N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

假单胞菌WBC—3甲基对硫磷水解酶性质的初步研究

从最近分离到的有机磷农药降解菌Pseudomonas sp．WBC—3中获得了甲基对硫磷水解酶（Methyl parathion hydrolase,MPH,EC3．1．8．3)。该酶在48h的培养物中分布比例分别为：上清液2．1％,胞内86．2％和胞间质11．7％,说明MPH为胞内酶。经过CM—sepharose Fast Flow阳离子交换层析,获得电泳纯的酶。SDS—PAGE和凝胶过滤层析表明,该酶为单体蛋白,分子量约为34kD。动力学分析显示该酶为非特异性有机磷降解酶,但最适底物为甲基对硫磷。在pH9～12范围,酶表现较高活力水平,最高活力的反应温度为40℃。根据各类金属离子和鳌合剂对酶活的影响,推测MPH为金属酶。     

黄鳝碱性磷酸酶的分离纯化及其部分性质研究

经Tris-HCl缓冲液(pH8.6)抽提,正丁醇处理,30%-75%硫酸铵分级沉淀分离,DEAE-Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从黄鳝内脏组织中分离纯化出电泳纯的碱性磷酸酶。该酶提纯倍数为564倍,比活力达到3015U/mg。酶学性质和动力学性质研究表明,该酶催化磷酸苯二钠的水解反应,最适pH值为10.2,pH小于7和大于12均不稳定;最适温度为40℃,温度高于50℃不稳定;米氏常数Km值为1.17mmo1/L。金属离子对该酶的催化活力有不同的影响,K+对该酶活力无影响,Mg2+对该酶有激活作用,Zn2+对该酶有抑制作用。       

工程菌酯酶B1的纯化及性质研究

工程菌酯酶B1降解酶经硫酸铵分级沉淀、DEAE SepharoseCL 6B离子交换柱层析、SephadexG 1 5 0凝胶过滤 ,得到了分离纯化 ,SDS PAGE鉴定为单一组分。经 1 2 .5 %SDS PAGE法测得分子量约为 5 6KD ,提纯倍数为33.3,收率为 1 7.9%,比活力为 74 .7U/mg。该酶的最佳作用条件是 37℃ ,pH =7.0 ,该酶作用于α 乙酸萘酯的Km为1 .35× 1 0 -3 mM ,Vmax为 2 .7× 1 0 4μ/ml。酶在pH6～ 9范围内较稳定 ,重金属Cu2 对该酶具有明显的促进作用 ,而SDS对酶具有抑制作用 ,对有机磷农药对硫磷 (1 6 0 5 )的降解率在 90min内达 91 .5 %。     

毛霉脂肪酶的研究

从土样中分离筛选到一株脂肪酶菌株—毛霉(Mucor sp.)M₂,其优化后的培养基组成(%);黄豆粉4.0、蔗糖0.5、橄榄油1.0、硫酸铵0.1、磷酸氢二钾0.2硫酸镤0.01、pH自然。产酶最适条件：初始pH6.5、培养温度28℃、培养周期96h.该酶最适作用温度50℃、最适pH8.0、pH稳定范围为7.0～10.0,Fe²⁺、Ca²⁺、Mg²⁺、K⁺对酶有激活作用。     

芽孢杆菌WS-3L淀粉酶的纯化和特性的研究

芽孢杆菌WS-3L产生的α-淀粉酶AmyL经过多步纯化,酶的回收率为15.5%,比活提高了345倍.该淀粉酶能够有效水解淀粉生成麦芽寡糖.酶的最适反应温度为45℃,最适反应pH为6.5,在pH 7.0～8.0,40℃以下酶活较稳定;离子Cu2 、NH4 、Ag 、Hg 和EDTA、SDS对酶活力有显著抑制作用,而其他一些常见金属离子如Na 、K 则对酶活影响不大.AmyL对可溶性淀粉、直链淀粉、支链淀粉的Km值和Vmax分别为2.81 mg/mL、8.37 mg/mL、1.80 mg/mL和11.67 μmol/(min·mL)、10.00μmol/(min·mL)、13.33 μmol/(min·mL),表明支链淀粉是该酶的理想水解底物.玉米淀粉对AmyL有很高的吸附率,预示这可以作为酶快速固定的一个简易方法应用到实际生产中.     

Personalised Mechanical Properties of Scoliotic Vertebrae Determined In Vivo Using Tomodensitometry

This in vivo study investigated the mechanical properties of apical scoliotic vertebrae using computed tomography (CT) and finite element (FE) meshing. CT examination was performed on seven scoliotic girls. FE meshing of each vertebral body allowed automatic mapping of the CT scan and the visualisation of the bone density distribution. Centroids and mass centres were compared to analyse the mechanical properties distribution. Compared to the centroid, the mass centre migrated into the concavity of the curvature. The three vertebrae of a same patient had the same body migration behaviour because they were located at the curvature apex. This observation was verified in the coronal plane, but not in the sagittal plane. These results represent new data over few geometrical analyses of scoliotic vertebrae. Same in vivo personalisation of mechanical properties should be performed on intervertebral discs or ligaments to personalise stiffness properties of the spine for the biomechanical modelling of human torso. Moreover, do this mechanical deformation of scoliotic vertebrae, that appears before the vertebral wedging, could be a predictive tool in scoliosis treatment?     

Complex rhamnolipid mixture characterization and its influence on DPPC bilayer organization

Rhamnolipids (RL) are one of the most important classes of biosurfactants produced by microorganisms using a wide range of carbon sources, from a simple carbon source like glucose to complex wastes such as the used cooking oils used in this work. The objective of this work was to learn about the rhamnolipid–phospholipid dipalmitoyl phosphatidyl choline (DPPC) molecular interactions through the behaviour observed in the neat products and four RL/DPPC mixtures. Size and z-potential were used to characterize the size and the charge of the vesicles, and small angle X-ray scattering (SAXS) was used to measure the vesicle bilayer characteristics, and the release of carboxyfluorescein to study the bilayer disrupting effect promoted by rhamnolipids. The results show that rhamnolipids are disposed in ordered bilayers with long repeating distances, which are stabilized by the charging of the bilayer and also by a strong fluidity of the bilayers. The ability of rhamnolipids to increase the fluidity of DPPC bilayers may be related with the strong haemolytic power of these molecules.     

Dispersal,establishment and survival of Ceriops tagal propagules in a north Australian mangrove forest

Studies of the ecology of mangroves show that a wide variety of factors, including salinity, desiccation, disturbance, competition and predation, may affect the distribution and abundance of species. Field studies were done to examine the relative importance of several of these factors in the establishment and early survival of Ceriops tagal, a species common in mid-to high-shore regions of mangrove forests in northern Australia. The fate of marked and tethered propagules was followed to estimate the range of dispersal and the intensity of predation. Propagules were artificially planted under different thicknesses of shade cloth (none, 30%, 80%) and in different habitats (clearing, forest, clearing-forest fringe) to examine the effects of light and soil conditions on survival and growth. Results suggested that dispersal was very limited: only 9% of marked propagules were ever found more than 3 m from the parent tree. Losses to predators were great, with 83% of tethered propagules being damaged or consumed within 3 months. On average, 56% of planted propagules survived for at least 6 weeks and 76% of these initiated growth. Survival in clearings was lower than in other habitats, with 29% fewer surviving six weeks and 48% fewer surviving 15 months. The growth of seedlings was correlated with soil temperature, but the effects of treatments were complex. Overall, results indicated that poor dispersal and establishment were the main factors likely to limit the colonisation and population growth of this species. Received: 12 September 1995/Accepted: 14 June 1996     

Isolation and characterization of microcin E 492 fromKlebsiella pneumoniae

The production of a dialysable peptidic antibacterial named microcin E492 by the strain of faecal originKlebsiella pneumoniae RYC492 has previously been reported. In this paper, a procedure to extract this antibiotic from liquid cultures of the producer strain is described. This method was based in the quantitative retention of the microcin on the hydrophobic matrix Bondapak C18 and led to highly active pigment- and salf-free concentrates appropriate for further purification by high pressure liquid chromatography. The characterization of purified preparations indicated that microcin E492 was a basic and hydrophobic peptide with an apparent molecular mass of about 5,000, acid- and heat-resistant and much more active in minimal than in rich medium. These properties are discussed with regard to the likely ecological role of the microcin in the microbial ecosystem of the intestine.Abbreviations AU Antibiotic Unit - CFU Colony-forming units - HPLC High Pressure Liquid Chromatography - Mr Relative molecular mass - RP Reversed phase - TEAP Triethylamine-phosphoric acid