甲基对硫磷水解酶的重组表达及其纯化和性质研究

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Escherichia coli BL21(DE3)中,经IPTG诱导表达,得到C末端含有6个寡聚组氨酸的甲基对硫磷水解酶,用NiNTA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pH86～8.8,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;1mmol/L EDTA·Na2+几乎不影响酶的活性,而10mmol/L EDTA·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pH86。25℃时,该酶对甲基对硫磷的米氏常数Km为（68.6 ± 5.1）μmol/L,kcat为（45 ± 6 ）S-1;对乙基对硫磷的米氏常数Km为（59.5 ± 6.0）μmol/L,kcat为（8 ± 1） S-1。Kcat/Km表明甲基对硫磷水解酶对甲基对硫磷的催化效率更高。     

脯氨酰内肽酶培养条件的优化及高密度发酵

基因工程菌E.coliBL21/pGEMPEP可以组成型表达重组的点状产气单胞菌脯氨酰内肽酶(PEP),但培养条件极大地影响着酶的产量,为了获得高效表达,首先测定了工程菌表达PEP的稳定性并考察了培养温度、pH、发酵时间、碳源、氮源、无机盐等对产酶的影响,得到了优化的发酵条件,L9(3⁴)正交试验进一步明确了摇床转速、培养温度、pH值、培养时间对产酶量的影响都有高度的统计学意义。在此基础上利用NBSBioFlo3000型5L自控发酵罐进行了高密度、高表达发酵、经20h培养,最终菌体密度达OD₆₀₀60(相当于干菌体225g/L),PEP表达量为28%,每升发酵液中含PEP酶315g。     

塔拉单宁水解酶产生条件的研究

微生物通过发酵产酶可以将植物单宁降解成小分子酚类化合物或其衍生物,但培养条件对其产酶影响很大。论文采用固态培养法,对黑曲霉产生塔拉单宁水解酶的条件进行了研究。结果表明,当培养液中塔拉单宁浓度为75 g/L、葡萄糖浓度为3 g/L、(NH4)2SO4浓度为0.2 g/L2、50 mL锥形瓶装液量为25 mL、惰性载体用量为5.6%(w/v)、起始pH为5.5、接种量为12%(v/v)、30℃培养72 h时,该黑曲霉产生的塔拉单宁水解酶活力可达到44.29 U/mL,是其自然条件下酶活力(24.09 U/mL)的1.84倍;没食子酸产率达到79.3%。研究结果对于揭示塔拉单宁生物降解的机理具有一定的参考价值。     

甲基对硫磷水解酶参与催化相关结构的研究

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Escherichia coli DH5α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NFDA1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。     

重组大肠杆菌高密度发酵生产RGD-TRAIL的条件优化

本文以一株产RGD-TRAIL的重组大肠杆菌为研究对象,在10L发酵罐中考查了诱导温度、pH值、溶氧、流加葡萄糖对重组大肠杆菌生长和RGD-TRAIL蛋白表达的影响。结果表明:诱导温度25℃,pH值控制7.0,溶氧控制30%,以5g·L~(-1)·h~(-1)流速流加葡萄糖最有利于菌体生长和蛋白表达,菌体收率和RGD-TRAIL产量分别达到45.99g·L~(-1)和160.2mg·L~(-1)。     

甲基对硫磷水解酶的酿酒酵母表面展示及在甲基对硫磷降解中的应用

PCR扩增假单胞菌WBC-3的甲基对硫磷水解酶基因,插入表面展示质粒pYD1的多克隆位点,构建pYD1-MPH重组质粒。重组质粒转化酿酒酵母EBY100,2%半乳糖诱导甲基对硫磷水解酶表达,并利用免疫荧光检测甲基对硫磷水解酶在酿酒酵母细胞表面的表达展示。研究了表面展示甲基对硫磷水解酶的酶学性质和酵母工程菌对水体中甲基对硫磷的降解效果。结果表明成功构建具有全细胞甲基对硫磷水解酶催化活性的酵母工程菌,经2%半乳糖诱导48 h,表面展示甲基对硫磷水解酶比酶活力为18.2 U/mg细胞干重。表面展示甲基对硫磷水解酶的最适作用pH为9.5,最适作用温度为30℃,在p H4.0-10.5之间和45℃以下稳定性较好,Mn2+、Co2+、Zn2+、Ca2+、Hg2+、K+、Ni2+对表面展示甲基对硫磷水解酶活性有激活作用,Na+、Fe3+、Ag+对展示酶活力有抑制作用。工程菌在1 h内对淡水中20 mg/L的甲基对硫磷的降解率在80%以上。     

复合诱变筛选变细胞壁水解酶木霉素株及产酶条件的优化

利用复合诱变技术,筛选到一株产几丁质酶和钱聚糖酶能力较强的绿色木霉菌株（Trichodermaviride)LD-18。并对其产酶条件进行了优化,发现在麸皮：诱导物：麦秸粉＝2：2：7的固体培养基上,以4g/L(NH4)2SO4为氮源,起始pH8.0经25摄氏度培养72h,产生的真菌细胞壁水解酶,用以溶解食用菌,黑曲霉等丝状菌丝体的细胞壁,制备原生质体,效果较优。     

甲基对硫磷水解酶OPHC2的序列分析及结构预测

将氨基酸的多序列局部比对、基于结构的序列比对和氨基酸替换忍耐性分析等方法联合起来对甲基对硫磷水解酶OPHC2进行了序列分析,确定出4组保守性功能序列。根据其高级结构发现这类酶具有典型的α/β（TIM）折叠桶结构,由8个折叠结构构成活性中心区域,并且这4组保守序列都位于这8个折叠结构上。通过对这4组保守序列的进一步分析发现当一个序列中同时含有它们时,这个序列为甲基对硫磷水解酶的可能性大于60％。     

产腈水解酶基因工程菌的产酶诱导条件及培养基优化

本文通过对产酶诱导条件及发酵培养基进行优化,成功提高了产腈水解酶基因工程菌E. coli BL21(DE3)-pETNYNit的产酶水平。研究结果显示,最佳发酵培养基为：葡萄糖0.2%、甘油0.7%(v/v)、蛋白胨1.2%、酵母膏0.8%、NaCl 0.3%、(NH4)2SO40.3%、NH4Cl 0.13%、Na2 HPO4·12H2 O 1.04%、KH2 PO40.39%、MgSO4·7H2 O 0.03%,pH 7.2。最佳产酶诱导条件为：发酵4 h时加入0.5 mmol/L IPTG,然后在28℃、240 r/min下诱导腈水解酶基因表达14 h~16 h。采用优化方案,重组菌产酶水平可提升至0.9~1×105 U,与野生菌株的产酶水平相比,提高幅度超过50%。同时重组菌培养仅需24 h,培养周期缩短超过50 h。     

腈水解酶psn基因工程菌的发酵及产酶条件优化

来自恶臭假单胞菌的腈水解酶具有高效催化3-氰基吡啶产烟酸的能力,对表达该酶的基因psn进行发酵和产酶条件优化,通过对C源、N源、磷酸盐、金属离子、温度、诱导剂浓度和诱导时间进行单因素考察,获得最适培养基条件(g/L):葡萄糖5、蛋白胨15、酵母粉5、(NH4)2SO45、K2HPO424.5、KH2PO45.76、MgSO40.48;最佳诱导条件:培养2.5 h后添加IPTG诱导,浓度0.2 mmol/L,诱导温度30℃。在该条件下培养,重组大肠杆菌的腈水解酶比酶活可达到45.67 U/mL,比优化前提高了2.26倍。在此基础上,于5 L发酵罐上进行C、N源的补料研究,获得最适分批补料策略,发现其腈水解酶活力可达到75.40 U/mL,是优化前的3.74倍。     

A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase

A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.     

Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene

AIMS: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications. METHODS AND RESULTS: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants. CONCLUSIONS: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.     

Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3

Methyl parathion hydrolase (MPH, E.C.3.1.8.1), isolated from the soil-dwelling bacterium Pseudomonas sp. WBC-3, is a Zn(II)-containing enzyme that catalyzes the degradation of the organophosphate pesticide methyl parathion. We have determined the structure of MPH from Pseudomonas sp. WBC-3 to 2.4 angstroms resolution. The enzyme is dimeric and each subunit contains a mixed hybrid binuclear zinc center, in which one of the zinc ions is replaced by cadmium. In both subunits, the more solvent-exposed beta-metal ion is substituted for Cd2+ due to high cadmium concentration in the crystallization condition. Both ions are surrounded by ligands in an octahedral arrangement. The ions are separated by 3.5 angstroms and are coordinated by the amino acid residues His147, His149, Asp151, His152, His234 and His302 and a water molecule. Asp255 and a water molecule serve to bridge the zinc ions together. MPH is homologous with other metallo-beta-lactamases but does not show any similarity to phosphotriesterase that can also catalyze the degradation of methyl parathion with lower rate, despite the lack of sequence homology. Trp179, Phe196 and Phe119 form an aromatic cluster at the entrance of the catalytic center. Replacement of these three amino acids by alanine resulted in a significant increase of K(m) and loss of catalytic activity, indicating that the aromatic cluster has an important role to facilitate affinity of enzyme to the methyl parathion substrates.     

Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates

Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.     

Development of microsatellite markers for Pythium helicoides

Pseudomonas sp. strain WBC-3 utilizes methyl parathion ( O,O -dimethyl O - p -nitrophenol phosphorothioate) or para -nitrophenol as the sole source of carbon, nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had been characterized previously and found to be located on a typical class I composite transposon that comprised IS 6100 (Tn mph ). In this study, the transposability of this transposon was confirmed by transposition assays in two distinct mating-out systems. Tn mph was demonstrated to transpose efficiently in a random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia sp. U2 by sequence analysis of the Tn mph insertion sites, both exhibiting MPH activity. The linkage of the mph -like gene with IS 6100 , together with the transposability of Tn mph , as well as its capability to transpose in other phylogenetically divergent bacterial species, suggest that Tn mph may contribute to the wide distribution of mph -like genes and the adaptation of bacteria to organophosphorus compounds.     

Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology

Aims: To evaluate the influence of environmental parameters on the production of antibiotics (xenocoumacins and nematophin) by Xenorhabdus nematophila and enhance the antibiotic activity. Methods and Results: Response surface methodology (RSM) was employed to study the effects of five parameters (the initial pH, medium volume in flask, rotary speed, temperature and inoculation volume) on the production of antibiotics in flask cultures by X. nematophila YL001. A 2^5?1‐factorial central composite design was chosen to explain the combined effects of the five parameters and to design a minimum number of experiments. The experimental results and software‐predicted values of production of antibiotics were comparable. The statistical analysis of the results showed that, in the range studied, medium volume in flask, rotary speed, temperature and inoculation volume had a significant effect (P < 0·05) on the production of antibiotics at their individual level, medium volume in flask and rotary speed showed a significant influence at interactive level and were most significant at individual level. The maximum antibiotic activity was achieved at the initial pH 7·64, medium volume in 250 ml flask 25 ml, rotary speed of 220 rev min^?1, temperature 27·8°C and inoculation volume of 15·0%. Maximum antibiotic activity of 331·7 U ml^?1 was achieved under the optimized condition. Conclusions: As far as known, there are no reports of production of antibiotic from X. nematophila by engineering the condition of fermentation using RSM. The results strongly support the use of RSM for fermentation condition optimization. The optimization of the environmental parameters resulted not only in a 43·4% higher antibiotic activity than unoptimized conditions but also in a reduced amount of the experiments. The chosen method of optimization of fermentation condition was efficient, relatively simple and time and material saving. Significance and Impact of the Study: This study should contribute towards improving the antibiotics activity of X. nematophila. Integrated into a broader study of the impact of environmental factors on the production of antibiotic, this work should help to build more rational control strategy, possibly involving scale‐up of production of antibiotics by X. nematophila.     

Cross-linked enzyme crystals of organophosphate hydrolase for electrochemical detection of organophosphorus compounds

Cross-linked enzyme crystals of organophosphate hydrolase (CLEC-OPH) prepared from crude recombinant E. coli cell lysate was used for the development of an electrochemical biosensor for the detection of organophosphate pesticides. CLEC-OPH showed an increased V _max of 0.721 U mg protein⁻¹ and a slightly lower K _m of 0.083 mM on paraoxon compared to the crude enzyme, resulting in an improved catalytic efficiency (k _cat/K _m = 4.17 × 10⁵ M⁻¹ min⁻¹) with a remarkable increase on thermostability. An amperometric biosensor was constructed based on glutaraldehyde and albumin cross-linkage of CLEC-OPH with carbon nanotubes. The sensor exhibited greater sensitivity and operational stability with a lower limit of detection when compared with a sensor using an equivalent loading of crude OPH in a non-crystal form. The application of crude enzyme-based CLEC would offer a simple and economical approach for the fabrication of efficient electrochemical biosensors.     

海藻糖生产过程中产酶发酵条件的研究

研究了产酶的培养基组分和比例以及最佳培养条件对微球菌生产麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)的影响,得到最优培养基组成为：葡萄糖2．0％,酵母膏2．0％,蛋白胨1．0％,磷酸氢二钾0．1％,硫酸镁0．05％;优化后的培养条件为：以15％的接种量接种至250mL的锥形瓶中,装液量为50mL,初始pH值7．5～8．5,培养温度为30℃,摇床培养4d。经优化后菌体干重由原来的1．938g／L增加到18．5g／L,生物量几乎增长了10倍;而酶活也由原来的30．64U／g增加到206．11U／g,酶活提高了接近7倍。     

Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter

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.