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Article ; Online: On the Protein Fibrillation Pathway

Jelica Milošević / Radivoje Prodanović / Natalija Polović

Molecules, Vol 26, Iss 4, p

Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy

2021 Volume 970

Abstract: Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods ... ...

Abstract	Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.
Keywords	ATR FTIR ; oligomer intermediates ; amyloid fibrillation ; HEWL ; secondary structure perturbation ; Organic chemistry ; QD241-441
Subject code	540 ; 500
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Gheorghita Menghiu / Vasile Ostafe / Radivoje Prodanović / Rainer Fischer / Raluca Ostafe

International Journal of Molecular Sciences, Vol 22, Iss 3041, p

2021 Volume 3041

Abstract: Chitinases catalyze the degradation of chitin, a polymer of N -acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of ... ...

Abstract	Chitinases catalyze the degradation of chitin, a polymer of N -acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A ( chiA ) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d- N , N ′, N ″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.
Keywords	FACS ; protein engineering ; error-prone PCR ; mutants ; bactericidal effect ; improved enzymes ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	540
Language	English
Publishing date	2021-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: In Silico and In Vitro Inhibition of SARS-CoV-2 PL pro with Gramicidin D

Sara Protić / Nevena Kaličanin / Milan Sencanski / Olivera Prodanović / Jelena Milicevic / Vladimir Perovic / Slobodan Paessler / Radivoje Prodanović / Sanja Glisic

International Journal of Molecular Sciences, Vol 24, Iss 1955, p

2023 Volume 1955

Abstract: Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This ... ...

Abstract	Finding an effective drug to prevent or treat COVID-19 is of utmost importance in tcurrent pandemic. Since developing a new treatment takes a significant amount of time, drug repurposing can be an effective option for achieving a rapid response. This study used a combined in silico virtual screening protocol for candidate SARS-CoV-2 PL pro inhibitors. The Drugbank database was searched first, using the Informational Spectrum Method for Small Molecules, followed by molecular docking. Gramicidin D was selected as a peptide drug, showing the best in silico interaction profile with PL pro . After the expression and purification of PL pro , gramicidin D was screened for protease inhibition in vitro and was found to be active against PL pro . The current study’s findings are significant because it is critical to identify COVID-19 therapies that are efficient, affordable, and have a favorable safety profile.
Keywords	anti SARS-CoV-2 ; PL pro ; COVID-19 ; gramicidin D ; PL pro candidate inhibitor ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	333
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination

Ivana Đukić / Nevena Kaličanin / Milan Sencanski / Snezana B. Pajovic / Jelena Milicevic / Jelena Prljic / Slobodan Paessler / Radivoje Prodanović / Sanja Glisic

Frontiers in Bioscience-Landmark, Vol 28, Iss 1, p

2023 Volume 8

Abstract: Background: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are ... ...

Abstract	Background: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. Methods: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. Results: L-arginine was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents.
Keywords	anti sars-cov-2 ; mpro ; covid-19 ; arginine ; vitamin c/arginine combination ; mpro candidate inhibitors ; Biochemistry ; QD415-436 ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	IMR Press
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Identification of SARS‐CoV‐2 Papain‐like Protease (PLpro) Inhibitors Using Combined Computational Approach**

Dr. Milan Sencanski / Dr. Vladimir Perovic / Dr. Jelena Milicevic / Dr. Tamara Todorovic / Dr. Radivoje Prodanovic / Dr. Veljko Veljkovic / Dr. Slobodan Paessler / Dr. Sanja Glisic

ChemistryOpen, Vol 11, Iss 2, Pp n/a-n/a (2022)

2022

Abstract: Abstract In the current pandemic, finding an effective drug to prevent or treat the infection is the highest priority. A rapid and safe approach to counteract COVID‐19 is in silico drug repurposing. The SARS‐CoV‐2 PLpro promotes viral replication and ... ...

Abstract	Abstract In the current pandemic, finding an effective drug to prevent or treat the infection is the highest priority. A rapid and safe approach to counteract COVID‐19 is in silico drug repurposing. The SARS‐CoV‐2 PLpro promotes viral replication and modulates the host immune system, resulting in inhibition of the host antiviral innate immune response, and therefore is an attractive drug target. In this study, we used a combined in silico virtual screening for candidates for SARS‐CoV‐2 PLpro protease inhibitors. We used the Informational spectrum method applied for Small Molecules for searching the Drugbank database followed by molecular docking. After in silico screening of drug space, we identified 44 drugs as potential SARS‐CoV‐2 PLpro inhibitors that we propose for further experimental testing.
Keywords	drug repurposing ; ISM ; molecular docking ; Papain-like protease ; SARS-CoV-2 ; Chemistry ; QD1-999
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	Wiley-VCH
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

Kovačević, Gordana / Raluca Ostafe / Ana Marija Balaž / Rainer Fischer / Radivoje Prodanović

The Society for Biotechnology, Japan Journal of bioscience and bioengineering. 2019 Jan., v. 127, no. 1

2019

Abstract: Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems ...

Abstract	Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting.
Keywords	DNA libraries ; biosensors ; catalytic activity ; directed evolution ; enzymatic reactions ; enzyme activity ; flow cytometry ; fluorescence ; fluorescent labeling ; glucose oxidase ; green fluorescent protein ; industry ; microbial fuel cells ; mutants ; protein synthesis ; screening ; yeasts
Language	English
Dates of publication	2019-01
Size	p. 30-37.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2018.07.002
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Drug Delivery

Marija Gavrovic-Jankulovic / Radivoje Prodanovic

Journal of Biomaterials and Nanobiotechnology, Vol 02, Iss 05, Pp 614-

Plant Lectins as Bioadhesive Drug Delivery Systems

2011 Volume 621

Abstract: Selective targeting of drugs to the proposed site of action provides therapeutic advantages such as reduced toxicity and smaller dose levels. Despite a huge progress made in drug design and delivery systems, many challenges still have to be solved. Small ...

Abstract	Selective targeting of drugs to the proposed site of action provides therapeutic advantages such as reduced toxicity and smaller dose levels. Despite a huge progress made in drug design and delivery systems, many challenges still have to be solved. Small therapeutic drugs always have the potential to pass into the kidneys and be excreted from the body. The use of macromolecular constructs (carriers) that allow longer circulation times, contribute to improved chemical functionality and more precise drug delivery is an attractive alternative option. Bioadhesive systems which will utilize intense contact to increase the drug concentration gradient could be an attractive approach. Because of their specific carbohydrate-binding, lectins can interact with glycoconjugates present on the epithelial cells that line all of the organs exposed to the external environment. The unique carbohydrate specificities of plant lectins can facilitate mucoadhesion and cytoadhesion of drugs. As immunostimulatory molecules with an adjuvant effect plant lectins can also be employed in vaccine development.
Keywords	Drug Delivery ; Epithelium ; Plant Lectin ; Polymers ; Vaccine ; Biotechnology ; TP248.13-248.65 ; Chemical technology ; TP1-1185 ; Technology ; T ; DOAJ:Biotechnology ; DOAJ:Life Sciences ; DOAJ:Biology and Life Sciences
Subject code	303
Language	English
Publishing date	2011-12-01T00:00:00Z
Publisher	Scientific Research Publishing
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries

Radivoje Prodanović / W. Lloyd Ung / Karla Ilić Đurđić / Rainer Fischer / David A. Weitz / Raluca Ostafe

Molecules, Vol 25, Iss 2418, p

2020 Volume 2418

Abstract: Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening ... ...

Abstract	Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant k cat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.
Keywords	fluorescent label ; sorting ; protein engineering ; enzyme optimization ; Organic chemistry ; QD241-441
Language	English
Publishing date	2020-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

Marija Blažić / Ana Marija Balaž / Olivera Prodanović / Nikolina Popović / Raluca Ostafe / Rainer Fischer / Radivoje Prodanović

Applied Sciences, Vol 9, Iss 7, p

2019 Volume 1413

Abstract: Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher ...

Abstract	Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.
Keywords	cellobiose dehydrogenase ; resazurin ; fluorescent assay ; flow cytometry ; yeast surface display ; Technology ; T ; Engineering (General). Civil engineering (General) ; TA1-2040 ; Biology (General) ; QH301-705.5 ; Physics ; QC1-999 ; Chemistry ; QD1-999
Subject code	570
Language	English
Publishing date	2019-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Chemical Modification of Glycoproteins’ Carbohydrate Moiety as a General Strategy for the Synthesis of Efficient Biocatalysts by Biomimetic Mineralization

Marija D. Stanišić / Nikolina Popović Kokar / Predrag Ristić / Ana Marija Balaž / Milan Senćanski / Miloš Ognjanović / Veljko R. Đokić / Radivoje Prodanović / Tamara R. Todorović

Polymers, Vol 13, Iss 3875, p

The Case of Glucose Oxidase

2021 Volume 3875

Abstract: Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to encapsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure ... ...

Abstract	Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to encapsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure electrostatic interactions between the protein’s surface and the positively charged Zn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modification of surface amino acid residues can lead to a rapid biocomposite crystal formation. However, a chemical modification of carbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the present study, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidation of GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule, from −10.2 to −36.9 mV, as shown by zeta potential measurements and native PAGE electrophoresis. Biomineralization experiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability of the ZIF-8 biocomposites. Periodate oxidation of carbohydrate components for glycoproteins can serve as a facile and general method for facilitating the biomimetic mineralization of other industrially relevant glycoproteins.
Keywords	metal–organic frameworks ; ZIF-8 ; biomimetic mineralization ; biocomposites ; biocatalysts ; Organic chemistry ; QD241-441
Subject code	540
Language	English
Publishing date	2021-11-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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