摘要
【目的】异硫代氰酸盐(Isothiocyanates,ITCs)具有抗癌和植物杀虫剂的作用,为解决天然硫苷酶提取复杂、费用昂贵等问题,【方法】本研究采用RT-PCR法扩增硫苷酶的关键基因MYR1,通过构建硫苷酶表达载体,并在毕赤酵母中进行体外诱导表达,以获得外源硫苷酶。将从榨菜叶中提取的总RNA,通过Sal I酶切,插入p PIC9K-S表达载体构建重组子。在YPD培养基中菌体悬浮培养至OD600为1左右,更换BMMY培养基进行诱导表达72 h。【结果】提取菌液进行SDS-PAGE电泳检测分析,发现表达蛋白的分子量为90 KDa左右,经诱导表达,Ni柱纯化后,得到纯度为85%的硫苷酶融合蛋白,产率约850 U/L。硫苷酶融合蛋白以sinigrin为底物,运用HPLC测得蛋白的比活力约等于0.003 U/μl。【结论】采用在毕赤酵母中进行体外诱导表达,以获得外源硫苷酶,对硫苷酶活性最佳条件进行筛选和确认,为扩增和签定硫苷酶活性等技术提供重要参考。
[ Objective] Isothiocyanates （ITCs） have potential as anti-cancer drugs and plant insecticides. The present paper aims to improve the expensive and challenging extraction of naturally occurring myrosinase. [ Method] MYR1 encoding myrosinase in tumorous stem mustard was amplified using RT-PCR, cloned into the pPIC9K-S expression vector, and recombinantly expressed in Pichia pastoris. Mycelia were cultured in YPD medium and expression was induced by replacing with BMMY medium for 72 hours. [ Result] SDS-PAGE revealed a protein of 90 kDa, and myrosinase was isolated using immobilized metal affinity chromatography to a purity of 85 % and an overall yield of 850 U/ l. With sinigrin as substrate, the specific activity of the myrosinase fusion protein was 0. 003 U/μl as measured by HPLC. [ Conclusion] Cloned into the pPIC9K-S expression vector, and recombinantly expressed in Pichia pastoris, using isothiocyanates from tumorous stem mustard as standards, enzymatic conditions were investigated to identify the optimum. This study can be provide important references for amplifying and signing enzyme activity of thioglycoside.
出处
《西南农业学报》
CSCD
北大核心
2017年第4期744-749,共6页
Southwest China Journal of Agricultural Sciences
基金
重庆市教委项目"茎瘤芥(榨菜)中硫苷生成异硫氰酸酯酶解条件优化及抗肿瘤的体外试验研究"(KJ1601223)
