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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #2)
Mining Dictyoglomus turgidum for Enzymatically Active Carbohydrases by Phillip Brumm; Spencer Hermanson; Becky Hochstein; Julie Boyum; Nick Hermersmann; Krishne Gowda; David Mead (pp. 205-214).
The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidum does not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.
Keywords: Dictyoglomus turgidum ; Biomass; Genome screening; Thermophilic cellulase; Hemicellulase; Glucosidase; Enzyme discovery
Statins: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) Reductase Inhibitors Demonstrate Anti-Atherosclerotic Character due to Their Antioxidant Capacity by Mohan-Kumari H. Puttananjaiah; Mohan Appasaheb Dhale; Vaishali Gaonkar; Shradha Keni (pp. 215-222).
Atherosclerosis is a chronic inflammatory disease of multiple etiologies. It is associated with the accumulation of oxidized lipids in arterial lesions leading to coronary heart disease. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (commonly known as statins) are widely used in cardiovascular disease prevention to lower the cholesterol. The antioxidant activity of HMG-CoA reductase inhibitors was studied by lipid peroxidation inhibition assay, DPPH, and hydroxyl radical scavenging-activity methods. The lovastatin (93%) and simvastatin (96%) showed significant action of lipid peroxidation inhibition compared to other HMG-CoA reductase inhibitors. The DPPH radical and hydroxyl radical scavenging activity of simvastatin was 38% and 33%, respectively. The oxidative modification of serum lipid due to reactive oxygen species causes atherosclerosis. This study revealed the importance of lovastatin and simvastatin to prevent oxidative stress-related cardiovascular diseases.
Keywords: HMG-CoA reductase inhibitors; Statins; Antioxidant; Lipid peroxidation; DPPH; Hydroxyl radical
Investigation of Biosurfactant-Producing Indigenous Microorganisms that Enhance Residue Oil Recovery in an Oil Reservoir After Polymer Flooding by Yue-Hui She; Fan Zhang; Jing-Jing Xia; Shu-Qiong Kong; Zheng-Liang Wang; Fu-Chang Shu; Ji-Ming Hu (pp. 223-234).
Three biosurfactant-producing indigenous microorganisms (XDS1, XDS2, XDS3) were isolated from a petroleum reservoir in the Daqing Oilfield (China) after polymer flooding. Their metabolic, biochemical, and oil-degradation characteristics, as well as their oil displacement in the core were studied. These indigenous microorganisms were identified as short rod bacillus bacteria with white color, round shape, a protruding structure, and a rough surface. Strains have peritrichous flagella, are able to produce endospores, are sporangia, and are clearly swollen and terminal. Bacterial cultures show that the oil-spreading values of the fermentation fluid containing all three strains are more than 4.5 cm (diameter) with an approximate 25 mN/m surface tension. The hydrocarbon degradation rates of each of the three strains exceeded 50%, with the highest achieving 84%. Several oil recovery agents were produced following degradation. At the same time, the heavy components of crude oil were degraded into light components, and their flow characteristics were also improved. The surface tension and viscosity of the crude oil decreased after being treated by the three strains of microorganisms. The core-flooding tests showed that the incremental oil recoveries were 4.89–6.96%. Thus, XDS123 treatment may represent a viable method for microbial-enhanced oil recovery.
Keywords: Polymer flooding; Indigenous microorganisms; Biosurfactant; Crude oil degradation; Microbial-enhanced oil recovery
Utilization of Horticultural Waste for Laccase Production by Trametes versicolor Under Solid-State Fermentation by Fengxue Xin; Anli Geng (pp. 235-246).
Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.
Keywords: Horticultural waste; Trametes versicolor ; Laccase; Solid-state fermentation; Decolorization
Structural and Thermal Investigations of Biomimetically Grown Casein–Soy Hybrid Protein Fibers by Thomas Balasingh Sudha; Palanisamy Thanikaivelan; Meiyazhagan Ashokkumar; Bangaru Chandrasekaran (pp. 247-257).
A hybrid protein fiber from different protein sources such as casein and soybean using wet-spinning technique was prepared. The casein/soybean hybrid fibers were synthesized at different weight ratios such as 100/0 (casein), 75/25, 50/50, 25/75, and 0/100 (soy) and characterized. Electron microscopic analysis confirmed the growth of pure and hybrid fibers and shows an increased surface roughness as the soy concentration increases in the hybrid fibers. Infrared spectra did not exhibit any significant changes in the functional groups between pure and hybrid fibers. X-ray diffraction pattern indicates slight increase in the diffraction peak values of hybrid fibers compared with the neat fibers. Thermal analyses show a moderate increase in the thermal stability of hybrid fibers when compared with the pure fibers. These results implicitly indicate that the casein and soy proteins are homogeneous in the hybrid fiber form. It has been demonstrated that the hybrid fiber with ≥50 wt.% casein content exhibits better morphology and increased thermal stability, which has scope for application in technical and medical industries.
Keywords: Fiber; Spinning; Thermal stability; Structure; Composite; Electron microscopy
Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine by Leman Tarhan; Hulya Ayar-Kayali (pp. 258-267).
Phenylalanine dehydrogenase (l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K m values of the native and the immobilized l-PheDH were determined as K m Phe = 0.118, 0.063 mM and K m NAD + = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD+ with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.
Keywords: Phenylketonuria; Phenylalanine dehydrogenase; Flow-injection system; Packed bed reactor; Immobilization
Galanthamine and Related Alkaloids Production by Leucojum aestivum L. Shoot Culture using a Temporary Immersion Technology by Ivan Ivanov; Vasil Georgiev; Milen Georgiev; Mladenka Ilieva; Atanas Pavlov (pp. 268-277).
The process of galanthamine and related alkaloids production by Leucojum aestivum shoot culture in a temporary immersion system was studied. It was established that temporary immersion approach is prospective for development of a biosynthetic process for obtaining valuable Amaryllidaceae alkaloids. Both immersion frequency and temperature had significant effect on biomass accumulation and the yields of galanthamine and related alkaloids. The maximal yield of galanthamine was achieved at the cultivation of L. aestivum shoot culture in temporary immersion RITA® system at immersion frequency 15 min flooding and 8 h stand-by periods, at 26 °C. Data on the relationships in the biological system “Nutrient medium–L. aestivum shoot culture–galanthamine” are presented as well.
Keywords: Galanthamine; Lycorine; Norgalanthamine; Leucojum aestivum ; Shoots; Amaryllidaceae; Temporary immersion system
Co-culture Based Blood-brain Barrier In Vitro Model, a Tissue Engineering Approach using Immortalized Cell Lines for Drug Transport Study by Zhiqi Zhang; Anthony J. McGoron; Eric T. Crumpler; Chen-Zhong Li (pp. 278-295).
This study evaluated the feasibility of using commercially available immortalized cell lines in building an in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Astrocytes-derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel biomimetic basement membrane for the endothelial cells to form tight junctions. Trans-Endothelial Electrical Resistance (TEER) and solute mass transport studies quantitatively evaluated the tight junction formation. Immuno-fluorescence microscopy and Western blot analysis qualitatively verified the expression of occludin, one of the tight junction proteins on the samples. Experimental data from a total of 13 experiments conclusively showed that the novel BBB in vitro co-culture model with aECM (CO + aECM) is promising in terms of establishing tight junction formation represented by TEER values, transport profiles, and tight junction protein expression when compared with traditional co-culture (CO) model setup or the endothelial cells cultured alone (EC). In vitro colorimetric sulforhodamine B (SRB) assay also revealed that the “CO + aECM” samples resulted in less cell loss on the basal sides of the insert membranes than traditional co-culture models. Our novel approach using immortalized cell lines with the addition of aECM was proven to be a feasible and repeatable alternative to the traditional BBB in vitro modeling.
Keywords: Blood-brain barrier; Co-culture; In vitro; Transport; Drug delivery; Membrane impedance; Western blot; Tight junction; Occludin
Targeted Delivery using Immunoliposomes with a Lipid-Modified Antibody-Binding Protein by Eiry Kobatake; Ryo Yamano; Masayasu Mie (pp. 296-303).
A recombinant antibody-binding protein originating from streptococcal protein G was modified with lipid in a site-directed manner by genetic engineering. The resulting lipoprotein was incorporated into the surface of liposomes by simple mixing. Immunoliposomes were then prepared by binding anti-IgG antibodies molecules onto the surface of proteoliposome via the lipid-anchored streptococcal protein G. Either small fluorophores or fluorescently labeled proteins were encapsulated into prepared immunoliposomes, and these molecular tracers could be delivered into cells whose surfaces were marked with specific antibodies.
Keywords: Immunoliposome; Protein G; Drug delivery system; Antibody-binding protein; Lipid-modified protein
Immobilization of a Recombinant Esterase from Lactobacillus plantarum on Polypropylene Accurel MP1000 by Deise Juliana Kolling; Willian Alexandre Suguino; Fábio Cristiano Angonesi Brod; Ana Carolina Maisonnave Arisi (pp. 304-312).
A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained 91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V max and higher K m than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse.
Keywords: Esterase; Lactobacillus plantarum ; Immobilization; Accurel MP1000; Recombinant protein; Lactic acid bacteria
The Optimization of Dilute Acid Hydrolysis of Cotton Stalk in Xylose Production by Ozlem Akpinar; Okan Levent; Şeyda Bostanci; Ufuk Bakir; Levent Yilmaz (pp. 313-325).
Cotton stalk, a lignocellulosic waste material, is composed of xylose that can be used as a raw material for production of xylitol, a high-value product. There is a growing interest in the use of lignocellulosic wastes for conversion into various chemicals because of their low cost and the fact that they are renewable and abundant. The objective of the study was to determine the effects of H2SO4 concentration, temperature, and reaction time on the production of sugars (xylose, glucose, and arabinose) and on the reaction by-products (furfural and acetic acid). Response surface methodology was used to optimize the hydrolysis process in order to obtain high xylose yield and selectivity. The optimum reaction temperature, reaction time, and acid concentration were 140 °C, 15 min, and 6%, respectively. Under these conditions, xylose yield and selectivity were found to be 47.88% and 2.26 g g−1, respectively.
Keywords: Xylose; Xylitol; Cotton stalk; Optimization