A novel salicylaldehyde dehydrosenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6

A novel salicylaldehyde dehydrogenase involved in catabolism of naphthalene from Pseudomonas putida ND6, NahV, has been identified. NahV exhibited lower identity in amino acid sequence with the classic salicylaldehyde dehydrogenase, NahF, from P. putida ND6. This is the first report of an isofunctional enzyme of bacterial salicylaldehyde dehydrogenase. Comparison of Km and Vmax values of NahV and NahF demonstrated that NahF has a more efficient catalytic reaction than NahV, while NahV has much higher affinity for salicylaldehyde and NAD^＋. Both enzymes exhibited broad substrate specificities and catalyzed the oxidation of salicylaldehyde, 5-chlorosalicylaldehyde, formaldehyde, m-nitrobenzaldehyde, o-nitrobenzaldehyde, o-methoxybenxaldehyde, glutaraldehyde, caprylic aldehyde, and glyoxal. However, the relative rates at which the substituted analogs are transformed differ considerably. NahV activity could be enhanced by Fe^2＋, Cu^2＋ and Zn^2＋; whereas NahF activity could only be stimulated by Fe^2＋, NahF is more stable than NahV at elevated temperatures. Dot-blot hybridization analyses showed that nahF-like genes occurred in all naphthalene-degradation bacteria isolated in this study, whereas nahV-like genes were present in only some naphthalene-degrading bacteria.     

StyS激酶自磷酸化对Pseudomonas putida B3中靛蓝生物合成的调节作用

在恶臭假单胞菌Pseudomonas putida B3中,靛蓝生物合成关键酶基因styAB上游存在一个二元调控系统StyS-StyR。StyS蛋白属于激酶家族,是磷酸化信号转导的重要媒介,但激酶StyS的自磷酸化传导机制对靛蓝生物合成的调节作用尚未探明,因此,研究以Pseudomonas putida B3中激酶蛋白StyS作为研究对象,发现了两个磷酸化结合位点,构建了磷酸化位点突变株。通过分析野生菌株P. putida B3、styS基因缺失菌株P. putida B3-ΔstyS、styS基因回补菌株P. putida B3-ΔstyS-8和磷酸化位点突变株P. putida B3-ΔstyS-1之间的靛蓝产量及酶活发现,P. putida B3产量为10.14mg/L,P. putida B3-ΔstyS-8产量为3.63mg/L,磷酸化位点突变菌株无激酶活性且失去了产生靛蓝的能力。结果表明,StyS自磷酸化作用在靛蓝发酵体系中起重要作用。研究结果将为靛蓝生物合成途径的进一步研究提供参考。     

恶臭假单胞菌由辛酸积累PHA的初步研究

恶臭假单胞菌可以利用辛酸为唯一碳源合成中链聚 3 羟基烷酸 (mclPHA)。在摇瓶条件下 ,对辛酸钠浓度、初始 pH、限制营养因子、通气量对菌体细胞生长和PHA积累影响进行了研究。结果表明 ,对菌体生长而言 ,辛酸钠的最适浓度和最适初始 pH分别为 1.2～ 1.3%和 7.0 ,限制NH 4 可以获得PHA的最大积累量 ,高达细胞干重的 74 %。限制通气量有利于PHA合成     

多孔陶粒固定化微生物效果及扫描电镜观察

采用0.2 mol/L和1.0 mol/L的HCl以及0.2 mol/L和1.0 mol/L的NaOH、铁盐和铝盐,分别对陶粒进行改性,将未处理陶粒和改性陶粒分别在30℃、60 r/min条件下吸附恶臭假单胞菌,通过测定不同时间上清液OD600值来比较它们的吸附效果,并用扫描电镜对吸附效果进行观察.结果表明:低浓度酸处...     

基于BP神经网络和析因设计的土壤中EE2和BPA超声辅助生物降解模型

以碳源添加量、 氮源添加量、 恶臭假单胞菌接种量、 超声时间和降解时间作为输入变量, 以雌激素乙炔基雌二醇（EE2）和双酚A（BPA）的降解率为输出变量, 构建土壤中超声辅助的EE2和BPA降解BP神经网络模型. 利用BP神经网络模型预测雌激素超声辅助降解析因设计的响应值, 并通过析因设计分析影响雌激素微生物降解的主效应及交互作用, 进而求解土壤中EE2和BPA的最优降解条件为10%的碳源添加量、 10%的氮源添加量、 接种量为20 mL、 超声时间为1 min和降解时间为168 h. 结果表明: BP神经网络模型的相关系数为0.952 5和0.983 1, 模拟效率系数（NSC）为0.956 5和0.957 2, 模型具有较准确的预测功能; 碳源添加量×氮源添加量和碳源添加量×恶臭假单胞菌接种量在雌激素降解过程中发挥协同作用, EE2和BPA最大降解率分别为87.13%和69.27%; 分析雌激素的有机碳标化系数 (lg K_oc)及其半衰期的结果表明, EE2和BPA的降解效果与其移动性成正比, 与持久性成反比.     

固定化恶臭假单胞菌细胞生产丙烯酰胺的研究

本文以海藻酸钙包埋法制备固定化恶臭假单胞菌细胞,以分批反应、间歇反应及连续柱式反应转化0.3mol/l 丙烯晴溶液来生产丙烯酰胺.丙烯晴溶液用0.05mol/l tris—HCl 缓冲液调至pH 值8.0,控制温度为5℃.间歇反应在恒温水浴摇床上以100r/min 振荡进行,13h 后丙烯酰胺浓度积累为30%,丙烯晴的转化率在95%以上.连柱柱式反应在带夹套的串联柱式反应器中进行,以每克湿固定化细胞每小时1 ml 的流速输入底物溶液,可有95%以上的丙烯晴被转化.酶柱实测半衰期达1200 h 以上.反应产物经分离提取后得到白色针状晶体,经红外光谱等鉴定,确认它为丙烯酰胺.     

Pseudomonas putida KT2442制备中长链聚羟基烷酸酯

初步探讨了利用Pseudomonas putida KT2442制备中长链聚羟基烷酸酯(MCL-PHAs)的工艺,考察了不同碳源对菌体生长和产物合成的影响.在2.5 L发酵罐上采用高密度细胞培养制备以油酸为碳源,细胞干重达120g/L,MCL-PHAs质量浓度达40g/L,产物质量分数为34%,生产速率为0.965 g·L-1·h-1;以玉米油水解物为碳源,细胞干重达105g/L,MCL-PHAs达28g/L,产物质量分数为26.5%,生产速率为0.667 g·L-1·h-1.以玉米油水解物为碳源所得细胞浓度、产物浓度与以油酸为碳源所得结果比较接近,有利于降低生产成本.     

A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6

A novel salicylaldehyde dehydrogenase involved in catabolism of naphthalene from Pseudomonas putida ND6, NahV, has been identified. NahV exhibited lower identity in amino acid sequence with the classic salicylaldehyde dehydrogenase, NahF, from P. putida ND6. This is the first report of an isofunctional enzyme of bacterial salicylaldehyde dehydrogenase. Comparison of Km and Vmax values of NahV and NahF demonstrated that NahF has a more efficient catalytic reaction than NahV, while NahV has much higher affinity for salicylaldehyde and NAD . Both enzymes exhibited broad substrate speci- ficities and catalyzed the oxidation of salicylaldehyde, 5-chlorosalicylaldehyde, formaldehyde, m-nitrobenzaldehyde, o-nitrobenzaldehyde, o-methoxybenxaldehyde, glutaraldehyde, caprylic aldehyde, and glyoxal. However, the relative rates at which the substituted analogs are transformed differ considerably. NahV activity could be enhanced by Fe2 , Cu2 and Zn2 ; whereas NahF activity could only be stimulated by Fe2 . NahF is more stable than NahV at elevated temperatures. Dot-blot hybridization analyses showed that nahF-like genes occurred in all naphthalene-degradation bacteria isolated in this study, whereas nahV-like genes were present in only some naphthalene-degrading bacteria.     

恶臭假单胞菌萘质粒ⅠNL基因的克隆及酶切图谱

恶臭假单胞菌（ＰｓｅｕｄｏｍｏｎａｓｐｕｔｉｄａＧ７）携带的质粒ＮＡＨ７，经限制性内切酶ＥｃｏＲＩ切割后，产生含有萘降解酶全部基因（２４，６ｋｂ）的片段，此片段插入到载体ｐＵＣ１１９的多克隆位点，构成了ｐＫＡＯ质粒，此质粒经ＨｐａＩ，ＥｃｏＲＩ酶切获得的５．１ｋｂ片段亚屯隆到ｐＵＣ１１９中，衍生出ｐＮＮ１质粒，绘制出内切酶图谱。从ｐＮＮＩ质粒上切下含目的基因ｎａｈＩ、ｎａｈＮ和ｎａｈＬ的ｋｐｎＩ－ｋｐｎＩ片段（２．３ｋｂ）．正向插入载体ＢｌｕｅｓｃｒｉｐｔⅡＳＫ＋中获得重组质粒ｐＮＮ２，反向插入为ｐＮＮ３，将其转化到大肠杆菌ＸＬ１－Ｂｌｕｅ中，目的基因得以大量增殖。