Characterization and functional improvement of novel enzymes involved in the desulfi dibenzothiophene in microorganisms

微生物脱硫二苯并噻吩新型酶的表征和功能改进

• 批准号: 13650857
• 负责人: IZUMU Yoshikazu
• 金额: $ 2.3万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650857/
• 关键词: dibenzothiophen (DBT); DBT-desulfurizing microorganism; DszB; HBSPiデスルフィナーゼ; HBPSiデスルフィナーゼ; DszC; ジベンゾチオフェン; 脱硫菌; DszA; フラビンレダクターゼ; Rhodococcus erythropolis; 好熱性Bacillus; モノオキシゲナーゼ

Organic sulfur compounds are found in fossil fuels, the combustion of which causes serious environmental problems, such as acid rain. At the refinery, hydrodesulfurization is currently performed to remove sulfur compounds form fossil fuels. However, it is difficult to remove polycyclic sulfur compounds. As legislative limits on sulfur emissions have become tighter, the need to remove polycyclic sulfur compounds form fuel has become more pressing. Dibenzothiophene (DBT) is considered as a model polycyclic sulfur compound contained in fossil fuels. We have reported that some bacteria such as Rhodococcus erythropolis D-1 utilize DBT as a sole source of sulfur without breaking its carbon-carbon backbone through the sulfur-specific pathway. In this pathway, DBT is oxidized to DBT sulfone via DBT sulfoxide by DszC, DBT sulfone is converted to 2-hydroxybiphenyl 2-sulfinic acid (HBPSi) by DszA, and HBPSi is desulfurized to 2-hydroxybiphenyl by DszB. Flavin reductase is necessary for monooxygen … More ase reactions by DszC and DszA.The research results of this study are summarized as follows.(1) We purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22 kDa subunits. The purified enzyme was characterized: 1) NADH-dependent reduction of flavin mononucleotide (FMN), the Km values for NADH and FMN (208 and 10.8 μM. respectively), the optimal temperature and optimal pH (35^。C and 6.0, respectively), and the heat stability (30% activity retaining at 80^。C for 30 minutes). The flavin reductase gene was amplified with primers designed by using a DszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichiacoli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.(2) DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherick coexpression with chaperonin genes, groEL/groES. The recombinant DszB was purified to homogeneity and character was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfurbond of H2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes. Less

有机硫化合物存在于化石燃料中，其燃烧会造成严重的环境问题，如酸雨。在炼油厂，目前进行加氢脱硫以去除化石燃料中的硫化合物。然而，多环硫化合物的去除是困难的。随着法律对硫排放的限制越来越严格，从燃料中去除多环硫化合物的需求变得更加迫切。二苯并噻吩（DBT）被认为是化石燃料中含有的典型多环硫化合物。我们已经报道了一些细菌，如红红红球菌D-1利用DBT作为硫的唯一来源，而不会通过硫特异性途径破坏其碳-碳主链。在该途径中，DBT被DszC通过DBT亚砜氧化为DBT砜，DBT砜被DszA转化为2-羟基联苯2-亚磺酸（HBPSi）， HBPSi被DszB脱硫为2-羟基联苯。黄素还原酶是DszC和DszA进行单氧反应所必需的酶。本研究的研究结果总结如下：(1)在含DBT作为唯一硫源的培养基中，对红杉D-1黄素还原酶进行了纯化和鉴定。纯化的黄素还原酶不含显色辅助因子，分子量为86 kDa，有4个相同的22 kDa亚基。结果表明：1)NADH依赖性还原黄素单核苷酸（FMN）， NADH和FMN的Km值分别为208和10.8 μM。)，最佳温度和最佳pH值(35^。和热稳定性(30%的活性保持在80^。C 30分钟)。利用红血球R. IGTS8的DszD设计引物扩增黄素还原酶基因，并在大肠杆菌中过表达。过表达菌株粗提物的比活性约为野生型菌株的275倍。(2)红红红球菌KA2-5-1与伴侣蛋白基因groEL/groES在Escherick共表达中过量产生DszB。重组产物DszB经纯化后均质性良好，认为是一种新型酶（HBPSi脱硫酶），它可以在不需要其他蛋白质组分和辅酶的帮助下，特异性地裂解H2-HBP和亚硫酸盐离子的碳硫键。少

项目成果

Yoshikazu Izumi: "Purification and characterization of enzymes involved in desulfurization of dibenzothiophene in fossil fuels"Journal of Molecular Catatysis B. Enzymatic. 11,2. 1061-1064 (2001)

Yoshikazu Izumi：“化石燃料中二苯并噻吩脱硫涉及的酶的纯化和表征”分子催化 B. 酶学杂志。

T. Matsubara, T. Ohshiro, Y. Nishina and Y. Izumi: "Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1"Applied and Environmental Microbiology. 67(2). 1179-118

T. Matsubara、T. Ohshiro、Y. Nishina 和 Y. Izumi：“红平红球菌 D-1 参与二苯并噻吩脱硫过程中黄素还原酶的纯化、表征和过表达”应用和环境微生物学。

Yoshikazu Izumi: "A novel enzyme, 2-hydroxybiphenyl-2'-sulfinate desulfinase (DszB) from dibenzothiophene desulfurizing bacterium, Rhodococcus erythropolis KA2-5-1 : Gene overexpression and enzyme characterization"Biochimica et Biophysica Acta. 1598. 122-

Yoshikazu Izumi：“来自二苯并噻吩脱硫细菌红平红球菌 KA2-5-1 的新型酶 2-羟基联苯-2-亚磺酸盐脱硫酶 (DszB)：基因过表达和酶表征”Biochimica et Biophysicala Acta。

Yoshikazu Izumi: "A Novel enzyme, 2-hydroxybiphenyl-2'-sulfinate desulfinase (DszB) from dibenzothiophene desulfurizing bacterium, Rhodococcus erythropolis KA2-5-1: Gene overexpression and enzyme characterization"Biochemica et Biophysica Acta. 1598. 122-1

Yoshikazu Izumi：“来自二苯并噻吩脱硫细菌红平红球菌 KA2-5-1 的新型酶 2-羟基联苯-2-亚磺酸盐脱硫酶 (DszB)：基因过表达和酶表征”Biochemica et Biophysica Acta。

Yoshikazu Izumi: "Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1"Appl. Environ. Microbiol.. 67.1. 1179-1184 (2001)

Yoshikazu Izumi：“红平红球菌 D-1 参与二苯并噻吩脱硫过程中黄素还原酶的纯化、表征和过表达”Appl。