摘要
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。
Objective To explore the production and properties ofmatallothionein （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 （HOo）, DPPH radicals （DPpH·）, and superoxide anion radicals （O · ）. 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 cysteineand one molecule of histidine. The eliminating action of C. kyusyuensis Zn-MT for HOo, DPPH·, and O· was better than that of glutathione, and the eliminating action was different in O · 〉 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 simi
出处
《中草药》
CAS
CSCD
北大核心
2013年第21期2997-3003,共7页
Chinese Traditional and Herbal Drugs
基金
山东省自然科学基金联合专项资助(ZR2010CL005)
烟台市科技发展计划项目资助(2011069)
关键词
九州虫草
金属硫蛋白
分离纯化
氨基酸组成
抗氧化活性
Cordyceps kyusyuensis （Fr.） Link
matallothionein
separation and purification
amino acid composition
anti-oxidative activity
