Stable degradation of benzoate by<Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> C302 immobilized in alginate and polyurethane

Benzoate produced from the degradative pathways of various aromatic chemicals is generally recognized as a pollutant compound. However, various bacterial strains isolated as benzoate degraders have exhibited certain limits to their functions, including a loss of viability and degradability when cultivated in a broth medium for a longer time. Accordingly, immobilization techniques have been utilized to overcome such problems, and the current study examined the use of alginate and polyurethane for immobilizingKlebsiella oxytoca C302 to extend its viability and degradability of benzoate. The organism was well encapsulated by both matrices and the immobilized cells showed a high stability as regards their viability and degradability of 2 mM benzoate in a MM2 broth medium during cultivation for longer than 60 h in a semicontinuous batch system.     

Biochemical and structural characterization of the paralogous benzoate CoA ligases from Burkholderia xenovorans LB400: defining the entry point into the novel benzoate oxidation (box) pathway

Xenobiotic aromatic compounds represent one of the most significant classes of environmental pollutants. A novel benzoate oxidation (box) pathway has been identified recently in Burkholderia xenovorans LB400 (referred to simply as LB400) that is capable of assimilating benzoate and intimately tied to the degradation of polychlorinated biphenyls (PCBs). The box pathway in LB400 is present in two paralogous copies (boxM and boxC) and encodes eight enzymes with the first committed step catalyzed by benzoate CoA ligase (BCL). As a first step towards delineating the biochemical role of the box pathway in LB400, we have carried out functional studies of the paralogous BCL enzymes (BCLM and BCLC) with 20 different putative substrates. We have established a structural rationale for the observed substrate specificities on the basis of a 1.84 A crystal structure of BCLM in complex with benzoate. These data show that, while BCLM and BCLC display similar overall substrate specificities, BCLM is significantly more active towards benzoate and 2-aminobenzoate with tighter binding (Km) and a faster reaction rate (Vmax). Despite these clear functional differences, the residues that define the substrate-binding site in BCLM are completely conserved in BCLC, suggesting that second shell residues may play a significant role in substrate recognition and catalysis. Furthermore, comparison of the active site of BCLM with the recently solved structures of 4-chlorobenzoate CoA ligase and 2, 3-dihydroxybenzoate CoA ligase offers additional insight into the molecular features that mediate substrate binding in adenylate-forming enzymes. This study provides the first biochemical characterization of a Box enzyme from LB400 and the first structural characterization of a Box enzyme from any organism, and further substantiates the concept of distinct roles for the two paralogous box pathways in LB400.     

尼泊尔水东哥的化学成分研究

从尼泊尔水东哥树皮的95%乙醇提取物中首次分离到12个化合物,应用波谱方法或与已知品对照的手段鉴定为auranamide(1)、aurantiamide benzoate(2)、齐墩果酸(3)、β-谷甾醇(4)、β-胡萝卜甙(5)、乌苏酸(6)、2α,3α-二羟基-12-烯-28-乌苏酸(7)、2α,3β,24-三羟基-12-烯-28-乌苏酸(8)、(2S,3S,4R,10E)-2-[(2′R)-2′-hydroxytetracosanoylamino]-10-octadecene-1,3,4-triol(9)、2α,3α,24-三羟基-12-烯-28-齐墩果酸(10)、2α,3β-二羟基-12-烯-28-乌苏酸(11)和2α,3α,24-三羟基-12-烯-28-乌苏酸(12)。     

亚高山草甸华灰早熟禾对气候变化的响应

利用亚高山草甸华灰早熟禾生长发育的定位观测资料和相应的气象观测资料,分析了1985—2005年甘肃省玛曲县气候变化对牧草生长发育的影响.结果表明,研究期间,玛曲县降水量的年际变化呈下降趋势,其线性拟合倾向率为-9.895 mm·(10 a)^-1,降水量存在3 a的周期变化;气温的年际变化呈上升趋势,其线性拟合倾向率为0.341 ℃·(10 a) ^-1;华灰早熟禾生长季的干燥指数呈显著上升趋势,其线性拟合倾向率为0.036·(10 a)^-1,20世纪90年代初至2005年明显趋于干旱化.华灰早熟禾返青到籽实成熟约需140～150 d,期间对≥0 ℃积温、降水量、日照时数的要求分别为1000 ℃～1200 ℃、400～450 mm和1000～1100 h;华灰早熟禾在返青后54 d开始由缓慢生长转为迅速生长阶段,返青后第80天的生长速度达最大,返青后104 d开始由迅速生长又转为缓慢生长.受气候变暖的影响,华灰早熟禾抽穗期、开花期、成熟期、黄枯期每10年分别提前15 d、7～8 d、8～9 d、3 d.研究区的气候变化使该区华灰早熟禾产量变幅明显加大,产量的不稳定性增加.     

西花蓟马田间种群对常用杀虫剂的抗性现状及防治对策

【目的】西花蓟马Frankliniella occidentalis在中国是一种严重危害温室蔬菜的入侵害虫。本研究旨在了解该害虫在中国的抗药性现状,为防治该害虫提供理论支持。【方法】采用Munger cell法测定了北京,山东寿光和青岛以及云南晋宁和呈贡等5个地区西花蓟马田间种群对多杀菌素、毒死蜱、阿维菌素、甲维盐、氟氯氰菊酯、溴虫腈、灭多威、吡虫啉和啶虫脒9种杀虫剂的抗药性水平,同时利用这些田间种群测定了多功能氧化酶抑制剂胡椒基丁醚（PBO）、谷胱甘肽S 转移酶抑制剂顺丁烯二酸二乙酯（DEM）和羧酸酯酶抑制剂三丁基三硫磷酸酯（DEF）对多杀菌素、吡虫啉和甲维盐的增效作用。【结果】生物测定结果表明,北京、晋宁及呈贡种群分别对多杀菌素产生了34.45, 47.45和64.45倍的高水平抗性;晋宁种群对灭多威和甲维盐分别产生了16.58和11.03倍的中等水平抗性;呈贡种群对甲维盐、啶虫脒、吡虫啉、阿维菌素、溴虫腈分别产生了24.17, 21.69, 20.05, 16.45和10.31的中等水平抗性;青岛种群对啶虫脒和吡虫啉产生了17.70和12.49倍的中等水平抗性;寿光种群没有对任何杀虫剂产生高等或中等水平抗性。增效剂生物测定结果表明,对于吡虫啉和甲维盐,多功能氧化酶抑制剂PBO在所有田间种群上均有显著的增效作用。谷胱甘肽S-转移酶抑制剂DEM在呈贡、寿光和青岛种群中对吡虫啉存在显著增效作用;在北京、呈贡和寿光种群中,DEM对甲维盐存在显著增效作用。羧酸酯酶抑制剂DEF在呈贡、晋宁和青岛种群中对吡虫啉存在显著增效作用;在北京、呈贡和晋宁种群中,DEF对吡虫啉存在显著增效作用。但所有增效剂在各田间种群中对多杀菌素均无显著增效作用。【结论】结果提示：在使用多杀菌素防治西花蓟马时,应与其他杀虫剂轮换使用;此外,可通过添加酶抑制剂来增强甲维盐和吡虫啉对西花蓟马的防效。     

3种药剂对广东地区草地贪夜蛾3龄幼虫室内毒力测定及其田间药效评价

为明确几种药剂对广东地区草地贪夜蛾Spodoptera frugiperda幼虫的室内毒杀作用及田间防治效果,合理使用化学农药对草地贪夜蛾进行科学防治提供理论依据.本研究采用浸叶法测定了氯虫苯甲酰胺、高效氯氟氰菊酯和甲氨基阿维菌素苯甲酸盐对草地贪夜蛾3龄幼虫的室内毒杀作用;采用茎叶喷雾法,测定了 200克/升氯虫苯甲酰...     

广西回迁粘虫产卵能力及其对11种杀虫剂敏感性测定

为广西粘虫预测预报和科学使用化学农药提供参考依据,本研究于2015年10月上旬田间采集回迁至广西的粘虫,带回室内观察其产卵能力,并采用浸叶法测定粘虫对11种杀虫剂的敏感性。结果表明回迁粘虫的平均蛹历期、产卵前期和产卵期分别为10.30 d、7.13 d和7.50 d,羽化率为52.15%,雌雄虫性比为1∶1.31,单雌平均产卵量为894.81粒,平均卵孵化率为91.49%,雌雄虫平均寿命分别为14.00 d和11.81 d。杀虫剂敏感性测定结果表明,氯虫苯甲酰胺、甲维盐、茚虫威和阿维菌素对粘虫3龄幼虫LC_(50)值分别为0.24、1.16、3.88和5.67 mg/L,在11种杀虫剂中毒力较高。因此,建议在每年10月中下旬可诱捕粘虫成蛾,11月上旬诱卵;粘虫大爆发时可轮换使用氯虫苯甲酰胺、甲维盐、茚虫威和阿维菌素进行防治。     

Purification and Characterization of a Novel Extracellular Lipase Catalyzing Hydrolysis of Oleyl Benzoate from Acinetobacter nov. sp. Strain KM109

A new lipase (OBase) which efficiently hydrolyzes oleyl benzoate (OB) was found in the culture supernatant of Acinetobacter nov. sp. strain KM109, a new isolate growing in a minimum medium containing OB as the sole carbon source. OBase was purified to homogeneity with 213-fold purification and 0.8% yield. The molecular weight was estimated to be 62,000±1,000 by SDS-PAGE under denatured-reduced conditions and to be 50,000±1,000 by gel-filtration HPLC under native conditions; these findings indicate that OBase is a monomeric enzyme. The optimum temperature and pH of OBase were about 45°C and pH 8. Temperature and pH stabilities were at or lower than 35°C and in a range of pH 6-8, respectively. Purified OBase preferentially hydrolyzed p-nitrophenyl benzoate (pNPB) over p-nitrophenyl acetate (pNPA) or p-nitrophenyl caproate (pNPC) [pNPB/pNPA=20 and pNPB/pNPC=5.4], indicating that OBase has a high affinity for benzoyl esters. Partial amino-acid sequences of OBase fragments obtained after lysyl endopeptidase treatment showed no similarity with known proteins.     

HPLC‐PDA‐ORD Bioassay of S‐(+) and R‐(−) Clopidogrel on Rat Dried Blood Spots

A simple and rapid chiral high‐performance liquid chromatography (HPLC) method was developed and validated for bioanalysis of clopidogrel enantiomers on rat dried blood spots (DBS). Clopidogrel enantiomers were extracted from DBS using ethanol: methanol (80:20, v/v) and separated on a Chiralcel OJ‐H column containing cellulose tris (4‐methly benzoate) as a polysaccharide stationary phase using n‐hexane–ethanol‐diethylamine (70:30, 0.1 v/v) as a mobile phase at a flow rate of 1.0 mL/min. The detection was carried out at 220 nm using a photodiode array (PDA) detector while the elution order of the enantiomers was determined by a polarimeter connected to PDA in series. The effect of hematocrit on extraction of clopidogrel enantiomers from DBS was evaluated and no interference from endogenous substances was noticed. The overall accuracy of (R) and (S) enantiomers of clopidogrel from DBS were 91.6 and 89.2%, respectively. The calibration curves were linear over the concentration range of 1–500 µg/mL for both enantiomers. The results show that the method is specific, precise, and reproducible (intra‐ and interday precision relative standard deviations (RSDs) <10.0%). The stability of racemic clopidogrel was performed under all storage conditions and the results were found to be well within the acceptance limits. Chirality 26:102–107, 2014.© 2014 Wiley Periodicals, Inc.