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A thermostable and organic solvent-tolerant bacterial laccase from Bacillus pumilus ARA has been expressed heterologously and characterized, which shows potential decolorization capacity to various types of industrial synthetic dyes. The optimal temperature and pH were 85 °C and 3.5, respectively, while the purified recombinant laccase B.P.Lacc was stable under 55-75 °C and pH 5.0-8.0 conditions. The apparent kinetic parameters Km and Vmax of B.P.Lacc for ABTS as the substrate were 0.33 mM and 32.4 U/mg, respectively. Ethanol (1%, v/v) and methanol (2%, v/v) could stimulate the enzyme activity. The recombinant laccase retained over 95% of its initial activity in 10% (v/v) methanol. The optimal expression conditions for the laccase production of B.P.Lacc in LB medium were obtained: induction temperature of 25 °C, 0.4 mM Cu2+, and 1.0 mM IPTG added into the culture. After 5 h, the final laccase production was 1283 U/mL. Moreover, the laccase activity increased to 4822 U/mL after follow-up 2 h stationary cultivation, with about a 3.76-fold increase. The purified B.P.Lacc was able to efficiently decolorize synthetic dyes combined with mediators. Adding 1.0 mM ABTS, more than 90% of BRRB was decolorized by the enzyme, whether at pH 4.0 or pH 7.9. The outstanding enzymatic properties suggested that B.P.Lacc may be suitable for a wide application in future biodegradation fields.

Berberine (BBR), a well-known alkaloid, exhibits various pharmacological activities, especially hypolipidemic activity, which has attracted much interest from medicinal chemists in the past decade. However, little progress was made on the structural modification of BBR for improving lipid-lowering activity, mainly due to its unclear biological target and low safety. In this study, a new scaffold of 7,9-disulfatetrahydroberberine was discovered unexpectedly, provided with extremely low cytotoxicity. Hence, a novel series of highly safe 7,9-disulfatetrahydroberberines were designed, synthesized, and evaluated for their hypolipidemic activities. In order to investigate the significance of the 9-position substituent, another new series of 7-sulfatetrahydroberberines were designed and synthesized. Lipid-lowering experiments showed that among these compounds, 5f exhibited the best lipid-lowering activity based on two cell models, 3T3-L1 cells and HepG2 cells. Compared with the blank control, the inhibition rate of compound 5f against total cholesterol was over 60%, the inhibition rate against triglyceride was over 70%, the inhibition rate against low-density lipoprotein cholesterol was approximately 75%, and the inhibition rate against high-density lipoprotein cholesterol was close to 50%, which were far superior to the positive control BBR. This result also verified the feasibility of the development of BBR as a lipid-lowering drug via disubstituted modification at the 7- and 9-position.

The development of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is an ongoing and challenging research field. However, the dynamic motion of the binding site of EGFR has not been accurately depicted, hindering the improvement of EGFR TKI. For this reason, about 33 protein complexes (32 EGFR proteins plus 1 ErbB4 protein) were carefully curated and subsequently studied for dynamic movements of their binding sites by molecular dynamics simulations in this study. The analysis of root mean square deviation (RMSD) revealed that T790M mutation can make an impact on dynamic motion of binding sites; the RMSD value of the EGFR binding site was unrelated to inhibitory activity. The analysis of the radius of gyration (Rg) revealed that T790M can slightly shrink the value of Rg, thereby influencing the shape of the EGFR binding site. More interestingly, the Rg value can exhibit weak correlation with inhibitory activity of most inhibitors. The relationship between Rg and biological activity deserve our serious interest since the best scoring function, Xscore, cannot distinguish highly active EGFR inhibitors. The root mean square fluctuation (RMSF) analysis of key residues derived from binding sites indicated that the most flexible residue was ASP800 with a large RMSF value against the steady residue ALA743 with a small RMSF value, and two other residues (MET793 and LEU844) were supposed to be involved with molecular recognition. In short, the obtained results would be more effective for guiding the development of a novel EGFR kinase inhibitor.