【摘要】 谷胱甘肽S-转移酶Zeta类(GSTZ)是一种重要的多功能酶,与细胞生化代谢、环境净化等密切相关。将拟南芥、甘蓝型油菜品系陕2B与垦C1的GSTZ基因克隆到大肠杆菌-酿酒酵母穿梭表达载体pYES2的多克隆位点,筛选到重组子后,提取重组质粒并将其转入酿酒酵母营养缺陷型菌株INVSc1细胞中,经SC-U培养基选择得到重组酵母Y2At、Y2BnB和Y2BnC。重组酵母在含棉子糖和半乳糖的诱导培养基中,表达出了具有二氯乙酸脱氯活力的谷胱甘肽S-转移酶Zeta类,且主要以可溶状态存在于酵母细胞中。不同碳源比较发现,使用半乳糖为唯一碳源时,与棉子糖和半乳糖共同使用相比,酵母生长虽受到轻微影响,但表达的GSTZ比活力几乎不受任何影响。0~96h诱导时间的优化实验表明,36 h诱导下呈现最高比活力。同时也对不同GSTZ的Km值进行了比较。更多还原

【Abstract】 The zeta class of glutathione S-transferase(GSTZs),which is an important multifunctional enzyme,relates to the cell metabolism and contamination elimination.The GSTZ genes from Arabidopsis thaliana L.and Brassica napus L.cv.Shan 2B and Ken C1 were cloned into the multi-cloning site of the shuttle expression vector pYES2.After the recombinants were obtained,the recombinant plasmids were isolated and introduced into the defective mutant INVSc1 of Saccharomyces cerevisiae.Then the recombinant strains Y2At,Y2BnB and Y2BnC were obtained after cultured on SC-U selective plates.When induced in the medium containing galactose and maltose,the recombinant yeast expressed as active GSTZs showing the dichloroacetic acid dechlorinating activity,which existed in the yeast cell as a soluble state.The comparison of different carbon sources showed that sucrose and glucose significantly exhibited the expression of GSTZ gene;glycerol somewhat affected the growth of yeast but increased the specific activity of GSTZ by 17%;and galactose slightly affected the yeast growth with no affection to the activity of GSTZ.Zero to ninety-six hrs induction experiments showed that specific activity of GSTZ in recombinant yeast reached highest when induced for 36 hours.The specific activity of AtGSTZ,BnGSTZ-B and BnGSTZ-C was 5.3 U/mg,4.3 U/mg and 0.3 U/mg,respectively.The values are lower than that expressed in the E.coli and wheat-sperm cell-free protein synthesis system.However,the relative activity of three sources was similar in E.coli and wheat cell free system.The K_m value of GSTZ genes from different sources was 0.59 mmol/L and 0.79 mmol/L for AtGSTZ and BnGSTZ-B,respectively,suggesting the GSTZ enzyme from Abrabidopsis thaliana has higher affinity to DCA than that from Brassica napus.更多还原