Pseudomonas sp.L19抗除草剂AHAS基因的克隆、表达及特性

乙酰羟酸合酶(AHAS)是磺酰脲类、咪唑啉酮类、三唑嘧啶类、磺酰胺类和嘧啶水杨酸类等乙酰羟酸合酶抑制剂类除草剂的作用靶标,获得能抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值.从长期使用甲磺隆的农田土壤中分离到1株对乙酰羟酸合酶抑制剂类除草剂有广谱抗性的菌株L19,根据表型特征、生理生化特性和16S rDNA序列系统发育分析,将其鉴定为假单孢菌属(Pseudomonas sp.).利用PCR技术从Pseudomonas sp.L19的总DNA中克隆AHAS基因,氨基酸序列比对结果表明,L19与对除草剂敏感菌株P.putida KT2440的AHAS调节亚基氨基酸序列完全相同,而催化亚基有4个氨基酸位点不同:[Val 29→Ala(L19→KT2440),Pro93→Ser,Val 345→Ala,Pro 484→Arg].分别将菌株L19与KT2440的AHAS催化亚基克隆到pET29a(+)的多克隆位点,构建了表达载体pET-L19AHASc和pET-KT2440AHASc,通过镍柱亲和层析纯化得到带有组氨酸标签的乙酰羟酸合酶.抗性试验结果表明菌株L19的乙酰羟酸合酶对四大类乙酰羟酸合酶抑制剂类除草剂的抗性均要强于KT2440的乙酰羟酸合酶,对甲磺隆、咪唑乙烟酸、唑嘧磺草胺和嘧草醚的抗性倍数分别达到53.6、9.3、8.2和9.5倍.菌株L19的乙酰羟酸合酶比活力、对ThDP和Mg2+的Kc值相应地比KT2440乙酰羟酸合酶的要低;而对底物丙酮酸钠的米氏常数Km值要比KT2440乙酰羟酸合酶的要高近1倍.     

秸秆与氮肥配施对辽西旱区土壤酶活性与土壤养分的影响

通过田间试验研究了玉米（Zea mays L.）秸秆与氮肥配施对耕层土壤酶活性及土壤养分的影响。试验设4个秸秆还田量水平,2个施氮量水平。结果表明：在秸秆配施氮肥条件下,耕层土壤中性磷酸酶、脲酶、转化酶和过氧化氢酶活性以及有机质和全氮质量分数均表现为随着玉米秸秆还田量的增加而提高,而硝态氮（3NO-N）和铵态氮（＋4NH-N）质量分数则表现为随着玉米秸秆还田量的增加而减少,4种土壤酶活性与土壤有机质和全氮质量分数均呈显著正相关,与土壤硝态氮和铵态氮质量分数则呈显著负相关。玉米秸秆还田量9 000 kg.hm-2配施氮肥量420 kg.hm-2是辽西风沙半干旱区效果较好的技术措施。     

生物膜系统中部分反硝化实现特性

以移动床生物膜反应器（moving-bed biofilm reactor,MBBR）为例,考察生物膜系统中部分反硝化NO₂^--N积累特性,并通过耦合厌氧氨氧化验证生物膜系统中部分反硝化耦合厌氧氨氧化（partial denitrification with anaerobic ammonium oxidation,PD+ANAMMOX）工艺的可行性.结果表明,在C/N为3.0,填充率为20%的条件下,经过40 d的富集培养,实现部分反硝化,NO₂^--N积累率达（69.38±3.53）%;接种生物膜NO₃^--N还原酶（nitrate reductase,NAR）活性为0.03 μmol·（min·mg）^-1,NO₂^--N还原酶（nitrite reductase,NIR）活性为0.18 μmol·（min·mg）^-1,富集培养后生物膜NAR活性增至0.45 μmol·（min·mg）^-1,NIR活性降至0.02 μmol·（min·mg）^-1,从酶学角度验证了部分反硝化实现;高通量测序结果显示,Thauera属从0.3%增加至37.27%,在微生物群落中占主导地位,该菌属被认为是部分反硝化过程的主要功能菌.随后与厌氧氨氧化耦合,出水总氮达（6.41±1.50） mg·L^-1,总氮去除率达（88.16±2.71）%,证明了生物膜系统中PD+ANAMMOX的可行性及稳定性.     

六价铬还原细菌Bacillus cereus S5.4还原机理及酶学性质研究

从宝钢电镀污泥中分离得到1株六价铬还原细菌Bacillus cereus S5.4,在液体LB培养基中培养72　h完全还原2　mmol/L Cr⁶⁺.测定该菌株六价铬还原后细胞内外六价铬和总铬浓度,检测细胞各组分六价铬还原能力,并结合扫描电镜分析六价铬还原前后细胞形态的变化.结果表明,细菌的细胞壁膜能阻止六价铬进入细胞,是六价铬发生还原的主要场所,其通透性的改变将影响六价铬还原酶的作用;该菌株六价铬还原酶为非分泌型,在细菌细胞内侧发生作用.测定六价铬还原酶活性和稳定性：其最适温度范围25～37℃,最适pH 7,Cu2＋有增强六价铬还原酶活性的作用;在37℃,该菌株六价铬还原酶K_m为125.61 μmol/L,V_max为7.68 nmol/(min·mg).     

秦岭中段撂荒地植被恢复过程中土壤微生物代谢特征

明确撂荒地植被恢复过程中土壤微生物养分限制特征及其驱动机制对揭示土壤养分循环和维持生态系统稳定具有重要意义.以秦岭中段不同年限撂荒地为研究对象,通过测定土壤理化性质、5种胞外酶活性[β-1,4-葡萄糖苷酶(BG)、纤维二糖水解酶(CBH)、β-1,4-N-乙酰基氨基葡萄糖苷酶(NAG)、亮氨酸氨基肽酶(LAP)和酸性磷...     

Measurement of the biodegradation of starch-based materials by enzymatic methods and composting

The aim of this study was to evaluate the suitability of in vitro enzymatic methods for assaying the biodegradability of new starch-based biopolymers. The materials studied included commercial starch-based materials and thermoplastic starch films prepared by extrusion from glycerol and native potato starch, native barley starch, or crosslinked amylomaize starch. Enzymatic hydrolysis was performed using excessBacillus licheniformis -amylase andAspergillus niger glucoamylase at 37°C and 80°C. The degree of degradation was determined by measuring the dissolved carbohydrates and the weight loss of the samples. Biodegradation was also determined by incubating the samples in a compost environment and measuring the weight loss after composting. The results indicated that the enzymatic method is a rapid means of obtaining preliminary information about the biodegradability of starch-based materials. Other methods are needed to investigate more accurately the extent of biodegradability, especially in the case of complex materials in which starch is blended with other polymers.     

The Role of a Salt Marsh Plant on Trace Metal Bioavailability in Sediments. Estimation By Different Chemical Approaches * (7 pp)

Goal, Scope and Background The presence or absence of vegetation can condition sediment characteristics. The main aim of this work was to investigate the influence of the sea rush Juncus maritimus on metal (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) availability to organisms living on or in estuarine sediments, from Douro River (NW Portugal), by comparing the characteristics and chemical behaviour of rhizosediments (collected within the plant assemblage) and those of sediment (collected around the plant). In order to evaluate whether and how sediment characteristics condition the role of plants on metal availability, sandy and muddy sediments colonised by J. maritimus were studied in parallel. Methods Metal availability was estimated by enzymatic digestion with pepsin (ED), which may provide an estimate of metal availability to organisms living at estuarine sediments. Nevertheless, since no consensus exists yet on the most suitable methodologies to estimate metal bioavailability in sediments, two more conventional approaches, BCR sequential extraction (SE) and AVS/SEM model, were also used, in parallel, and the information these approaches provided was compared with that provided by ED. Total-recoverable metal contents were determined by atomic absorption spectrophotometry after sediment digestion using a high-pressure microwave system. Results and Discussion Plants could concentrate metals around its roots and rhizomes. In addition, they were capable of oxidizing (release of oxygen by the roots) the anaerobic medium surrounding their roots in muddy sediment (reducing AVS). As sulphide oxidation renders metals (Cd, Cu, Ni, Pb and Zn) into more soluble forms, according to the AVS/SEM model, metals from muddy sites would be more available in rhizosediment than in sediment. The SE approach led to a similar conclusion. Nevertheless, the results provided by ED pointed at opposite conclusion, particularly for Cd and Zn, indicating less availability at rhizosediments than in the surrounding sediment. ED results were interpreted as a consequence of an enrichment of the rhizosediment in organic ligands exuded by the roots or liberated by dead plants. The effect of complexation of metals by organic compounds, which ED could not decompose/dissolve, seemed to overcome that caused by sediment oxidation. In general, a comparison of the information about metal availability by ED, SE, AVS/SEM, showed that it did not always match and in few cases it was even contradictory. Conclusion and Outlook Therefore, a thorough evaluation of the metal availability in sediments requires a combination of different chemical approaches, so as to take into consideration differences in ways of organism exposure (interstitial water and/or ingestion of sediment particles). - * The basis of this peer-reviewed paper is a presentation at the 9th FECS Conference on 'Chemistry and Environment', 29 August to 1 September 2004, Bordeaux, France.     

硫循环的酶促反应机制及湖泊环境效应

在湖泊沉积物表层存在着强烈的生物作用 ,使SO2 -4 转变成S2 -,然后和Fe2 + 结合而固定在沉积物中 ,这种SO2 -4 的清除机制具有重要的环境意义。本文论述了H2 S的产生和各种形态硫转变成SO2 -4 的酶促反应机制 ,并阐述了不同微生物电子传递载体、最终电子受体和电子进入传递链的部位的差异以及生成ATP的数目和环境效应的差异。     

Enzymatic hydrolysis of banana stems (Musa acuminata): Optimization of process parameters and inhibition characterization

In current work, an optimum solid loading (solid: liquid = 1:20), pH (4.8), temperature (50°C), and enzyme dosing of 20 filter paper unit (amount of enzyme required to release 1 µmol of glucose as reducing sugar from filter paper in per mL per minute) were enumerated for enzymatic hydrolysis of banana stem using cellulase from Trichoderma reesei. Further, inhibition study on enzymatic hydrolysis of banana stem was investigated by the supplementation of monosaccharides (glucose, galactose, mannose, xylose, and arabinose), disaccharide (cellobiose), and inhibitors (acetic acid and furfural obtained from pre-enzymatic hydrolysis steps). Glucose and cellobiose showed inhibitory effect on enzymatic hydrolysis of pretreated banana stem at or above 8 g/L while galactose, mannose, and xylose showed a significant inhibitory effect at or above 4 g/L. Instead of inhibition, arabinose enhanced the enzymatic hydrolysis with increase in total reducing sugars. Acetic acid did not show any significant inhibition while furfural inhibited the system at a comparative low concentration of 2 g/L. Further, scanning electron microscopy analysis was performed to investigate the difference in ultra-structural morphology of raw biomass, pretreated biomass, and biomass obtained after enzymatic hydrolysis.     

膨化预处理玉米秸秆的还原糖酶解工艺

应用膨化技术对玉米秸秆进行预处理.在单因素实验基础上,以还原糖转化率为影响值设计正交实验,研究温度、pH、液固比、酶浓度及酶解时间五因素对纤维素酶解过程的影响.得出玉米秸秆糖化酶解最佳工艺条件为:温度48℃,pH 4.5,液固比8:1,酶浓度36.0 U/g,酶解时间25 h；在此工艺条件下还原糖转化率达到28.98％.扫描电镜表征对比观察可看出,膨化后玉米秸秆纤维素酶解充分；结合红外光谱对各组分特征基团分析表明,膨化后的玉米秸秆酶解的纤维素基团特征峰变化更为明显.