ISOLATION OF PROTEIN FROM THE SPINE VENOM OF PTEROIS VOLITANS FOUND IN THE INDONESIAN OCEAN, USING A HEATING PROCESS, FOR ANTICANCER, ANTIRETROVIRAL, ANTIBACTERIAL, AND ANTIOXIDANT ASSAYS

Objective: This research investigates the antibacterial, anticancer, antioxidant, and antiretroviral activities of the lionfish spine poison extract. Methods: Isolation and purification of the phospholipase A2 (PLA2) protein obtained from the spine poison were conducted through the following stages, including, extraction of the venom by sonication, heating, and purification using gradual saturation levels of ammonium sulfate. Furthermore, the purity and concentration of PLA2 were analyzed using the Lowry test and Marinetti’s method, respectively, while its protein content was ascertained through SDS-PAGE. Toxicity was then evaluated employing the brine shrimp lethality test (BSLT), and its anticancer activity was assessed in human cervical carcinoma cells (HeLa cells). Finally, its antioxidant, antibacterial, and antiretroviral activities were analyzed using the DPPH method, agar diffusion test against Salmonella sp. and E. coli, and SRV-2 and RT-qPCR tests, respectively. Results: The protein demonstrated 37.79% inhibition for anticancer activity, IC50 1312 ppm for antioxidant activity, 98.81%, and 89.28% inhibition of E. coli and Salmonella sp. respectively for antibacterial activity and 98.13% inhibition for antiretroviral activity. Conclusion: It can be concluded that lionfish (Pterois volitans) has the potential to be developed as an antioxidant, anticancer, antibacterial, and antiretroviral agent. Furthermore, the pharmacological activity of its spine venom was determined by isolating PLA2 protein from its extract, using an optimum heating temperature of 70 °C and an ammonium sulfate saturation level of 80%.

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The influence of heating process on anticancer activity of Pterois volitans (red lionfish) venom extraction against human cervical carcinoma cell

10.1063/1.5139359 ◽

2019 ◽

Author(s):

Andy Noorsaman Sommeng ◽

Mustika Sari ◽

Mikael Januardi Ginting ◽

Muhamad Sahlan ◽

Heri Hermansyah ◽

...

Keyword(s):

Anticancer Activity ◽

Cervical Carcinoma ◽

Carcinoma Cell ◽

Heating Process ◽

Pterois Volitans ◽

Human Cervical Carcinoma ◽

Red Lionfish

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190416144650 ◽

2019 ◽

Vol 20 (6) ◽

pp. 497-505 ◽

Cited By ~ 1

Author(s):

Abeer M. Abd El-Aziz ◽

Mohamed A. Shaker ◽

Mona I. Shaaban

Keyword(s):

Gold Nanoparticles ◽

Lipolytic Activity ◽

Market Demand ◽

Biotechnological Applications ◽

Lipase Gene ◽

Expression Plasmid ◽

E Coli ◽

Nickel Affinity Chromatography ◽

Recombinant Production ◽

Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

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Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213104224 ◽

2020 ◽

Vol 20 (8) ◽

pp. 970-981

Author(s):

Hamed A. Ghramh ◽

Essam H. Ibrahim ◽

Mona Kilnay

Keyword(s):

Anticancer Activity ◽

Cancer Cells ◽

Biological Activities ◽

Sugar Content ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bioactive Molecules ◽

Sds Page ◽

Splenic Cells ◽

Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

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Design and Manufacturing of a Disposable, Cyclo-Olefin Copolymer, Microfluidic Device for a Biosensor †

Sensors ◽

10.3390/s19051178 ◽

2019 ◽

Vol 19 (5) ◽

pp. 1178 ◽

Cited By ~ 2

Author(s):

Jorge Prada ◽

Christina Cordes ◽

Carsten Harms ◽

Walter Lang

Keyword(s):

Microfluidic Device ◽

Heating Temperature ◽

Low Cost ◽

Reaction Chamber ◽

Microfluidic System ◽

Heating Process ◽

Design And Manufacturing ◽

On Chip ◽

Working Parameters ◽

Auto Fluorescence

This contribution outlines the design and manufacturing of a microfluidic device implemented as a biosensor for retrieval and detection of bacteria RNA. The device is fully made of Cyclo-Olefin Copolymer (COC), which features low auto-fluorescence, biocompatibility and manufacturability by hot-embossing. The RNA retrieval was carried on after bacteria heat-lysis by an on-chip micro-heater, whose function was characterized at different working parameters. Carbon resistive temperature sensors were tested, characterized and printed on the biochip sealing film to monitor the heating process. Off-chip and on-chip processed RNA were hybridized with capture probes on the reaction chamber surface and identification was achieved by detection of fluorescence tags. The application of the mentioned techniques and materials proved to allow the development of low-cost, disposable albeit multi-functional microfluidic system, performing heating, temperature sensing and chemical reaction processes in the same device. By proving its effectiveness, this device contributes a reference to show the integration potential of fully thermoplastic devices in biosensor systems.

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The Food Anti-Microbials β-Phenylethylamine (-HCl) and Ethyl Acetoacetate Do Not Change during the Heating Process

Antibiotics ◽

10.3390/antibiotics10040418 ◽

2021 ◽

Vol 10 (4) ◽

pp. 418

Author(s):

Shelley M. Horne ◽

Angel Ugrinov ◽

Birgit M. Prüβ

Keyword(s):

Microbial Activity ◽

Food Processing ◽

Ethyl Acetoacetate ◽

Mass Spectra ◽

Heating Process ◽

Toxic Compounds ◽

Minimal Inhibitory Concentrations ◽

E Coli ◽

Cooking Process ◽

Early Retention

β-Phenylethylamine hydrochloride (PEA-HCl) and ethyl acetoacetate (EAA) are anti-microbials with applications in food processing. As food anti-microbials, the compounds will have to withstand the cooking process without changing to toxic compounds. With this Communication, we address the question of whether PEA and EAA are altered when heated to 73.9 °C or 93.3 °C. A combination of gas chromatography and mass spectrometry was used to analyze solutions of PEA(-HCl) or EAA in beef broth or water. In addition, the anti-microbial activity of PEA-HCl and EAA was compared between heated and unheated samples at a range of concentrations. The gas chromatograms of PEA(-HCl) and EAA showed one peak at early retention times that did not differ between the heated and unheated samples. The mass spectra for PEA and EAA were near identical to those from a spectral database and did not show any differences between the heated and unheated samples. We conclude that PEA(-HCl) and EAA formed pure solutions and were not altered during the heating process. In addition, the anti-microbial activity of PEA-HCl and EAA did not change after the heating of the compounds. Regardless of temperature, the minimal inhibitory concentrations (MICs) for PEA-HCl were 20.75 mmol mL−1 for Escherichia coli and Salmonella enterica serotype Typhimurium. For EAA, the MICs were 23.4 mmol mL−1 for E. coli and 15.6 mmol mL−1 for S. enterica.

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Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2008001000020 ◽

2008 ◽

Vol 43 (10) ◽

pp. 1405-1411 ◽

Cited By ~ 6

Author(s):

Paula Radaelli ◽

Thor Vinícius Martins Fajardo ◽

Osmar Nickel ◽

Marcelo Eiras ◽

Gilvan Pio-Ribeiro

Keyword(s):

Virus Disease ◽

Disease Diagnosis ◽

Coat Proteins ◽

Grapevine Virus ◽

Western Blots ◽

E Coli ◽

Sds Page ◽

Grapevine Virus B ◽

Polyclonal Antisera ◽

Infected Grapevine

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

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ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF COCOA POD THAT ASSOCIATED IN MALTODEXTRIN IN VARIOUS CONCENTRATION

AGROLAND The Agricultural Sciences Journal ◽

10.22487/j24077593.2018.v5.i2.12188 ◽

2018 ◽

Vol 5 (2) ◽

pp. 123

Author(s):

Asriani Hasanuddin ◽

Chairil Anwar ◽

Marhawati Mappatoba ◽

Hafsah Hafsah

Keyword(s):

Antimicrobial Activity ◽

Antioxidant Activity ◽

Antioxidant Activities ◽

Diffusion Method ◽

Natural Food ◽

Skin Extract ◽

E Coli ◽

Dpph Method ◽

Antioxidant And Antimicrobial Activity ◽

Peel Extract

Cocoa pod extract ((Theobroma cacao L.) has antioxidant and antimicrobial activity that has the potential as a natural food preservative. However, in its use the cocoa fruit skin extract has a disadvantage because the short shelf time and its application to food are limited, efforts are needed to prevent damage and extend shelf life, one of the efforts that can be done is by encapsulating the extract.This study aims to determine the antibacterial activity and antioxidant encapsulation of cocoa peel extract, this study begins with the extraction of cocoa pods with ethanol solvent by comparing cocoa pods : solvent 1: 4 The skin of cacao cocoa fruit used is yellow harvested cocoa fruit, then chopped and dried to form flour.The sample is extracted by maceration with ethanol solvent Antioxidant test is done by DPPH method, while antibacterial test is carried out by the well diffusion method. This study used a completely randomized design method (CRD) with 5 treatments using a maltodextrin concentration of 20% (M1); 30% (M2); 40% (M3); 50% (M4) and 60% (M5). The results showed that the treatment gave the highest yield in the treatment of 60% maltodextrin concentration (M5), while the highest antioxidant activity was obtained in the treatment of 20% maltodextrin (M1) with IC50 75.98 µg / mL and the treatment with the lowest antioxidant activity was obtained at treatment of 60% maltodextrin concentration (M5) with IC50 value 114.89 µg / mL. While for the antimicrobial activity also obtained with the same results, namely treatment of 20% (M1) obtained a higher inhibition diameter compared to treatment at 30%; 40%; 50% and 60% for all types of bacteria. The inhibition diameter in the treatment of the concentration of maltodextrin 20% (M1) for E. coli bacteria is between 4.12 mm - 10.95 mm, Salmonella sp is 2.85 mm - 8 , 25 mm and for Staphylococcusaureus of 5.15 mm - 13.90 mm and the lowest inhibition diameter was obtained in the treatment of 60% maltodextrin concentration (M5) for E. coli bacteria of between 2.0 mm - 4.79 mm, Salmonella sp of 1.15 mm - 4.35 mm and for Staphylococcusaureusat 2.76 mm - 5.17 mm.This study concluded that the encapsulation of cocoa peel extract using 20% maltodextrin had the highest antioxidant and antimicrobial activity when compared with other treatments namely 30% concentration; 40%; 50% and 60% but for the treatment of 20% and 30% there is no difference. Ethanol extract of cocoa pods can be made in the form of encapsulates which are very likely to be used as natural preservatives.

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DEVELOPMENT OF RECOMBINANT PEPTIDE TECHNOLOGY WITH ANTIMICROBIAL PROPERTIES OF A BROAD ACTION SPECTRUM

Science Evolution ◽

10.21603/2500-1418-2017-2-2-3-14 ◽

2017 ◽

pp. 3-14

Author(s):

Alexander Prosekov ◽

Olga Babich ◽

Irina Milenteva ◽

...

Keyword(s):

Molecular Weight ◽

Ammonium Sulfate ◽

Action Spectrum ◽

Antimicrobial Properties ◽

Hplc Method ◽

Salting Out ◽

Antimicrobial Spectrum ◽

Recombinant Peptide ◽

Isolation And Purification ◽

Protein Nature

Bacteriocins are antibacterial, mainly complex, substances of protein nature. The promising strains producing bacteriocins used in the food industry are lactic acid microorganisms. This study examines the development of a technology for the production of a recombinant peptide with broad-spectrum antimicrobial properties. An important step is the isolation and purification of the recombinant peptide. It has been proved that the highest antimicrobial activity is manifested by a recombinant peptide isolated by a method based on salting out with ammonium sulfate. During the purification of the recombinant bacteriocin preparation, three kinds of columns were used. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. Based on the results of determining the molecular weight and purity of the recombinant bacteriocin, it was found that the molecular weight of the recombinant bacteriocin having the amino acid sequence: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXATGGH GGNHCCGMSRYIQGIPDFLRGYLHGISSANKHKKGRL, is 13 kDa. A technology for the preparation of a broad-action antimicrobial spectrum peptide has been developed. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain of Escherichia coli BL21DE3; separation of biomass from the nutrient medium; precipitation of bacteriocins by ammonium sulfate; centrifugation; washing the precipitate; centrifugation at 4200 rpm and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags of polymeric and combined materials; storage at a temperature of 18±2°C for 12 months.

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CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

Indonesian Journal of Agricultural Science ◽

10.21082/ijas.v16n1.2015.31-38 ◽

2015 ◽

Vol 16 (1) ◽

pp. 31

Author(s):

Kusdianawati Kusdianawati ◽

Apon Zaenal Mustopa ◽

Suharsono Suharsono ◽

Bugi Ratno Budiarto ◽

Fatimah Fatimah ◽

...

Keyword(s):

Escherichia Coli ◽

Lactobacillus Plantarum ◽

Proteolytic Enzymes ◽

Leader Peptide ◽

Human Consumption ◽

Mature Peptide ◽

Expression And Purification ◽

Food Preservative ◽

E Coli ◽

Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

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