TY  - CONF
AU  - Matijašević, Danka
AU  - Pavlović, Vladimir
AU  - Kljajević, Nemanja
AU  - Gardijan, Lazar
PY  - 2024
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2367
AB  - In last decades, bacterial resistance to first choice
antibiotics has been drastically increasing, therefore,
the research of new antimicrobial substances
is of great importance. This rising problem
with bacterial resistance to existing antibiotics
affects not only the health care institutes but also
food plants. S. aureus and S. enteritidis pose a
number of challenges to the food industry and
cause foodborne illness in humans. In addition,
due to their favourable elemental composition,
oyster mushrooms (Pleurotus sp.) are a reservoir
of bioactive compounds that give them remarkable
antibacterial potential. P. ostreatus is of
great economic importance and is the second
most cultivated edible mushroom, therefore information
about its possible targets on bacterial
cells is of great importance for use as a dietary
supplement or medicinal purposes. The results of
the antibacterial assay showed that tested bacterial
strains were susceptible to the methanol extract of P. ostreatus (PoME), while microbicidal
activity was only detected against Gram-positive
bacteria. Scanning electron microscopy (SEM)
micrographs suggested that extract acted on
cytoplasmic membrane of S. aureus, while the
cell envelope of S. Enteritidis was the most likely
target. Natural extracts may outperform individual
bioactive compounds due to the synergistic
interaction between the metabolites, which can
enhance the effects of the individual components.
Extracts rich in antibacterials are emerging
as alternatives to synthetic antibiotics in the food
and health sectors. Among these, crude mushroom
extracts are particularly sought after for
their diverse bioactive ingredients, as they can
combat resistant strains of bacteria due to their
different targets and modes of action. PoME can
be used as an effective antimicrobial agent, suitable
for applications aiming to eradicate foodborne
pathogens, thus enhancing food safety.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
T1  - MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM
EP  - 37
SP  - 37
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2367
ER  - 

@conference{
author = "Matijašević, Danka and Pavlović, Vladimir and Kljajević, Nemanja and Gardijan, Lazar",
year = "2024",
abstract = "In last decades, bacterial resistance to first choice
antibiotics has been drastically increasing, therefore,
the research of new antimicrobial substances
is of great importance. This rising problem
with bacterial resistance to existing antibiotics
affects not only the health care institutes but also
food plants. S. aureus and S. enteritidis pose a
number of challenges to the food industry and
cause foodborne illness in humans. In addition,
due to their favourable elemental composition,
oyster mushrooms (Pleurotus sp.) are a reservoir
of bioactive compounds that give them remarkable
antibacterial potential. P. ostreatus is of
great economic importance and is the second
most cultivated edible mushroom, therefore information
about its possible targets on bacterial
cells is of great importance for use as a dietary
supplement or medicinal purposes. The results of
the antibacterial assay showed that tested bacterial
strains were susceptible to the methanol extract of P. ostreatus (PoME), while microbicidal
activity was only detected against Gram-positive
bacteria. Scanning electron microscopy (SEM)
micrographs suggested that extract acted on
cytoplasmic membrane of S. aureus, while the
cell envelope of S. Enteritidis was the most likely
target. Natural extracts may outperform individual
bioactive compounds due to the synergistic
interaction between the metabolites, which can
enhance the effects of the individual components.
Extracts rich in antibacterials are emerging
as alternatives to synthetic antibiotics in the food
and health sectors. Among these, crude mushroom
extracts are particularly sought after for
their diverse bioactive ingredients, as they can
combat resistant strains of bacteria due to their
different targets and modes of action. PoME can
be used as an effective antimicrobial agent, suitable
for applications aiming to eradicate foodborne
pathogens, thus enhancing food safety.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health",
title = "MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM",
pages = "37-37",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2367"
}

Matijašević, D., Pavlović, V., Kljajević, N.,& Gardijan, L.. (2024). MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
Serbian Society for Microbiology., 37-37.
https://hdl.handle.net/21.15107/rcub_imagine_2367

Matijašević D, Pavlović V, Kljajević N, Gardijan L. MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health. 2024;:37-37.
https://hdl.handle.net/21.15107/rcub_imagine_2367 .

Matijašević, Danka, Pavlović, Vladimir, Kljajević, Nemanja, Gardijan, Lazar, "MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM" in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health (2024):37-37,
https://hdl.handle.net/21.15107/rcub_imagine_2367 .

TY  - CONF
AU  - Malešević, Milka
AU  - Ćurčić, Jovana
AU  - Gardijan, Lazar
AU  - Obradović, Mina
AU  - Stanisavljević, Nemanja
AU  - Pantić, Milena
AU  - Matijašević, Danka
PY  - 2024
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2381
AB  - Pseudomonas aeruginosa has been recognized as
a priority pathogen by World Health Organization,
due to the emergence of multidrug-resistant
(MDR) strains. Thus, new treatment options
such as antivirulence strategy is urgently needed.
This strategy is based on the disruption of quorum
sensing (QS) activity of this pathogen. The
focus of this research was to explore the anti-QS
activity of four selected medicinal mushrooms
(Lentinula edodes, Cantharellus cibarius, Trametes
versicolor and Pleurotus ostreatus) extracts on
MDR clinical isolate P. aeruginosa MMA83. Another
aim was to check their cytotoxicity on Caenorhabditis
elegans AU37 (glp-4(bn2) I; sec-1(km4).
Among three types of mushroom extracts - hot
water polysaccharide extracts (WPE), hot alkali
polysaccharide extracts (APE) and methanol
extracts (Met), APE extracts downregulated all
tested QS and virulence factors genes of P. aeruginosa
MMA83. The most prominent effect was
observed for C. cibarius APE extract, lowering
expression from 2-fold (for lasI gene) to 20-fold
for lasB gene. Extracts didn’t show cytotoxic effect
on C. elegans. The efficacy of APE extracts
in lowering the expression of QS and virulence
factors genes of P. aeruginosa MMA83 indicate
that these extracts can reduce pathogenicity of
P. aeruginosa. Also, they possess one of the desirable
biotechnology features – the absence of
cytotoxicity. Anti-QS and antivirulence effect of
APE extracts on P. aeruginosa envisages these extracts
as the promising therapeutic candidates
for the development of next-generation antivirulence
agents.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
T1  - MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE
EP  - 145
SP  - 145
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2381
ER  - 

@conference{
author = "Malešević, Milka and Ćurčić, Jovana and Gardijan, Lazar and Obradović, Mina and Stanisavljević, Nemanja and Pantić, Milena and Matijašević, Danka",
year = "2024",
abstract = "Pseudomonas aeruginosa has been recognized as
a priority pathogen by World Health Organization,
due to the emergence of multidrug-resistant
(MDR) strains. Thus, new treatment options
such as antivirulence strategy is urgently needed.
This strategy is based on the disruption of quorum
sensing (QS) activity of this pathogen. The
focus of this research was to explore the anti-QS
activity of four selected medicinal mushrooms
(Lentinula edodes, Cantharellus cibarius, Trametes
versicolor and Pleurotus ostreatus) extracts on
MDR clinical isolate P. aeruginosa MMA83. Another
aim was to check their cytotoxicity on Caenorhabditis
elegans AU37 (glp-4(bn2) I; sec-1(km4).
Among three types of mushroom extracts - hot
water polysaccharide extracts (WPE), hot alkali
polysaccharide extracts (APE) and methanol
extracts (Met), APE extracts downregulated all
tested QS and virulence factors genes of P. aeruginosa
MMA83. The most prominent effect was
observed for C. cibarius APE extract, lowering
expression from 2-fold (for lasI gene) to 20-fold
for lasB gene. Extracts didn’t show cytotoxic effect
on C. elegans. The efficacy of APE extracts
in lowering the expression of QS and virulence
factors genes of P. aeruginosa MMA83 indicate
that these extracts can reduce pathogenicity of
P. aeruginosa. Also, they possess one of the desirable
biotechnology features – the absence of
cytotoxicity. Anti-QS and antivirulence effect of
APE extracts on P. aeruginosa envisages these extracts
as the promising therapeutic candidates
for the development of next-generation antivirulence
agents.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health",
title = "MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE",
pages = "145-145",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2381"
}

Malešević, M., Ćurčić, J., Gardijan, L., Obradović, M., Stanisavljević, N., Pantić, M.,& Matijašević, D.. (2024). MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
Serbian Society for Microbiology., 145-145.
https://hdl.handle.net/21.15107/rcub_imagine_2381

Malešević M, Ćurčić J, Gardijan L, Obradović M, Stanisavljević N, Pantić M, Matijašević D. MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health. 2024;:145-145.
https://hdl.handle.net/21.15107/rcub_imagine_2381 .

Malešević, Milka, Ćurčić, Jovana, Gardijan, Lazar, Obradović, Mina, Stanisavljević, Nemanja, Pantić, Milena, Matijašević, Danka, "MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE" in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health (2024):145-145,
https://hdl.handle.net/21.15107/rcub_imagine_2381 .

TY  - JOUR
AU  - Malešević, Milka
AU  - Gardijan, Lazar
AU  - Miljković, Marija
AU  - O'Connor, Paula M
AU  - Mirković, Nemanja
AU  - Jovčić, Branko
AU  - Cotter, Paul D
AU  - Jovanovic, Goran
AU  - Kojić, Milan
PY  - 2023
UR  - https://doi.org/10.1093/lambio/ovad004
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1828
AB  - Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.
T2  - Letters in Applied Microbiology
T2  - Letters in Applied MicrobiologyLetters in Applied Microbiology
T1  - Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants
IS  - 2
SP  - ovad004
VL  - 76
DO  - 10.1093/lambio/ovad004
ER  - 

@article{
author = "Malešević, Milka and Gardijan, Lazar and Miljković, Marija and O'Connor, Paula M and Mirković, Nemanja and Jovčić, Branko and Cotter, Paul D and Jovanovic, Goran and Kojić, Milan",
year = "2023",
abstract = "Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.",
journal = "Letters in Applied Microbiology, Letters in Applied MicrobiologyLetters in Applied Microbiology",
title = "Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants",
number = "2",
pages = "ovad004",
volume = "76",
doi = "10.1093/lambio/ovad004"
}

Malešević, M., Gardijan, L., Miljković, M., O'Connor, P. M., Mirković, N., Jovčić, B., Cotter, P. D., Jovanovic, G.,& Kojić, M.. (2023). Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants. in Letters in Applied Microbiology, 76(2), ovad004.
https://doi.org/10.1093/lambio/ovad004

Malešević M, Gardijan L, Miljković M, O'Connor PM, Mirković N, Jovčić B, Cotter PD, Jovanovic G, Kojić M. Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants. in Letters in Applied Microbiology. 2023;76(2):ovad004.
doi:10.1093/lambio/ovad004 .

Malešević, Milka, Gardijan, Lazar, Miljković, Marija, O'Connor, Paula M, Mirković, Nemanja, Jovčić, Branko, Cotter, Paul D, Jovanovic, Goran, Kojić, Milan, "Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants" in Letters in Applied Microbiology, 76, no. 2 (2023):ovad004,
https://doi.org/10.1093/lambio/ovad004 . .

TY  - CONF
AU  - Malešević, Milka
AU  - Stanisavljević, Nemanja
AU  - Rašić, Slađan
AU  - Vukotić, Goran
AU  - Gardijan, Lazar
AU  - Obradović, Mina
AU  - Kojić, Milan
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2150
AB  - Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term
effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated.
Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region
(mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library.
Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead
to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive
changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment
had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment
cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly
reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey.
Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome
EP  - 112
SP  - 112
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2150
ER  - 

@conference{
author = "Malešević, Milka and Stanisavljević, Nemanja and Rašić, Slađan and Vukotić, Goran and Gardijan, Lazar and Obradović, Mina and Kojić, Milan",
year = "2023",
abstract = "Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term
effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated.
Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region
(mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library.
Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead
to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive
changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment
had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment
cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly
reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey.
Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome",
pages = "112-112",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2150"
}

Malešević, M., Stanisavljević, N., Rašić, S., Vukotić, G., Gardijan, L., Obradović, M.,& Kojić, M.. (2023). Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 112-112.
https://hdl.handle.net/21.15107/rcub_imagine_2150

Malešević M, Stanisavljević N, Rašić S, Vukotić G, Gardijan L, Obradović M, Kojić M. Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:112-112.
https://hdl.handle.net/21.15107/rcub_imagine_2150 .

Malešević, Milka, Stanisavljević, Nemanja, Rašić, Slađan, Vukotić, Goran, Gardijan, Lazar, Obradović, Mina, Kojić, Milan, "Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):112-112,
https://hdl.handle.net/21.15107/rcub_imagine_2150 .

TY  - JOUR
AU  - Slavić, Marinela Šokarda
AU  - Kojić, Milan
AU  - Margetić, Aleksandra
AU  - Stanisavljević, Nemanja
AU  - Gardijan, Lazar
AU  - Božić, Nataša
AU  - Vujčić, Zoran
PY  - 2023
UR  - https://www.sciencedirect.com/science/article/pii/S0141813023029501
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2066
AB  - α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60–80 °C and is active and stable over a wide pH range (4.0–9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.
T2  - International Journal of Biological Macromolecules
T1  - Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications
SP  - 126055
VL  - 249
DO  - 10.1016/j.ijbiomac.2023.126055
ER  - 

@article{
author = "Slavić, Marinela Šokarda and Kojić, Milan and Margetić, Aleksandra and Stanisavljević, Nemanja and Gardijan, Lazar and Božić, Nataša and Vujčić, Zoran",
year = "2023",
abstract = "α-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60–80 °C and is active and stable over a wide pH range (4.0–9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry.",
journal = "International Journal of Biological Macromolecules",
title = "Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications",
pages = "126055",
volume = "249",
doi = "10.1016/j.ijbiomac.2023.126055"
}

Slavić, M. Š., Kojić, M., Margetić, A., Stanisavljević, N., Gardijan, L., Božić, N.,& Vujčić, Z.. (2023). Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications. in International Journal of Biological Macromolecules, 249, 126055.
https://doi.org/10.1016/j.ijbiomac.2023.126055

Slavić MŠ, Kojić M, Margetić A, Stanisavljević N, Gardijan L, Božić N, Vujčić Z. Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications. in International Journal of Biological Macromolecules. 2023;249:126055.
doi:10.1016/j.ijbiomac.2023.126055 .

Slavić, Marinela Šokarda, Kojić, Milan, Margetić, Aleksandra, Stanisavljević, Nemanja, Gardijan, Lazar, Božić, Nataša, Vujčić, Zoran, "Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications" in International Journal of Biological Macromolecules, 249 (2023):126055,
https://doi.org/10.1016/j.ijbiomac.2023.126055 . .

TY  - CONF
AU  - Gardijan, Lazar
AU  - Kojić, Milan
AU  - Jovanović, Goran
AU  - Malešević, Milka
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/804
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2125
AB  - Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactisbv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage andfoodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU onits antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBUwere selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/mlto 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureusATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)and 30°C (L. lactis and L. monocytogenes).Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBUvariants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the correspondingpositions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU
EP  - 123
SP  - 123
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2125
ER  - 

@conference{
author = "Gardijan, Lazar and Kojić, Milan and Jovanović, Goran and Malešević, Milka",
year = "2023",
abstract = "Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactisbv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage andfoodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU onits antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBUwere selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/mlto 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureusATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)and 30°C (L. lactis and L. monocytogenes).Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBUvariants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the correspondingpositions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU",
pages = "123-123",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2125"
}

Gardijan, L., Kojić, M., Jovanović, G.,& Malešević, M.. (2023). Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 123-123.
https://hdl.handle.net/21.15107/rcub_imagine_2125

Gardijan L, Kojić M, Jovanović G, Malešević M. Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:123-123.
https://hdl.handle.net/21.15107/rcub_imagine_2125 .

Gardijan, Lazar, Kojić, Milan, Jovanović, Goran, Malešević, Milka, "Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):123-123,
https://hdl.handle.net/21.15107/rcub_imagine_2125 .

TY  - JOUR
AU  - Stanisavljević, Nemanja
AU  - Malešević, Milka
AU  - Rašić, Slađan
AU  - Vukotić, Goran
AU  - Gardijan, Lazar
AU  - Obradović, Mina
AU  - Kojić, Milan
PY  - 2023
UR  - https://doi.org/10.1080/00218839.2023.2244710
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2063
AB  - In the current study, honey bees’ diet was supplemented with spores of Brevibacillus laterosporus BGSP11 at concentration of 108 CFU/ml in sucrose solution and its short-term effects on their micro- and mycobiota have been analyzed using Illumina MiSeq sequencing. Obtained results indicate that this treatment does not lead to potentially harmful changes in the bacterial microbiome of worker bees, slightly affecting the composition of core microbiota. Moreover, several potentially beneficial changes have been observed. The treatment has led to a significant increase in the abundance of Snodgrassella alvi, and species from Lactobacillus and Bifidobacterium genera which play important roles in protection against several honey bee pathogens. Simultaneously, B. laterosporus enriched diet have led to almost complete eradication of Enterobacteriaceae family, the taxon that contains several putative pathogen species. On the other hand, the treatment affected mycobiota more profoundly, which was expected considering the greater instability compared to microbiota. Although the observed changes in honey bee mycobiome cannot be considered a priori beneficial or harmful, since the interaction between the bee and its mycobiome has not been sufficiently studied, certain beneficial consequences of the treatment have been observed. They are primarily reflected in the reduction of phytopathogenic fungi that can affect the organoleptic and techno-functional characteristics of honey. In addition, before introducing B. laterosporus in beekeeping practice as a biological agent for pathogen control it is necessary to perform more thorough studies of the impact on the honey bee microbiome, immune system, physiology and economic characteristics of honey bee colonies.
T2  - Journal of Apicultural Research
T1  - Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study
DO  - 10.1080/00218839.2023.2244710
ER  - 

@article{
author = "Stanisavljević, Nemanja and Malešević, Milka and Rašić, Slađan and Vukotić, Goran and Gardijan, Lazar and Obradović, Mina and Kojić, Milan",
year = "2023",
abstract = "In the current study, honey bees’ diet was supplemented with spores of Brevibacillus laterosporus BGSP11 at concentration of 108 CFU/ml in sucrose solution and its short-term effects on their micro- and mycobiota have been analyzed using Illumina MiSeq sequencing. Obtained results indicate that this treatment does not lead to potentially harmful changes in the bacterial microbiome of worker bees, slightly affecting the composition of core microbiota. Moreover, several potentially beneficial changes have been observed. The treatment has led to a significant increase in the abundance of Snodgrassella alvi, and species from Lactobacillus and Bifidobacterium genera which play important roles in protection against several honey bee pathogens. Simultaneously, B. laterosporus enriched diet have led to almost complete eradication of Enterobacteriaceae family, the taxon that contains several putative pathogen species. On the other hand, the treatment affected mycobiota more profoundly, which was expected considering the greater instability compared to microbiota. Although the observed changes in honey bee mycobiome cannot be considered a priori beneficial or harmful, since the interaction between the bee and its mycobiome has not been sufficiently studied, certain beneficial consequences of the treatment have been observed. They are primarily reflected in the reduction of phytopathogenic fungi that can affect the organoleptic and techno-functional characteristics of honey. In addition, before introducing B. laterosporus in beekeeping practice as a biological agent for pathogen control it is necessary to perform more thorough studies of the impact on the honey bee microbiome, immune system, physiology and economic characteristics of honey bee colonies.",
journal = "Journal of Apicultural Research",
title = "Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study",
doi = "10.1080/00218839.2023.2244710"
}

Stanisavljević, N., Malešević, M., Rašić, S., Vukotić, G., Gardijan, L., Obradović, M.,& Kojić, M.. (2023). Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study. in Journal of Apicultural Research.
https://doi.org/10.1080/00218839.2023.2244710

Stanisavljević N, Malešević M, Rašić S, Vukotić G, Gardijan L, Obradović M, Kojić M. Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study. in Journal of Apicultural Research. 2023;.
doi:10.1080/00218839.2023.2244710 .

Stanisavljević, Nemanja, Malešević, Milka, Rašić, Slađan, Vukotić, Goran, Gardijan, Lazar, Obradović, Mina, Kojić, Milan, "Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study" in Journal of Apicultural Research (2023),
https://doi.org/10.1080/00218839.2023.2244710 . .

TY  - JOUR
AU  - Novović, Katarina
AU  - Malešević, Milka
AU  - Dinić, Miroslav
AU  - Gardijan, Lazar
AU  - Kojić, Milan
AU  - Jovčić, Branko
PY  - 2022
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1525
AB  - Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358.
PB  - MDPI, Basel
T2  - International Journal of Molecular Sciences
T1  - RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum
IS  - 15
VL  - 23
DO  - 10.3390/ijms23158232
ER  - 

@article{
author = "Novović, Katarina and Malešević, Milka and Dinić, Miroslav and Gardijan, Lazar and Kojić, Milan and Jovčić, Branko",
year = "2022",
abstract = "Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358.",
publisher = "MDPI, Basel",
journal = "International Journal of Molecular Sciences",
title = "RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum",
number = "15",
volume = "23",
doi = "10.3390/ijms23158232"
}

Novović, K., Malešević, M., Dinić, M., Gardijan, L., Kojić, M.,& Jovčić, B.. (2022). RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum. in International Journal of Molecular Sciences
MDPI, Basel., 23(15).
https://doi.org/10.3390/ijms23158232

Novović K, Malešević M, Dinić M, Gardijan L, Kojić M, Jovčić B. RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum. in International Journal of Molecular Sciences. 2022;23(15).
doi:10.3390/ijms23158232 .

Novović, Katarina, Malešević, Milka, Dinić, Miroslav, Gardijan, Lazar, Kojić, Milan, Jovčić, Branko, "RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum" in International Journal of Molecular Sciences, 23, no. 15 (2022),
https://doi.org/10.3390/ijms23158232 . .

TY  - JOUR
AU  - Novović, Katarina
AU  - Malešević, Milka
AU  - Gardijan, Lazar
AU  - Kojić, Milan
AU  - Jovčić, Branko
PY  - 2022
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1572
AB  - The rapid and appropriate response of Pseudomonas spp. to environmental fluctuations has been enabled by numerous signal transduction regulatory systems. Regulatory systems in Pseudomonas aeruginosa are organized in a complex network which provides quick and fine-tuned cellular response through regulation of virulence and antibiotic resistance determinants production. Mobile integrons represent genetic elements included in the rapid dissemination of multiple antibiotic resistance determinants. The key factor of integron dynamics is enzyme integrase. So far, global regulators LexA, RpoS and PsrA have been recognized as regulators of the intI1 transcription. In this study, we discovered novel activator of the intI1 transcription, sensor kinase RclS, in Pseudomonas putida WCS358. This regulation is limited to stationary growth phase and appears to be indirect, at least through regulation of the rpoS expression. Sensor kinase RclS is a part of novel three-component system Rcl (Roc-like) together with two response regulators, RclR and RclA. RclS acted as a negative regulator of the rclA transcription, while the role in the rclR transcription regulation could not be defined. The RclSAR regulatory system seems to be a part of complex intI1 regulatory network which includes major stress response (SOS and RpoS) regulons.
PB  - Elsevier, Amsterdam
T2  - Research in Microbiology
T1  - Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase
IS  - 1-2
VL  - 173
DO  - 10.1016/j.resmic.2021.103885
ER  - 

@article{
author = "Novović, Katarina and Malešević, Milka and Gardijan, Lazar and Kojić, Milan and Jovčić, Branko",
year = "2022",
abstract = "The rapid and appropriate response of Pseudomonas spp. to environmental fluctuations has been enabled by numerous signal transduction regulatory systems. Regulatory systems in Pseudomonas aeruginosa are organized in a complex network which provides quick and fine-tuned cellular response through regulation of virulence and antibiotic resistance determinants production. Mobile integrons represent genetic elements included in the rapid dissemination of multiple antibiotic resistance determinants. The key factor of integron dynamics is enzyme integrase. So far, global regulators LexA, RpoS and PsrA have been recognized as regulators of the intI1 transcription. In this study, we discovered novel activator of the intI1 transcription, sensor kinase RclS, in Pseudomonas putida WCS358. This regulation is limited to stationary growth phase and appears to be indirect, at least through regulation of the rpoS expression. Sensor kinase RclS is a part of novel three-component system Rcl (Roc-like) together with two response regulators, RclR and RclA. RclS acted as a negative regulator of the rclA transcription, while the role in the rclR transcription regulation could not be defined. The RclSAR regulatory system seems to be a part of complex intI1 regulatory network which includes major stress response (SOS and RpoS) regulons.",
publisher = "Elsevier, Amsterdam",
journal = "Research in Microbiology",
title = "Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase",
number = "1-2",
volume = "173",
doi = "10.1016/j.resmic.2021.103885"
}

Novović, K., Malešević, M., Gardijan, L., Kojić, M.,& Jovčić, B.. (2022). Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase. in Research in Microbiology
Elsevier, Amsterdam., 173(1-2).
https://doi.org/10.1016/j.resmic.2021.103885

Novović K, Malešević M, Gardijan L, Kojić M, Jovčić B. Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase. in Research in Microbiology. 2022;173(1-2).
doi:10.1016/j.resmic.2021.103885 .

Novović, Katarina, Malešević, Milka, Gardijan, Lazar, Kojić, Milan, Jovčić, Branko, "Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase" in Research in Microbiology, 173, no. 1-2 (2022),
https://doi.org/10.1016/j.resmic.2021.103885 . .

TY  - JOUR
AU  - Gardijan, Lazar
AU  - Miljković, Marija
AU  - Obradović, Mina
AU  - Borović, Branka
AU  - Vukotić, Goran
AU  - Jovanović, Goran
AU  - Kojić, Milan
PY  - 2022
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1603
AB  - Aims The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results To improve the pMAL expression vector, we introduced the His(6) tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His(6)-Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His(6) tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His(6) and His(6)-enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human beta-defensin. Conclusions We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Significance and Impact of the Study Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.
PB  - Wiley, Hoboken
T2  - Journal of Applied Microbiology
T1  - Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides
EP  - 1013
IS  - 2
SP  - 1001
VL  - 133
DO  - 10.1111/jam.15623
ER  - 

@article{
author = "Gardijan, Lazar and Miljković, Marija and Obradović, Mina and Borović, Branka and Vukotić, Goran and Jovanović, Goran and Kojić, Milan",
year = "2022",
abstract = "Aims The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results To improve the pMAL expression vector, we introduced the His(6) tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His(6)-Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His(6) tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His(6) and His(6)-enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human beta-defensin. Conclusions We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Significance and Impact of the Study Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.",
publisher = "Wiley, Hoboken",
journal = "Journal of Applied Microbiology",
title = "Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides",
pages = "1013-1001",
number = "2",
volume = "133",
doi = "10.1111/jam.15623"
}

Gardijan, L., Miljković, M., Obradović, M., Borović, B., Vukotić, G., Jovanović, G.,& Kojić, M.. (2022). Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides. in Journal of Applied Microbiology
Wiley, Hoboken., 133(2), 1001-1013.
https://doi.org/10.1111/jam.15623

Gardijan L, Miljković M, Obradović M, Borović B, Vukotić G, Jovanović G, Kojić M. Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides. in Journal of Applied Microbiology. 2022;133(2):1001-1013.
doi:10.1111/jam.15623 .

Gardijan, Lazar, Miljković, Marija, Obradović, Mina, Borović, Branka, Vukotić, Goran, Jovanović, Goran, Kojić, Milan, "Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides" in Journal of Applied Microbiology, 133, no. 2 (2022):1001-1013,
https://doi.org/10.1111/jam.15623 . .