An effective immobilized haloalkane dehalogenase DhaA from Rhodococcus rhodochrous by adsorption, crosslink and PEGylation on meso-cellular foam.

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 100 mg/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, 0.588 mM) than DhaA (0.905 mM), 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 5 h, 87.3% in 3 M urea solution for 1 h, 25.9% at pH 3.0, and 51.8% at room temperature for 30 days. Thus, our study was expected to develop an effective immobilized DhaA for practical application.

Characterization of FM2382 from Fulvimarina manganoxydans sp. Nov. 8047 with potential enzymatic decontamination of sulfur mustard.

Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min. The catalytic activity of FM2382 was activated by Co2+ and Mg2+, and inhibited by Zn2+, Cu2+ and Fe3+. Furthermore, site-specific mutagenesis proved that D34, K207 D232, D237 were amino acid residues related to the catalytic activity of SM. In conclusion, we found a thermostable haloacid dehalogenases (HAD) family dehalogenase showing SM-degradation activity, which may be useful for practical application in the future.

PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA.

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.