Purification and characterization of metallothionein in Cordyceps hawkesii mycelia

Abstract:

[Objective] To produce and characterize metallothionein in Cordyceps hawkesii mycelia under the zinc ion stress. [Methods] C. hawkesii achieved maximum mycelia of 12.2 g/L in a fermenter as Zn2+ up to 18 g/L. Pure C. hawkesii mycelia Zn-metallothionein was obtained with Sephadex G-50 and desalting Sephadex G-25 gel filtration, dried by freeze-drying. The methods of bradford and silver saturation with atomic absorption spectrometry analysis were used to separately determine the protein content and MT content. The methods of Ellman’s reagent colorimetry and atomic flame absorption spectrometry were used to determine the sulfhydryl content and the number of zinc atoms, respectively. The molecular weight of C. hawkesii mycelia Zn-metallothionein was determined by electron spray mass spectrometry (ESMS). Automatic amino acid analyzer measured amino acid composition. The antioxidant activity was identified by scavenging capacity of hydroxyl radicals, DPPH radicals and superoxide anion radicals. [Results] The Zn-metallothionein reached 15.3 mg/g mycelium (wet weight) after 64 h of fermentation. The molecular weight of Zn-metallothionein was 7 680 Da. One molecule Zn-metallothionein contained 18 mercapto groups and combined 4 zinc atoms. Amino acid composition analysis shows that each molecule of the protein contained 60 molecules of amino acids, which contained 15 molecules of cysteine, and contained three molecules of histidine and one molecule of aromatic amino acid. The antioxidant activity was slightly stronger than glutathione and weaker than mammal metallothionein. [Conclusion] C. hawkesii can synthesize Zn-metallothionein under Zn2+ stress and the structure was similar to mammalian metallothioneins.