Production and properties of matallothionein in Cordyceps kyusyuensis under Zn2+ stress / 中草药

ABSTRACT

Objective: To explore the production and properties of matallothionein (MT) in Cordyceps kyusyuensis under the zinc ion (Zn2+) stress. Methods: By adding ZnSO4 to the culture, the production regularity of MT in C. kyusyuensis under Zn2+ stress was explored. Ultrasonic cell disruption, crude extraction, and gel column chromatography were employed to isolate and purify the MT. The methods of Coomassie brilliant blue, electron spray mass spectrometry (ESMS), Ellman's reagent colorimetry, atomic flame absorption spectrometry, silver saturation method with atomic absorption spectrometry analysis, and amino acid analyzer were used to determine the purity, molecular weight, sulfhydryl content, zinc content, MT content, and amino acid composition, respectively. In addition, the anti-oxidative activity was identified by scavenging capacity of hydroxyl radicals (HO·), DPPH radicals (DPPH·), and superoxide anion radicals (O2·̄). Results: The contents of Zn and MT in mycelia increased strikingly with the increase of Zn2+ concentration (0-15 g/L) in the culture medium. In 10 L fermenter, MT achieved the maximum yield up to 14.335 mg/g mycelium (wet weight) with Zn concentration of 15 g/L in 56 h, and the biomass reached 16.921 g/L. The C. kyusyuensis Zn-MT was obtained with Sephadex G-50 and Sephadex G-25 gel filtration, and dried by vacuum freeze-drying. The molecular weight of the protein was 6 750, one molecule Zn-MT binded six zinc atoms and contained 56 molecules of amino acid, which contained 20 molecules of cysteine and one molecule of histidine. The eliminating action of C. kyusyuensis Zn-MT for HO·, DPPH·, and O2·̄ was better than that of glutathione, and the eliminating action was different in O2·̄ > DPPH· > HO·. The concentration of half clearance rate was 71.49, 80.58, and 100.65 mg/L. Conclusion: According to ultraviolet scan pattern analysis, protein column chromatogram analysis, physicochemical properties, and anti-oxidative activity of C. kyusyuensis Zn-MT, it is similar with the characteristics of standard animal MT, and a certain concentration of zinc sulfate liquid fermentation of C. kyusyuensis could induce the synthesis of Zn-MT, which could provide the reliable theoretical basis for the industrial production.