The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identiWed. To further conWrm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was puriWed to homogeneity by metal-aYnity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the puriWed recombinant MPH indicated that a signal peptide of the Wrst 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the puriWed mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel Wltration of the puriWed mature recombinant MPH revealed that the MPH was a monomer.

The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.

分离到一株假单胞菌（Pseudomonassp.）P3，该菌能够以对硝基苯酚为唯一碳源和氮源进行生长。在有外加氮源的条件下，P3降解对硝基苯酚并在培养液中积累亚硝酸根。P3有比较广泛的底物适应性，对多种芳香族化合物都有降解能力。不同金属离子对P3降解对硝基苯酚有不同的作用。葡萄糖的存在对P3降解对硝基苯酚无明显促进作用，而微量酵母粉可以大大促进P3对硝基苯酚的降解。以P3为受体菌，通过接合转移的手段将甲基对硫磷水解酶基因mpd克隆至P3菌中，获得了表达甲基对硫磷水解酶活性的基因工程菌PM，PM能够以甲基对硫磷为唯一碳源进行生长。工程菌PM具有较高的甲基对硫磷降解活性及稳定性。

A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli.

用PCR方法获得甲基对硫磷水解酶编码基因，构建了重组表达质粒pET29a-mpd，将其转化至EscherichiacoliBL21（DE3）中，经IPTG诱导表达，得到C2末端含有6个寡聚组氨酸的甲基对硫磷水解酶，用Ni2NTA亲和层析纯化得到具有活性的甲基对硫磷水解酶。测定了环境因素对酶活性的影响及酶动力学参数。甲基对硫磷水解酶水解甲基对硫磷时，最适pH8.6～8.8，最佳反应温度15℃；Mn2+、Zn2+、Cu2+可使酶活性增加15%～20%，Ca2+、Mg2+微弱地促进酶的作用，Ni2+对酶活性几乎无影响；1mmolPLEDTA•Na2+几乎不影响酶的活性，而10mmolPLEDTA•Na2+对甲基对硫磷水解酶有较强的抑制作用。甲基对硫磷水解酶水解乙基对硫磷时，最适pH816。25℃时，该酶对甲基对硫磷的米氏常数Km为（68.6±511）μmolPL，kcat为（45±6）S-1；对乙基对硫磷的米氏常数Km为（59.5±6.0）μmolPL，kcat为（8±1）S-1。kcatPKm表明甲基对硫磷水解酶对甲基对硫磷的催化效率更高。

Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host’s chromosome.

α-L-Rhamnosidases play important roles in the metabolism of plant cell walls, glycosides and bacterial biofilms. This enzyme is also used industrially for debittering citrus fruits by releasing rhamnose from the plant flavonoid naringin. Bacillus sp. GL1 α-l-rhamnosidase (RhaB) is a member of glycoside hydrolase (GH) family 78. Native and selenomethionine-derivative enzymes were crystallized at 293 K by hanging-drop vapour diffusion with polyethylene glycol 8000 as a precipitant. This is the first report of the crystallization of a family GH78 enzyme.

以短短小芽孢杆菌B15的总DNA为模板，利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列，测序分析后提交GenBank，登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHI和PstI酶切位点，将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK，插入片段置于该载体中mpd基因的上游，并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751（pP15MK），在短短小芽孢杆菌启动子和信号肽元件的带动下，mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达，表达产物结合在细胞膜上；发酵液在48h酶活达到最高值7.79U/mL，是出发菌株邻单胞菌M6表达量的8.1倍。

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.

甲基对硫磷水解酶（MPH）是一种新的有机磷水解酶。将完整的甲基对硫磷水解酶基因（mpd）构建入pUC19载体，使得mpd基因以自身的启动子在EscherichiacoliDH5中表达并得到了纯化。金属螯合实验发现MPH的活性不受金属螯合剂1，10菲啉的影响；但用电感耦合等离子发射光谱测定其金属含量显示MPH是金属酶，1mol酶中结合了2mol的Zn2+为确定参与MPH催化活性的必需氨基酸，用化学修饰剂碳化二亚胺、二乙基焦磷酸酯、磷酸吡哆醛和丁二酮处理MPH，然后检测其残余酶活力，结果表明天冬氨酸、谷氨酸、赖氨酸和精氨酸残基与酶的催化活性无关；而二乙基焦磷酸酯对组氨酸侧链的化学修饰引起酶活性的大幅度的下降，其对酶活性的抑制率达到9.6h-1，说明组氨酸是酶活力所必需的基团。这些结果为进一步研究酶的结构及对酶进行分子改造提供了必要的基础数据。