Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E. coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27. 5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC2) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5-10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.TUBITAK and Izmir Institute of Technolog

Identification and characterization of novel thermostable ?-amylase from Geobacillus sp. GS33

In this study, the heterologous expression and biochemical characterization of a thermostable ?-amylase from Geobacillus sp. GS33 was investigated. The recombinant ?-amylase was overexpressed in Escherichia coli BL21 (?DE) and purified via anion exchange and size-exclusion chromatography. The purified ?-amylase had a molecular weight of about 60 kDa, and was active in a broad range of pH 3–10 and temperature (40–90 °C) with maximum activity at pH 7–8 and 60 °C. The enzyme retained 50% residual activity at 65 °C, but only 20% at 85 °C after 16 h. At pH 9 and pH 7, the residual activity at 65 °C was 50% and 30%, respectively. The enzyme was remarkably activated by Co2+, Ca2+, Mg2+, PMSF, DTT, and Triton X-100, but partially inhibited by Cu2+, methanol, hexane, ethanol, acetone, SDS, and Tween 20. A molecular phylogeny analysis showed that the enzyme's amino acid sequence had the closest connection with an ?-amylase from Geobacillus thermoleovorans subsp. stromboliensis nov. 3D-structure-based amino acid sequence alignments revealed that the three catalytic residues (D217, E246, D314) and the four Ca2+ ion coordination residues (N143, E177, D186, H221) were conserved in ?-amylase from Geobacillus sp. GS33. The temperature stability and neutral pH optimum suggest that the enzyme may be useful for industrial applications. © 2020 Elsevier B.V.The authors would like to thank Biotechnology & Bioengineering Research Center at ?zmir Institute of Technology for the facilities and technical support

BODIPY-conjugated chitosan nanoparticles as a fluorescent probe

Recently, development of fluorescent nanoparticle-based probes for various bioimaging applications has attracted great attention. This work aims to develop a new type fluorescent nanoparticle conjugate and evaluate its cytotoxic effects on A549 and BEAS 2B cell lines. Throughout the study, ionically crosslinked chitosan nanoparticles (CNs) were conjugated with carboxylated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-COOH). The results of conjugates (BODIPY-CNs) were investigated with regard to their physic-chemical, optical, cytotoxic properties and cellular internalization. The morphology of BODIPY-CNs was found to be spherical in shape and quite uniform having average diameter of 70.25 ± 11.99 nm. Cytotoxicty studies indicated that although BODIPY-COOH itself was quite toxic on both A549- and BEAS 2B-treated cells, CNs increased the cell viability of both cell lines via conjugation to BODIPY-COOH fluorescent molecule up to 67% for A549 and 74% for BEAS 2B cells. These results may suggest a possible utilization of the new fluorescent nanoparticle-based probe for bioimaging in biology and medicine

In vitro evaluation of doxorubicin-incorporated magnetic albumin nanospheres

Magnetic albumin nanospheres that incorporate doxorubicin (M-DOX-BSA-NPs) were prepared previously by our research group to develop magnetically responsive drug carrier system. This nanocarrier was synthesized as a drug delivery system for targeted chemotherapy. In this work, cytotoxic effects of doxorubicin (DOX)-loaded/unloaded or magnetic/non-magnetic nanoparticles and free DOX against PC-3 cells and A549 cells were determined with the MTT test and the results were compared with each other. DOX-loaded magnetic albumin nanospheres (M-DOX-BSA-NPs) were found more cytotoxic than other formulations. The quantitative data obtained from flow cytometry analysis further verified the higher targeting and killing ability of M-DOX-BSA-NPs than free DOX on both of the cancer cell lines. Additionally, the results of cell cycle analysis have showed that M-DOX-BSA-NPs affected G1 and G2 phases. Finally, cell images were obtained using spin-disk confocal microscopy, and cellular uptake of M-DOX-BSA-NPs was visualized. The findings of this study suggest that M-DOX-BSA-NPs represent a potential doxorubicin delivery system for targeted drug transport into prostate and lung cancer cells. In this study, we found that M-DOX-BSA-NPs provide many advantages as targeted drug delivery, enhanced drug killing ability and bioavailability based on cytotoxicity, flow cytometry, and confocal microscopy image results

Regio- and stereo-chemical ring-opening reactions of the 2,3-epoxy alcohol derivative with nucleophiles: Explanation of the structures and C-2 selectivity supported by theoretical computations

The ring-opening reactions of (1aS,2S,6bR)-5-ethyl-2-hydroxyhexahydro-4H-oxireno[2,3-e]isoindole-4,6(5H)-dione were investigated under very mild and nonchelated conditions. C-2 selective ring-opening products were obtained with nucleophilic additions such as Cl-, Br- and N-3(-). The exact configuration of (3aS,4R,5R,6S,7aS)-5-chloro-2-ethyl-4,6-dihydroxyhexahydro-1H-isoindole-1,3(2H)-dione was determined by X-Ray diffraction analysis which was obtained from the reaction of epoxy alcohol with HCl . On the other hand, theoretical computations were carried out to explain the regioselectivity in the ring opening reaction of epoxy alcohols. The results showed that the ring-opening reaction of both epoxy alcohols proceeds in a kinetically controlled manner and regioselectivity occurs depending on the transition state. (c) 2022 Published by Elsevier B.V

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies

Abstract Proteins are important targets in cancer research because malignancy is associated with defects in cell protein machinery. Protein profiling is an emerging independent subspecialty of proteomics that is rapidly expanding and providing unprecedented insight into biological events. Quantitative assessment of protein levels in hematologic malignancies seeks a comprehensive understanding of leukemiaassociated protein patterns for use in aiding diagnosis, follow-up treatment, and the prediction of clinical outcomes. Many recently developed high-throughput proteomic methods can be applied to protein profiling. Herein the importance of protein profiling, its exploitation in leukemia research, and its clinical usefulness in the treatment and diagnosis of various cancer types, and techniques for determining changes in protein profiling are reviewed. (Turk J Hematol 2011; 28: 1-14) Key words: Hematologic malignancies, leukemia, proteomics, protein profiling Received: November 24, 2010 Accepted: January 19, 2011 Özet Malignitelerde proteinlerin hücresel mekanizmalarında meydana gelen bozukluklardan dolayı, proteinler kanser araştırmaları için önemli hedeflerdir. Proteomiksin alt uzmanlık dalı olarak ortaya çıkan ve bağımsız bir alan olan protein profilleme biyolojik olaylara farklı bir bakış açısı sağlamak amacıyla hızla gelişmektedir. Hematolojik malignitelerdeki protein düzeylerinin kantitatif olarak değerlendiril-mesi, teşhise yardımcı olması, tedavinin izlenmesi ve klinik sonuçların tahmininde mükemmel bir yaklaşım olması nedeni ile lösemi ile ilgili protein modellerinin kapsamlı bir şekilde incelenmesini amaçlamaktadır. Son dönemlerde geliştirilen yüksek verimli yöntemler protein profillemede kullanıla-bilir. Bu makalede, protein profillemenin önemi, lösemi araştırmalarındaki rolü, çeşitli kanser tiplerinin tanısı ve tedavisi için klinik kullanımları ve protein profilindeki değişikliklerin belirlenmesinde kullanılan teknikler değerlendirilmiştir

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7M CaCl 2 solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.Izmir Institute of Technology Research Foundatio

A thermophilic α-l-Arabinofuranosidase from Geobacillus vulcani GS90: heterologous expression, biochemical characterization, and its synergistic action in fruit juice enrichment

α-l-Arabinofuranosidases with an orchestral action of xylanolytic enzymes degrades the xylan in plant cell wall. In this study, heterologous expression, biochemical characterization, and synergistic action of α-l-Arabinofuranosidase from previously identified.Geobacillus vulcani GS90 (GvAbf) was investigated. The recombinant α-l-Arabinofuranosidase was overexpressed in Escherichia coli BL21 (λDE) and purified via His-tag Ni-affinity and size-exclusion chromatography. Optimum activity of the purified α-l-Arabinofuranosidase was obtained at pH 5 and at 70 °C. The GvAbf was active in a broad pH and temperature ranges; pH 4–9 and 30–90 °C, respectively. In addition, it retained most of its activity after an hour incubation at 70 °C and remained relatively stable at pH 3–6. GvAbf was quite stable against various metal ions. The kinetic parameters of GvAbf was obtained as Vmax and Km; 200 U/mg and 0.2 mM with p-nitrophenyl-α-l-arabinofuranoside and 526 U/mg and 0.1 mM with sugar beet arabinan, respectively. The synergistic action of GvAbf was studied with commercially available xylanase on juice enrichment of apples, grapes, oranges, and peaches. The best juice enrichment in terms of clarity, reducing sugar content, and yield, was achieved with GvAbf and xylanase together compared to treatment with xylanase and GvAbf alone in all fruits. The treatment with GvAbf and xylanase together lead to an increased juice yield by 26.56% (apple), 30.88% (grape), 40.00% (orange) and 32.20% (peach) as well as having a significant effect on juice clarity by an increase of % transmittance 47.26, 25.98, 41.77, and 44.97, respectively. The highest reducing sugar level of fruit juices also obtained with GvAbf and xylanase together compared to treatment with xylanase and GvAbf alone in all types of fruits. GvAbf and xylanase together as simultaneous synergistic manner may have an exciting potential for application in fruit juice processing

Enhanced thermostability of the immobilized thermoalkalophilic esterase onto magnetic-cornstarch nanoparticle

The immobilization of the biocatalysts onto magnetic nanoparticles has been extensively applied as the external magnetic field facilitates the enzyme recovery from the reaction mixture. In the present study, glutaraldehyde-modified magnetite-cornstarch nanoparticles (MCNs) were successfully synthesized, elaborately characterized by ZetaSizer and surface-enhanced Raman spectroscopy, and used for the immobilization of a thermoalkalophilic esterase from Geobacillus sp. The optimal immobilization conditions were obtained at 65 degrees C, 2:3 molar ratios of Fe2+:Fe3+, and 1 g cornstarch resulted in approximately 90 nm magnetic particles in size. Also, immobilization yield and immobilization efficiency of the esterase were found as 74% and 82%, respectively. Scanning electron microscopy micrographs showed that MCNs were uniform, spherical in shape, and well dispersed and esterase immobilized MCNs displayed similar morphology as free MCNs. The maximum activity of free and immobilized esterase was obtained at 65 degrees C and pH 9. Immobilization onto glutaraldehyde-modified MCNs significantly enhanced the esterase thermostability. Additionally, the immobilized esterase kept its residual activity of 75% after three sequential cycles, suggesting that it has favorable operational stability

A novel thermostable xylanase from Geobacillus vulcani GS90: Production, biochemical characterization, and its comparative application in fruit juice enrichment

Xylanases have great attention to act as a potential role in agro-industrial processes. In this study, production, characterization, and fruit juice application of novel xylanase from thermophilic Geobacillus vulcani GS90 (GvXyl) were performed. GvXyl was purified via acetone precipitation and gel-filtration chromatography. The results showed that GvXyl had 1,671.4 U/mg of specific activity and optimally worked at pH 8 and 55°C. It was also active in a wide pH (3–9) and temperature (30–90ºC) ranges. GvXyl was highly stable at 90ºC and relatively stable at pH 3–9. The kinetic parameters of GvXyl were obtained as Km, Vmax, and kcat; 10.2 mg/ml, 4,104 µmol min?1 mg?1, and 3,542.6 s?1, respectively. GvXyl had higher action than commercial xylanase in fruit juice enrichment. These results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability. Practical applications: Polysaccharides include starch, pectin, and hemicellulose create problems by lowering fruit juice quality in beverages. To overcome this problem, various clarification processes might be applied to natural fruit juices. Even though chemicals are widely used for this purpose, recently enzymes including xylanases are preferred for obtaining high-quality products. In this study, we reported the production and biochemical characterization of novel thermostable xylanase from thermophilic G. vulcani GS90 (GvXyl). Also, apple and orange juice enrichment were performed with the novel xylanase to increase the quality in terms of yield, clarity, and reducing sugar substance. The improved quality features of apple and orange juices with GvXyl was then compared to commercially available ?-1,4-xylanase. The results revealed that GvXyl might possess a potential influence in fruit juice processing because of its high specific activity and great thermal stability. © 2021 Wiley Periodicals LLC.The authors thank Biotechnology & Bioengineering Research Center at ?zmir Institute of Technology for the facilities and technical support