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

有机硫化合物存在于化石燃料中，其燃烧会导致严重的环境问题，如酸雨。在该炼油厂，目前正在进行加氢脱硫，以从化石燃料中去除硫化物。然而，脱除多环含硫化合物是很困难的。随着立法对硫排放的限制变得更加严格，从燃料中去除多环硫化合物的必要性变得更加紧迫。二苯并硫(DBT)被认为是化石燃料中含有的一种典型的多环硫化合物。我们已经报道了一些细菌，如红球红球菌D-1，利用DBT作为硫的唯一来源，而不通过硫特有的途径破坏其碳-碳骨架。在该反应中，DBT被DszC氧化成DBT砜，DBT砜被DszA氧化成2-羟基联苯2-亚磺酸(HBPSi)，HBPSi被DszB还原成2-羟基联苯。黄素还原酶是单氧…所必需的DszC和DszA还进行了更多的酶反应。本研究的研究结果总结如下：(1)在以DBT为唯一硫源的培养条件下，从红城红曲霉D-1中分离纯化了黄素还原酶。纯化的黄素还原酶不含生色辅因子，分子量为86 kDa，有4个相同的22 kDa亚基。结果表明：1)依赖NADH还原黄素单核苷酸，NADH和FMN的Km值分别为2 0 8和10 8μM，最适温度和最适pH分别为35℃和35℃；C和6.0)和热稳定性(30%的活性保持在80℃)。C 30分钟)。以红城红曲霉IGTS8的DszD为模板设计引物，扩增出黄素还原酶基因，并在大肠杆菌中高效表达。高效表达菌株粗提液中的比活力约为野生型菌株的275倍。(2)利用Escherick共表达的方法，高效表达了DBT脱硫菌红球红球菌Ka2-5-1的DszB。对重组DszB进行了纯化，鉴定为一种新的酶(HBPSi脱硫酶)，该酶能在不依赖任何其他蛋白质组分和辅酶的情况下，特异性地裂解H2-HBP和亚硫酸盐离子的碳硫键。较少

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