In vitro production of recombinant ergokonin protein and its application in the polylactic acid film to control Aspergillus niger and Penicillium notatum
Subject Areas : Food Microbiology
Mohammad javad Akbarian Meymand
1
(Ph.D. student, Grape Environmental Science Department, Research Institute for Grapes and Raisin (RIGR), Malayer University, Iran.)
Arash Babaei
2
(Assistant Professor, Biology Department, Faculty of Sciences, Malayer University, Malayer, Iran.)
Keywords: Gene expression, Antimicrobial active packaging, Antifungal combination, Poly-lactic acid film, Ergoconin,
Abstract :
Background & Objectives: Fungal is one of the most important causes of food spoilage. The use of antimicrobial active packaging increases food safety and shelf life. Hence, this study aimed to produce antifungal compounds (Ergoconin) and its application in the polylactic acid film to reduce the fungal contamination of Aspergillus niger and Penicillium notatum. Materials & Methods: To produce the Ergoconin recombinant protein, the target gene was entered into the pET21 expression vector, and the recombinant vector was transferred to Escherichia coli BL21 (DE3) as host of expression. Gene expression was evaluated by SDS-PAGE, western blot and dot blot. To purify the recombinant protein, a chromatography column was used. Poly-lactic acid bioactive films were prepared by casting method and adding different amounts of Ergoconin (0, 0.25, 0.5, and 0.75%) to 4% wt Poly-lactic acid solution. To investigate the antifungal effect of the poly-lactic acid film, Disc diffusion was used. Results: Ergokonin gene was replicated by specific primers. The expression of recombinant protein was performed by SDS-PAGE and confirmed by dot and western blot. Ergoconin compound had an antifungal effect on both fungi. The inhibition zone diameter for both fungi increased with increasing Ergoconin concentration. The antimicrobial film had a more antifungal effect on Penicillium notatum. Conclusion: Ergoconin destroys fungi by preventing the synthesis of glucan in the wall of fungi. Therefore, Poly-lactic acid film containing Ergoconin can be used as antimicrobial active packaging to increase food safety.
production, applications, nanocomposites, and release studies comper. Rev Food Sci Food Saf.
2010; (9): 552-571.
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nanocomposites. preparation, characterization, and properties. Macromol. 2002; 35(8):
3104-3110.
3. Almasi H, Ghanbarzadeh B, Dehghannia J. Film nanocomposite (acid lactic) poly of properties
nanofibers cellulose modified containing. Poly Sci and Technol. 2014; 6(26): 485-497.
4. Ivana DR, Milan S, Olgica DS, Marina DT, Ljiljana R, Čomic AM. Anti Aspergillus properties
of different extracts from selected plants. Afr J Microbiol. 2011; 5(23): 3986–3990 .
5. Arnoult M, Dargent E, Mano JF. Mobile amorphous phase fragility in semi-crystalline
polymers: Comparison of PET and PLLA. Polymer. 2007; 48: 1012-1019.
6. Salehi Z, Kheirkhah B, Masoumalinejad Z, Zinatizadeh M R. Identification of aflR gene in
aflatoxigenic Aspergillus isolated from pistachio kernel of Sirjan Region using molecular
method. Iran J Med Microbiol. 2018; 12(1): 43-50.
7. Prapagdee B, Kuekulvong Ch, Mongkolsuk S. Antifungal potential of extracellular
metabolites produced by Streptomyces hyroscopicus against phytopathogenic fungi. Int J
Biol Sci. 2008; 4: 330-337.
8. Kezuk Y, Ohishi M, Itoh Y, Watanabe J .Structural studies of a two-domain chitinase from
Streptomyces griseus HUT6037. J HIV Mol Biol. 2006; 358, 472-484.
9. Vicente MF, Cabello A, Platas G, Basilio A, Dõ Âez MT, Dreikorn S. Antimicrobial activity of
ergokonin A from Trichoderma longibrachiatum. J App Microbiol. 2001; 91, 806-813.
10. Shekarfrosh Sh, Azizi Shirazi A, Vali A. The effect of packaging on some of the microbial,
chemical and physical properties of Oncorhynchus mykiss kept in the refrigerator. Fisheries Sci
Technol. 2014; 3(4): 31-42.
11. Salmieri S, Islam F, Khan RA, Hossain FM, Ibrahim HM, Miao C, Hamad WY, Lacroix M.
Antimicrobial nanocomposite films made of poly (lactic acid)–cellulose nanocrystals (PLA–
CNC) in food applications-part B: effect of oregano essential oil release on the inactivation of
Listeria monocytogenes in mixed vegetables. Cellulose. 2014; 21(6): 4271-4285.
12. Plisky S, Perlińska-Lenart U, Górka-Nieć W, Graczyk S, Antosiewicz B, Zembek P,
Palamarczyk G, Kruszewska JS. Overexpression of erg20 gene encoding farnesyl
pyrophosphate synthasehas contrasting effects on activity of enzymes of the dolichyl and
sterolbranches of mevalonate pathway in Trichoderma reesei. Gene. 2014; 544(2):114-122
13. Collins T, Azevedo-silva J, Costa A, Branca F, Machado R, Casal M. Batch production of a silk
-elastin-like protein in E. coli BL21(DE3): key parameters for optimization. Microb Cell Fact.
2013; 1: 12-21.
14. Ausubel FM, Brent R, Kingston R, Moore DD, Seidman JG, Smith JA, Struh L. Current
protocols in molecular biology. New York. 2002; 102-109.
15. Atehortúa A. Cost-effectiveness analysis of diagnosis of duchenne/becker muscular dystrophy
in Colombia. Value Health Reg Issues. 2018;17: 1-6
16. Silveria MF. Active film incorporated with Sorbic acid on pastry dough conservation. Food
Control. 2007; 18: 1063-1067.
17. Kezuk Y, Ohishi M, Itoh Y, Watanabe J. Structural studies of a two-domain chitinase fromStreptomyces griseus HUT6037. HIV Mol Biol. 2006; 358: 472-484.
18. Vicente MF, Cabello A, Platas G, Basilio A, Dõ Âez MT, Dreikorn S. Antimicrobial activity of
ergokonin A from Trichoderma longibrachiatum. App Microbiol. 2001; 91: 806-813.
19. Erdohan ZÖ, Çam B, Turhan KN. Characterization of antimicrobial polylactic acid based films.
J Food Eng. 2013; 119(2): 308-315.
20. Abdolshahi A. Antifungal properties of gelatin-based coating containing mannoprotein from
Saccharomyces cerevisiae on Aspergillus flavus growth in Pistachio. J Mazandaran Univ Med
Sci. 2016; 26(139): 93-102.
21. Toufiq N. Improved antifungal activity of barley derived chitinase I gene that overexpress a
32 kDa recombinant chitinase in Escherichia coli host. Braz J Microbiol .2018; 49:
414-421.
22. Ariannejad H. Cloning, over expression and characterization of alkalin phytase enzyme in
Escherichia Coli. J Agr Biotechnol. 2013; 5: 1-15.
23. Liu H, Naismith JH. A simple and efficient expression and purification system using two
newly constructed vectors. Protein Expr Purif. 2009; 63(2): 102-111.
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production, applications, nanocomposites, and release studies comper. Rev Food Sci Food Saf.
2010; (9): 552-571.
2. Suprakas SR, Pralay M, Masami O, Kazunobu Y, Kazue U. New polylactide/layered silicate
nanocomposites. preparation, characterization, and properties. Macromol. 2002; 35(8):
3104-3110.
3. Almasi H, Ghanbarzadeh B, Dehghannia J. Film nanocomposite (acid lactic) poly of properties
nanofibers cellulose modified containing. Poly Sci and Technol. 2014; 6(26): 485-497.
4. Ivana DR, Milan S, Olgica DS, Marina DT, Ljiljana R, Čomic AM. Anti Aspergillus properties
of different extracts from selected plants. Afr J Microbiol. 2011; 5(23): 3986–3990 .
5. Arnoult M, Dargent E, Mano JF. Mobile amorphous phase fragility in semi-crystalline
polymers: Comparison of PET and PLLA. Polymer. 2007; 48: 1012-1019.
6. Salehi Z, Kheirkhah B, Masoumalinejad Z, Zinatizadeh M R. Identification of aflR gene in
aflatoxigenic Aspergillus isolated from pistachio kernel of Sirjan Region using molecular
method. Iran J Med Microbiol. 2018; 12(1): 43-50.
7. Prapagdee B, Kuekulvong Ch, Mongkolsuk S. Antifungal potential of extracellular
metabolites produced by Streptomyces hyroscopicus against phytopathogenic fungi. Int J
Biol Sci. 2008; 4: 330-337.
8. Kezuk Y, Ohishi M, Itoh Y, Watanabe J .Structural studies of a two-domain chitinase from
Streptomyces griseus HUT6037. J HIV Mol Biol. 2006; 358, 472-484.
9. Vicente MF, Cabello A, Platas G, Basilio A, Dõ Âez MT, Dreikorn S. Antimicrobial activity of
ergokonin A from Trichoderma longibrachiatum. J App Microbiol. 2001; 91, 806-813.
10. Shekarfrosh Sh, Azizi Shirazi A, Vali A. The effect of packaging on some of the microbial,
chemical and physical properties of Oncorhynchus mykiss kept in the refrigerator. Fisheries Sci
Technol. 2014; 3(4): 31-42.
11. Salmieri S, Islam F, Khan RA, Hossain FM, Ibrahim HM, Miao C, Hamad WY, Lacroix M.
Antimicrobial nanocomposite films made of poly (lactic acid)–cellulose nanocrystals (PLA–
CNC) in food applications-part B: effect of oregano essential oil release on the inactivation of
Listeria monocytogenes in mixed vegetables. Cellulose. 2014; 21(6): 4271-4285.
12. Plisky S, Perlińska-Lenart U, Górka-Nieć W, Graczyk S, Antosiewicz B, Zembek P,
Palamarczyk G, Kruszewska JS. Overexpression of erg20 gene encoding farnesyl
pyrophosphate synthasehas contrasting effects on activity of enzymes of the dolichyl and
sterolbranches of mevalonate pathway in Trichoderma reesei. Gene. 2014; 544(2):114-122
13. Collins T, Azevedo-silva J, Costa A, Branca F, Machado R, Casal M. Batch production of a silk
-elastin-like protein in E. coli BL21(DE3): key parameters for optimization. Microb Cell Fact.
2013; 1: 12-21.
14. Ausubel FM, Brent R, Kingston R, Moore DD, Seidman JG, Smith JA, Struh L. Current
protocols in molecular biology. New York. 2002; 102-109.
15. Atehortúa A. Cost-effectiveness analysis of diagnosis of duchenne/becker muscular dystrophy
in Colombia. Value Health Reg Issues. 2018;17: 1-6
16. Silveria MF. Active film incorporated with Sorbic acid on pastry dough conservation. Food
Control. 2007; 18: 1063-1067.
17. Kezuk Y, Ohishi M, Itoh Y, Watanabe J. Structural studies of a two-domain chitinase fromStreptomyces griseus HUT6037. HIV Mol Biol. 2006; 358: 472-484.
18. Vicente MF, Cabello A, Platas G, Basilio A, Dõ Âez MT, Dreikorn S. Antimicrobial activity of
ergokonin A from Trichoderma longibrachiatum. App Microbiol. 2001; 91: 806-813.
19. Erdohan ZÖ, Çam B, Turhan KN. Characterization of antimicrobial polylactic acid based films.
J Food Eng. 2013; 119(2): 308-315.
20. Abdolshahi A. Antifungal properties of gelatin-based coating containing mannoprotein from
Saccharomyces cerevisiae on Aspergillus flavus growth in Pistachio. J Mazandaran Univ Med
Sci. 2016; 26(139): 93-102.
21. Toufiq N. Improved antifungal activity of barley derived chitinase I gene that overexpress a
32 kDa recombinant chitinase in Escherichia coli host. Braz J Microbiol .2018; 49:
414-421.
22. Ariannejad H. Cloning, over expression and characterization of alkalin phytase enzyme in
Escherichia Coli. J Agr Biotechnol. 2013; 5: 1-15.
23. Liu H, Naismith JH. A simple and efficient expression and purification system using two
newly constructed vectors. Protein Expr Purif. 2009; 63(2): 102-111.
