Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Water Concentration and Activity Effects on Aminoacylase in Aqueous/Organic One-Liquid-Phase Systems

Aminoacylase has been employed as a model system to study its catalytic properties at low water concentrations/water activities with different water-miscible organic cosolvents. Cosolvents assayed were alcohols and polyols with pure logarithm of the partition coefficient (log P) values, on the standard water/octanol system, ranging between -5.2 and 0.24.

Experimental hydrolysis equilibrium constants (K_app), at a constant water concentration, decreased with the fall in log P of the cosolvent, as well as with reduction of the water concentration/water activity, as would be expected. The enzyme hydrolytic and synthetic activities, measured at a constant water concentration/water activity value, followed a sigmoidal dependence on log P of the cosolvent employed when the water concentration or water activity values were lower than 50% (w/w) or 0.66, respectively. This became a hyperbolic relationship at higher water concentration/water activity values. A linear relationship between the logarithm of the limiting water activity necessary to maintain enzyme activity and log P was obtained. Both hydrolytic and synthetic activities were suppressed for water activities higher than 0.66 and cosolvents with log P lower than -1.6.     

水在有机介质酶催化反应中的作用

本文针对有机介质酶催化反应中水的作用,结合最新研究进展作了综合评述。指出水在有机介质中对维持酶的活力构象起到“润滑剂”作用,水活度是衡量水作用的有效参数。分析了酶的水化程度、水化方式、有机溶剂及固定化载体对酶活力的影响及其作用机理,并对反应过程中水的控制问题进行了讨论。     

Modification of the Microenvironment of Enzymes in Organic Solvents. Substitution of Water by Polar Solvents

Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.     

有机相酶促酯化反应中水分调控技术的研究

在有机相酶促反应中,水含量是影响酶活力的关键因素.对异辛烷/正辛醇体系中柱状假丝酵母脂肪酶催化萘普森酯化反应中的水分调控技术进行了研究. 结果表明：水合盐对——Na₂SO₄·10H₂O/Na₂SO₄对系统水分的变化具有有效的缓冲作用;非极性硅藻土吸附固定酶,使之对水分的敏感性得到缓解;另外,加入分子筛去除副产物——“水”可促进酯化过程的进行.     

The water-dependence of the catalytic activity of bilirubin oxidase suspensions in low-water systems.

We investigated the enzymic activity of bilirubin oxidase when it is suspended as a lyophilized powder in a low-water system. The enzyme required buffer salts and a source of water to show activity. This study investigated the complete range of water thermodynamic activity (a(w)) by combining the use of salt hydrates and two-phase systems with concentrated solutes in the aqueous phase. When free water was added, activity reached a maximum at a defined water content, but this maximum increased with buffer content, suggesting that there was competition for water with the buffer salts from which the enzyme was lyophilized. Alternatively, a range of salt hydrates was used, each able to fix the water activity (a(w)) at a different value. By providing water to the organic solvent phase in this way, the dependency of enzyme activity upon a(w) was investigated and shown to be independent of buffer concentration. However, the optimum a(w) was uncertain because the available a(w) range for salt hydrates is < or = 0.90. Investigation of the remaining water activity range was made possible by using an a(w) depressor (sorbitol) to lower the a(w) of a two-phase system. The optimum a(w) for the bilirubin oxidase activity in this two-phase system was a(w) = 0.936, independent of buffer concentration. The study therefore confirmed the need to control the water 'available' to low-water systems and the dependence of enzyme activity on water thermodynamic activity (a(w)) not water content.     

Algal Biomass as Potential Biosorbent for Reduction of Organic Load in Gray Water and Subsequent Reuse: Effect on Seed Germination and Enzyme Activity

Biosorptive treatment using algal biomass of Rhizoclonium riparium was proposed for higher-loading gray water for its effective utilization. A batch sorption study was conducted using composite wastewater having a wide range of initial chemical oxygen demand (COD) values (2400–44,800 mg/L). The study showed an optimum dose of 5 g/L of biosorbent resulted in 96% reduction of COD for wastewater with an initial COD of 10,500 mg/L. The equilibrium isotherm data at different temperatures were fitted to linear and nonlinear isotherms. Biosorption kinetics was studied by various kinetic models. Chemical composition and surface morphology of the biosorbent were characterized by infrared, x-ray diffraction, and scanning electron microscopy techniques before and after biosorption. Applicability of the biosorbent-treated wastewater in agricultural uses was explored by studying the effect of untreated and treated wastewater on the germination of two seeds, i.e., Vigna radiata and Lens esculenta, and subsequently the activity of peroxidase (POD) enzymes were studied to understand the toxicity in plants. The study revealed that compared with seeds treated with different dilutions of untreated wastewater, germination (%) was significantly higher for biosorbent-treated wastewater, with a reduced level of POD activity, indicating positive response of the seeds towards the biosorptive treatment.     

Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic (liquid/solid) media

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (a_w = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high a_w (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration. The mass of the enzyme preparation was shown to be a parameter affecting the capacity of the lipase to produce enough water in its immediate environment. The lack of activity observed for a small quantity of enzyme was eliminated by addition of heat-denaturated lipase.     

The Influence of Organic Matter and pH on DDT Aqueous Solubility

We have studied the concentrations of DDT in ground water samples at field locations with DDT-polluted topsoil and concentrations and solubility in samples prepared from deionized water with different types and concentration of organic acids. The solubility of DDT increased with increasing concentration of humic acid when the pH of the samples was low (adjusted to about 5.5). The effect flutters in the humic acid concentration range from 200 to 300?mg/L, in accordance with humic acid hydrophobicity, operationally measured as liquid surface tension. The findings correspond to trends previously reported in the literature. The trend of increasing solubility was not found using fulvic acid or low-molecular-weight aliphatic acids. No trend was found adding humic acid without adjusting the pH. The mechanism of enhanced solubility due to humic compounds can explain relatively high levels of DDT in ground water. The ground water samples, however, had a moderately high concentration of maximum 6?µg/L compared with a maximum of about 2300?µg/L in the water samples with humic acid in pure water.     

圆柏属常绿木本植物叶片水分、渗透调节物质的季节变化与抗冷冻性的关系

以祁连圆柏（Sabina przewalskii）和圆柏（Sabina chinensis）为材料,研究了圆柏属植物的抗冷冻性与叶片水分和渗透调节物质季节变化的关系。结果表明,随秋季气温下降,叶片组织相对含水量（RWC）和自由水含量（FWC）下降,束缚水含量（BWC）、脯氨酸（Pro）、游离氨基酸（FAA）、可溶性蛋白质（SP）和可溶性糖（SS）含量增加,翌年春季气温回升,相对含水量和自由水含量增加,束缚水含量下降,有机渗透调节物再次增加。有机渗透调节物在秋末冬初及初春的增加正好与圆柏属植物抗冻锻炼及脱冻恢复生长的时间相吻合,因而是植物抗冻及脱冻适应的重要生理响应,在降低细胞冰点、防止细胞结冰引起的膜机械伤害、抑制膜脂过氧化、保护膜稳定性方面具有重要作用。祁连圆柏的相对含水量和自由水含量均低于圆柏,3种有机渗透调节物含量均高于圆柏,表明祁连圆柏在抗冷冻性诱导中具有更广泛的适应性策略。     

四棱豆(Psophocarpus tetragonolobus)酰脲相对丰度与固氮酶活力的关系

四棱豆根瘤固氮酶活力日变化呈双峰曲线,两个峰分别出现在14:30和20:00;其固氮酶活力日变化与叶片酰脲含量变化的相关系数为0.67,且与根、茎、叶的酰脲相对丰度(URA)相关系数分别为0.59,0.61,0.76。在个体发育过程中,根瘤固氮酶活力与叶片酰脲含量以及酰脲相对丰度之间呈极显著相关。贮存在30℃条件下5 h不影响植物材料的酰脲相对丰度。四棱豆叶片酰脲相对丰度可作为估价根瘤固氮酶活力的一个指标。     

Use of Fenton's Reagent for the Degradation of TCE in Aqueous Systems and Soil Slurries

Fenton's reaction is comprised of hydrogen peroxide (H₂O₂) catalyzed by iron, producing the hydroxyl radical (·OH), a strong oxidant. ·OH in turn may react with H₂O₂ and iron and is capable of destroying a wide range of organic contaminants. In this laboratory study, Fenton's reaction was observed in aqueous and soil slurry systems using trichloroethylene (TCE) as the target contaminant, with the goal of maximizing TCE degradation while minimizing H₂O₂ degradation. Fenton's reaction triggers a complex matrix of reactions involving ·OH, H₂O₂, iron, TCE, and soil organics. In soil slurries with a high fraction of organic carbon (f_OC), iron tends to sorb to soil organics and/or particles. In aqueous systems the optimal ratio of H₂O₂:Fe²⁺:TCE to degrade TCE in a timely fashion, minimize costs, and minimize H₂O₂ degradation is 300?mg/L: 25?mg/L: 60?mg/L (19:1:1 molar ratio), while soil slurries with a f_OC up to approximately 1% and a soil:water ratio of 1:5 (weight ratio) require about ten times the amount of H₂O₂, the optimal ratio being 3000?mg/L: 5?mg/L: 60?mg/L (190:0.2:1 molar ratio). TCE degradation rates were observed to decrease in soil slurries with higher f_OC because of competition by soil organic matter, which appears to act as a sink for ·OH. H₂O₂ degradation rates tended to increase in soil slurries with higher f_OC, most likely due to increased demand for ·OH by soil organics, increased available iron and other oxidation processes.     

小球藻病毒基因调控序列在大肠杆菌和真核藻中的调控活性研究

以小球藻病毒腺嘌呤甲基转移酶基因(amt)和主要外壳蛋白VP54基因的5′上游调控序列构建大肠杆菌和真核藻转化载体。以P_RP_L及CaMV35S启动子为阳性对照,研究了小球藻病毒来源的两种调控序列在E.coli和真核藻细胞中的启动活性。发现P_AMT在4种E.coli菌株中都具有极强的调控活性,启动Luc基因表达而产生的酶活性高于P_RP_L 50～400倍;P_VP54在DH5α中也具有较强的启动活性。同时PAMT在两种小球藻中启动GUS基因瞬时表达的能力也明显高于CaMV35S启动子,表明它们有可能在真核藻类遗传转化中具有很好的应用前景。     

盐旱胁迫对3种新疆造林树木种子萌发的影响

以3种新疆常见造林树木白榆、大叶白蜡、梭梭种子为材料,通过测定不同土壤含盐量(0.1%～0.5%)和含水量(9%～23%)复合胁迫作用下种子的发芽率、发芽指数,以探讨3种种子萌发对盐分和干旱胁迫的响应特征.结果显示:(1)梭梭种子对盐和干旱胁迫的适应范围最广,大叶白蜡次之,然后是白榆.(2)在土壤含盐量不超过0.2%情况下,土壤含水量达到17%以后,3种树木种子发芽率都在50%以上;而当土壤含盐量为0.3%时,3树种种子仅在23%含水量下有较高发芽率;0.4%土壤含盐量条件下,只在含水量为23%时,梭梭、大叶白蜡萌发率能达到50%以上;0.5%土壤含盐量时,仅梭梭种子萌发率达到50%.(3)白榆、大叶白蜡、梭梭的耐旱临界值分别为15.1%、10.7%、9.6%,耐盐临界值分别为0.34%、0.41%、1.03%.研究表明,盐旱胁迫存在明显互作效应,盐旱复合胁迫对种子萌发的危害性明显强于单一的盐胁迫或旱胁迫.     

Leaf Litter as a Source of Dissolved Organic Carbon in Streams

Dissolved organic carbon (DOC) is an abundant form of organic matter in stream ecosystems. Most research has focused on the watershed as the source of DOC in streams, but DOC also comes from leaching of organic matter stored in the stream channel. We used a whole-ecosystem experimental approach to assess the significance of leaching of organic matter in the channel as a source of DOC in a headwater stream. Inputs of leaf litter were excluded from a forested Appalachian headwater stream for 3 years. Stream-water concentration, export, and instream generation of DOC were reduced in the litter-excluded stream as compared with a nearby untreated reference stream. The proportion of high molecular weight (HMW) DOC (more than 10,000 daltons) in stream water was not altered by litter exclusion. Mean DOC concentration in stream water was directly related to benthic leaf-litter standing stock. Instream generation of DOC from leaf litter stored in the stream channel contributes approximately 30% of daily DOC exports in this forested headwater stream. This source of DOC is greatest during autumn and winter and least during spring and summer. It is higher during increasing discharge than during base flow. We conclude that elimination of litter inputs from a forested headwater stream has altered the biogeochemistry of DOC in this ecosystem. Received 2 September 1997; accepted 27 January 1998.     

The Use of Probabilistic Risk Assessment in Establishing Drinking Water Quality Objectives

There has been a trend in recent years toward the use of probabilistic methods for the analysis of uncertainty and variability in risk assessment. By developing a plausible distribution of risk, it is possible to obtain a more complete characterization of risk than is provided by either best estimates or upper limits. We describe in this paper a general framework for evaluating uncertainty and variability in risk estimation and outline how this framework can be used in the establishment of drinking water quality objectives. In addition to characterizing uncertainty and variability in risk, this framework also facilitates the identification of specific factors that contribute most to uncertainty and variability. The application of these probabilistic risk assessment methods is illustrated using tetrachloroethylene and trihalomethanes as examples.     

Control of the flux to arginine in Neurospora crassa: de-repression of the last three enzymes of the arginine pathway

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold.By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.     

Tellurium-Induced Alterations in 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue-Specific Regulation of Cholesterol Synthesis

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.