Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain
Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84...
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| Format: | Article |
| Language: | English |
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Serbian Chemical Society
2020-01-01
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| Series: | Journal of the Serbian Chemical Society |
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| Online Access: | http://www.doiserbia.nb.rs/img/doi/0352-5139/2020/0352-51391900058B.pdf |
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| author | Balaž Ana Marija J. Blažić Marija B. Popović Nikolina Prodanović Olivera L. Ostafe Raluca V. Fischer Rainer Prodanović Radivoje M. |
| author_facet | Balaž Ana Marija J. Blažić Marija B. Popović Nikolina Prodanović Olivera L. Ostafe Raluca V. Fischer Rainer Prodanović Radivoje M. |
| author_sort | Balaž Ana Marija J. |
| collection | DOAJ |
| description | Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZα plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON172049, Grant no. ON173017 and Grant no. III46010] |
| first_indexed | 2024-12-11T14:35:45Z |
| format | Article |
| id | doaj.art-7223b2c76da444f6b9aeddd16b64c4a8 |
| institution | Directory Open Access Journal |
| issn | 0352-5139 1820-7421 |
| language | English |
| last_indexed | 2024-12-11T14:35:45Z |
| publishDate | 2020-01-01 |
| publisher | Serbian Chemical Society |
| record_format | Article |
| series | Journal of the Serbian Chemical Society |
| spelling | doaj.art-7223b2c76da444f6b9aeddd16b64c4a82022-12-22T01:02:12ZengSerbian Chemical SocietyJournal of the Serbian Chemical Society0352-51391820-74212020-01-01851253510.2298/JSC190320058B0352-51391900058BExpression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strainBalaž Ana Marija J.0Blažić Marija B.1Popović Nikolina2Prodanović Olivera L.3Ostafe Raluca V.4Fischer Rainer5Prodanović Radivoje M.6Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, SerbiaInstitute of Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, SerbiaFaculty of Chemistry, University of Belgrade, Belgrade, SerbiaInstitute for Multidisciplinary Studies, University of Belgrade, Belgrade, SerbiaMolecular Evolution Protein Engineering and Production facility (MEPEP), Purdue University, West Lafayette, IN, USAIndiana Bioscience Research Institute, Single Cell Analytics Center, Indianapolis, IN, USAFaculty of Chemistry, University of Belgrade, Belgrade, SerbiaProduction of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZα plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON172049, Grant no. ON173017 and Grant no. III46010]http://www.doiserbia.nb.rs/img/doi/0352-5139/2020/0352-51391900058B.pdfmutant proteinsyeastprotein purificationkinetic characterization |
| spellingShingle | Balaž Ana Marija J. Blažić Marija B. Popović Nikolina Prodanović Olivera L. Ostafe Raluca V. Fischer Rainer Prodanović Radivoje M. Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain Journal of the Serbian Chemical Society mutant proteins yeast protein purification kinetic characterization |
| title | Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain |
| title_full | Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain |
| title_fullStr | Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain |
| title_full_unstemmed | Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain |
| title_short | Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain |
| title_sort | expression purification and characterization of cellobiose dehydrogenase mutants from phanerochaete chrysosporium in pichia pastoris km71h strain |
| topic | mutant proteins yeast protein purification kinetic characterization |
| url | http://www.doiserbia.nb.rs/img/doi/0352-5139/2020/0352-51391900058B.pdf |
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