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›› 2008, Vol. 8 ›› Issue (1): 125-129.

• 生化工程专栏 • 上一篇    下一篇

脱硫菌Rhodococcus sp.LY822专一性脱硫活性及相关基因的研究

白雪晶,熊小超,姜声华,刘会洲,李信   

  1. 中国农业科学院研究生院生物化学与分子生物学实验室
  • 出版日期:2008-02-20 发布日期:2008-02-20

Study on Specific Desulfurization Activity and Related Genes of Rhodococcus sp. LY822

BAI Xue-jing,xiong-xiao-chao,JIANG Sheng-hua,LIU Hui-zhou,LI Xin   

  1. Graduate School of Chinese Academy of Agricultural Sciences
  • Online:2008-02-20 Published:2008-02-20

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摘要： 以专一性脱硫菌Rhodococcus sp. LY822质粒DNA为模板，利用已知的脱硫基因序列设计引物，PCR扩增得到了3个脱硫基因片段dszA, dszB和dszC. 构建了相应的表达质粒pETA, pETB和pETC，在转化大肠杆菌BL21(DE3)中表达，得到了dszA, dszB和dszC基因的融合表达产物. SDS-PAGE分析结果显示，蛋白带分子量分别为50, 40和45 kDa. 3种重组菌BL21(DE3)(pETA), BL21(DE3)(pETB)和BL21(DE3)(pETC)的无细胞粗提液(2 mL)的活性检测结果表明，DszC酶的粗提液反应30 min能够将0.02 mmol/L二苯并噻吩完全转化为二苯并噻吩砜，DszA酶的粗提液能够将0.01 mmol/L二苯并噻吩砜完全转化为羟基联苯亚磺酸盐，DszA和DszB酶的粗提液联合作用能够将0.01 mmol/L二苯并噻吩砜完全转化为2-羟基联苯，证明LY822对二苯并噻吩的降解符合专一性脱硫的"4S途径".

关键词: 二苯并噻吩, 生物脱硫, 脱硫基因, 红球菌

Abstract: Desulfurization related genes of a dibenzothiophene desulfurizing bacterium Rhodococcus sp. LY822 were separately amplified via polymerase chain reaction with specific primers based on the related sequences of Rhodococcus erythropolis IGTS8, and dszA, dszB and dszC were cloned. Three expression plasmids, pETA, pETB and pETC, were constructed and transformed into E. coli BL21 strain. After the isopropyl-beta-d-thiogalactopyranoside induction with them, dszA, dszB and dszC were expressed effectively in the recombinant E. coli BL21 strain. The SDS-PAGE results indicated that the molecular weights of desulfurization related genes expression products were about 50, 40 and 45 kDa. Desulfurization activity analysis showed that BL21(DE3)(pETC) cell-free extracts could convert 0.02 mmol/L DBT into DBTO2. BL21(DE3)(pETA) cell-free extracts could convert 0.01 mmol/L DBTO2 into HBPS. BL21(DE3)(pETA) and BL21(DE3)(pETB) cell-free extracts could convert 0.01 mmol/L DBTO2 into 2-HBP. It could be concluded that Rhodococcus sp. LY822 could specially break the C?S bond of dibenzothiophene and convert dibenzothiophene into 2-hydrobenzophene by "4S" biodesulfurization pathway.

Key words: dibenzothiophene, biodesulfurization, dszABC, Rhodococcus