TY  - JOUR
AU  - Šokarda Slavić, Marinela
AU  - Kojić, Milan
AU  - Margetić, Aleksandra
AU  - Stanisavljević, Nemanja
AU  - Gardijan, Lazar
AU  - Božić, Nataša
AU  - Vujčić, Zoran
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6458
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.
PB  - Elsevier
T2  - International Journal of Biological Macromolecules
T1  - Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications
VL  - 249
SP  - 126055
DO  - 10.1016/j.ijbiomac.2023.126055
ER  - 

@article{
author = "Šokarda Slavić, Marinela 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.",
publisher = "Elsevier",
journal = "International Journal of Biological Macromolecules",
title = "Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications",
volume = "249",
pages = "126055",
doi = "10.1016/j.ijbiomac.2023.126055"
}

Šokarda Slavić, M., 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
Elsevier., 249, 126055.
https://doi.org/10.1016/j.ijbiomac.2023.126055

Šokarda Slavić M, 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 .

Šokarda Slavić, Marinela, 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  - JOUR
AU  - Šokarda Slavić, Marinela
AU  - Kojić, Milan
AU  - Margetić, Aleksandra
AU  - Ristović, Marina
AU  - Pavlović, Marija
AU  - Nikolić, Stefan
AU  - Vujčić, Zoran
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6518
AB  - The combination of b-oligosaccharides from enzymatically hydrolysed barley b-glucan has attracted interest
recently due to its positive effects on human health. This study aimed to assess the impact of the
endo-b-1,3-1,4-glucanase enzyme from Bacillus subtilis 168 on improving the nutritional and bioactive
properties of barley b-glucan. A new procedure for the isolation of b-glucan was developed, at a lower
temperature (45 °C), enabling purity from starch contamination, without affecting the yield (6 g b-glucan
from 100 g of barley flour). The endo-b-1,3-1,4-glucanase is cloned into E. coli pQE_Ek enables the high
production and purification (82% yield, 1.8 mg mL
 1 and 440 U mg
 1) of an enzyme identical to the
natural one (25.5 kDa). The enzymatic reaction showed high efficiency of b-glucan degradation by recombinant
enzyme, giving a mixture of products (of which 3-O-b-cellobiosyl-D-glucose and 3-O-b-cellotriosyl-
D-glucose are the most abundant), the reduction of viscosity (17%) and increase in antioxidant capacities
by 15.2%, 30.9% and 44.0% assessed by ABTS, DPPH and ORAC, respectively. These results indicate
the possible application of endo-b-1,3-1,4-glucanase enzyme in improving the properties of barley bglucan
used as functional foods.
PB  - Wiley
T2  - International Journal of Food Science and Technology
T1  - Improvement of nutritional and bioactive properties of barley β-glucan-based food products using Bacillus subtilis 168 endo-β-1,3-1,4-glucanase
DO  - 10.1111/ijfs.16647
ER  - 

@article{
author = "Šokarda Slavić, Marinela and Kojić, Milan and Margetić, Aleksandra and Ristović, Marina and Pavlović, Marija and Nikolić, Stefan and Vujčić, Zoran",
year = "2023",
abstract = "The combination of b-oligosaccharides from enzymatically hydrolysed barley b-glucan has attracted interest
recently due to its positive effects on human health. This study aimed to assess the impact of the
endo-b-1,3-1,4-glucanase enzyme from Bacillus subtilis 168 on improving the nutritional and bioactive
properties of barley b-glucan. A new procedure for the isolation of b-glucan was developed, at a lower
temperature (45 °C), enabling purity from starch contamination, without affecting the yield (6 g b-glucan
from 100 g of barley flour). The endo-b-1,3-1,4-glucanase is cloned into E. coli pQE_Ek enables the high
production and purification (82% yield, 1.8 mg mL
 1 and 440 U mg
 1) of an enzyme identical to the
natural one (25.5 kDa). The enzymatic reaction showed high efficiency of b-glucan degradation by recombinant
enzyme, giving a mixture of products (of which 3-O-b-cellobiosyl-D-glucose and 3-O-b-cellotriosyl-
D-glucose are the most abundant), the reduction of viscosity (17%) and increase in antioxidant capacities
by 15.2%, 30.9% and 44.0% assessed by ABTS, DPPH and ORAC, respectively. These results indicate
the possible application of endo-b-1,3-1,4-glucanase enzyme in improving the properties of barley bglucan
used as functional foods.",
publisher = "Wiley",
journal = "International Journal of Food Science and Technology",
title = "Improvement of nutritional and bioactive properties of barley β-glucan-based food products using Bacillus subtilis 168 endo-β-1,3-1,4-glucanase",
doi = "10.1111/ijfs.16647"
}

Šokarda Slavić, M., Kojić, M., Margetić, A., Ristović, M., Pavlović, M., Nikolić, S.,& Vujčić, Z.. (2023). Improvement of nutritional and bioactive properties of barley β-glucan-based food products using Bacillus subtilis 168 endo-β-1,3-1,4-glucanase. in International Journal of Food Science and Technology
Wiley..
https://doi.org/10.1111/ijfs.16647

Šokarda Slavić M, Kojić M, Margetić A, Ristović M, Pavlović M, Nikolić S, Vujčić Z. Improvement of nutritional and bioactive properties of barley β-glucan-based food products using Bacillus subtilis 168 endo-β-1,3-1,4-glucanase. in International Journal of Food Science and Technology. 2023;.
doi:10.1111/ijfs.16647 .

Šokarda Slavić, Marinela, Kojić, Milan, Margetić, Aleksandra, Ristović, Marina, Pavlović, Marija, Nikolić, Stefan, Vujčić, Zoran, "Improvement of nutritional and bioactive properties of barley β-glucan-based food products using Bacillus subtilis 168 endo-β-1,3-1,4-glucanase" in International Journal of Food Science and Technology (2023),
https://doi.org/10.1111/ijfs.16647 . .

TY  - CONF
AU  - Pavlović, Marija
AU  - Šokarda Slavić, Marinela
AU  - Kojić, Milan
AU  - Ristović, Marina
AU  - Momčilović, Miloš
AU  - Drulović, Nenad
AU  - Vujčić, Zoran
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7136
AB  - Pectinases are widely used in the fruit juice industry for clarification, liquefaction and stabilization of juices. Primarily, these enzymes are responsible for the breakdown of long and complex molecules in the fruit pulp (pectin). Unlike the combination of polygalacturonase (PG) and pectin esterase (PE) commonly found in commercial products, pectin lyase (PNL) is the only known pectinase capable of breaking down highly esterified pectins (such those found in fruits) into short molecules via a β-elimination mechanism without producing methanol. Methanol is toxic and can be harmful to health. Commercial preparations with PNL as the major component are preferable in juices and wine processing because it avoids the production of methanol, precipitation of pectin partially de-esterified with endogenous calcium, and damage of the volatile ester content responsible for the specific aroma of various fruits. This study reports the first cloning and characterization of a PNL gene, ppr, from Bacillus velezensis, and the deduced amino acid sequence is compared with those of other lyases. In this work, the gene encoding the pectin lyase (PNL; EC 4.2.2.10) from Bacillus velezensis was successfully expressed under optimized conditions for high soluble protein expressions as soluble PNL-6His in E. coli M15[pREP4]. After expression, purification of 6xHis-labeled PNL was performed according to QIAGEN instructions. IMAC fractions were analyzed using SDS-PAGE. After purification, 3-fold purification was observed with a yield of 82%. In this study, we report for the first time a method to express PNL in E. coli and purify it as a pure native enzyme. The cloned PLN was used to clarify and liquefy apple and orange juices and to improve the flavor of these juices. In this way, pulp waste after juice extraction is reduced. Methanol content was determined by gas-liquid chromatography and was not detectable in the clarified juices.
PB  - Elsevier
C3  - Book of Abstracts - 3rd Food Chemistry Conference, 10-12 October 2023, Dresden, Germany
T1  - Highly efficient clarification of fruit juices with new pectin lyase from Bacillus velezensis
SP  - P3.015
UR  - https://hdl.handle.net/21.15107/rcub_cer_7136
ER  - 

@conference{
author = "Pavlović, Marija and Šokarda Slavić, Marinela and Kojić, Milan and Ristović, Marina and Momčilović, Miloš and Drulović, Nenad and Vujčić, Zoran",
year = "2023",
abstract = "Pectinases are widely used in the fruit juice industry for clarification, liquefaction and stabilization of juices. Primarily, these enzymes are responsible for the breakdown of long and complex molecules in the fruit pulp (pectin). Unlike the combination of polygalacturonase (PG) and pectin esterase (PE) commonly found in commercial products, pectin lyase (PNL) is the only known pectinase capable of breaking down highly esterified pectins (such those found in fruits) into short molecules via a β-elimination mechanism without producing methanol. Methanol is toxic and can be harmful to health. Commercial preparations with PNL as the major component are preferable in juices and wine processing because it avoids the production of methanol, precipitation of pectin partially de-esterified with endogenous calcium, and damage of the volatile ester content responsible for the specific aroma of various fruits. This study reports the first cloning and characterization of a PNL gene, ppr, from Bacillus velezensis, and the deduced amino acid sequence is compared with those of other lyases. In this work, the gene encoding the pectin lyase (PNL; EC 4.2.2.10) from Bacillus velezensis was successfully expressed under optimized conditions for high soluble protein expressions as soluble PNL-6His in E. coli M15[pREP4]. After expression, purification of 6xHis-labeled PNL was performed according to QIAGEN instructions. IMAC fractions were analyzed using SDS-PAGE. After purification, 3-fold purification was observed with a yield of 82%. In this study, we report for the first time a method to express PNL in E. coli and purify it as a pure native enzyme. The cloned PLN was used to clarify and liquefy apple and orange juices and to improve the flavor of these juices. In this way, pulp waste after juice extraction is reduced. Methanol content was determined by gas-liquid chromatography and was not detectable in the clarified juices.",
publisher = "Elsevier",
journal = "Book of Abstracts - 3rd Food Chemistry Conference, 10-12 October 2023, Dresden, Germany",
title = "Highly efficient clarification of fruit juices with new pectin lyase from Bacillus velezensis",
pages = "P3.015",
url = "https://hdl.handle.net/21.15107/rcub_cer_7136"
}

Pavlović, M., Šokarda Slavić, M., Kojić, M., Ristović, M., Momčilović, M., Drulović, N.,& Vujčić, Z.. (2023). Highly efficient clarification of fruit juices with new pectin lyase from Bacillus velezensis. in Book of Abstracts - 3rd Food Chemistry Conference, 10-12 October 2023, Dresden, Germany
Elsevier., P3.015.
https://hdl.handle.net/21.15107/rcub_cer_7136

Pavlović M, Šokarda Slavić M, Kojić M, Ristović M, Momčilović M, Drulović N, Vujčić Z. Highly efficient clarification of fruit juices with new pectin lyase from Bacillus velezensis. in Book of Abstracts - 3rd Food Chemistry Conference, 10-12 October 2023, Dresden, Germany. 2023;:P3.015.
https://hdl.handle.net/21.15107/rcub_cer_7136 .

Pavlović, Marija, Šokarda Slavić, Marinela, Kojić, Milan, Ristović, Marina, Momčilović, Miloš, Drulović, Nenad, Vujčić, Zoran, "Highly efficient clarification of fruit juices with new pectin lyase from Bacillus velezensis" in Book of Abstracts - 3rd Food Chemistry Conference, 10-12 October 2023, Dresden, Germany (2023):P3.015,
https://hdl.handle.net/21.15107/rcub_cer_7136 .

TY  - CONF
AU  - Tomić, Katarina
AU  - Šokarda Slavić, Marinela
AU  - Kojić, Milan
AU  - Stanisavljević, Nemanja
AU  - Nikolić, Stefan
AU  - Vujčić, Zoran
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/5917
AB  - One of the most abundant natural polymers with multidimensional and multifaceted application is starch. Due to energy fuel sustainability concern, the world is focusing on renewable energy including energy from renewable biological materials like starch1. The importance of the enzymatic hydrolysis of granular starch below the temperature of gelatinization has been well recognized, mainly due to energy savings and the effective utilization of biomass, which reduces the overall cost of starch processing2. A new α-amylase gene (Amy35) was cloned from newly isolated thermophilic Anoxybacillus sp. ST4 and expressed in Escherichia coli. The purified recombinant α-amylase had an wide pH optimum range from 4.5 to 8.5 and optimum temperature of 75°C. The enzyme retained 95% of its activity after 3h of incubation at 50 and 60°C. Hydrolysis rates of potato, horseradish and corn starches, at 1% concentration were 20, 70 and 65%, respectively, in a period of 16 h. Analysis of the enzyme properties proved its high efficacy for the digestion of diverse raw starches below gelatinization temperature and, therefore, its potential commercial value for use as an industrial enzyme.
PB  - University of Belgrade - Faculty of Chemistry
PB  - Serbian Biochemical Society
C3  - Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia
T1  - Cloning and characterization of new raw starch digestion α-amylase from thermophilic Anoxybacillus sp.
SP  - 147
UR  - https://hdl.handle.net/21.15107/rcub_cer_5917
ER  - 

@conference{
author = "Tomić, Katarina and Šokarda Slavić, Marinela and Kojić, Milan and Stanisavljević, Nemanja and Nikolić, Stefan and Vujčić, Zoran",
year = "2022",
abstract = "One of the most abundant natural polymers with multidimensional and multifaceted application is starch. Due to energy fuel sustainability concern, the world is focusing on renewable energy including energy from renewable biological materials like starch1. The importance of the enzymatic hydrolysis of granular starch below the temperature of gelatinization has been well recognized, mainly due to energy savings and the effective utilization of biomass, which reduces the overall cost of starch processing2. A new α-amylase gene (Amy35) was cloned from newly isolated thermophilic Anoxybacillus sp. ST4 and expressed in Escherichia coli. The purified recombinant α-amylase had an wide pH optimum range from 4.5 to 8.5 and optimum temperature of 75°C. The enzyme retained 95% of its activity after 3h of incubation at 50 and 60°C. Hydrolysis rates of potato, horseradish and corn starches, at 1% concentration were 20, 70 and 65%, respectively, in a period of 16 h. Analysis of the enzyme properties proved its high efficacy for the digestion of diverse raw starches below gelatinization temperature and, therefore, its potential commercial value for use as an industrial enzyme.",
publisher = "University of Belgrade - Faculty of Chemistry, Serbian Biochemical Society",
journal = "Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia",
title = "Cloning and characterization of new raw starch digestion α-amylase from thermophilic Anoxybacillus sp.",
pages = "147",
url = "https://hdl.handle.net/21.15107/rcub_cer_5917"
}

Tomić, K., Šokarda Slavić, M., Kojić, M., Stanisavljević, N., Nikolić, S.,& Vujčić, Z.. (2022). Cloning and characterization of new raw starch digestion α-amylase from thermophilic Anoxybacillus sp.. in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia
University of Belgrade - Faculty of Chemistry., 147.
https://hdl.handle.net/21.15107/rcub_cer_5917

Tomić K, Šokarda Slavić M, Kojić M, Stanisavljević N, Nikolić S, Vujčić Z. Cloning and characterization of new raw starch digestion α-amylase from thermophilic Anoxybacillus sp.. in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia. 2022;:147.
https://hdl.handle.net/21.15107/rcub_cer_5917 .

Tomić, Katarina, Šokarda Slavić, Marinela, Kojić, Milan, Stanisavljević, Nemanja, Nikolić, Stefan, Vujčić, Zoran, "Cloning and characterization of new raw starch digestion α-amylase from thermophilic Anoxybacillus sp." in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia (2022):147,
https://hdl.handle.net/21.15107/rcub_cer_5917 .