Antimicrobial Activity of Divaricatic Acid Isolated from the Lichen Evernia mesomorpha against Methicillin-Resistant Staphylococcus aureus.

One hundred and seventy seven acetone extracts of lichen and 258 ethyl acetate extracts of cultured lichen-forming fungi (LFF) were screened for antimicrobial activity against Staphylococcus aureus and Enterococcus faecium using a disk diffusion method. Divaricatic acid was isolated from Evernia mesomorpha and identified by LC-MS, ¹H-, 13C- and DEPT-NMR. Purified divaricatic acid was effective against Gram + bacteria, such as Bacillus subtilis, Staphylococcus epidermidis, Streptococcus mutans, and Enterococcus faecium, with the minimum inhibitory concentration (MIC) values ranging from 7.0 to 64.0 µg/mL, whereas vancomycin was effective in the MICs ranging from 0.78 to 25.0 µg/mL. Interestingly, the antibacterial activity of divaricatic acid was higher than vancomycin against S. epidermidis and E. faecium, and divaricatic acid was active against Candida albicans. In addition, divaricatic acid was active as vancomycin against S. aureus (3A048; an MRSA). These results suggested that divaricatic acid is a potential antimicrobial agent for the treatment of MRSA infections.

Improvement of enzyme activity of ß-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues.

ß-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG2-6, BG2-10, and BG5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG5-28 was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6 and BG2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BGwt (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k cat/K m) of BG5-28 were 1.92- and 2.12-fold greater than those of BGwt at 40 °C, respectively. The catalytic efficiency of BG5-28 increased to 3.09-fold that of BGwt at 60 °C. These increases in the thermostability and catalytic efficiency of BG5-28 might be useful for the hydrolysis of ß-glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α6/α6-double-barrel structure is essential for enzyme activity.