Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1
Chitinases are glycosyl hydrolases, that cleave the β-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls...
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Serbian Chemical Society
2022-01-01
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| Series: | Journal of the Serbian Chemical Society |
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| Online Access: | http://www.doiserbia.nb.rs/img/doi/0352-5139/2022/0352-51392200017M.pdf |
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| author | Menghiu Gheorghita Prodanović Radivoje Blažić Marija Mincea Manuela Moraru Cristina Ostafe Vasile |
| author_facet | Menghiu Gheorghita Prodanović Radivoje Blažić Marija Mincea Manuela Moraru Cristina Ostafe Vasile |
| author_sort | Menghiu Gheorghita |
| collection | DOAJ |
| description | Chitinases are glycosyl hydrolases, that cleave the β-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes – such as chitooligosaccharides with vast importance in the medical and biofuels industry – lead to the necessity of creating chitinases using different strains of organisms. In this paper, the chiA gene from the Bacillus licheniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahistidine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chitinase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression continued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), as a heterogeneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cerevisiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl β-D-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed. |
| first_indexed | 2024-04-13T10:40:23Z |
| format | Article |
| id | doaj.art-8070b066406b4d2fb36fd261b75a3622 |
| institution | Directory Open Access Journal |
| issn | 0352-5139 1820-7421 |
| language | English |
| last_indexed | 2024-04-13T10:40:23Z |
| publishDate | 2022-01-01 |
| publisher | Serbian Chemical Society |
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| series | Journal of the Serbian Chemical Society |
| spelling | doaj.art-8070b066406b4d2fb36fd261b75a36222022-12-22T02:49:56ZengSerbian Chemical SocietyJournal of the Serbian Chemical Society0352-51391820-74212022-01-0187667769210.2298/JSC210913017M0352-51392200017MNon-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1Menghiu Gheorghita0https://orcid.org/0000-0002-6523-5738Prodanović Radivoje1https://orcid.org/0000-0003-4662-1825Blažić Marija2https://orcid.org/0000-0003-0857-1708Mincea Manuela3Moraru Cristina4https://orcid.org/0000-0001-6202-8736Ostafe Vasile5Advanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Timisoara, RomaniaFaculty of Chemistry, University of Belgrade, Belgrade, SerbiaFaculty of Chemistry, University of Belgrade, Belgrade, SerbiaAdvanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Timisoara, RomaniaAdvanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Timisoara, RomaniaAdvanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Timisoara, RomaniaChitinases are glycosyl hydrolases, that cleave the β-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes – such as chitooligosaccharides with vast importance in the medical and biofuels industry – lead to the necessity of creating chitinases using different strains of organisms. In this paper, the chiA gene from the Bacillus licheniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahistidine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chitinase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression continued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), as a heterogeneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cerevisiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl β-D-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed.http://www.doiserbia.nb.rs/img/doi/0352-5139/2022/0352-51392200017M.pdfchitinolytic enzymesmolecular cloningdot blottingfluorescent assayglycosylation |
| spellingShingle | Menghiu Gheorghita Prodanović Radivoje Blažić Marija Mincea Manuela Moraru Cristina Ostafe Vasile Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 Journal of the Serbian Chemical Society chitinolytic enzymes molecular cloning dot blotting fluorescent assay glycosylation |
| title | Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 |
| title_full | Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 |
| title_fullStr | Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 |
| title_full_unstemmed | Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 |
| title_short | Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1 |
| title_sort | non conventional expression of recombinant chitinase a originated from bacillus licheniformis dsm8785 in saccharomyces cerevisiae invsc1 |
| topic | chitinolytic enzymes molecular cloning dot blotting fluorescent assay glycosylation |
| url | http://www.doiserbia.nb.rs/img/doi/0352-5139/2022/0352-51392200017M.pdf |
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