Zheng_2019_Int.J.Biol.Macromol_125_1016

Haloalkane dehalogenase DhaA catalyzes the hydrolysis of halogenated compounds by cleavage of the carbon-halogen bond. However, DhaA suffers from poor environmental stability and difficult recovery, which significantly increase the cost of DhaA. Here, an effective enzyme immobilization strategy was developed to overcome the disadvantages of DhaA. DhaA was physically absorbed with amine-functionalized meso-cellular foam (MCF). The MCF-absorbed DhaA (MD) was intermolecularly crosslinked with 8-arm PEG Nhydroxysuccinimide ester and then PEGylated by maleimide-thiol chemistry. DhaA from Rhodococcus rhodochrous was absorbed at a loading capacity of 100mg/g in MD. The bulk crystallinity and morphology of MCF were largely maintained. The immobilized DhaA (MD-P1-P2) showed a lower Michaelis constant (Km, 0.588mM) than DhaA (0.905mM), along with an extremely low leaching ratio of DhaA (1.1%) from MCF. MD-P1-P2 exhibited a high stability in the extreme environmental conditions, as reflected by the remaining activity of 99.8% in 40% (v/v) DMSO for 5h, 87.3% in 3M urea solution for 1h, 25.9% at pH3.0, and 51.8% at room temperature for 30days. Thus, our study was expected to develop an effective immobilized DhaA for practical application.