title: Co-immobilization of PEGylated Aspergillus flavipes L-methioninase with glutamate dehydrogenase: a novel catalytically stable anticancer consortium.
abstract: Aspergillus flavipes L-methioninase (AfMETase) exhibits reliable pharmacokinetic properties and anticancer potency in vitro[10]. To maximize its therapeutic efficiency as protection against in vivo proteolysis, reduction of antigenicity and hyperammoniemia, the enzyme was PEGylated and coupled with glutamate dehydrogenase (GDH). The highest degree of PEGylation was measured at 40-50/1 molar ratio of PEG to AfMETase, with a lower mobility on SDS-PGE, compared to the native AfMETase. The activity of free AfMETase was reduced to 66.2% and further to 50% upon PEGylation and GDH conjugation, respectively. The highest degree of surface NH2 modification of AfMETase-GDH co-immobilizates (65%), was reported using 300 mM glutaraldehyde, with 31% methionine conversion. Using L-cysteine and L-methionine as active site protectors, the activity of PEG-AfMETase and PEG-AfMETase-GDH was increased by 14.4 and 32.9-fold, respectively. At 45°C, PEG-AfMETase, PEG-AfMETase-GDH and AfMETase-GDH conjugate have a T1/2 10.3, 8.5 and 7.6 h, inactivation rate (Kr) 0.021, 0.03 and 0.016 min, with 2.0, 1.65 and 1.47-fold stabilization, respectively. Kinetically, the three immobilizates have a relatively similar Km values for L-methionine (7.4-7.9 mM), with lower affinity to homocysteine and cysteine, with stability to PLP-enzyme inhibitors (propargylglycine and hydroxylamine), indicating the protective effect by PEG moieties on the enzyme structure. Also, the three immobilizates exhibited improved stability against proteolysis in vitro, comparing to free AfMETase.
authors.affiliation: Microbiology Department, Faculty of Science, Zagazig University, Egypt. Electronic address: ash.elsayed@gmail.com.
authors.forename: Ashraf S A
authors.initials: AS
authors.lastname: El-Sayed
authors.forename: Hend
authors.initials: H
authors.lastname: Ibrahim
authors.forename: Mahmoud Z
authors.initials: MZ
authors.lastname: Sitohy
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001230:Aspergillus / Q000201:enzymology*; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D020134:Catalytic Domain; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011092:Polyethylene Glycols; D005969:Glutamate Dehydrogenase; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase
keywords: AfMETase; AfMETase–GDH; PEG–AfMETase; PEG–AfMETase–GDH