Studies on the structural characterization and antimicrobial activities of Zn-MT-like proteins induced from Chlorella vulgaris

Chlorella vulgaris was cultivated for five days under the stress of 60 μmol/L of Zn²⁺ (ZnCl₂). After harvest by centrifugation, the algal cells were washed with EDTA and water, and were homogenized by a supersonic cell disintegrator. After centrifugation, the supernatant, a crude protein-containing extract, was separated and purified with a gel filtration column (Sephadex G-75, 3.5 i.d.×80 cm) and a desalting gel filtration column (Sephadex G-25, 1.5 i.d. × 30 cm). The obtained Zn-binding proteins were characterized with several analytical methods. Results showed that the molecular weight of the proteins measured with tricine-SDS-PAGE was about 8.2 ku, and the cysteine content of the proteins was 15.4%. In addition, the characteristic ultraviolet spectrum of the proteins was found to be similar to that of the standard MTs from rabbit liver. Therefore, the Zn-binding proteins were referred as metallothionein-like (Zn-MT-like) proteins. Based on the agar hole pervasion method, the activities of anti-bacterial and antifungal of Zn-MT-like proteins were tested. The results showed that Zn-MT-like proteins strongly exhibited the antimicrobial activities against gram-positive bacteria (including Staphylococcus aureus and Bacillus subtilus), Penicillium chrysogenum and Aspergillus niger, but was insensitive to Escherichia coli and brewer’s yeast. The diameters of inhibition zone for 1 mg/mL of Zn-MT-like protein were 13.2, 17.4, 7.2 and 9.8 mm for S. aureus, B. subtilus, P. chysogenum and A. niger, respectively. However, no anti-microbial activity was found for the standard Zn-MTs from rabbit liver. The antimicrobial activities of Zn-MT-like proteins from C. vulgaris may be helpful for the further comprehensive development and utilization of the alga.