Improving Degradation of Polycyclic Aromatic Hydrocarbons by Bacillus atrophaeus Laccase Fused with Vitreoscilla Hemoglobin and a Novel Strong Promoter Replacement

SIMPLE SUMMARY: Polycyclic aromatic hydrocarbons (PAHs) are a large group of persistent organic pollutant chemicals that have significant carcinogenic effects on humans and animals. Degradation of PAHs by microbial laccase is a low-cost and high-efficiency pipeline, but the catalytic activity and expression level for most of the native bacterial laccases are low, limiting their wide use in industry. A recombinant enzyme called LacH5 showed high-efficiency PAH degradation activity, and its degradation activity was further improved by fusion expression and the expression level was augmented by a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for PAH degradation and the development of a new candidate laccase for PAH biodegradation. ABSTRACT: Laccases catalyze a variety of electron-rich substrates by reducing O(2) to H(2)O, with O(2) playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, lach5, was identified from Bacillus atrophaeus through sequence-based screening. LacH5 was engineered for modification by fusion expression and promoter replacement. Results showed that the purified enzyme LacH5 exhibited strong oxidative activity towards 2,2’-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) under optimum pH and temperature conditions (pH 5.0, 60 °C) and displayed remarkable thermostability. The activity of the two fusion enzymes was enhanced significantly from 14.2 U/mg (LacH5) to 22.5 U/mg (LacH5-vgb) and 18.6 U/mg (Vgb-lacH5) toward ABTS after LacH5 fusing with Vitreoscilla hemoglobin (VHb). Three of six tested polycyclic aromatic hydrocarbons (PAHs) were significantly oxidized by two fusion laccases as compared with LacH5. More importantly, the expression level of LacH5 and fusion protein LacH5-vgb was augmented by 3.7-fold and 7.0-fold, respectively, by using a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for improving the activity and expression level of bacterial laccases, and these strategies can be extended to other laccases and multicopper oxidases.