title: Immobilization on macroporous resin makes E. coli RutB a robust catalyst for production of (-) Vince lactam.
abstract: A novel (+) γ-lactamase gene (rutB) was cloned from Escherichia coli JM109 and expressed in E. coli BL21 (DE3), and the recombinant protein was characterized. The optimal conditions for the enzyme were pH 7.0 and temperature 30 °C, which indicated that it was a mesophilic protein. The free purified enzyme was deactivated when incubated at 50 °C for 30 min. However, the k cat value of RutB at its optimal temperature was about 2.5 times that of the archaeal enzyme from Sulfolobus sofataricus at its optimal temperature (85 °C). After immobilization on macroporous resin using glutaraldehyde cross-linkage, the thermostability of the crude enzyme was greatly enhanced and the deactivating temperature was raised to 70 °C. After immobilization, the minimal substrate inhibition concentration for RutB also improved from 0.75 to 1.5 M. The optimal concentrations of immobilized enzyme and substrate were determined to be 250 mg/ml and 1.5 M, when the initial reaction velocity was the response variable in batch transformations. This immobilization of RutB on macroporous resins provides another feasible approach for the preparation of optically active Vince lactam. As a member of the isochorismatase superfamily, RutB was demonstrated to be another typical γ-lactamase that showed catalytic promiscuity.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No 1 Beichen West Road, Chaoyang, Beijing, 100101, People's Republic of China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.forename: Junge
authors.initials: J
authors.lastname: Zhu
authors.forename: Sheng
authors.initials: S
authors.lastname: Wu
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007769:Lactams / Q000378:metabolism*; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D007769:Lactams; D006867:Hydrolases; C064012:isochorismatase; D000581:Amidohydrolases; C401943:gamma-lactamase