title: Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support.
abstract: Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-aminocephalosporanic acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the β-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA's oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the β-subunit of CCA from the support.
authors.affiliation: School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China, luohui@ustb.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Luo
authors.forename: Huan
authors.initials: H
authors.lastname: Zhao
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Chang
authors.forename: Qixin
authors.initials: Q
authors.lastname: Wang
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006038:Glyoxylates / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D004800:Enzymes, Immobilized; D006038:Glyoxylates; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C070399:glyoxyl agarose; D011107:Polylysine; C025163:cephalosporin C; D012685:Sepharose; D010399:Penicillin Amidase