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目的探讨Zn2+胁迫下九州虫草Cordyceps kyusyuensis金属硫蛋白(MT)的产生及性质。方法以培养基中添加ZnSO4的方式,研究九州虫草在Zn2+胁迫下MT的产生规律。通过超声波细胞破碎、提取和凝胶柱色谱法对MT进行分离纯化,利用考马斯亮蓝法进行纯度鉴定。用电喷雾质谱法测定MT的相对分子质量,以Ellman’s方法测定巯基的量,以原子火焰吸收法测定Zn的量,以Ag饱和分析法结合原子吸收光谱(AAS)测定MT的量,用氨基酸自动分析仪对Zn-MT进行氨基酸组成分析。通过对羟基自由基(HO)、DPPH自由基(DPPH)和超氧阴离子自由基(O2)的清除率实验探讨九州虫草MT的抗氧化活性。结果菌丝体中Zn和MT的量随着培养基中Zn2+质量浓度(0~15 g/L)的增加而增加。在10 L发酵罐培养时,培养基中Zn2+质量浓度为15 g/L,培养时间为56 h,金属硫蛋白的量为14.335 mg/g菌丝体(湿质量)。对九州虫草的菌体蛋白通过2次凝胶柱色谱,冷冻干燥得到九州虫草MT,电喷雾质谱法测得其相对分子质量为6 750,每分子Zn-MT氨基酸总量为56个,含有20个半胱氨酸,含极少量的芳香族氨基酸,可结合6个Zn原子。九州虫草Zn-MT对于HO、DPPH、O2有较好的清除作用,清除效果依次为O2>DPPH>HO,其半数清除率质量浓度(IC50)分别为71.49、80.58、100.65 mg/L。结论通过紫外扫描图谱分析、蛋白柱色谱图谱分析、九州虫草Zn-MT理化性质以及抗氧化活性的测定,可以判断其与标准的兔肝MT非常相似,进一步可以确定通过九州虫草菌丝体液态发酵法可生产MT,以期为工业生产提供可靠的理论依据。
Aim To investigate the production and properties of metallothionein (MT) in Cordyceps kyusyuensis strain under Cordyceps kyllosis under Zn2 + stress. Methods The addition of ZnSO4 to the medium was used to study the production of MT in Cordyceps kyushuensis under Zn2 + stress. The MT was isolated and purified by ultrasonic cell disruption, extraction and gel column chromatography, and the purity was identified by Coomassie brilliant blue method. The relative molecular mass of MT was determined by electrospray ionization mass spectrometry. The amount of thiol was determined by Ellman’s method. The amount of Zn was determined by flame atomic absorption spectrometry. The amount of MT was determined by Ag saturation analysis and atomic absorption spectrometry (AAS) Analyzer for Zn-MT amino acid composition analysis. The anti-oxidative activity of Cordyceps militaris MT was studied by scavenging hydroxyl free radical (HO), DPPH free radical (DPPH) and superoxide anion free radical (O2). Results The amount of Zn and MT in mycelia increased with the increase of Zn2 + concentration (0 ~ 15 g / L) in the culture medium. In the 10 L fermenter, the concentration of Zn2 + in the medium was 15 g / L, the incubation time was 56 h, and the amount of metallothionein was 14.335 mg / g mycelia (wet mass). Cordyceps kyushu bacterial protein by 2 gel column chromatography, lyophilized to obtain Kyushu Cordyceps MT, electrospray ionization mass spectrometry measured relative molecular mass of 6 750, the total amount of Zn-MT amino acids per molecule 56, containing 20 A cysteine, with a very small amount of aromatic amino acids, can bind 6 Zn atoms. The Cordyceps kyushuensis Zn-MT had better scavenging activities on HO, DPPH and O2, and the scavenging effects were O2> DPPH> HO. The half-value clearance rates (IC50) were 71.49, 80.58 and 100.65 mg / L, respectively. Conclusion UV-Vis spectroscopy, protein column chromatogram analysis, the physical and chemical properties of Zn-MT of Kyushu Cordyceps and the determination of anti-oxidative activity can be judged to be very similar to the standard rabbit liver MT and further confirmed by the liquid fermentation of Cordyceps kyushuii Law can produce MT, in order to provide a reliable theoretical basis for industrial production.