title: Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio.
abstract: d-Amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H2O2during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H2O2from one catalytic site to the other, enabling efficient decomposition of the generated H2O2. The reduced cofactors of the DAAO subunits were reoxidized by the evolved molecular oxygen around. Kinetics analysis showed that the d-alanine substrate follows Michaelis-Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4-fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3 ± 2.7%. The encapsulated DAAO&CAT has retained 98.1 ± 3.1% and 94.9 ± 2.9% of the activity of free DAAO&CAT at 30 and 40 °C, respectively.
authors.affiliation: Department of Biochemical Engineering, Beijing University of Chemical Technology , Beijing, 100029, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Du
authors.affiliation: Department of Biochemical Engineering, Beijing University of Chemical Technology , Beijing, 100029, China.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Biochemical Engineering, Beijing University of Chemical Technology , Beijing, 100029, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Biochemical Engineering, Beijing University of Chemical Technology , Beijing, 100029, China.
authors.forename: Wei
authors.identifier: 0000-0002-7046-6469
authors.initials: W
authors.lastname: Feng
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D002374:Catalase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D019154:Protein Splicing; D021122:Protein Subunits
chemical_list: D010455:Peptides; D021122:Protein Subunits; D006861:Hydrogen Peroxide; D002374:Catalase; D000594:Amino Acid Oxidoreductases