Keywords: Quartz crystal nanobalance; Net analyte signal; Homovanillic acid; Vanillylmandelic acid; HLA/GO method
Controlled Production of Fructose by an Exoinulinase from Aspergillus Ficuum by T. Mutanda; B. Wilhelmi; C. G. Whiteley (65-77).
An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg−1(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K m and V max values were 4.75 mM and 833.3 μmol min−1 ml−1, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.
Keywords: Fructose; Fructose synthesis; Exoinulinase; Response surface methodology; Production
Improved Production of Raw Starch Degrading Enzyme by Aspergillus oryzae F-30 Using Methyl Glucoside Sesqui-Stearate by Hai-Yan Sun; Lu Wang; Jian-Wen Liu; Ming Peng (78-84).
The effect of methyl glucoside sesqui-stearate (MGS) on the production of raw starch degrading enzyme (RSDE) by Aspergillus oryzae F-30 was studied in this paper. The activity of RSDE formed by Aspergillus oryzae F-30 was enhanced dramatically by the addition of MGS to the medium. As a result, with the addition of 1.5 g MGS in 1 L basal medium, RSDE activity and productivity were 107 U mL−1 and 1.49 U mL−1 h−1, 4.3-fold and 7.1-fold greater than the values obtained in the basal medium, respectively. The effect of MGS on the synthesis of RSDE by Aspergillus oryzae F-30 was also studied on a molecular level. It was observed that the regulation of RSDE synthesis in Aspergillus oryzae F-30 occurs at both transcriptional and translational level and the enzyme synthesis was provoked by the addition of MGS at transcriptional level.
Keywords: Aspergillus oryzae ; Methyl glucoside sesqui-stearate; Raw starch degrading enzyme; Transcriptional level; Translational level
A Novel β-mannanase with High Specific Activity from Bacillus circulans CGMCC1554: Gene Cloning, Expression and Enzymatic Characterization by Peilong Yang; Yanan Li; Yaru Wang; Kun Meng; Huiying Luo; Tiezheng Yuan; Yingguo Bai; Zhichun Zhan; Bin Yao (85-94).
A DNA fragment of 2,042 bp containing a novel β-mannanase gene, man5A, was identified from the genome of the mannan-degrading bacterium Bacillus circulans CGMCC1554. The open reading frame of man5A comprised 978 bp encoding a protein of 326 amino acids with a predicted molecular weight of 32 kDa. The amino acid sequence of the encoded mannanase, MAN5A, showed the highest identity (78.5%) to β-mannanases belonging to glycosyl hydrolases family 5. The gene man5A was efficiently expressed in Escherichia coli and Pichia pastoris with the highest activity of 541 U/ml in a 3-L fermenter. Recombinant MAN5A purified from E. coli had a high specific activity of 4,839 U/mg, which is much higher than that of enzymes that showed high sequence identity. The enzyme showed maximum activity at pH 7.6 and 60 °C and resistance to trypsin. After hydrolysis of LBG, oligomannosides accounted for 76% of the hydrolysis products. All these properties collectively make MAN5A a better candidate than current mannanases for use in the food and feed industry.
Keywords: β-Mannanase; Bacillus circulans ; Expression; Characterization; High specific activity
AnSBBR Applied to Organic Matter and Sulfate Removal: Interaction Effect Between Feed Strategy and Cod/Sulfate Ratio by Gregor F. Friedl; Gustavo Mockaitis; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat; Eugênio Foresti (95-109).
A mechanically stirred anaerobic sequencing batch reactor containing anaerobic biomass immobilized on polyurethane foam cubes, treating low-strength synthetic wastewater (500 mg COD L−1), was operated under different operational conditions to assess the removal of organic matter and sulfate. These conditions were related to fill time, defined by the following feed strategies: batch mode of 10 min, fed-batch mode of 3 h and fed-batch mode of 6 h, and COD/[SO4 2−] ratios of 1.34, 0.67, and 0.34 defined by organic matter concentration of 500 mg COD L−1 and sulfate concentrations of 373, 746, and 1,493 mg SO4 2− L−1 in the influent. Thus, nine assays were performed to investigate the influence of each of these parameters, as well as the interaction effect, on the performance of the system. The reactor operated with agitation of 400 rpm, total volume of 4.0 L, and treated 2.0 L synthetic wastewater in 8-h cycles at 30 ± 1°C. During all assays, the reactor showed operational stability in relation to the monitored variables such as COD, sulfate, sulfide, sulfite, volatile acids, bicarbonate alkalinity, and solids, thus demonstrating the potential to apply this technology to the combined removal of organic matter and sulfate. In general, the results showed that the 3-h fed-batch operation with a COD/[SO4 2−] ratio of 0.34 presented the best conditions for organic matter removal (89%). The best efficiency for sulfate removal (71%) was accomplished during the assay with a COD/[SO4 2−] ratio of 1.34 and a fill time of 6 h. It was also observed that as fill time and sulfate concentration in the influent increased, the ratio between removed sulfate load and removed organic load also increased. However, it should be pointed out that the aim of this study was not to optimize the removal of organic matter and sulfate, but rather to analyze the behavior of the reactor during the different feed strategies and applied COD/[SO4 2−] ratios, and mainly to analyze the interaction effect, an aspect that has not yet been explored in the literature for batch reactors.
Keywords: Sulfate removal; Feed strategy; Immobilized biomass; AnSBBR; Mechanical agitation
Ex Vitro Expansion of Human Placenta-Derived Mesenchymal Stem Cells in Stirred Bioreactor by Yanqiu Yu; Kun Li; Chunyu Bao; Tianqing Liu; Yunan Jin; Haiqin Ren; Wei Yun (110-118).
The high demand of human placenta-derived mesenchymal stem cells (hPDMSCs) for therapeutic applications requires reproducible production of large numbers of well-characterized cells under well-controlled conditions. However, no method for fast hPDMSCs proliferation has yet been reported. In the present study, the feasibility of using a stirred bioreactor system to expand hPDMSCs was examined. hPDMSCs were cultured either in stirred bioreactors or in tissue culture flasks (T-flasks) for 5 days. Total cell density and several parameters of physical microenvironments were monitored in the two culture systems every 24 h. The maintenance of the antigenic phenotype of hPDMSCs before and after culturing in the stirred bioreactor system was cytometrically assessed. Data suggested that the physical microenvironment in the stirred bioreactors was much more favorable than that of the T-flasks. At the end of 144 h culturing, the total cell number was increased 1.73 times from the T-flasks to the stirred bioreactors. In addition, hPDMSCs could maintain their antigenic phenotype when cultured in stirred bioreactors. These results provide the initial assessment for large-scale hPDMSCs production using suspension culture bioreactors.
Keywords: Mesenchymal stem cells; Stirred bioreactor; Cell expansion; Tissue engineering; Regeneration medicine
Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived Metschnikowia reukaufii and its Expression in E. coli by Jing Li; Zhenming Chi; Zhiqiang Liu; Lixi Yue; Ying Peng; Lin Wang (119-132).
Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.
Keywords: Aspartic protease; Gene cloning; Expression; Metschnikowia reukaufii ; Milk clotting activity
Enhanced Production of Poly (γ-glutamic acid) from Bacillus licheniformis NCIM 2324 by Using Metabolic Precursors by Ishwar B. Bajaj; Rekha S. Singhal (133-141).
The aim of the present work was to study the effect of addition of different amino acids and tricarboxylic acid cycle intermediates as metabolic precursors on the production of poly (γ-glutamic acid) (PGA) by Bacillus licheniformis NCIM 2324. A maximum yield of 35.75 g/l was obtained with the medium supplemented with 0.5 mM l-glutamine and 10 mM α-ketoglutaric acid as compared to 26.12 g/l PGA achieved with the control in the absence of metabolic precursors. Addition of precursors also enhanced the utilization of l-glutamic acid during fermentation.
Keywords: Poly (γ-glutamic acid); Fermentation; Bacillus licheniformis ; Precursors; α-Ketoglutaric acid
Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of Bacillus cereus C7 by Sanghamitra Dutta; Lalitagauri Ray (142-154).
A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.
Keywords: Lipase; Bacillus cereus ; Alkaline; Thermostable; Production; Characterization
Taxonomic Identification and Use of Free and Entrapped Cells of a New Mycobacterium sp., Strain Spyr1 for Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) by E. Karabika; A. Kallimanis; A. Dados; G. Pilidis; C. Drainas; A. I. Koukkou (155-167).
A polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strain Spyr1 was isolated from Greek creosote polluted soil by an enrichment method using pyrene as sole carbon and energy source. Spyr1 was identified as Mycobacterium sp. based on 16S rDNA analysis and it was capable of degrading pyrene, fluoranthene, fluorene, anthracene, and acenaphthene. The effect of entrapment in glass beads and alginate/starch mixtures on the survival and pyrene degradation ability of Spyr1 cells in liquid suspensions and soil microcosms was tested and compared with that of freely suspended cells. In general, free cells showed higher degradation of pyrene and other PAH than immobilized cells. However, immobilized cells could better tolerate PAH and they maintained their viability and PAH degradation capability for at least 1 year after storage at 4 °C. Entrapped cells in glass beads exhibited better pyrene biodegradation performance than alginate/starch entrapped cells in liquid suspensions and could be used effectively for at least ten repeated cycles. Alginate/starch entrapped cells exhibited better yields than glass beads entrapped cells for removing pyrene as well as mixtures of PAH in soil microcosms.
Keywords: Biodegradation; Entrapment; Mycobacterium sp.; Soil bacteria; PAH
Co-Expression of Recombinant Nucleoside Phosphorylase from Escherichia coli and its Application by Chongtao Ge; Liming OuYang; Qingbao Ding; Ling Ou (168-177).
The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved. In the transglycosylation reaction, yield of 2,6-diaminopurine-2’-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.
Keywords: Nucleoside phosphorylase; Co-expression; Escherichia coli
Engineering of Cysteine Residues Leads to Improved Production of a Human Dipeptidase Enzyme in E. coli by Ronan O’Dwyer; Rafia Razzaque; Xuejun Hu; Susan K. Hollingshead; J. Gerard Wall (178-190).
Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human β-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem β-lactam antibiotics and can cleave dipeptides containing amino acids in both d- and l-configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.
Keywords: Cysteine; Disulfide bridge; E. coli ; Mammalian dipeptidase; Periplasm; Recombinant protein
Enhancement of L-Asparaginase Production by Isolated Bacillus circulans (MTCC 8574) Using Response Surface Methodology by M. Hymavathi; T. Sathish; Ch. Subba Rao; R. S. Prakasham (191-198).
L-asparaginase production was optimized using isolated Bacillus circulans (MTCC 8574) under solid-state fermentation (SSF) using locally available agricultural waste materials. Among different agricultural materials (red gram husk, bengal gram husk, coconut, and groundnut cake), red gram husk gave the maximum enzyme production. A wide range of SSF parameters were optimized for maximize the production of L-asparaginase. Preliminary studies revealed that incubation temperature, moisture content, inoculum level, glucose, and L-asparagine play a vital role in enzyme yield. The interactive behavior of each of these parameters along with their significance on enzyme yield was analyzed using fractional factorial central composite design (FFCCD). The observed correlation coefficient (R 2) was 0.9714. Only L-asparagine and incubation temperature, were significant in linear and quadratic terms. L-asparaginase yield improved from 780 to 2,322 U/gds which is more than 300% using FFCCD as a means of optimizing conditions.
Keywords: L-Asparaginase; Bacillus circulans ; Fractional factorial central composite design; Optimization; Red gram husk; Solid-state fermentation
Syntheses of Retinol Glycosides Using β-glucosidase in SCCO2 Media by Rajachristu Einstein Charles; Thangavel Ponrasu; Kadimi Udaya Sankar; Soundar Divakar (199-207).
β-Glucosidase isolated from sweet almond catalyzed syntheses of water soluble retinol glycosides were carried out in SCCO2 media with carbohydrates—D-glucose 2, D-galactose 3, D-mannose 4, D-fructose 5, and D-sorbitol 6. Retinol glycosides yields were in the 9–34% range. Reaction with D-fructose 5 gave a highest yield of 34%. Excellent regioselectivity was observed with D-mannose 4 and D-sorbitol 6 which gave exclusively C1β-mannoside and C1-D-sorbitolide.
Keywords: β-glucosidase; Regioselectivity; Retinol; Retinol glycoside; Supercritical carbon dioxide media (SCCO2); Enzymatic catalysis
Circulating IgSF Proteins Inhibit Adhesion of Antibody Targeted Microspheres to Endothelial Inflammatory Ligands by Matthew B. Kerby; Jane C. Urban; Lea Mouallem; Anubhav Tripathi (208-220).
Proposed methods for detecting circulatory system disease include targeting ultrasound contrast agents to inflammatory markers on vascular endothelial cells. For antibody-based therapies, soluble forms of the targeted adhesion proteins of the immunoglobulin superfamily (IgSF) reduce adhesion yet were left unaccounted in prior reports. Microspheres labeled simply with a maximum level of antibodies can reduce the diagnostic sensitivity by adhering to proteins expressed normally at a low level, while sparsely coated particles may be rendered ineffective by circulating soluble forms of the targeted proteins. A new microdevice technique is applied to simultaneously measure the adhesion profile to a series of IgSF-protein-coated surfaces. In this investigation, we quantify the in vitro binding characteristics of 5-μm microspheres to oriented intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein-coated surfaces in the presence of human serum at physiological concentrations. Defined regions of a slide were coated with recombinant chimeric Fc-human ICAM-1 and VCAM-1 in variable ratios but constant total concentration. Monoclonal human anti-ICAM-1 or anti-VCAM-1 antibodies in competition with non-binding mouse anti-rabbit antibodies coat the microsphere surface at a constant surface density with variable yet controlled surface activities. Using multiple slide surface IgSF protein and microsphere antibody concentrations, an adhesion profile was developed for the microspheres with and without IgSF proteins from human serum, which demonstrated that exposure to serum reduced microsphere binding, on average, more than 50% compared to the no-serum condition.. The serum effects were limited to antibodies on the microsphere, since binding inhibition was reversed after rinsing serum from the system and fresh antibody-coated microspheres were introduced. This analysis quantifies the binding effects of soluble IgSF proteins from human serum on antibody-based targeted ultrasound detection and drug delivery methods.
Keywords: Immunoglobulin superfamily; IgSF; Human serum; Contrast agents; ICAM-1; VCAM-1; Adhesion; Microparticle
Proteomics and Detection of Uromodulin in First-time Renal Calculi Patients and Recurrent Renal Calculi Patients by Lau Wai-Hoe; Leong Wing-Seng; Zhari Ismail; Gam Lay-Harn (221-232).
Renal calculi disease or known as kidney stone disease is the most common urological disorder in both men and women, although it is more prevalent in men. The lifetime chance for an individual to develop renal calculi is ~10% whereas the risk of recurrence in a 10-year period is 74%. Therefore, a diagnostic tool for screening or detecting renal calculi is greatly needed. In this study, we analyze urinary protein profiles from patients with renal calculi for the first time (RC), healthy subjects (HS), and patients with recurrent renal calculi (RRC) to identify a biomarker for detecting the disease. Urinary proteins were isolated by salt precipitation and the proteins resolved by SDS-PAGE. Target proteins were analyzed with LC/MS/MS. Thirty-two proteins were identified from healthy subjects and patients. Uromodulin was the most abundant urinary protein in HS but was a very faint band if detected at all from those that formed renal calculi for the first time (p < 0.05). Yet the excreted levels of urinary uromodulin in RRC were similar to those of the HS suggesting that uromodulin is a reliable biomarker for only RC. In addition, a few immunoglobulins that were commonly found in the urine of both RC and RRC, which include Ig alpha heavy chain 1, Ig gamma-2 c region, Ig gamma-3 heavy chain disease protein, Ig heavy chain variable region, Ig heavy constant region gamma 4, and Ig heavy chain. Ig heavy chain Fab frag and antibody a5b7 chain B may serve as potential biomarkers for renal calculi disease.
Keywords: Renal calculi disease; Biomarker; Proteomics; SDS-PAGE; LC/MS/MS
Screening and Selection of Marine Isolate for l-Glutaminase Production and Media Optimization Using Response Surface Methodology by Padma V. Iyer; Rekha S. Singhal (233-250).
The current work details the screening of about 400 marine isolates from various marine niches, from which one isolate was finally selected based on the productivity of glutaminase (71.23 U/l). Further, biochemical identification tests and 16S rRNA sequencing identified this isolate to be Providencia sp. This isolate was taken up for further media optimization studies by using one-factor-at-a-time approach and subsequently by response surface methodology. A face centered central composite design was employed to investigate the interactive effects of four variables, viz., concentrations of glucose, methionine, urea, and succinic acid on glutaminase production. A significant influence of urea on glutaminase production was noted. Response surface methodology showed that a medium containing (g/l) glucose 10.0, urea 5.15, methionine 3.5, succinic acid 6.0, ammonium sulfate 2.5, and yeast extract 6.0 to be optimum for the production of glutaminase. The applied methodology was validated using this optimized media and enzyme activity 119 ± 0.12 U/l and specific activity of 0.63 U/mg protein after 28 h of incubation at 25 °C was obtained.
Keywords: Glutaminase; Response surface methodology; Marine isolates; Screening
Dried–Reswollen Immobilized Biocatalysts for Detoxification of Organophosphorous Compounds in the Flow Systems by Elena N. Efremenko; Ilya V. Lyagin; Fatima M. Plieva; Igor Yu. Galaev; Bo Mattiasson (251-260).
New immobilized biocatalysts based on polypeptides containing N- or C-terminal polyhistidine sequences and possessing organophosphorus hydrolase activity were investigated for detoxification of organophosphorous neurotoxic compounds in the flow systems. The biocatalysts were revealed to have a high catalytic activity within wide pH and temperature ranges 7.5–12.5 °C and 15–65 °C, respectively. The immobilized biocatalysts can be dried and reswollen before use with 92–93% catalytic activity remaining after drying and rehydration procedures. The half-lives of the biocatalysts under wet and dry storage conditions were 420 and 540 days, respectively.
Keywords: Organophosphorus hydrolase; Polyhistidine sequence; Dried/reswollen immobilized biocatalyst; Organophosphorous compound
Direct Observation of Single Molecule Conformational Change of Tight-Turn Paperclip DNA Triplex in Solution by Ching-Ping Liu; Ming-Tsai Wey; Chia-Ching Chang; Lou-Sing Kan (261-269).
DNA triplex modulates gene expression by forming stable conformation in physiological condition. However, it is not feasible to observe this unique molecular structure of large molecule with 54 oligodeoxynucleotides directly by conventional nuclear magnetic approach. In this study, we observed directly single molecular images of paperclip DNA triplexes formation in a buffer solution of pH 6.0 by atomic force microscopy (AFM). Meanwhile, a diffuse “tail” of unwound DNA was observed in pH 8.0 solution. This designable approach in visualizing the overall structures and shapes of oligo-DNAs at the single molecular level, by AFM, is applicable to other biopolymers as well.
Keywords: DNA triplex; Atomic force microscopy; Tight-turn paperclip DNA; Single molecule image
Purification and Characterization of Pectin Lyase Produced by Aspergillus terricola and its Application in Retting of Natural Fibers by Sangeeta Yadav; Pramod Kumar Yadav; Dinesh Yadav; Kapil Deo Singh Yadav (270-283).
An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal strain number MTCC 7588 has been used as source for pectin lyase production. The extracellular pectin lyase was purified to homogeneity from the culture filtrate of A. terricola by ion exchange and gel filtration chromatography. The determined molecular weight was 35 ± 01 kDa. The K m and k cat (turnover) values of the purified enzyme at 37 °C using citrus pectin as the substrate were found to be 1.0 mg/ml and 110.0 s−1, respectively. The pH and temperature optima of the enzyme were 8.0 and 50 °C, respectively. The retting ability of the purified pectin lyase for natural fibers viz. Cannabis sativa and Linum usitatissimum has been demonstrated for the first time.
Keywords: Pectin lyase; Retting; Aspergillus terricola ; Cannabis sativa ; Crotalaria juncea ; Linum usitatissimum
Isolation and Difference in Anti-Staphylococcus aureus Bioactivity of Curvularin Derivates from Fungus Eupenicillium sp. by Lian Wu Xie; Yong Chang Ouyang; Kun Zou; Guang Hua Wang; Min Jie Chen; Hui Min Sun; Shi Kun Dai; Xiang Li (284-293).
With the anti-microbial and anti-tumor composite screening model, bioassay-guided fractionation led to the isolation of two structurally related bioactive compounds, curvularin and αβ-dehydrocurvularin, from ethyl acetate extract of Eupenicillium sp. associated with marine sponge Axinella sp. Further study on the structure–activity relationship demonstrated that both compounds exhibited differences in bioactive profiles which are highly associated with their minor structural differences. Both curvularin and αβ-dehydrocurvularin have similar level of anti-fungal and anti-tumorous activity, while αβ-dehydrocurvularin is active against Staphylococcus aureus with a minimal inhibitory concentration of 375 μg/ml but curvularin does not. No detectable activity against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa exists for both compounds. It is suggested that the partial planar backbone structure, due to the conjugation of π electrons in the presence of a 3,4-double bond and the carbonyl group at position C-2 in αβ-dehydrocurvularin, acts as a key factor for the inhibition of S. aureus, a Gram-positive low G + C bacteria that are often the hospital-acquired and/or community-acquired pathogen.
Keywords: Curvularin; αβ-Dehydrocurvularin; Anti-Staphylococcus aureus ; Bioassay-guided isolation