title: Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.
abstract: Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. jianhui18686@163.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Jian
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. wyw@jlu.edu.cn.
authors.forename: Yingwu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. by0330@126.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. ryong_li@163.com.
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. gaorj@jlu.edu.cn.
authors.forename: Renjun
authors.initials: R
authors.lastname: Gao
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D019149:Bioreactors; D002384:Catalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005285:Fermentation; D005998:Glycine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D012822:Silicon Dioxide / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D000447:Aldehydes; D004800:Enzymes, Immobilized; C509969:SBA-15; C032736:N-formylglycine; D012822:Silicon Dioxide; D006867:Hydrolases; D005998:Glycine
keywords: Sulfolobus tokodaii; dehalogenase; formylglycine-generating enzyme; immobilization